EPA/600/R-93/217
November 1993
Research Perspectives for
Dolphin Mortalities in
North America
by
Dr. Gregory Bossart
Miami Seaquarium, Miami
Miami, Florida
Dr. David Busbee
Texas A&M University
College Station, Texas
Dr. Caret Lahvis
University of Maryland
School of Medicine
Baltimore, Maryland
Dr. Graham A.J. Worthy
Texas A&M University
Galveston, Texas
Prepared by
National Oceanic and Atmospheric Administration
National Marine Fisheries Service
Office of Protected Resources
1335 East-West Highway
Silver Spring, Maryland 20910
and
U.S. Environmental Protection Agency
Center for Marine & Estuarine Disease Research
Environmental Research Laboratory
Office of Research and Development
Gulf Breeze, Florida 32561
Printed on Recycled Paper
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DISCLAIMER
Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
11
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PREFACE
The Environmental Protection Agency's Center for Marine & Estuarine Disease Research
(CMED) was created in 1990 when the First Gulf Breeze Symposium on Marine and Estuarine
Disease Research was convened. The symposium emphasized the need for scientific coordination
and sound, comprehensive programs with consistent funding for aquatic disease research.
Eighteen presentations during the symposium were published in a book (Pathobiology of Marine
and Estuarine Organisms), edited by Drs. J.A. Couch and J.W. Fournie (CRC Press Advances
in Fisheries Science Series, 1993).
Since then, CMED has relied on internal, extramural and collaborative projects to address
priority areas of aquatic animal disease. In most cases, these projects involve laboratory and field
research, but CMED has also sponsored meetings and workshops to strengthen communication
and collaboration among scientists.
The Second Gulf Breeze Symposium was co-sponsored by CMED and the National Marine
Fisheries Service, Office of Protected Resources, in late 1992 to address questions of dolphin
mortalities in North America. The symposium allowed more than 40 participants to focus on
current scientific information, research strategies, and research needs related to deaths of
bottlenose dolphins Tursiops truncatus. Presentations from the second symposium and several
invited manuscripts will be featured in the publication (Dolphins: Factors in Morbidity and
Mortality) to be edited by Drs. R. Haebler (EPA) and A. Hohn (NMFS).
During the second symposium, four study groups were formed to address research questions
related to (1) pathology and disease, (2) pollution analyses and biomarkers of exposure, (3)
physiology and biomarkers of effects and (4) stranding and sampling logistics for bottlenose
dolphins. The responses and recommendations summarized by group leaders are presented in this
document.
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CONTENTS
Preface
Introduction
Pathology and Disease
Pollution of Analyses/Biomarkers of Exposure
Physiology/Biomarkers of Effects
Stranding and Sampling Logistics
111
1
2
7
14
19
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Introduction
Dolphin strandings are not recent
occurrences; they were recorded by
Aristotle at least 2300 years ago. Public
interest in dolphin and other marine
mammal strandings has intensified during
the past century and numerous theories
have been generated concerning their
causes. Recently there has been an
increase in reported marine mammal
mortality events throughout the world. A
mortality event is generally considered to
be an unusually large number of animals
dying, beach-cast or floating, over a
relatively short period of time in a
restricted geographic area. Large-scale
dolphin mortalities have included bottle-
nose dolphins (Tursiops truncatus) along
the U.S. mid-Atlantic coast in 1987-1988
and two separate mortality events in the
Gulf of Mexico in 1990 and 1992. Addi-
tionally, over 1000 dead striped dolphins
(Stenella coeruleoalbd) were recovered
from the Mediterranean Sea in 1990-1991.
Stranded dolphins may represent only a
fraction of the population affected.
Possible causes for unusual dolphin
mortalities include fishery-bycatch,
pathogens and pollution. Pollution can be
directly responsible for mortalities or can
indirectly alter behavioral or immuno-
logical responses, increasing susceptibility
to predators and infectious diseases.
Investigations to link dolphin mortality
events with potential causes must include
thorough examination of stranded dolph-
ins and consideration of several interacting
factors. Knowledge of life history data
(age, reproductive status, etc.) can be
important in interpreting clinical data.
Gross and histological pathology initiated
before deterioration of tissues can provide
the best insight to potential trauma and
disease. Microbial cultures can help to
determine whether the microorganisms
present caused the mortality, or merely
represent an opportunistic infection of a
dying individual. Tissue samples can be
analyzed and, if properly interpreted, may
indicate the type and degree of exposure
to toxicants. Physiological and immuno-
logical evaluations may also indicate
changes associated with disease or intoxi-
cation prior to death.
Post-mortem analyses must be
supported by knowledge of the normal
range of microbial, toxicant, physiological,
and immunological characteristics of living
dolphins. Due to the protection afforded
dolphins as marine mammals, such knowl-
edge can only be gained through non-
lethal sampling of dolphins in aquaria or
capture-release studies. Such studies not
only support post-mortem analyses, but
may also lead to prediction of compro-
mised conditions that may lead to mortal-
ity in free-ranging populations.
Three groups of participants at the
Dolphin Mortality Conference posed and
addressed several questions related to
investigations of disease and contaminant
exposure and their physiological effects on
dolphins. A fourth group examined the
ability of stranding and sampling programs
to respond to scientific needs.
1
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Pathology and Disease
Group Leader: Gregory Bossart
Lee'Courtney
Jack Fournie
Thomas Lipscomb
William Medway
Robin Overstreet
Esther Peters
Yvonne Schulman
Diane Sips
Michael Walsh
Remarks
Largely because of dolphin mortality
events along the mid-Atlantic coast of the
U.S. and the Gulf of Mexico, a Federal
marine mammal die-off response plan was
instituted. This plan included an "unusual
mortality" task group to investigate events
where large numbers of mortalities occur-
red in a short period of time and where
mortalities appeared unique or alarming.
Improvements have also been, made in
regional marine mammal stranding
networks. But despite organizational
progress and advancement of the general
biologic knowledge of marine mammals,
there is a paucity of data on diseases and
associated pathologic lesions in dolphins;
consequently, understanding of the causes
and mechanisms of diseases is very
limited.
In considering diseases and pathology
of free-ranging dolphins, it is important
that each mortality event be approached
as a unique incident to be accompanied by
a systematic gathering of relevant environ-
mental, biological, clinical, and pathologi-
cal data. The time-honored systematic
approach to the pathologic basis of disease
must be continually emphasized in charac-
terizing dolphin mortalities; the concept
that should be held foremost is that
pathologic investigations are studies in
clinical medicine to address altered
structure and function. This systematic
approach must include a logical and
stepwise plan of collection, documentation
and storage of antemortem clinicopatho-
logic data, must employ standardized gross
necropsy and tissue collection procedures,
and must provide for histopathologic
evaluation of freshly-fixed tissues and
subsequent studies in specialized disci-
plines such as immunology, virology, and
toxicology.
The scope of these pathologic studies
should be expanded to investigate the
mechanisms of marine mammal diseases
and to characterize distributions, origins,
morphologic changes, and resultant clini-
cal symptoms. Attention should be direct-
ed to the pathophysiologic mechanisms
leading to morphologic changes and to the
clinical implications of these cellular and
organ changes. Causes of dolphin mortal-
ities may be revealed only if diseases are
recognized as dynamic processes influ-
enced by abiotic and biotic environmental
factors, individual susceptibility, and
population interactions.
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Question 1
Do gross, histological and clinical
(hematological, serological, serum
chemical, and microbiological)
databases exist for "normal" dolphins?
Response
A need exists to widen the data base
of gross, histologic, and clinical parameters
for "normal" or "healthy" dolphins.
Presently, clinical and histological data
from normal dolphins are either inadequ-
ate or difficult to access and retrieve.
Clinical data can vary tremendously
among studies performed at oceanaria or
by stranding personnel. For example,
peripheral blood studies could range from
standard blood cell counts to in-depth
studies involving viral serologic or
immunologic parameters. Gross necropsy
quality (i.e., completeness, lesion
description, reporting protocols, etc.)
depends on the experience of the facility
prosector and histopathologic evaluation
may depend on the experience and
commitment of the pathologist reading the
tissues. Access and retrieval of these data
may be problematic since each facility has
its.own program and procedures.
Recommendations
1. A focused project should be
implemented through the Armed
Forces Institute of Pathology (AFIP)
to establish a complete histologic set
of tissues from 12 "normal" Atlantic
bottlenose dolphins (Tursiops
truncates). Code 1 (live) or Code 2
(freshly-dead) animals from
incidental takes or human interact-
ions are needed to ensure quality
specimens and a "normal" status.
This will require coordination with
regional stranding coordinators,
National Marine Fisheries Service
(NMFS) observer programs, or other
sources.
2. The AFIP would subsequently act as
a central tissue repository for
wet/paraffin-embedded tissues (for
light microscopy) and resin-
embedded tissues (for electron
microscopy) from both normal and
stranded dolphins.
3. A reference set of normal tissues
should be incorporated into a
histological atlas for this species.
4. Future reference tissues from other
dolphin species should be organized
and archived in a similar fashion.
Question 2
Are present descriptions of pathology
well-defined for infectious, non-
infectious and parasitic diseases?
Response
Investigations of disease and
mortality in captive and stranded dolphins
have resulted in an initial list of
pathogenic microorganisms, parasitic
infections, and some nutritional disorders.
However, even though primary morpho-
logic diagnoses of disease states have been
defined, the etidlogic diagnoses remain
obscure (viruses, intoxications, etc.). It is
not uncommon to describe histopathologic
lesions for which the etiology cannot be
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determined; hence, emphasis is placed on
reporting morphologic diagnoses and the
systematic classification of lesions. This is
especially true for viral infections, due in
large part to the lack of specific dolphin
cell lines generally necessary for viral
isolation. Similar problems also exist in
suspected intoxications due to lack of
baseline tissue concentration data and
specific toxic effects data in the target
species.
Recommendations
1. Provide a mechanism to advise the
NMFS on the various elements that
should be incorporated into the
marine mammal data base.
Implementation of such a data base
could be aided by a computer
network.
2. IJnitiate a multi-disciplinary approach
that would widen the data base
through correlation of clinical data
(hematology, serology, serum
chemical) with gross, histological and
microbial data. This should be
coordinated so that all regions use
similar protocols.
Question 3
Can present pathology databases help
in determining the causes of (free-
ranging) dolphin mortalities?
Response
The present data bases, if consoli-
dated into a usable format, could help in
determining the causes of mortality. .For
example, hematologic, serum analyte, and
microbiologic data already-collected and
recorded from "healthy" dolphins could be
collated from various facilities to generate
a baseline data bank. Likewise,
pathologic lesions already described for
individual cases might be categorized by
specific morphologic diagnoses to give a
better picture of disease incidence in the
study population. Stockpiled serum
samples at various oceanaria should be an
invaluable source for determining
seroprevalance of known infectious agents
(e.g., viruses, bacteria, protozoans, fungi)
in a population.
Recommendations
1. Current databases could be impro-
ed by (a) increasing the amount and
type of data collected, (b) concen-
trating on live or freshly-dead
animals, (c) incorporating concurrent
comparative studies with "healthy"
dolphins and (d) standardizing
necropsy protocols and tissue
submission forms.
2. Databases from different facilities
should be consolidated and made
more accessible to scientists.
3. Photography should be used more
often for documentation.
Question 4
Are captive dolphin studies useful for
understanding mortalities of free-
ranging dolphins?
Response
Captive dolphins are a useful resource
for understanding mortalities in free-
ranging dolphins. Captive dolphins can
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provide information essential to develop
techniques and understand mechanisms of
disease even though etiologies may differ
from free-ranging dolphins. It is believed
that abundant information is available
(e.g., hematologic, serum chemical), but
not in a standardized format or a central
location. Sampling procedures used on
captive animals should be adaptable for
use on free-ranging dolphins.
Recommendations
1. Coordinate/standardize protocols
and data collection with cooperating
marine mammal facilities.
2. Establish blood serum banks for
captive and stranded dolphins; this is
a valuable resource for insight to
mortalities of free-ranging dolphins.
3. Support collaborations with marine
mammal facilities to use captive
dolphins for developing new
sampling techniques and procedures.
4. The condition of free-ranging
dolphins could be better evaluated if
NMFS observers on fishing vessels
were trained to collect samples from
dolphins caught in nets.
Question 5
Are logistical procedures (stranding
networks, sampling protocols, tissue
banks, etc.) adequate and realistic for
successful pathologic evaluations?
Response
There appears to be a positive evo-
lution toward optimizing data collection
and improving logistical procedures for
pathologic evaluations. This is partly due
to the development of a national die-off
response plan,improvement of the regional
stranding networks, and development of a
marine mammal tissue bank to evaluate
anthropogenic toxins and biotoxins. For
example, standard protocols have been or
are being developed for performing gross
necropsies and collecting tissue samples
for histopathologic, microbiologic, and
toxicologic evaluations. Also, pathologists
are being consulted and are participating
in necropsies more frequently.
To identify and investigate future
mortality events more effectively, studies
must be designed and conducted to
advance the knowledge of the etiology of
dolphin disease (i.e., infective pathogenic
agents as well as metabolic, genetic, and
neoplastic disease). These advances will
also require use of code 1 or 2 animals
and more complex sampling protocols
necessary for proper specimen evaluation.
The concept of specimen retrieval
from tissue banks is a potentially valuable
tool for broadening the pathologic data
base. Currently, tissue banks for
histopathologic or serologic evaluation are
either not developed or poorly organized
for retrieval purposes.
Recommendations
1. Code 1 or 2 dolphins should be the
highest priority and the scientific
investigation should follow a tiered
progression that incorporates the
classical characterization of disease
states: (a) clinical data collection,
e.g., hematologic or serum analysis,
(b) complete gross and histopatho-
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logic evaluation, and (c) any evaluat-
ions for specialized disciplines (e.g.,
virology, toxicology, immunology).
2. When possible, veterinarians or other
trained personnel should conduct
necropsies for optimal quality of
samples and reporting. This will
require participation by laboratories
and veterinarians hi all regions that
in contact with the stranding net-
work.
3. Databases should be expanded
through establishment and/or
improvement of tissue banks that are
accessible to scientists.
Question 6
How should epizootics, especially
those related to unusually high
dolphin mortalities, be tracked and
investigated?
Response
Response to unusual dolphin mortality
events is now reasonably well coordinated
in the sense of a multi-disciplinary
scientific response. For the response to be
successful, the NMFS must be notified of
the mortality event in a timely manner.
Live "healthy" and "sick" dolphins should
be sampled during an epizootic. This
approach was applied recently after of the
1992 mortality in Texas.
Success of the 1992 Texas capture set
a precedent in live dolphin captures,
involving the live-capture and release of
36 bottlenose dolphins (Tursiops truncatus)
from the region where mortality occurred.
During the brief holding period, each
dolphin was given a complete physical
examination. Blood samples were
withdrawn and later evaluated for
standard hematologic and serum analyte
determinations, as well as specialized
serologic and imrnunologic studies.
Sampling of free-ranging wildlife
populations is frequently used with
terrestrial wildlife species in a disease
outbreak to monitor antemortem clinical
data and obtain tissue samples not
influenced by postmortem autolysis. This
methodology can provide important data,
including disease etiology, disease
pathogenesis, disease incidence, and other
epidemiologic characteristics. This is
perhaps the first time capture/release
techniques was used in U.S. dolphin
mortality studies.
Recommendations
1. Time is a critical factor in an
epizootic. Although live captures
have been successfully implemented
in an epizootic (see above), live
capture/sampling techniques and
permit mechanisms must be assessed
continually for rapid response.
2. Physiological measurements of live
dolphins should be improved and be
comparable to measurements of
stranded or moribund animals.
3. Euthanasia of "healthy" dolphins
should be a last resort; current
NMFS requirements for special
authorization should be retained.
Euthanasia should be performed by
a veterinarian only when a
consensus of experts agrees that all
options have been exhausted.
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Pollution Analyses/Biomarkers of Exposure
Group Leader: David Busbee
Paul Becker
Larry Flood
Doug Kuehl
Remarks
The organohalogens (OH), aromatic
hydrocarbons (AH), polynuclear aromatic
hydrocarbons (PAH), and toxic metals
include several broad groups of chemicals
extensively used in industrial processes or
arising from the recombination of fossil
fuel wastes. These compounds are widely
disseminated as both terrestrial and
aquatic pollutants and their environmental
distribution has been clearly identified by
sedimentation analysis to coincide with the
worldwide growth and development of the
chemical industry. The stability and
lipophilicity of the organic chemicals and
some metals (e.g., mercury) result in their
tendency to be concentrated in fatty
tissues, leading to their bioaccumulation
up the food chain. Detectable concen-
trations of a variety of the OH/AH/PAH
and metals have been identified in the
tissues of both terrestrial and aquatic
animals. Public awareness, of and concern
for, the potentially adverse human health
and environmental effects resulting from
exposure to these chemicals has increased
significantly with reports of agricultural
and occupational exposures, a series of
PCB poisonings in the Orient leading to
the onset of immunodeficiency diseases,
Keith Miles
Geoff Scott
John Stein
and the sequelae of immunotoxic effects
of the PBB cattle feed contamination
disaster in Michigan.
Shallow bays and estuaries in the
vicinity of chemical plant discharge/
shipping sites and large urban areas are
among the most heavily contaminated
coastal regions and in some instances have
been shown to constitute a potential threat
to the safety and well-being of humans
and animals in close proximity to the
polluted sites. Animals taken directly
from contaminated sites may have high
tissue concentrations of lipophilic
chemicals, as evidenced by PCB
concentrations as high as 600 ppm in fat
of beluga whales from the St. Lawrence
estuary. Marine mammals sampled in
virtually all oceans, including pristine
waters generally considered to be
uncontaminated, have alarmingly high, but
perhaps not acutely toxic, tissue residues
of organic and inorganic chemical
contaminants.
Although physiological consequences
of chronic cellular exposure to subacute
levels of chemicals and resultant
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bioaccumulation of contaminants relevant
to metabolism are unknown, elevated PCB
residues in tissues may initiate a state of
immunosuppression, while elevated levels
of some metals may contribute to neuro-
logical damage. Contaminant-initiated
immunosuppression may have contributed
to recent epizootic deaths of dolphins in
U.S. and the western Mediterranean,
where PCB levels as high as 3,000 ppm
were detected.
Enormous volumes of data collected
from almost all of the oceans show that
organohalogen and, to a lesser degree,
aromatic hydrocarbon residues are ele-
vated in tissues of marine mammals. The
fact that animals are exposed to these
compounds is clear. The effects of such
exposure and the synergism between
classes of pollutants that may be found as
tissue residues is unknown. Organo-
halogen pollutants may interact with a
series of cellular receptors to (a) induce
the synthesis of cytochrome P450 enzymes,
(b) alter immune system function, (c)
cause dermal lesions, (d) change hepatic
function, (e) modify endocrine profiles, (f)
decrease reproductive capacity, and (g)
initiate embryotoxic phenomena. Recent
data show that elevated levels of organo-
halogens such as PCBs and TCDD may
predispose animals to increased DNA
damage from subsequent exposure to
hydrocarbon contaminants by increasing
the induction of enzyme systems that
biotransform procarcinogenic compounds.
While data from these studies support the
proposal that hydrocarbon induction of
cytochrome P450 enzyme systems may
serve as an early warning indicator for
organic pollutants, the existence of
pollutants and the exposure of animals to
these pollutants is not the question.
Rather, the question is whether a suite of
biomarkers (physiological effects) can be
developed to monitor the health of
animals relative to their exposure states.
Examinations of subacute vs. acute
levels of toxicity of organic and inorganic
chemicals in mammals must consider the
stress-induced mobilization of chemicals
bioaccumulated in fat reserves and
increased concentrations of lipophilic
xenobiotic compounds in milk that would
be ingested by nursing offspring.
Question 1
What dolphin populations should be
evaluated to best understand pollution
exposure?
Response
Due to existing ancillary information,
the large volume of information available,
and the infrastructure for continued
collection of data, the Sarasota dolphin
population is a leading choice for study.
The Sarasota population resides in a
unique area with both industrially polluted
and reasonably clean regions; it has been
continuously monitored and studied for
many years.
At least one other large, well-
established and relatively stable popu-
lation should be studied. The Matagorda
Bay population could provide comparative
information, even though it is not yet well
characterized. Matagorda Bay receives
runoff from a highly cultivated area of
Texas via the Colorado, Navidad and
Lavaca Rivers, carrying a periodic heavy
load of insecticides and herbicides to
expose resident dolphins and their prey.
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The Matagorda Bay area also receives
industrially-derived pollutants from
chemical and petrochemical plants.
Identification and characterization efforts
have been initiated on this population.
A population of bottlenose dolphins
on the East Coast of Florida (Indian River
Lagoon) has been monitored since 1974,
with three mark and release studies
performed. This population should also
be considered for future study.
There was concern that a reference
population in an unpolluted area should
be identified. However, participants
agreed that such a population was not
known.
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Recommendations
1. Efforts should be made to .continue
comprehensive studies on the
Sarasota dolphin population while
attempting to characterize the
Matagorda Bay population for
comparative purposes.
2. A dolphin population from an
unpolluted site should be sought for
similar study. However, it is possible
that the Sarasota population may be
from a sufficiently clean environment
to be used as a reference site for
comparison with Matagorda Bay
dolphins.
Question 2
What dolphin tissues are best
sampled for analysis of xenobiotics
residues?
Response , s
Blubber was the first choice for
xenobiotic tissue analysis. A baseline
characterization of analytes in blubber
should be completed for comparison with
other tissues. However, sampling of
blubber could provide highly variable
aromatic hydrocarbon, organohalogen and
polycyclic aromatic hydrocarbon
(AH/OH/PAH) data unless the moisture
content and lipid profile of the blubber
are known. For live animals, the order of
tissue of preference is (a) blubber, (b)
milk in lactating females, (c) blood serum,
and (d) cellular blood components.
Necropsy samples of choice were (a)
blubber, (b) kidney, (c) liver, and (d) a
hematopoietic tissue, in order of
preference. It is noted that the team of
scientists for the National Marine
Mammal Tissue Bank, preferred liver over
kidney for contaminant analysis.
Recommendations
1. Establish baseline levels of analytes
in blubber,' milk of lactating females,
blood serum and cellular blood
components.
2. Develop methods to normalize
blubber residues that take into
consideration moisture content and
lipid profiles.
3. Life history information needs to be
collected whenever analyses of this
nature are performed; results may be
influenced by age, gender,
reproductive status, nutritional
status, etc.
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Question 3
Which biological or chemical analyses
are most relevant for determining
xenobiotic exposures?
Response
There are no unconditional methods to
quantify dolphin exposure to xenobiotics.
At this time, no publications indicated a
direct correlation between environmental
exposure and tissue residue levels, much
less a physiological change in the animals.
Residue analysis needs to be as inclusive
as possible, even though correlations
betwe'en tissue residues and mortalities
are limited to relatively few examples.
These include (a) fish in Lake Michigan,
(b) cormorants in Green Bay, and (c)
pinnipeds in the Baltic. Analyses of
organohalogen-induced cell function
changes might be best achieved in vitro.
There are limited studies of bio-
chemical markers of contaminant exposure
in dolphins. Early studies have shown
cytochrome P450 to be detectable in
dolphins, but no information is available
to indicate the relative degree of similarity
among dolphin cytochrome P450 systems
and other species. No existing data show
correlations between xenobiotic load in
dolphin tissues and the induction of
cytochrome P450s. Preliminary data
(unpublished, Busbee and Carvan)
indicate that monoclonal antibodies
against rat CYP1A1, which is induced by
exposure to OH and PAH, do not react
against dolphin cytochrome P450s, but that
cDNA probes developed from rats
hybridize with cetacean CYP1A1 mRNA.
Recommendations
1. Standardize the various
analytical/chemical methods using
reference standards obtained from
the National institute of Standards
and Technology.
2. Correlate the state of dolphin health
with tissue residue levels of OH,
AH, and PAH. The best measure of
in vivo health may be immune
system function.
3. Characterize the cytochrome P450
system in dolphins to better
understand its capacity to
biotransform contaminants: This
would entail (a) development of in
vitro cell lines, (b) development of
monoclonal antibodies specific to
dolphin cytochrome P450 isozymes,
(c) development of dolphin-specific
cDNA probes (or determination that
rat cDNA probes hybridize with
dolphin DNA or mRNA sequences),
and (d) application of molecular
detection techniques (e.g., cDNA
probes, antisense riboprobes).
4. In addition to cytochrome P450
induction studies, resident DNA
adduct levels and glutathione
depletion should be measured and
correlated with xenobiotic exposure.
Question 4
What are the preferred methods to
ensure analytical quality?
10
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Response
Question 5
Sampling of tissues from live-
capture and necropsied animals has been
addressed (Question #2). For each tissue
(blubber, milk, blood serum and cells),
samples should be analyzed for organo-
halogens by gas chromatography of organ-
ic solvent-extracted analytes. Inadvertent
bias in residue analyses should be rigor-
ously avoided. To that end, both gel
permeation and silica gel column sepa-
rations should be used to obtain both
hydrophilic and hydrophobic contami-
nants. Whatever methods of
OH/AH/PAH residue analysis are used,
the investigator needs to know the wet,
dry, and lipid weight of the sample. This
need stems from the fact that both the
water and fat content and type will differ
in blubber samples dependent on the
health and relative hydration of the
animal. Methods used for residue analysis
must be validated through reference stand-
ards for OH/AH/PAH to ensure quality
and comparability of residue quantitation.
Recommendations
1. Analysis of organohalogen residues
in marine mammal tissues has been
completed with techniques that
provide excellent reproducibility. All
analyses must include reference
materials to assess the accuracy of
the measurements and to allow
comparisons among different studies.
Marine mammal tissues are currently
being prepared as control materials.
2. Analyses for organohalogens should
allow for correlation among samples
with different wet/dry/lipid content.
What are the known effects of xeno-
biotics and heavy metals on other
animals and can this knowledge be
extrapolated to dolphins?
Response
The organohalogens (PCBs, TCDD,
TCDF, etc.) are known to cause immune
system dysfunction in rats, man, monkeys,
cattle, and avian species, and the assump-
tion is that they cause the same responses
in marine mammals. There are some sig-
nificant problems in interpretation of data
derived from animals exposed to complex
mixtures of pesticides, insecticides and
herbicides. Some of the compounds show-
ing up as environmental contaminants are
known to bind to the Ah receptor (induct-
ion of cytochrome P450s), whereas others
are known to act as xenobiotic steroids
and apparently bind to the cortisone type
and/or estrogen type receptors. Wasting
syndrome, weight loss, immune dysfunc-
tion (including thymic atrophy),
porphyrias, chloracne and dermal lesions
and fetotoxicity are all associated with
exposure to pollutant chemicals that bind
to Ah and steroid receptors. The precise
biochemical and physiological mechanisms
by which ligand binding to receptors
initiates toxic responses are not known.
Some of the OH, AH and PAH have
estrogenic activity and some have anti-
estrogenic activity. Care must be taken in
attempting to interpret data for animals
exposed to mixtures of contaminants.
Toxic metals analysis in cetaceans,
typically whales of a variety of species, has
not led to an overt correlation of toxicity,
11
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neoplasia, or death of animals with high
residue levels. It is not known whether
metal contamination is synergistic to
OH/AH/PAH toxicity.
Recommendations
1. Different potential surrogate species
should be evaluated in relation to
their responses to OH/AH/PAH
exposure. Mink and pinnipeds are
known to react adversely to OH, but
it is not known if these animals are
appropriate surrogates for dolphins.
2. Research on surrogate species should
include investigation of interactions
between OH/AH/PAH and heavy
metals.
Question 6
What in vitro methods are being
applied or could be applied to dolphin
mortality research?
Response
There is an immediate need for
studies on dolphin cells to determine
baseline data on cytochrome P450
induction and activity, DNA adduct levels
correlated with OH/AH/PAH tissue
residues, and glutathione depletion or
enhancement analyses. Such studies
would be best suited to a fetal derived cell
line, preferably of hepatocytes or
keratinocytes. A hepatocarcinoma line
would be less useful but more likely to be
obtained and more practical than a fetal
hepatocyte line. Only one of these cell
lines, a fetal keratinocyte line, is currently
available for study (originated by Busbee
and Carvan, Carvan Dolphin Kidney,
CDK). A series of immortalized CDK
derivatives have been initiated using
pSVS.neo, an SV40-derived plasmid that
expresses large T-cell antigen as the only
virally encoded protein in the host cell.
Additional immortal cell lines can be
initiated from tumor tissue; however,
tumor tissue from dolphins is not regularly
available.
Recommendations
1. Continue development of in vitro
fetal cell lines and/or immortalized
cell lines for use in toxicological
analyses. Cell lines of hepatocytes
and epithelioid cells should be a high
priority.
2. Initiate methods development for in
vitro determination of cytochrome
P450 induction, for evaluation of in
situ DNA adduct levels in stranded
animals or in capture-release
dolphins, and for assessing in situ
glutathione content.
3. Compare in vitro and in situ
techniques, especially with respect to
correlations with exposure to DNA
damaging agents.
4. Institute a strategy for recovery of
biopsied, rather than necropsied,
dolphin tumor tissue from which
immortal cell lines might be
initiated.
Question 7
Can dolphins be used as a "pollution
biomarker" with respect to potential
environmental health hazards?
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Response
Established biomarkers of pollutant
exposures that are pre-clinical or pre-acute
should be developed. Rather than trying
to establish the cause of a complex series
of morphological and physiological
changes in a morbid animal, there is a
need for identification of small, pre-
morbid changes associated with exposure
to organic and inorganic contaminants.
Recommendations
1. In vitro studies should be pursued to
develop the means to utilize dolphins
as biomarkers of pollution,,
2. Contaminant residue analyses of
tissues should be further evaluated
with regard to physiological factors (e.g.,
lipid content and lipid profiles) and life
history factors (e.g., age, gender, maturity,
reproductive status) to improve the
assessment of dolphin exposure to organic
and inorganic contaminants.
3. In vitro analyses should be pursued
to* determine the cellular responses
of dolphins to OH/AH/PAH
exposure.
4. Develop a reliable mechanism (such
as HLA typing for humans or PCR
and DNA fingerprinting) for
determining the genetic identity of
animals.
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Physiology/Biornarkers of Effects
Group Leader: Garet Lahvis
Michael Carvan
David Ferrick
Doretha Foushee
John McCarthy
Theo Colborn
William Fisher
Paul Klein
Jeffrey Stott
Remarks
It is possible to determine causes of
dolphin die-offs only if the biological
mechanisms that link cause and effect are
understood. By conducting non-invasive
evaluations of healthy dolphins and using
the vast amount of data generated from
studies of laboratory animals, causes of
mortality can be better understood. This
scientific approach requires expanded
capture/ release programs to characterize
the biology of living dolphins. Further,
this approach requires public and scientific
consensus that studies of laboratory
models are adequate alternatives to
invasive studies of dolphins to determine
effects of stressors (e.g., pollutants,
brevetoxins, viruses).
Three levels of investigation can link
dolphin mortality with potential causes.
Pathological studies define the final
disease state of dead dolphins. Physio-
logical dysfunctions responsible for the
diseased state present a second tier of
investigation. Competent pathological
studies can provide insight into this tier.
For example, studies of the pathology
ofstranded dolphins have indicated that
the diseased animals were heavily infected
with opportunistic bacterial species,
indicative of an underlying immune
(physiological) dysfunction. A third tier of
investigation involves determination of
causes of the dysfunction witnessed at the
physiological level. Possible causative
agent(s) that would be investigated at the
third level include pollutants, toxins, and
viruses.
Question 1
Is there reasonable understanding of
the biology of healthy dolphins at the
cellular and molecular levels of
organization?
Response
Very little research on dolphin
physiology has incorporated modern
techniques of cell and molecular biology.
Since dolphins (and all marine mammals)
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have become high-profile species, it is not
possible to sacrifice animals for study.
Consequently, research must be performed
on stranded (morbid or dead) dolphins or
on live dolphins using non-invasive
techniques in capture-release programs or
oceanaria. The value of studying morbid
or dead animals is limited, since most if
not all of the normal biological functions
of these animals is minimal or non-
existent. The understanding of most of
the cellular biology of these animals is
derived solely from capture-release
specimens. Interpretation of the data
gathered from such studies should also
consider information gained from studies
of non-marine laboratory mammals.
Recommendations
1. Strong support should be given to
capture-release programs because
these are the only source of
relatively healthy free-swimming
individuals for study.
2. Studies on captive dolphins should
be initiated where non-invasive
measurements are monitored for
variations related to season,
reproduction, feeding, and other
controllable or measurable factors.
3. Extrapolation of data from non-
marine laboratory mammals should
be emphasized in toxicological
studies, since dose-response studies
cannot be conducted on dolphins.
4. Training of NMFS observers on fish-
ing vessels to collect samples for
cellular and molecular study could
augment information collected from live
healthy animals.
Question 2
Which are the most important
physiological systems that could be
linked to dolphin mortalities?
Response
Many physiological systems are
important to understanding stress and
mortality in bottlenose dolphins. The
following were considered most sensitive
to environmental stressors:
Reproduction
Neurology and Behavior
Endocrine physiology
Immunology
Metabolism (bioenergetics, nutrition)
Renal physiology
Gastro-intestinal physiology
These systems have been shown to
be susceptible to stressors, such as
pollutants, in studies of other mammal
species. Polychlorinated biphenyls
(PGBs), for example, have been shown to
impair reproduction, behavior, endocrine
physiology, and immune function in rats.
Renal function can be severely impaired
when rodents are exposed to. methyl
mercury. Lead has dramatic effects on
behavior. Gastro-intestinal function can
be demonstrably perturbed by entero-
pathic bacterial species. Given the
constraints imposed by non-invasive
studies of dolphins and the high
probability that immune dysfunction plays
some role in unusual dolphin mortality
15
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events, understanding the immune system
and using it as an indicator of health
should be a high priority.
Recommendations
1. Develop and standardize new non-
invasive techniques to measure
elements of each of the seven
disciplines (above) considered most
sensitive to environmental stressors.
2. Pursue a better understanding of
immunological functions and
dysfunctions of dolphins.
Question 3
For the most critical physiological
systems, what technical hurdles limit
their use in determining causes of
dolphin mortalities?
Response
Most technical hurdles to
investigating critical physiological systems
stem from the necessity of obtaining
samples using non-invasive techniques.
Several non-invasive techniques are
currently available or under development:
Reproduction: genital size via
ultrasound; sperm viability from
sperm capture techniques; blood
hormone levels.
Neurology/Behavior: electro-
encephalograms with dermal patches;
neurotransmitters in serum;
behavioral observations using
ethograms.
Endocrine physiology: endocrine
biochemicals from blood serum.
Immunology: B and T lymphocyte
proliferation; cytotoxic T cell killer
assay; antibody production;
lymphocyte phenotype profiles;
macrophage function.
Metabolism: lipid levels, retinoic
acid, porphyria and fluid electrolytes
from serum; DNA adducts from
whole blood; blubber thickness using
ultrasound.
Renal physiology: standard assays
(BUN, creatinine) from blood and
urine.
Gastro-intestinal physiology:
digestive/absorptive efficiencies from
the stool.
Recommendations
1. Continue the development and
application of non-invasive
techniques toward understanding
dolphin immunological systems.
2. The continued development of non-
invasive techniques for physiological
measurements requires a coinciding
progression in sampling methodology
and quality assurance; quality
methods should be generated and
standardized for each new
physiological measurement.
3. Liquid nitrogen storage of live
samples should be included as an
essential element of archiving, as it
allows for supplementary analysis of
cellular function.
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Question 4
Recommendations
To what extent can we predict dolphin
biology and responses to stress from
existing laboratory models?.
Response
Information derived from other bio-
logical systems, such as rodent models,
should at least be used to prioritize areas
of research. In Question 2, specific tissues
were identified for study based on the
knowledge that such tissues in other
species were highly sensitive to certain
xenobiotics. Additionally, hypotheses can
be forwarded from existing laboratory
models. For example, the adverse effects
of 2,3,7,8-TCDD (dioxin) on immune
function in rodents has been extensively
documented. Therefore, similar studies on
dolphins could be initiated to determine
whether parallels exist. A prudent study
design would involve measurement of
immunological endpoints in dolphins
which are the most sensitive endpoints in
rodent laboratory models.
It is clear that while dolphins may be
similar to rodent models or humans in
general biological terms, reagents and
assays need to be developed to accommo-
date inherent species-specific differences.
For example, most mammalian proteins
retain highly conserved cross-species
functions, but they can exhibit significant
epitopic variability. It is possible then, to
use rodent data to predict the function of
various dolphin proteins but ultimately
species-specific monoclonal antibodies will
be needed to identify them.
1. Rodent models should be
emphasized as laboratory surrogates
for prioritizing and guiding dolphin
research; rodents are
phylogenetically related and an
enormous scientific foundation exists
at the cellular and molecular levels
for both normal and compromised
animals.
2. Reagents (monoclonal antibodies)
specific to dolphins must be
developed for application to relevant
investigations.
Question 5
Is there a research strategy that would
enhance the use of cellular and
molecular endpoints in determining
the cause(s) of mortalities?
Response
A good understanding of dolphin
physiology at the cellular and molecular
level will require sampling of healthy, or
living individuals. The simplest strategy is
to relate xenobiotic or stressor dose with
physiological dysfunction through animal
sacrifice and analysis, i.e., establish a
typical laboratory dose-response relation-
ship. But since dolphins are a protected
species and cannot be sacrificed, less
direct means must be used to obtain the
same information.
This is a situation similar to human
health concerns. For human health issues,
17
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medical science uses two major pathways
to establish information on healthy and
compromised individuals: laboratory
models (surrogate species, in vitro cell
culture), and non-invasive physiological
measurements. The same must be follow-
ed for dolphins, as has been described in
response to other questions above.
However, given the rapid decreases
in dolphin populations around the world,
the option of animal sacrifice may become
more attractive as determination of cause
becomes more pressing. The public and
scientific community would like to avoid
sacrifice of individuals, but when survival
of an entire population is at stake, indirect
methods may be insufficient.
Recommendations
1. Provide long-term support for
capture-release programs to establish
normal ranges of physiological
values; the sampling font approach
should be epidemiological rather
than opportunistic to insure
statistically relevant data.
2. Develop in vitro cell culture
techniques using dolphin tissue to
pursue xenobiotic dose-response
relationships.
3. Investigate similarities and differ-
ences of surrogate species, including
other marine mammals and rodents.
4. Establish criteria for limited sacrifice
of individuals for scientific study.
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Stranding and Sampling Logistics
Group Leader: Graham AJ. Worthy
Larry Hansen
Daniel Odell
Dean Wilkinson
Remarks
This working group examined Igistics
of stranding events and sampling required
for a proper scientific response. This
included consideration of the limitations
of current stranding networks consisting
primarily of lay volunteers. This working
group addressed questions pertaining to
sampling protocols and data forms,
administrative channels, sampling
techniques and volunteer and scientific
response to strandings. A summary of
recommendations follows:
1. Implement existing protocols and
make any organizational changes that
are needed for implementation.
2, Actively seek new participation by
laboratories, universities, and
government agencies to expand the
capabilities of some regions and to
initiate operations in others.,
3. Pursue and develop alternate funding
sources to supply networks.
4. Initiate a philosophical change from
a "volunteer" to a more "professional"
network and more active input of
NMFS laboratories in terms of time
and resources.
Aleta Hohn
Randall Wells
5. Develop a set of national priorities
for study in order to concentrate
efforts on certain questions such as
the development of cell culture lines
and standard histological sets or
obtaining baseline data on the
natural history of populations.
Question 1
How might current sampling protocols
and data forms be improved to
increase data collection and improve
scientific response?
Response
In terms of available protocols, the
recent release of a training handbook by
Dr. S. J. Geraci and V. Lounesburg
("Marine Mammals Ashore," Texas A&M
Sea Grant Publication) and the forensic
manual being prepared by Dr. S. Galloway
will more than adequately cover pro-
cedures required for a scientific response.
Improvement is needed more in use of the
protocols than the protocols themselves.
An upgrade in the level of expertise of
personnel in stranding networks required
for proper implementation and quality
assurance. The video produced by the
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Southeastern U.S. Marine Mammal
Stranding Network Video Guide to Record-
ing Marine Mammal Stranding Data will
aid immensely in training, but covers only
measurements and data required for the
basic stranding report and does not detail
forensic procedures. Current NMFS
scientific requirements are mirrored in
existing protocols but expectations should
be modified depending on the condition
code of the stranded animal; maximal
information can be derived from code 1
and 2 animals. These animals should be
transported to a facility staffed by well-
trained personnel. Quality control for all
samples rests solely with individual
researchers who request samples. They
alone are responsible for training network
personnel. Scientific needs will change
over time; the system should be flexible to
respond quickly to those needs.
Recommendations
1. Provide training to upgrade expertise
in stranding networks.
2. Attract new laboratories and agen-
cies into stranding network organi-
zations to expand the capacity and
upgrade scientific response.
Question 2
Can administrative channels be alter-
ed to foster a greater research effort?
Response
The changing focus toward better-
trained personnel may require initiation of
Letters of Authorization to denote differ-
ent levels of training. Network personnel
must agree to higher expectations
regarding data and tissue collections or be
denied a Letter of Authorization. A
Chain of Custody form is not desired and
should not be required under routine
conditions. A Chain of Custody procedure
should be implemented in unusual
mortality events, especially if there is a
possibility of future litigation. Transport
of collected tissues is prohibited without
authorization, which is generally granted
by NMFS with few (if any) restric tions.
Current NMFS policy that allows qualified
individuals access to tissue bank samples
for research or teaching is appropriate.
Recommendations
1. Strandings should be prioritized by
condition code to maximize data
collection for code 1 and 2 animals
and re,duce effort on animals of
higher codes due to the progressive
deterioration of tissues.
2. Letters of Authorization should
reflect training of stranding network
personnel and NMFS expectations.
The more demanding procedures
required for code 1 and 2 animals
should be met by trained personnel
only.
3. A policy pertaining to proper pro-
cedures for a Chain of Custody
should be developed by the Unusual
Mortality Event Working Group of
NMFS. Simple criteria to determine
cases that would benefit from a
Chain of Custody procedure should
also be generated.
4. A policy should be developed to
require data-sharing from sample end
users (scientists) relating to the
20
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results of analyses. The data should
be included in a federal repository
and made available to other
researchers. This requirement should
be similar to the policy of the Marine
Mammal Tissue Bank.
Question 3
How is the quality of sample collection
and storage assured?
Response
The quality of sample collection and
storage is unquestionably a direct function
of proper training for stranding network
volunteers and scientific personnel. All
samples must be properly identified with
a "field number assigned by the network
followed by proper curation and/or stor-
age. Effectiveness of the procedures can
be assessed only by end users, i.e., scien-
tists who ultimately derive a product. At
this time, end users have no formal means
to evaluate sampling procedures. A feed-
back mechanism needs to be formalized to
assure quality control and also provides a
means to modify procedures.
Recommendations
1. Ensure adequate materials and
training for stranding network
volunteers to assure quality of
samples.
2. Continue efforts to improve training
materials and training programs.
3. When possible, the American Society
of Mammalogists Curatorial Guide-
lines should be applied to storage of
samples.
4. Develop mechanisms to solicit
feedback from end-users on the
quality of samples and incorporate
this information into protocol
improvements.
Question 4
Do sampling techniques provide the
range and quality of materials required
for current and emerging research
efforts?
Response
There are increasing demands being
placed on network volunteers. Research-
ers must take into account the realities of
sampling and modify or simplify protocols
to be functional under a variety of con-
ditions. They should also be realistic
about the amount of time required for
their particular procedures in light of all
of the other sampling that network
personnel may be asked to perform.
Foreknowledge of sampling priorities can
alleviate many of the sampling problems.
Recommendations
1. Initiate pilot studies to assess the
changes which take place over time
with tissue deterioration and the
impacts (if any) of this on the
usability of tissues with changing
condition code of an animal. This
could be incorporated into studies
designed to characterize "normal"
dolphins.
2. Establish priorities for sampling so
that network personnel can first
satisfy those elements of highest
priority.
21
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3. The availability of material could be
greatly increased if NMFS observers
on fishing vessels were trained in
collection of life-history information
and specimens for analysis; free-
ranging dolphins are accidentally
caught in the fishing nets, but are
otherwise healthy and could augment
understanding of the "normal"
condition.
Question 5
How can the volunteer response be
improved to meet growing scientific
and monitoring needs?
Response
The stranding networks have been
relatively successful in reporting stranded
marine mammals. There are geographical
gaps in coverage of some segments of the
U.S. coastline and these are regions where
some investment of time and effort, partic-
ularly in developing new grass-root organi-
zations of volunteers, may yield some
positive results. Improvements in existing
networks are also required as higher
quality and different types of samples are
needed for developing research areas. In
terms of dealing with mass mortality
events, the Southeast Region of NMFS is
better equipped than a few years ago due
to the development of the Task Force, the
identification of key individuals with
specialized expertise, and the placement of
emergency response kits with both NMFS
laboratories and stranding network person-
nel in all of the major stranding areas.
Recommendations
1. Organize new volunteers to cover
gaps in the network reporting of
marine mammal strandings.
2. Institute a philosophical shift in
stranding network programs that
requires a higher level of expertise
for volunteers to enable higher
quality samples for research. This
should include the addition of better
trained volunteers at high quality
laboratory facilities, increased
training for current olunteers, and
movement toward a more professi-
onal network.
3. Implement a multi-tiered response
approach to address the problem of
coverage. Such a strategy could
include key "index" areas where there
is a very efficient coverage of the
beaches, a high recovery rate of fresh
specimens and high quality facilities
and personnel to examine animals.
Other areas will have a lower grade
of response and be used to monitor
trends in stranding rates and for
possible occurrence of unusual
mortality events.
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&U.S. GOVERNMENT PRINTING OFFICE: 1994 - 5SO-001/80332
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