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REPORT ON INVESTIGATION OF 1990 GULF OF MEXICO
BOTTLENOSE DOLPHIN STRANDINGS
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Larry J. Hansen,
Investigation Coordinator
National Oceanic and Atmospheric Administration
National Marine Fisheries Service
Southeast Fisheries Science Center
75 Virginia Beach Drive
Miami, FL 33149
November 1992
Contribution: MIA-92/93-21
REPORT ON INVESTIGATION OF 1990 GULF OF MEXICO
BOTTLENOSE DOLPHIN STRANDINGS
Edited by
Lany J. Hansen,
Investigation Coordinator
National Oceanic and Atmospheric Administration
National Marine Fisheries Service
Southeast Fisheries Science Center
75 Virginia Beach Drive
Miami, FL 33149
November 1992
Contribution: MIA-92/93-21
Table of Contents
Overview 1
Introduction 10
Section I: Hansen, LJ. - Stranding Rate and Trends 15
Section n: Hansen, LJ. - Age Structure 21
Section ni: Hansen, LJ. - Population Abundance and Strandings 24
Section IV: Barros, N3. - Food Habits 29
Section V: Blaylock, RA • Environmental Factors 35
Section VI: Tester, PA. - Pfaytoplankton Distribution 44
Section VII: Staff - Sunnnary of Brevnoodn Analysis 53
Section VIE: Varanasi, U^ ILL. Tilbuiy, D.W. Brown, MM- Krahn, CA. Wigren,
R.C Clark, S. Chan • Chemical Contaminants 56
Section DC: Staff - Summary of Available Pathology Reports 88
Section X: Hansen, LJ. • Limitations and Recommendations 90
Appendices:
I: Stranded bottienose dolphins documented during January-Jime,
1990, along the U^. Gulf of Mexico coast 95
11: Bottlenose dolphin strandings fay state in the northern Gulf of
Mexico, 1982-90 101
ni: Overall sex ratios and sex ratios for animal <140cm by year for
Januaiy-June Texas bottlenose dolphins strandings 102
IV: Report on aerial surveys of bottlenose dolphin abundance
conducted in near-and ofEshore waters off the Texas coast
during 1990 103
V: Oymnodinium breve presence/absence^ and quantitative cell
coimts^ 109
VL Contract report on brevetoxin analysis 113
VII: Results of analyses for metals and chlorinated hydrocarbons,
including quality assurance 133
Vni: Available rijpirnl necropsy and histopathology reports of
bottlenose dolphins stranded in the U^ Gulf of Mexico during
Januaiy-June, 1990 157
DC: Proposal outline for Southeast Fisheries Science Center
representative system for the Marine Mammal Stranding Network . 186
X: Report on the Southeast Hsheries Science Center Marine
Manunal Stranding Network Representative System Organizational
Workshop 203
PREFACE
This document was prepared as a source for complete information on the National
Marine Fisheries Service's (NMFS) investigation of an apparent anomalous mortality event
involving bottlenose dolphins during January-June, 1990, in the coastal northern and western
U.S. Gulf of Mexico. As such, it contains numerous tables, appendices, and other
information that might not be included in a paper prepared for pubhcation. The report was
reviewed by the NMFS Task Force on Unusual Marine Mammal Mortalities; the Service
wishes to thank them for taking the time to review such a large document and for providing
many useful comments and suggestions for improving the report.
OVERVIEW
The Southeast Fisheries Science Center (SEFC) coordinated a multi-disciplinary
investigation into the cause, extent, and potential effects of higher than previously reported
numbers of bottlenose dolphin {Tursiops truncams) strandings which occurred during 1990
in the northern Gulf of Mexico. The majority of the funding for this investigation was
provided by the National Marine Fisheries Service Office of Protected Resources. The
investigation of the Gulf strandings was organized into seven main themes. Four of the
themes were concerned with population biology and included: 1) stranding rate and trends;
2) population abundance and strandings; 3) age structure of stranded animals; and 4) food
habits. The other themes related to extent and causes of mortality, and included studies of:
5) environmental factors; 6) biotoxins; and 7) contaminants. Summaries of each theme are
provided in the overview; the complete reports on each theme are presented as separate
sections. The available pathology reports are summarized in Section DC
The SEFC took several steps to assist the Southeastern U.S. Stranding Network
(SEUS) in stranding reporting and recovery operations during the mortality investigation.
Communications were established with Network participants throughout the Gulf to
ascertain stranding rates, to alert participants to the ongoing event, and to request collection
of a standard set of tissue samples. SEFC personnel participated in stranding recovery
operations in Texas and Mississippi. Strandings along the Louisiana coast were documented,
and occasionally examined, by SEFC personnel onboard routine U.S. Coast Guard helicopter
flights. Strandings on the barrier islands of Mississippi were documented by SEFC pei^onnel
conducting offshore surveys for marine mammals. Small boat surveys, funded or conducted
by the SEFC, were conducted along the Texas and Alabama coasts to visually examine
bottlenose dolphin herds for the presence of "affected" animals. Authorization was obtained
to collect, hold, and examine afff-f red animals. However, no affected animals were observed
during the surveys.
The Texas Marine Mammal Stranding Network (TMMSN) was provided with
equipment and supplies to deal with the large numbers of animals stranding along the Texas
coast. Travel expenses were provided to veterinary pathologists and others assisting the
TMMSN. The SEFC also provided funds to cover costs for shipping specimens.
The Beaufort, Charleston, Galveston, Miami and Mississippi Laboratories of the
SEFC and the National Marine Fisheries Service Office of Protected Resources provided
support for the investigation. Additional support was provided by a variety of agencies,
institutions and other groups and individuals, including: the Armed Forces Institute of
Pathology; the University of Miami's Cooperative Institute for Marine and Atmospheric
Sciences; Environmental Protection Agency; Florida Department of Natural Resources;
Greenpeace; Kansas State University; Louisiana University Marine Consortium; Marine
Mammal Commission; National Oceanic and Atmospheric Administration Aircraft
Operations Center; Oak Ridge National Laboratories; Sea World Orlando; Smithsonian
Institution; Southeast U.S. Marine Mammal Stranding Network; Spring Hill College; Texas
A&M University; Texas Marine Mammal Stranding Network; US Coast Guard; and others.
This report provides a background on bottlenose dolphin strandings in the Gulf and
the various research activities pursued during the investigation. Reports on several research
activities are included as separate sections. The rationale for the research directions taken
are discussed, as are the difficulties encountered in executing the investigation. The results
of each research activity are summarized and discussed, and, when appropriate, integrated
with other research results for further discussion. Reconmiendations are given for improving
the SEFC's stranding investigation capabihties, along with steps taken to implement these
recommendations.
Stranding Rate and Trends
The mortality event investigated occurred during January through May, 1990.
Strandings from January through June, 1986-89, were compared to the January through
June, 1990, strandings. Strandings during 1986-89 for these months ranged from 122-174;
strandings totaled 367 during the same months in 1990. Compared to the 1986-89 average,
during 1990 the largest increase in sfrandings was observed in Alabama (9.68 fold) and the
lowest in Texas (1.80 fold). For the U.S. Gulf as a whole, January-Jime 1990 strandings
were 2.62 times the 1986-89 average and 2.1 times the prior maximum recorded.
The 1990 strandings generally followed the same seasonal pattern previously
observed, with a peak in sfrandings during March. The 1990 strandings during January-
March were from about 3 to 4 times greater than the 1986-89 averages. April 1990
strandings were about twice the prior 4-year average, and May and June 1990 strandings
were also about double, but were relatively low in number, 25 and 23, respectively.
Strandings throughout the Gulf during 1990 were the highest on record for all months except
August.
The sex ratio of the Texas, January- June 1990 sfrandings, was compared to that of
1984-89 Texas strandings. The sex was recorded for 400 animals that stranded during 1984-
89, and the sex ratio was 1.00:0.67, males to females. The sex ratio for 1990 was 1.00:0.98,
which still falls within the yearly ranges observed (1.00:0.34 to 0.85:1.00).
The length-frequency distribution of Texas, January-Jime 1990 strandings, was
compared to that of the Texas, January-June 1984-89, combined length frequency
distribution. Contingency table analysis indicated that the differences between the 1990 and
1984-89 distributions were statistically significant. The largest differences occurred in the
numbers of animals < 140cm, with proportionally half as many stranding during 1990 as
compared to 1984-89. About 30% of the January- June 1984-89 strandings were < 140cm;
during 1990 for these months only 15% were < 140cm. This is the lowest proportion
observed, except for 1985 (only 35 strandings were recovered during 1985, and only 4 were
< 140cm). The proportion of stranded animals measuring < 140cm has been decreasing since
1986.
Population Abundance and Trends
Estimates of bottlenose dolphin abundance from large-scale aerial surveys of the Gulf
of Mexico (Scott et al. 1989) were compared with historical stranding data. The available
population abundance estimates for the northwestern Gulf of Mexico indicate that
abundance is lowest in the winter, increases during the spring, and remains at about the
spring level through the summer and fall. Strandings in the northwestern Gulf peak from
February-April; it appears that strandings in the northwestern Gulf peak during a period
when abundance is increasing. However, if only the nearshore and inshore £ireas (waters
< 18.3m) are considered, strandings peak when abundance is at intermediate levels and
declining.
Available bottlenose dolphin abundance estimates for the northeastern Gulf of
Mexico indicate that the lowest abundance occurs during the summer, with the highest
abundance during the winter. Strandings for the northeastern Gulf peak diuing March and
April, which appears to be when abundance is declining. When only the nearshore and
inshore areas are considered, strandings peak when abundance appears to be declining.
Aerial surveys of portions of the northwestern Gulf were conducted during March and
June, 1990. The results of these surveys indicate that there may have been more animals
present in the inshore areas (waters to 18.3m) and offshore areas (waters 18.3m to 183m)
during 1990 than the previous sampling period (1983-84). These results are similar to the
findings of nearshore surveys conducted prior to the 1987-88 east coast dolphin dieoff, but
differ from the results of offshore surveys conducted during the 1987-88 dieoff which
indicated there was a 60% chance of a decline in the offshore stock abundance (Scott and
Bum 1987).
Age Structure of Stranded Animals
The age structure of the bottlenose dolphins stranded along the Texas coast was
examined to determine if was different during 1990 as compared to previous years. The
available sample of teeth from animals stranded during January- June during 1983-90 were
aged using standard techniques for bottlenose dolphins (e.g., see Hohn et al 1989). The
available sample consisted of 195 animals, of which 70 were obtained in 1990.
Comparison of the 1983-89 and 1990 aged samples indicated that the differences
observed were not statistically significant (Kolmogorov-Smimov two-sample test, DN=0.19,
p>0.10). However, further examination of the samples indicated that the 1983-89 aged
sampled was most likely biased, and that this likely bias resulted in under-representation of
young animals in the sample. For this reason, comparisons of the available age samples were
considered inconclusive. The length structure of the stranded animals, although only a gross
approximation of age, was not considered biased and indicated that proportionally fewer
younger animals (<1 year old) stranded during 1990 than during previous years.
Food Habits
Due to the implication of feeding habits in the recent mortalities of bottlenose
dolphins (Geraci 1989) and humpback whales (Geraci et al. 1989) along the eastern U.S.
coast, there was concern of a sirnilar occurrence in the Gulf of Mexico during 1990. This
study examined the food habits of bottlenose dolphins stranded along the coast of Texas
during January-April 1990 and compared the results with a previous study of bottlenose
dolphin stranded during 1986-87 in the same area (Barros and Odell 1990),
Entire stomachs were collected from 38 stranded bottlenose dolphins and frozen for
analysis. A total of 15,950 otohths, 1,681 squid beaks and remains of 59 crustaceans were
found, representing 7,109 fish, 915 squid and 59 crustaceans. Prey items averaged 219.1 and
prey taxa 9.4 per stomach. Altogether, 46 species of fish (from 11 families), 3 species of
cephalopods and 2 species of crustaceans were identified. Six prey species occurred in more
than 50% of the stomachs and accoimted for 57% of all prey. These species were: the
Atlantic croaker (Micropogonias undulatus), silver perch (Bairdiella chrysoura), brief squid
{Lolliquncula brevis), sand seatrout {Cynoscion arenarius) and an unidentified telelost.
Wet weight of the stomach contents (an indicator of stomach fullness), the numbers
of prey items and prey taxa in each stomach, and the categories of prey types (fish,
cephalopod, crustacean) were not significantly different from those reported in Barros and,
Odell (1990). The four most commonly and numerically important prey (M. undulatus, C.
arenarius, B. chrysoura, and L. brevis) were represented in both 1986-87 and 1990. The
results obtained in the present study show that bottlenose dolphins stranded during the 1990
Gulf of Mexico mortality event had a similar prey spectrum as dolphins stranded in previous
years, and suggest that the food habits of the dolphins stranded during 1990 were not
significantly different from 1986-87.
Environmental Factors
The Texas marine mammal stranding data base allowed analysis of bottlenose dolphin
stranding in relation to certain physical factors of the environment. Bottlenose dolphin
stranding records for the period January 1986-June 1990, were analyzed by linear regression
with monthly mean sea surface and air temperatures, salinity, and offshore transport.
Bottlenose dolphin stranding on the Texas Gulf Coast peaked significantly in March;
otherwise, stranding rates did not differ significantly among the months January-June. No
significant difference in stranding rates was detected among years; however, the relatively
low power of the ANOVA test suggested some undetected differences among years. Using
the upper 95% confidence interval on the 1986-1990 monthly stranding means as a criterion
for detecting significant differences, the number of strandings during January-March 1990
was significantly greater than during the preceding four years.
The slope of monthly stranding rates regressed against Texas coastal monthly mean
sea surface temperature was significant; however, a low correlation coefficient suggested that
there was no simple linear relationship. A negative exponential relationship between first
semester stranding rates and the preceding December-January mean sea surface
temperature was detected; the lowest January-December mean sea surface temperatures
preceded the highest January- June stranding incidence. A weak relationship between
dolphin strandings and air temperature for the same period was not significant. Sea surface
temperature anomaly data from NOAA's Oceanographic Monthly Summary for December
1989- January 1990 ranged from -04 to -2.0 °C The persistence of the negative anomaly
throughout the winter of 1989-90 suggested abnormally low sea surface temperatures. Mean
salinity varied significantly among months, among years, and among months within years;
however, there was no significant relationship between bottlenose dolphin stranding and
salinity. Monthly mean offshore transport varied significantly among years, but there was
no apparent relationship between monthly mean offishore transport and monthly mean
bottlenose dolphin stranding. Examination of seasonal stranding and offishore transport
suggested a weak, but significant, inverse relationship during the spring. Other than an
inverse relationship between winter sea surface temperatures and spring dolphin mortality
rate, these analyses detected no strong significant relationships between bottlenose dolphin
strandings and other enviromnental variables.
The association of low winter sea surface temperatures with an increased spring-time,
dolphin stranding rate suggests t ■- possibility of thermaUy-induced stress. Alternatively, the
observed association between v, .er sea surface temperature and spring bottlenose dolphin
stranding rates may be less dirt -. An estimated 2.7 million fish, of which approximately
2.6 million were striped mullet . Jugil cephalus), died in East Matagorda Bay after a severe
cold spell in December 1989. ^nd smaller kills occurred in Texas Bays from Sabine to
Laguna Madre Bay. It is possible that bottlenose dolphins were forced to switch to inferior
prey items because of unusual weather-related fish migration patterns or fish mortalities.
However, the available food habits study results do not indicate M. cephalus as a major prey
item in stranded dolphin stomachs.
The inverse relationship between spring bottlenose dolphin stranding and offshore
currents (Ekman transport), although not strong, may contribute to an increase in beach-cast
mortalities in the spring and thus, an apparent increase in mortality rate. An increased
nearshore occurrence of bottlenose dolphins, with normal mortality rates, during the spring
season could also contribute to an apparent increase in mortality.
Phytoplankton Distribution
Toxins produced by the dinoflagellate Gymnodinium breve were implicated as a
proximate cause of the mass mortality of bottlenose dolphins along the U.S. east coast
during 1987-88 (Geraci, 1989). For this reason, 123 phytoplankton samples from the Texas-
Louisiana offshore area were examined for the presence of the G. breve. Eighty percent of
these samples contained G. breve cells. Seventy samples from the upper half of the water
column were examined in detail, and quantitative counts confirmed that 94% contained some
G. breve cells while 65% contained >50 cells 1'^ (see Section IV, Figure 1). These
concentrations are far below those considered a "bloom" (>5 x 10^ cells 1'^). Comparative
samples fi-om other areas in the Gulf of Mexico suggest that G. breve concentrations in the
primary study area during the March 1990 sampling period were within normal background
levels but consistently higher than quantitative counts of samples from similar areas in the
northern Gulf of Mexico of from the primary study area later in the summer.
Discolored water patches noted during aerial observations of the primary study area
were blooms (approx. 1 x 10^ cells 1'^) of the dinoflagellate Noctihica spp. This genus is not
known to normally be toxic. However, it should be noted that a toxic dinoflagellate species,
Gonyaulax monalata, was found in elevated concentration near the Mississippi delta in late
summer 1990.
Brevetoxin Analysis
Brevetoxin is a neurotoxin produced by a toxic dinoflagellate, G. breve. Poisoning by
this toxin was believed to have caused the 1987-88 mass mortality of bottlenose dolphins
along the U.S. east coast. Because of the previous implication of brevetoxin as a cause of
mass mortalities of bottlenose dolphins, a total of 50 bottlenose dolphin liver samples were
analyzed for individual brevetoxins (40 samples from 1990 Gulf of Mexico samples, and 10
control samples). Toxicity was determined by several methods: 1) fish bioassay - Gambusia
affinis, fish death at a fixed interval indicates toxin present but does not necessarily indicate
brevetoxin; 2) HPLC separation of toxin fractions - HPLC separation provides a means to
confirm or deny the presence of brevetoxins in comparison to valid Pb Tx-standards; 3)
Radioimmunoassay provides a means to positively identify brevetoxin-like materials and is
sensitive to authentic Pb Tx-3.
Following the first thin-layer chromatography (TLC) plate, 33 of the 50 samples were
found non-toxic in the fish bioassay and were not tested further. Of the remaining 17
samples that tested positive in at least one fraction of the first TLC plate, nine had multiple
toxic fractions. Of the 17 samples, 12 tested negative by fish bioassay following the second
TLC plate. Of the five fi-actions found toxic after the second TLC separation, three were
judged to be in such limited quantity to preclude further TLC separation. The other two
retained toxicity after the third TLC separation.
The three toxic fractions of limited quantity were judged to contain less than 5ug
toxin/total original sample by HPLC; this was presimied to be a negative result. The other
two fractions, purified through the third TLC, appeared to contain Pb Tx-2 by HPLC
separation and co-elution. Radioimmunoassay was performed on these five fractions, using
tritiated Pb Tx-3 as the internal displacement standard. Based on this assay, the three
fractions purified through 2 TLC steps contained 10.2, 12.2, and 9.33 ng toxin/g liver; the two
fractions purified through 3 TLC steps contained 17 and 240ng toxin/g liver.
The process of extraction, purification, chromatographic separation, and
radioimmunoassay conducted on the 50 samples led to the conclusion that five of the
samples contained brevetoxin or some very similar toxin. Reported concenfrations in original
samples were calculated by proportion of sub-sampling at the various steps and were based
on "Pb Tx-3 equivalents" in the radioimmimoassay.
Of the five toxin-spiked control samples only one was detected as containing
brevetoxin; this sample was spiked with the largest amount of Pb Tx-3, 25ug. Two other
samples were spiked with 20 and 15ug of Pb Tx-3 respectively, but were not identified as
containing brevetoxin. PbTx-1 and PbTx-2 were also added to several of the samples; PbTx-1
is known to hydrolyze quite quickly. The fact that purified toxins "stick" to glass- and plastic-
ware may explain the low level of apparent spike of the liver samples. It is quite possible
that neither the PbTx-1 or PbTx-2 spikes were effective, or it is possible that they do not
effectively displace radio-labeled PbTx-3 in the radioimmimoassay.
Of the five carrier-spiked control samples (treated with MeOH only), three were
identified by the radioimmunoassay as containing brevetoxin. It is difficult to explain this
finding. The other two carrier-spiked samples were found to be negative when purified to
the second TLC step. It is possible that an interfering substance was removed in the early
cleanup phases of some of the controls and not in others.
The sample reported to contain the largest amount of brevetoxin, as determined by
radioimmunoassay, was one of the non-toxin (MeOH only) spiked control samples. The only
dolphin liver sample from the strandings that was identified as containing brevetoxin at all
stages contained 10.2 ng toxin/g liver. This level of toxin is considered to be very low.
The problems encountered in properly identifying the spiked and non-spiked control
samples raised serious questions concerning the efficacy of this assay method for detecting
brevetoxin in bottlenose dolphin liver samples. Certainly, the results of this brevetoxin
analysis and other studies which used the same assay methods (e.g. Geraci 1989) cannot be
considered conclusive. That is, based on the incorrect assay results of the control samples,
brevetoxin poisoning cannot be ruled out as a proximate cause or factor in the 1990
bottlenose dolphin strandings.
The author of the report suggests that samples should continue to be collected so that
assays for brevetoxin detection may be refined. His research group will be conducting
collaborative research on the assay of brevetoxins in marine animal tissues; this process
should assist in the further development and verification of the assays. A major difficulty in
establishing an assay of this type is obtaining a true "control liver" sample, known to be free
of toxins or other substances that interfere with the assay.
Contaminants
Tissues from a sub-set of the stranded bottlenose dolphins were examined to
investigate the possibility that environmental contaminants may have caused or contributed
to the observed strandings. Blubber and liver samples from 20 of the stranded bottlenose
dolphins were analyzed for chlorinated hydrocarbons (CHs). In addition, liver and kidney
samples of these dolphins were analyzed for certain metals.
The concentrations of mercury in the livers of 2 dolphins (114 and 117 jig/g or ppm
based on wet weight) were notably elevated and may be of toxicological concern. The
concentrations of total CHs in the 20 animals sampled varied widely; 3.0-190 ppm in blubber
and 0.5-58 ppm in liver with eight dolphins having levels of total CHs in blubber that were
greater than 50 ppm. Interestingly, relative levels of DDT, compared to the levels of DDE
(a metabolite of DDT), in three of the dolphins may indicate an exposure to relatively
recently released DDT.
The concentrations of CHs and certain metals, especially mercury and selenium, in
some of the dolphins were sufficiently high to warrant a more systematic study of
contaminant exposure of this species and of potential health effects due to this exposure. In
addition, special efforts are needed to investigate possible sources of certain toxic chemicals;
including CHs and aromatic hydrocarbons, by measuring parent compounds and their
metabolites in tissues and stomach contents of these animals.
Summary
The investigation did not provide any conclusive evidence of a single causal agent, or
multiple causal agents, for the increase in strandings. Available abundance estimates indicate
that bottlenose dolphin abundance may have been higher in the nearshore northwestern Gulf
during the spring and summer of 1990 than during 1984; however, the 1990 estimates are
limited in geographic scope and may not reflect the overall abundance patterns. No
statistically significant differences were found between the 1990 and the 1984-89 age
structure of the stranded animals. But, possible biases in the 1984-89 age sample were
identified and the age structure analysis must be considered inconclusive. The results of the
food habits analysis indicate that the bottlenose dolphins stranded during 1990 had a similar
prey spectrum as in previous years. The analysis of environmental factors (sea surface and
air temperatures, salinity, and offshore transport) detected a statistically significant inverse
8
relationship between the winter sea surface temperature and spring bottlenose dolphin
strandings. The winter 1989-90 sea surface temperatures were considered abnormally low
and suggest the possibility of thermally-induced stress. However, the stranding database
covers only 5 years and was insufficient to determine if the inverse relationship between
winter temperatures and spring strandings is consistent. The results of the brevetoxin analysis
were questionable and must be considered inconclusive, and although the phytoplankton
study determined that the brevetoxin producing organism was present, there is no
information available on the "normal" occurrence patterns of the organism within the
phytoplankton study area. The contaminant analyses indicated that although a few dolphins
had concentrations of contaminants at levels of possible toxicological concern, contaminant
concentrations in most of the dolphins were relatively low. Fewer than 3% of the stranded
dolphins received thorough pathological exams. As a result, essentially almost no
pathological information was available. Overall, none of the studies conducted provided
conclusive evidence of circumstances or agents which caused the observt.d increase in
strandings.
INTRODUCTION
The Southeast U.S. Marine Mammal Stranding Network (SEUS) was organized in
1977 to document and salvage marine mammal strandings along the U.S. coast from Texas
to Virginia (for a review of the network, see Odell, 1991). Most of the Network participants
are volunteers, and stranding reporting and salvage efforts vary considerably. For example,
salvage efforts range from nearly none along Louisiana to nearly 100% along Texas.
Although the salvage efforts are not consistent, stranding records may provide an index of
mortality rates and have been used to estimate the potential effects of anomalous mortality
events (Scott et al., 1988). Nearly all of the stranding reports and specimens used in the
investigation of 1990 strandings were provided by the participants of the SEUS. A list of
specimens collected during the mortality event imder investigation is provided in Appendix
I. The specimens are listed by stranding date, and information is provided on location, sex,
length, tissues collected, and analyses conducted.
The investigation was initiated due to reports that higher than normal numbers of
bottlenose dolphins were stranding along the U.S. Gulf coast. Begiiming in early 1990, a
number of stranding and mortality observations of bottlenose dolphins were made. There
was a mass dieoff of 23 dolphins in January in Matagorda Bay, Texas, that may have been
caused by unusually cold weather (Miller, 1991). A higher level of strandings than the prior
4-year average was observed in the Gulf along the coasts of Rorida, Alabama, Mississippi,
and Texas beginning in January. Floating dolphin carcasses were consistently observed during
January and February while NMFS observers were on transit to deep water aerial survey
study areas in the central northern Gulf of Mexico. As a result of all of these observations,
the SEFC notified the NMFS Office of Protected Resources and the MMC of the
observations and began intensified monitoring of the strandings and initiated an investigation
of the cause and extent of the strandings.
Higher munbers of dolphins than the prior 4-year average continued to strand from
the Florida panhandle to Texas during February and March, 1990. Strandings decreased to
average or below
average along the
Texas coast after
March, 1990, but
continued to occur
sporadically at
higher than
average levels
along the
Mississippi,
Alabama, and
Florida coasts
from February
through May,
TaMc L Northern Gulf of Mexico 1990 bottlenose dolphin strandings by slate and month.
MONTH
STATE
J
F
M
A
M
J
J
A
S
O
N
D
ALL
FL
10
12
12
6
4
9
7
3
3
6
3
11
86
AL
7
4
12
11
8
4
1
1
1
1
2
6
58
MS
8
5
24
7
8
5
9
4
7
2
3
2
84
LA
3
1
13
27
3
1
0
0
0
0
0
1
49
TX
42
40
59
16
2
4
2
2
4
9
13
8
201
All.
70
62
120
67
25
23
19
10
15
18
21
28
478
10
1990. It appeared that the mortality was within the prior 4-year average by the end of May,
1990. A total of 344 stranded bottlenose dolphins were recovered along the U.S. Gulf coast
during January-May, 1990. Investigators from Texas recovered 159 strandings during this
period, while strandings recovered in the other Gulf states ranged from 42-52. Strandings by
state and month are shown in Table I. Overall, the number of reported strandings for
January-May, 1990, in the U.S. Gulf of Mexico was about 2.5 times the 1986-89 average for
those months. By comparison, during the 1987-88 U.S. Atlantic coast dieoff over 700
bottlenose dolphins were reported stranded over a 10-month period and represented a 10-
fold increase over the prior 4-year average in strandings.
Between Januaiy-May, 1990, most of the dolphins recovered on a state by state basis
during the investigation stranded along the Texas coast. Approximately 166 dolphins were
reported stranded there from January- June, and 163 were recovered. Within January-March
and all states, the greatest number of recovered animals were from the Texas coast during
March (59), with January and February totals (also from Texas) of 42 and 40, respectively.
The totals for these months were the highest on record. Texas strandings for January were
4.4 times the 1986-89 Texas monthly average, February 2.3 times average, and March 1.5
times average. The cumulative Texas total for January-March, 1990, was 2.1 times average.
However, the Texas stranding recovery rate decreased after March, 1990, and Texas
strandings for April, 1990, were about 50% of average and the lowest total for April since
1986.
A total of 204 animals were recovered from January-June along the coasts of the
other Gulf states. These represented about 55% of the recovered strandings for that period.
A total of 57 dolphins were recovered in Mississippi, 52 in Florida, 48 in Louisiana, and 46
in Alabama. By month, most (24) of the Mississippi strandings were recovered during March,
most (12 each) of the Florida strandings during February and March, most (27) of the,
Louisiana strandings during April, and most (12) of the Alabama strandings during March.
Recovery effort in 1990 in Mississippi and Florida was relatively constant, but the stranding
recovery effort along Louisiana was not, because of USCG supported helicopter beach-
surveys conducted during March and April, 1990. Stranding recovery effort along the
Alabama coast was consistent January-March. Alabama stranding recovery efforts increased
significantly during the last half of April, primarily due to Greenpeace efforts to increase
stranding reporting and recovery along the Alabama coast.
It is likely that the effort expended on reporting and recovering of strandings was not
consistent between 1986-89 and 1990. The mass stranding of bottlenose dolphins in
Matagorda Bay, Texas during January 1990 was well publicized in Texas. The 1987-88 mass-
mortality of bottlenose dolphins on the east coast likely sensitized stranding networks and
various groups to strandings. The observed increase in stranding reports could thus be an
artifact of the influence of these events on reporting and recovery efforts.
The results of regular, controlled effort surveys for marine mammal and sea turle
strandings along the Texas coast provided a basis for obtaining an independent measure of
11
stranding rates along the Texas coast. These surveys were conducted by the SEFSC during
1988-90 along portions of the Texas coast. Bottlenose dolphin stranding data collected during
the beach surveys were examined to determine if the surveys detected an increase in
strandings between 1990 and 1988-89 during the months of January-April. The beach surveys
for 8 areas were examined; Table 2 lists the areas surveyed and the number of dolphin
strandings recorded.
TbUc 1. Texas beach surveys conduaed from 1988-90 (Januaiy-May) by area with reported bottlenose dolphin strandings.
1988
1989
1990
AREA
SURVEY
STRAND
SURVEY
STRAND
SURVEY
STRAND
Bolivar
5
0
11
9
9
7
Galveston
6
0
8
0
10
1
Bryan Beach
9
0
8
0
11
1
Sargent's Beach
10
1
8
5
11
2
East Matagorda Peninsula
10
4
8
4
11
3
Matagorda Island
10
4
4
2
10
9
Mustang Island
16
0
14
0
22
4
South Padre Island
20
0
21
0
1
0
TOTAL
86
9
82
20
85
27
The number of surveys per area varied yearly, but the total number of surveys per
year was similar, and ranged from 82-86. The number of miles of beach surveyed per year
was also similar, and ranged from 2388-2580 (Table 3). Compared to the 1988-89 average,
1990 strandings increased in 5 areas, decreased in 2 areas, and were unchanged in 1 area.
Overall, the number of strandings recorded during 1990 were about 1.9 times the 1988-89
average. The beach surveys provided a measure of the stranding rate; the 1990 stranding
rate (stranded dolphins per mile surveyed) was approximately 1.8 times the 1988-89 average.-
The number of bottlenose dolphin strandings reported by the Texas Marine Mammal
Stranding Network (TMMSN) for the beach survey areas showed an increase during 1990
of about 2.5 times the 1988-89 average (Table 4).
Potential biases were apparent in comparisons of trends in strandings between the
beach survey data and the TMMSN data. First, the yearly number of beach surveys by area
was not consistent. For instance, during the first five months of 1988 and 1989 about 20
surveys were conducted each year at South Padre Island, while during 1990 only one survey
was conducted (Table 3). Second, surveys were not conducted at an adequate frequency.
Surveys averaged about 2 per month (range, 1-4), but during peak stranding periods
bottlenose dolphins stranded about once every 3 days. Third, discrepancies between the data
sets indicated that under-reporting was occurring in both systems. The beach survey data
should be a temporal subset of the Network data, but in fact some of the beach survey areas
reported more strandings during 1988 and 1989 than the Network reported for those areas
(Table 4). The Network reported 45 dolphins stranded in the Galveston area during 1988
while the Galveston beach survey reported none that year. These discrepancies may indicate
under-reporting within each data set, and/or variations in geographic definitions. Overall, the
12
sample size of dolphin strandings by area from the beach surveys was too low to derive any
reliable inferences as to monthly trends or differences between areas. However, the beach
surveys did show an overaD increase during 1990 in the total number of stranded bottlenose
dolphins and in their stranding rate (1.8 times the 1988-89 average). The beach surveys
provided an independent measure of the number of strandings and the stranding rate and
confirmed the observations of the TMMSN.
Tabic 2 Toos beach turveys conducted from 198S-90 (Januaiy-May) by area reporting number of miles surveyed and number
ef stranded botilenose dolphins per mile surveyed.
1988
1989
1990
AREA
STRAND/
MILE
MILES
STRAND/
MILE
Mn RS
STRAND/
MILE
MILES
Bolivar
0
472
0.0177
508
0.0142
491
GaKeston
0
518
0
415
0.0022
449
Bryan Beach
0
45
0
51
0.0161
62
Sargent's Beach
0.0112
89
0.0532
94
0.0180
111
East Matagorda Peninsula
0.0160
250
0.0196
204
0.0107
280
Matagorda Island
0.0151
265
0.0134
149
0.0275
327
Mustang Island
0
341
0
367
0.0054
735
South Padre Island
0
600
0
600
0
30
TOTAL
00035
2580
0.0084
2388
0.0109
2485
Table 3. Numbers of bottlenose dolphins reponed stranded by the Tecas beach surveys and by the Marine Mammal Stranding
Network (MMSN) during January-Arril, 1988-90.
1988
1989
1990
AREA
MMSN
BEACH
MMSN
BEACH
MMSN
BEACH
Bolivar
G
0
16
9
32
7
Galveston
Bryan Beach
45
0
0
11
1
0
0
29
1
1
1
Sargent's Beach
1
3
5
4
2
East Matagorda Peninsula
4
7
4
32
3
Matagorda Island
4
6
2
11
9
Mustang Island
0
0
0
13
4
South Padre Island
A
0
3
0
4
0
TOTAL
•kj
9
57
20
126
27
13
Literature Cited
Miller, W.G. 1991. An investigation of dolphin (Tursiops tmncatus) deaths in East Matagorda
Bay, Texas, January 1990. Naval Ocean Systems Center, Code 5107, San Diego, CA
92152.
Odell, D.K. 1991. A review of the Southeastern United States Marine Mammal Stranding
Network: 1978-1987. Pages 19-24 in J.E. Reynolds III and D.K. Odell (eds.). Marine
mammal strandings in the United States. NOAA Tech. Rep. NMFS 98. 157 pp.
Scott, G.P., D.M. Bum and LJ. Hansen. 1988. The dolphin dieoff: long-term effects and
recovery of the population. Pages 819-823 in Proceedings of the Oceans '88
Conference, Baltimore, MD. IEEE Catalog No. 88-CH2585-8.
14
SECnON I
STRANDING RATE AND TRENDS
Lany J. Hansen
Southeast Fisheries Science Center
Miami Laboratory
75 Virginia Beach Drive
Miami, FL 33149
Methods
Since the mortality event under investigation appeared to have occurred between
January through May, 1990, the 1990 stranding records for January through June were
compared to those from prior years for the same months. Unless otherwise noted, references
to aimual or yearly strandings refer to strandings that occurred during January through June.
In those cases where the number of strandings were compared, strandings from 1986-89
were used for comparison (except when comparing the highest number stranded, then
records from 1984 on were used). This was done since stranding recovery effort in several
areas of the Gulf was probably relatively consistent during those years, although this effort
cannot be measured. Prior to 1986, stranding recovery effort was generally sporadic but was
assumed to have provided a random sample of the strandings.
For analytical purposes, in most cases the strandings were divided into two main
groups, Texas strandings and non-Texas strandings. This was done primarily because a
consistent stranding network has been operating on
the Texas coast beginning in about 1986, and the
yearly sample size from Texas was adequate for
analysis. The stranding networks in the remainder of
the Gulf vary in consistency, and none have adequate
yearly sample sizes. For these reasons, additional
analyses (sex and length structure) were conducted on
the Texas sample.
400
in MO
o
z
i 230
I I TEXAS
?77i LOUIStMM
Jga MtSSSSPPI
m ALABAMA
R^ FUJRIOA
200
Results and Discussion
The number of stranded bottlenose dolphins
examined between 1984-89 varied from 63 to 174
animals. During 1990, 361 stranded bottlenose
dolphins were examined. The number of animals
ISO
o
m too
SO
Figiirc 1: Bottlenose dolphin strandings
nonbern Gulf of Medcx). 1984-90.
in the
15
stranded for 1984-90 by state is illustrated in Figure 1 and listed in Appendix II. From 1984-
89, more animals were reported stranded aimually along the Texas coast than in the rest of
the U.S. Gulf. However, during 1990, the number of animals reported stranded on the Texas
coast was less than that reported in the remainder of the U.S. Gulf (161 vs 200). The rate
of increase in strandings during 1990, expressed as January through June 1990
strandings/prior 4-year average, varied by state (Appendix II). The highest increase was
observed in Alabama (9.68 times), and the lowest was observed in Texas (1.80 times). For
the U.S. Gulf as a whole, strandings during 1990 were 2.62 times the 1986-89 average.
Historically, strandings of bottlenose dolphins
in the U.S. Gulf of Mexico have shown a seasonal
trend. The greatest number of dolphins strand during
March, with February and April also showing an
increase over the rest of the year (Figure 2). Overall,
the 1990 strandings by month showed a pattern similar
to that previously observed. Some deviation from this
pattern was observed in Alabama, Lx)uisiana and
Texas. However, the stranding reporting and recovery
effort in Alabama and Louisiana was considerably
greater during 1990 than in prior years, and the
degree and duration of increases observed in those
states may, in part, be an artifact of increased effort.
The Texas strandings deviated from the 1984-89 trend
during January, with more strandings occurring during
that month than during February (Figure 3). However,
if the anomalous mortality that occurred in Matagorda
Bay, Texas, during January is excluded, the pattern is
similar to that previously observed. Although it may be
reasonable to exclude the Matagorda Bay strandings,
it should be noted that the 1990 pattern for the U.S.
Gulf exclusive of Texas shows the same pattern of a
higher proportion of strandings during January (Figure
4).
With the exception of August, strandings were
higher in all months during 1990, and were outside of
the upper 95% confidence interval about the mean for
the period 1986-89 (Figure 2), and were generally
greater than the maximum monthly reported for that
period. Strandings in Texas were above the 95%
confidence interval for January-March and October-
November (Figure 3). Strandings in the remainder of
the Gulf were above the 95% confidence interval for
all months except August and November (Figure 4).
1*0
120
100
BO
60
40
20
CULF OF MEXICO
• 1990
V 1984-89 HIGH
ERROR BARS 9SS
un^
J F M A M J
A S 0 N D
Figure 2 Comparison of northern Gulf of Mocioo
1990 monthly bottlenose dolphin strandings with
1986-89 mean, with 1986-89 95% conndence
interval High monthly from 1984-89 data.
70
60
50
40 -
30 -
20
10
JFMAMJJASONO
Figure 3: Comparison of Texas 1990 monthly
bottlenose dolphin strandings with 1986^
mean, showing 1986-89 95% confidence interval
High monthly from 1984-89 data.
During January-June 1990, Texas
TEXAS
• 1990
•
^ 19B4-89 HIGH
• ^
ERROR BARS 95X
.•7'
1
i
7
\
f
\
\ yC
■y
W^jf^S^
1
16
reported strandings were >1.5 times the previous high
for only January and February, whereas reported
strandings in the remainder of the Gulf were >U
times the previous high for January-May. Thus it
appears that most of the anomalous mortality
occurred outside of Texas, both in terms of duration
and numbers.
The increases in reported stranding rates
(strandings/month) in the Gulf were compared to the
increases observed during the 1987-89 bottlenose
dolphin dieoff along the U.S. east coast. Compared to
the prior 3-year average, the east coast dieoff
represented a 10 fold increase (Geraci 1989; Scott et
al. 1988) while in the Gulf a 2.6 times increase was
observed. On a state and month basis, increases along
70
60
50
40
30
20
10 -
NON-TEXAS
• 1990
^ 19B4-89 HIGH
ERROR BARS 95k
JFMAUJJASOND
Figure 4: Compahsoo of non-Texas 1990
monthly bottlenoae dolphin strandings with
1986-89 mean, tbowing 1986-89 95% confidence
intervals. High monthly from 1984-89 data.
the east coast were up to >40 times the average as compared to a maximum of about 12
times the average in the Gulf. Figure 5 illustrates the range of relative increases observed
in the Gulf and along the east coast by state and month in reference to prior 4-year averages
for each region. The total number of animals stranded during the east coast dieoff was about
twice that observed in the Gulf, but the east coast relative strandings were dramatically
higher.
so
X
2
20 ■
• ATUNTC 1987-08
A GULF tmo
The 1990 sex ratio of Texas strandings was
compared to that observed during 1984-89 for the
months January-June. Information on sex was used
only for specimens with length data. This was done to
avoid the possible bias that may exist in sexing animals
(i.e., it may be easier to sex males, even when more
than moderately decomposed). It was assumed that
specimens with length data would be reliably sexed.
During January-June 1984-89, sex and length was
recorded for a total of 334 animals, and the resulting
sex ratio was 1.00:0.69, males to females (range:
1.00:0.45 to 0.85:1.00; see Appendix III). Except for
1989, the available data suggest that there were more
strandings of males than females. The sex ratio for
1990 for the same months was 1.00:0.98, which falls
within the previously observed range. A z-test,
corrected for continuity (Snedecor and Cochran 1973),
was used to test the null hypothesis that there was no
significant difference between the proportions of males stranded during 1984-89 and during
1990. The results of this analysis indicated that the proportions of observed during 1984-89
and during 1990 were not significantly different (z = 1.545, p > 0.12).
P t , ^
S 0
D J F
IIONTH
Figiire 5: Bottlenose dolphin strandings by state
and month during the 1987-88 easi coast dieoff
and the 1990 nonhern Gulf of Modco mortality
event compared to prior 4-year averages.
17
During 1984-89, lengths were recorded for 431
Texas strandings during the months January-June. The
1984-89 length-frequency distribution (by 10cm
intervals) is shown in Figure 6 and the 1990 January-
June length-frequency distribution is shown in Figure
7. Based on the 1984-89 distribution pattern, the
length data was separated into three groups: < 140cm,
> 139cm to <230cm, >229cm. Contingency table
analysis indicated that the differences between the
1984-89 and the 1990 distributions were significant
(X^= 13.66, p<0.01). The largest differences are in the
first two groups, with proportionally half as many
< 140cm during 1990, and proportionally about 30%
fewer of > 139cm to <230cm. The proportion in the
last group, those > 229cm was nearly the same (1984-
89, 45%; 1990, 50%).
130-
180- 230-
280-
138
1BS 239
l£NCTM CM
289
Figmc 6: Length frequency distribution, based on
lOcm intervals, of 1984-89 January-June Texas
bottlenose dolphin strandings.
Historically, 88% of the yearly strandings of
animals < 140cm occurred during January- June; during
1990 over 95% stranded during those months. About
30% of the January-June 1984-89 strandings were
< 140cm. During 1990 for the same months, 15% of
the strandings were < 140cm. This is the lowest
proportion observed, except for 1985. However, only
35 strandings were recovered during 1985, and only 31
were measured (4 were < 140cm). The proportion of
stranded animals measuring < 140cm has been
decreasing since 1986 (Figure 8).
130-
180- 230-
280-
13S
189 239
LENGTH CM
289
Figure 7: Length frequency dislribution, based on
lOcm intervals, of Tecas 1990 January-June
bottlenoce dolphin strandings.
Figure & Comparison of proportions
by length (< 140cm and >- 140cm)
and year of Tescas January-June
1984-90 bottlenose dolphin
strandings.
Males accounted
for 58% of the Texas
strandings with sex and
length information
during January-June, 1984-89. During this same period, males
accounted for 73% of the strandings < 140cm. Overall, during
this period, the ratio of males to females has ranged from
1.00:0.45 to 0.85:1.00, while the ratio of males to females for
animals < 140cm has ranged from 1.00:0.18 to 1.00:0.55
(Appendix III). During January-June 1990, males accounted
for 68% of the strandings < 140cm. The ratio of males to
females during this period was 1.00:0.98 while the ratio of
males to females for animals < 140cm was 1.00:0.46. Although
the percentage of females < 140cm stranding during 1990 was
somewhat less than the 1984-89 percentage (5% vs 7%), the
18
percentage of males < 140cm stranding during 1990 was about half of those stranding during
1984-89 (11% vs 20%). It appears that the apparent decrease in the proportion of strandings
< 140cm was accompanied by a decrease in strandings of males < 140cm. This result could
reflect a decrease in natality rate, a decrease in mortality rates of animals (primarily males)
< 140cm, or some other factor, such as a change in distribution patterns of animals with
dependent calves or a differential bias between periods of study. A z-test, corrected for
continuity (Snedecor and Cochran 1973), was used to test the null hypothesis of no
difference between the proportions of males < 140cm stranded during 1984-89 and during
1990 (where the proportion is equal to (# males < 140cm)/(total males plus females)). The
results of this test indicated that 1984-89 and the 1990 proportions were significantly
different (z = 2.037, p < 0.04). A z-test was also applied to the proportions of females
< 140cm stranded during 1984-89 and during 1990, and the results indicated that the
difference in the proportions was not significant (z = 0.614, p > 0.51). However, the 1990
sample size for animals < 140cm was small (n = 19) and therefore the results of the z-test
analyses should be regarded with caution.
The sex ratio of animals stranding along the U.S. Gulf of Mexico coast outside of
Texas during January-June 1984-89 ranged from 1.00:0.50 to 0.87:100, males to females. The
overall sex ratio for this period was 1.00:0.91, males to females. The 1990 sex ratio for the
same months was 1.00:0.67, males to females. These ratios are opposite those observed for
Texas; the 1984-89 Texas ratio was 1.00:0.69 and the 1990 Texas ratio was 1.00:0.98, males
to females.
For the Gulf outside of Texas during January-June 1984-89, the overall proportion
of animals < 140cm was 0.30 and ranged yearly from 0.15 to 0.49. During 1990 for the same
area and months the proportion of animals < 140cm was 0.27, which is essentially the same
as the proportion during 1984-89. During January-June 1984-89, the proportion of males
< 140cm was 0.29, ranging yearly from 0.14 to 0.61, and the 1990 proportion was similar at
0.27. The proportion of females < 140cm for these months during 1984-89 was about 0.25,
ranging yearly from 0.00 to 0.37, while the 1990 proportion for the same months was about
0.19. TTiis contrasts with the changes observed for the Texas coast, where the proportion of
males < 140cm decreased while the proportion of females remained about the same.
However, as with the Texas strandings, the 1990 < 140cm sample was small and the changes
in the sex ratio may not be significant.
The stranding rate and trends could reflect a change or changes in factors which
cause carcasses to reach the beach, rather than a change in the mortality rate. The
bottlenose dolphin population in the northern Gulf of Mexico is conservatively estimated to
consist of 35,000 to 45,000 animals (Scott et al., 1989). The annual natural mortality rates
of bottlenose dolphins are beheved to range from 4% to 14% (Hersh, 1987, Wells and Scott,
1988). Using the estimated range of population size and the estimated range of natural
mortality, approximately 1,400 to 5,300 bottlenose dolphins deaths per year would be
expected in the northern Gulf of Mexico. If the these estimates are correct, only 2.8% to
12.7% of carcasses available to strand are observed. It is obvious that only a marginal
19
increase in the rate of beachings could result in a doubling to more than five times increase
in numbers recovered. It is generally assumed that the stranding recovery rate is an index
of the mortality rate. However, in a large complex system with a large population of animals
available to strand, the variability in the stranding rate may be more sensitive to factors
which cause carcasses to drift to the beach than to changes in the mortality rate.
Changes in the pattern and causes of mortality may be a more reliable indicator of
anomalous mortality events than increases (which do not deviate from the seasonal pattern)
in the number of stranded animals. Both of these conditions were evident during the 1987-88
east coast bottlenose dolphin dieoff (Geraci, 1989; Scott et al., 1988). The occurrence of the
1987-88 strandings deviated from the normal pattern, and the pathology associated with the
strandings was unusual. Neither of these conditions were evident in the 1990 Gulf of Mexico
bottlenose dolphin strandings.
Literature Cited
Geraci, J.R. 1989. Clinical investigation of the 1987-88 mass mortality of bottlenose dolphins
along the U.S. central and south Atlantic coast Final Report to NMFS, ONR, and
MMC.
Hersh, S.L. 1987. Mortality, natahty, migration and organismic growth rates of bottlenose
dolphins (Genus Tursiops): a review and management considerations. NMFS/SEFC
Miami, Contract Report No. 40-GENF-700715.
Scott, G.P., D.M. Bum and L.J. Hansen. 1988. The dolphin dieoff: long-term effects and
recovery of the population. Pages 819-823 in Proceedings of the Oceans '88
Conference, Baltimore, MD. IEEE Catalog No. 88-CH2585-8.
Scott, G.P., D.M. Bum, L.J. Hansen and R.E. Owen. 1989. Estimates of bottlenose dolphin
abundance in the Gulf of Mexico from regional aerial surveys. NMFS/SEFC Miami
Contribution No. CRD-88/89-07.
Snedecor, G.W. and W.G. Cochran. 1973, Statistical methods, 6'*' ed. Iowa State University
Press, Ames, Iowa. 593 pp.
Wells, R.S. and M.D. Scott. 1988. Estimating bottlenose dolphin population parameters from
individual identification and capture-release techniques. NMFS/SEFC Miami Contract
Report No. 50-WCNF-7-06083.
20
SECTION n
AGE STRUCTURE
Lany J. Hansen
Southeast Fisheries Science Center
Miami Laboratory
75 Virginia Beach Drive
Miami, FL 33149
Hersh (1988) found that the age structiire of bottlenose dolphins stranded during the
1987-88 U.S. east coast dieoff differed significantly from the available pre-dieoff composite
sample. Specifically, a significantly larger proportion of 5 to 9 year olds stranded during the
dieoff. This may have reflected that the hypothesized disease epidemic (Geraci, 1989) caused
proportionally higher than normal rates of mortality among an age group which usually
exhibits a relatively low mortality rate (Hersh, 1988).
Methods
An age analysis was conducted on the available sample of teeth from animals that
stranded along the Texas coast to evaluate the age structure of the 1990 strandings as
compared to the composite sample of previous years. The teeth were examined for growth
layer groups (GLG or GLGS; see Perrin and Myrick, 1980) by Ms. S. Fernandez, with
assistance from Dr. A. Hohn, a leading expert on techniques for aging odontocete cetaceans.
The methods used followed those detailed in Myrick et al (1983) as modified for bottlenose
dolphins (Hohn et aL 1989). The sample included animals that stranded from 1983-1990. Of
this sample, 195 stranded during January- June, of which 70 were from 1990.
Results and Discussion
The cumulative distributions of ages of the two samples (animals from 1983-89 and
animals from 1990) are shown in Figure 1. These distributions were compared using a
Kolmogorov-Smimov two-sample test. There were proportionally more young animals in the
1990 sample, but the results of this test indicate that the cumulative distributions were not
significantly different (DN=0.186, p>0.10).
The length data form a larger and probably more representative sample of the
stranded animals. A comparison of the cumulative distributions of the 1990 zmd 1983-89
length samples (Figure 2) indicates that proportionally more short (i.e., young) animals
stranded during 1983-89. The distributions were significantly different (DN=0.0987, p<0.04).
21
This trend was the opposite of that of the aged sample (This trend is also discussed in
Section II).
IJO •
,.^ '
o.t •
.'-^ y^
Oi ■
/'" ^^ 1
0.7 •
/ jT"
/ ItM
IM3-W
OJ.
r /
0.5.
1/
0.4 -
0 J ■
,^
^^
f r'
■^
ON • o.ias7
0.1 -
/
t > 0.104
0.1 -
1 r > I
20 13
CLBS
Figmc 1: Cumulative distributions of ages (id GLGS) of
Texas January-June 1983-89 and 1990 bottlenose dolphin
strandings.
1.0 -
o.t -
/^
S a* ■
f
1 0.7 .
f — itoo
1 0.4.
go..
0.3-
0.1 .
/^
ON . 0.0M7
> < 0.040
M no IM laa it* im 2i« zm
LEKTTM Ol
290 ]7s no 3ia
Fignre 2 Cumulative distributions of lengths of Tens
Januaiy-June 1983-89 and 1990 bottlenose dolphin
unndings.
The aged samples are a sub-sample of the stranded animals with length data. If the
aged sample is representative of the length sample, the length structure of the aged samples
should reflect that of the overall length samples. To examine this, the cumulative
distributions of the lengths of the 1983-89 and 1990 aged samples were compared to the
1983-89 and 1990 length samples, respectively (Figure 3-4). The results of the Kohnogorov-
Smimov two-sample tests of these distributions indicate that the while the 1990 aged sample
was not significantly different from the 1990 length sample (DN=0.105, p>0.59), the 1983-89
aged sample was significantly different from the 1983-89 length sample (DN=0.245, p<0.01).
1.0 ■
o.s -
/T"
1 0.S •
/ ,' loai-M
i 0 7 .
/ / — «i
s "■•■
/ ; MXD SMWJE
/ t
5 04.
^^y^ J
\ "-'■
— _^
y y OM . 0J4S0
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0.1 •
/ ,-'' » < OJ»l
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o.« ■
\ 0.0 ■
'</ mo
1 0.7 .
/ — *"•
S "^ '
A — *ca> SM»i£
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3 0 4 .
^l-''
g OJ-
^„^—' OM - O.I04«
o.i.
^^-
--=*^' » > 03M
0.1 ■
*S IIS ISO ISO IT! IM 111 130 230 270 l*a III
IINCTM Ol
Figure 3: Cumulative distributions all lengths and lengths
of aged sample ofToas 1983-89 January-June bottlenose
dolphin strandings.
M no ISO ISO
170 Ita 211 230 290 171 2M 3l«
UNCTM CH
Figiiic 4: Cumulative distributions
lengths of aged sample of Texas
bottlenose dolphin strandings.
of all lengths and
1990 January-June
These results indicate that the 1983-89 aged sample was biased in some way. It
appears that shorter (< 140cm) animals were under-represented in the 1983-89 aged sample.
The teeth of most of these shorter animals probably were not collected during 1983-89
because the teeth had not yet erupted.
22
Because the 1983-89 aged sample was probably not an unbiased sample, the age
structure of this sample most likely does not accurately reflect that of the animals stranded
during 1983-89. Therefore, the results of the age structure comparison between 1983-89 and
1990 should be considered inconclusive. Since there is no indication that the length samples
were biased, they can be used for a gross comparison of age structure. The 1983-89 and 1990
length samples were significantly different, and indicate that proportionally fewer younger
animals and more older animals stranded during 1990 than during 1983-89. A yearly
comparison of lengths demonstrates that the proportion of stranded animals < 140cm has
been decreasing since 1986 (see Section II, Figure 8).
Literature Cited
Geraci, J.R. 1989. Qinical investigation of the 1987-88 mass mortality of bottlenose dolphins
along the U.S. central and south Atlantic coast. Final Report to NMFS, ONR, and
MMC.
Hersh, S.L. 1988. Age class distribution of bottlenose dolphins stranded during the east coast
die-off of 1987/1988. NMFS/SEFC Miami, Contract Report No. 45-WCNfF-800633.
Hohn, A.A., M.D. Scott, R.S. Wells, J.C. Sweeney and A.B. Irvine. 1989. Growth layers in
teeth from known-age, free-ranging bottlenose dolphins. Marine Mammal Science,
5(4):315-342.
Myrick, A.C., Jr., A.A. Hohn, TA.. Sloan, M. Kimura and D.D. Stanley. 1983. Estimating age
of spotted and spinner dolphins {Stenella attenuata and Stenella longirostris) from
teeth. NOAA Tech. Rep. NMFS 30. 17 pp.
Perrin, W.F., and A.C. Myrick, Jr., eds. 1980. Age determination of toothed whales and
sirenians. Reports of the International Whaling Commission, Special Issue 3. 229 pp.
23
SECTION m
POPULATION ABUNDANCE AND STRANDINGS
Lany J. Hansen
Southeast Fisheries Science Center
Miami Laboratory
75 Virginia Beach Drive
Miami, FL 33149
Methods
Estimates of bottlenose dolphin abundance from large-scale aerial surveys of the Gulf
of Mexico (Scott et al. 1989) were compared with historical stranding data. Seasonal
estimates were available for fall (September-October 1983), winter (January-February 1984),
spring (April-May 1984), and summer (July-August 1984) for the northwestern Gulf. For the
northeastern Gulf, seasonal estimates were available for fall (September-October 1985),
winter (January-February 1986), and simimer (June-August 1985). The estimates were
stratified by the following zones: bay (embayments and inshore of barrier islands), inshore
(seaward of the coast or embayment boundaries to the 18.3m isobath), and offshore (18.3m
isobath to 9.3km seaward
of the 182.9m isobath).
The area surveyed is
shown in Figure 1.
M
JOT<
•• 1
Kw tr* 10^
*
■w •»■» *rm »rw
— t ■
ir«
^
^
.--'
^
^
^^
*% f
/•
054
c^rcr^^co \ ^^ J
jrii
-1-
v ^^
■7
*
Vs'.-^i
An aerial survey of
a portion (Block 154) of
the northwestern Gulf
inshore zone along Texas
and Louisiana was
conducted during March,
1990, in response to the
1990 anomalous mortality
event (see Appendix IV
for details). This area, and another adjacent portion of the inshore zone (Blocks 152 and 153)
and an adjacent portion of the offshore zone (Block 054 and B) were surveyed during June,
1990, in response to the MEGABORG oil spill (see Appendix IV). The estimated abundance
for these areas sampled in 1990 was compared to the survey results from during 1984.
Fignrc 1: Aerial »urvcy blocks of the northern Gulf of Mexico used during 1983-86 NMFS
regional surveys for bottlenose dolphins (Scott et al^ 1989), and blocks surveyed dunng
1990 (152, 153. 154, 054, B).
24
Results and Discussion
The 1983-84 abundance estimates of
all zones combined for the northwestern
Gulf were lowest in the winter (January-
February), increased during the spring
(April-May), and remained at that level
through the summer and fall (Figure 2).
The Texas strandings provide the best
sample for comparison with the
northwestern Gulf abundance estimates.
The peak in strandings occurs from
February-April, with monthly levels
approximately equal throughout the rest of
the year (Section 1, Figure 2). Thus, if the
abundance estimates represent the normeil,
seasonal trends, strandings occur during a
period when abundance is apparently
increasing.
The trend in 1985-86 abundance
estimates of all zones combined for the
northeastern Gulf of Mexico differed from
that of the northwestern Gulf, with the
lowest seasonal abundance in the summer
rather than the winter (Figure 3). In fact,
the highest seasonal abundance in the
northeastern Gulf occurred during the
winter. Strandings for the northeastern Gulf
peak during March and April (Section I,
Figure 4), which would correspond to
spring, however a spring abundance
estimate is not available. Based on the
winter and summer abundance estimates
for all zones, it appears that the peak in
strandings occurs while there is an apparent
declining trend in abundance. This is the
opposite of the trend in the northwestern
Gulf.
UJ
o
i
z
r>
CD
<
20
18
16
14 -
12 -
10 -
8 -
6 -
4-
O BAY
V INSHORE
D OFFSHORE
A ALL ZONES
ff
f
V 4
O^r ^
-9-r
w S S F
-I — I — I I I — I — i—T — I — I — \ — r I I
WSSF WSSF
ZONE AND SEASON
WSSF
Fignne 2 Estimates o( seasonal bottlenose cSolphin abundance in
the northwestern Gulf of Mexico (Scott et al., 1989). Error bais
represent 95% confldenoe intervals.
o
I
z
CD
<
. 1
50 -
45 -
40 -
O BAY
V INSHORE
D OFFSHORE
£, ALL ZONES
1
35 -
i
i
30 -
A
25 -
I
1
20 -
■
1
i
1
15 -
1
1
I-
10 -
5 -
0 o6 ^
1 1 1 1 1 1 1 1 1 1 1
— I—
1 1 1 1
— r—
WSSF WSSF WSSF
ZONE AND SEASON
WSSF
Fignre 3: Estimates of seasonal bottlenose dolphin abundance in
the nonheastetn Gulf of Mexico (Soott et aL, 1989). Error bart
represent 95% confidence intervals.
Another aspect of the abundance
estimates to consider is the likelihood of whether or not animals which die in the different
zones will eventually strand. Mead (1979) discussed factors which affect the discovery of
cetacean strandings. He stated that "virtually all cetaceans" are negatively buoyant. Dolphins
25
which die and sink in waters deep enough to keep decomposition gases in solution should
not strand. He also suggested that, because of increased exposure to scavengers and
predators, the greater the distance from shore at which an animal dies or is debilitated, the
less likely the animal will strand. Because of these factors, it is probably reasonable to
assume that most of the animals which strand, died or became debilitated within the bay and
inshore zones.
In the northwestern Gulf, the trends in the bay and inshore zones abundance
estimates have a different pattern than that of all zones combined, which essentially reflects
the trend of the offshore zone. In the inshore zone, fall had the highest estimated
abundance, summer the lowest, and winter and spring had intermediate levels. Using a 95%
c.i. criterion, the inshore summer estimated abundance was significantly different from the
other seasons, but the other seasons were not significantly different from each other. In the
bay zone, winter and fall had the lowest estimated abimdance, with spring and summer
having approximately equal, but higher levels. The bay winter estimate was significantly
lower than the other seasons, but the other seasons were not significantly different from each
other (95% c.i. criterion). If the bay and inshore zones are combined, the apparent trend
follows that of the inshore zone. In any case, if the trend in the estimated seasonal
abundance does reflect the normal pattern, strandings in the northwestern Gulf peak when
abundance in the bay and nearshore zones combined is at intermediate levels and is
declining.
The trends in estimated seasonal abundance in the northeastern Gulf for all zones
combined also reflect that of the offshore zone. However, the trend in the inshore zone is
similar, with the lowest estimated
abundance occurring during the summer
and winter. Estimated abundance in the bay
zone seems to be constant. Keeping in mind
that no spring abundance estimate is
available and that seasonal estimates of
abundance may not be statistically
significant, strandings peak in the
northeastern Gulf while the abundance in
the bay and nearshore zones combined
appears to be declining, which was similar
to the pattern in the northwestern Gulf.
The 1990 point abundance estimates
for Block 154 were higher than the 1984
point estimates for winter, spring, and fall
(Figure 4). Based on the 95% c.i. criterion,
the 1990 March estimate was significantly
different from only the 1984 spring
estimate, while the 1990 June estimate was
iiW -
450-
O 1984
400 -
V 1990
^ 350 -
o
i^ 300-
UJ
o 250-
\
r
z 200 -
" 150 -
T y
f
100 -
{ ^
I
50 -
1
6
1 1
?
JAN
MAR
MAY
JUN
SURVEY MONTH
AUG
Figure 4: Estimates of bottlenose dolphin abundance for aerial
survey Block 154 (sec Figure 1 for location) (Scolt et al., 1989,
and Mullin, this volume). Error bars represent 95% confidence
intervals.
26
significantly different from the 1984 spring and summer estimates. Based on these surveys,
it appears that there may have been more animals in this area during spring and summer
of 1990 than during the 1984 sampling period.
The estimated abundance for Block 153 was also higher for June, 1990, than for the
spring and summer 1984 estimates (Figure 5). This is consistent with the pattern observed
in Block 154. The estimated abundance for the offshore Block 054 for June, 1990, was
significantly higher than the spring 1984 estimate (95% c.i. criterion), and was higher but not
significantly different from the the summer 1984 estimate (Figure 6). In fact, the June, 1990,
estimate was about 20 times higher than the spring 1984 estimate. Similarly, the summer
1984 estimate was about 11 times higher than the spring 1984 estimate. This could indicate
that the pattern observed in 1984 (higher
summer abundance in Block 054) was
evident again in 1990. The 1990 estimates
for these adjacent blocks (153, 154, and
054) were consistently higher than the
estimates from 1983-84. It is important to
note that the boundaries between the
inshore and offshore blocks were not based
on any known distribution patterns of the
Gulf bottlenose dolphins. The available
survey results indicate that the distribution
of these dolphins was more or less
continuous from the shore out to at least
the seaward boundary of the offshore
zones.
90 -
80 -
O 1984
V 1990
TO-
GO •
50 -
40 -
30 -
' r
20-
10 -
6
1 1
o
MAY
JUN
JUL
SURVEY MONTH
Figmc S: Estimaies of boKlenose dolphin abundance for aerial
The results of these aerial surveys
were similar to those conducted prior to the
1987-88 east coast bottlenose dolphin
dieoff. Keinath and Musick (1988) reported
that the results of nearshore aerial surveys
along the Virginia coast suggested
bottlenose dolphin densities were higher
during the dieoff than previous years (1980-
86). However, Scott and Bum (1987)
reported that their analysis of surveys
conducted during the dieoff along New
Jersey and Virginia out to the 1000 fathom
isobath indicated there was a 60% chance
of decline in the offshore stratum
abundance ind'jx, as compared to the 1980-
81 abundance index. Data were insufficient
to evaluate the nearshore stratum (Scott
survey
MuUin,
intervals
Blodc 153 (see Figure 1 for location) (Scott et al., 1989;
this volume). Error ban represent 95% confidence
z
3
70 -
O 1984
V 1990
60 -
50 H
40 -
'
r
X -
■
20 -
<
)
10 -
■^
.
9 ,
JUN
SURVEY MONTH
JUL
Figure 6: Estimates of bottlenoae dolphin abundance for aerial
survey Block 054 (see Figure 1 for location) (Scott cl aL, 1989;
Mullin, this volume). Error bar* represent 95% confidence
intervals.
27
and Bum, 1987). Although the information is limited, it appears that anomalous monalities
of bottlenose dolphins during 1987-88 and during 1990 may have occurred when apparent
densities in nearshore areas were higher than previously observed, but the pattern in
offshore densities was not similar.
Literature Cited
Mead, J.G. 1979. An analysis of cetacean strandings along the eastern coast of the United
States. Pages 54-71 in J.B. Geraci and DJ. StAubin (eds.). Biology of marine
mammals: insights through strandings. NTIS No. PB-293 890. 343 pp.
Keinath, J.A., and J.A. Musick. 1988. Population trends of the bottlenose dolphin {Tursiops
tmncatus) in Virginia, 1980-87. NfMFS/SEFC Miami, Contract Report No. 40-GENF-
800564.
Scott, G.P., and D.M. Bum. 1987. The potential impact of the 1987 mass mortahty on the
Mid- Atlantic offshore stock of bottlenose dolphins. NMFS/SEFC Miami, Contribution
No. ML-CRD-87/88-10.
Scott, G.P., D.M. Bum, LJ. Hansen and R.E. Owen. 1989. Estimates of bottlenose dolphin
abundance in the Gulf of Mexico from regional aerial surveys. NMFS/SEFC Miami
Contribution No. CRD-88/89-07.
28
SECTION IV
FOOD HABITS
Nelio B. Barros
Division of Biology and Living Resources
Rosensnel School of Marine and Atmospheric Science
University of Miami
Miami, FL 33149
An unusual mortality of bottlenose dolphins in the Gulf of Mexico (primarily along the
Texas coast) took place during early 1990. Due to the implication of food habits in the
recent mortalities of bottlenose dolphins (Geraci 1989) and humpback whales (Geraci et al.
1989) in the eastern coast of the United States, there was concern of a similar occurrence
in the Gulf of Mexico during 1990. This study analyzes the food habits of bottlenose dolphins
stranded along the coast of Texas during January- April 1990 and compares the results with
a previous study in the same area (Barros and Odell 1990). Samples obtained from 26
dolphins stranded in Matagorda Bay in late January 1990 were not available for examination
and were not included in this study.
Methods
Samples (entire stomachs) were collected from 38 stranded dolphins and frozen for
analysis (see Appendix I for details on stranding date, location, sex, age, etc.). This sample
was comprised of all the whole stomachs that were coUected and frozen; it was not possible
to determine if the sample was representative of the animals that died during January-April
1990. One stomach was empty and the remaining 37 had food matter (Table 1). In this
sample, dolphins ranged in size (total length) from 160 to 269 cm. Fish otoliths, squid beaks
and shrimp rostra and/or telsons were the structures utilized in prey identification, following
methodology outlined in Barros and Odell (1990).
Results and Discussion
Wet weight of the 37 stomach contents ranged from 1 to 3382 g (Table 1), with a mean
of 237.95 g per stomach. When only stomachs with contents weighing 10 g or more are
considered (see Barros and Odell 1990) this figure increases to 398.95 g (n= 22). The
number of prey items per stomach averaged 219.14 (SD= 287.80, n= 37) and number of
prey taxa 9.41 (SD= 5.49, n= 37) (Table 1). A total of 15,950 fish otoUths (13,816 sagittae,
2,124 lapilli, 10 asterisci), 1,681 (890 upper and 791 lower) squid beaks, and remains
(rostra/telsons) of 59 crustaceans were found in all stomachs, representing 7,109 fish, 915
squid and 59 crustaceans. The categories of prey type were: fish only (F): 9 (24%); fish and
29
cephalopod (F,C): 23 (62%); fish, cephalopod, crustacean (F,C;K): 4 (11%), and fish and
crustacean (F,K): 1 (3%) (Table 1). Altogether, 46 species offish (19 of which could not be
identified) distributed in 11 families, and 3 species of cephalopods and 2 species of
crustaceans were identified (Table 2). Six prey species occurred in more than 50% of the
stomachs: the Atlantic croaker (Micropogonias undulatus), silver perch (Bairdiella chrysoura),
brief squid (LoUiguncula brevis), sand seatrout (Cynoscion arenarius), and a unidentified
teleost, and accounted for 57% of all prey taken. The family Sciaenidae was the most
important fish family, accounting for 64% of all fish prey. Cephalopods of the family
Loliginidae dominated this category, numbering 914 of the 915 specimens found. Shrimp of
the genus Penaeus comprised 58 of the 59 crustaceans identified in all stomachs.
A comparison of these results with the study of Barros and Odell (1990) (Table 3)
indicates that bottlenose dolphins in this study did not differ significantly in their food habits.
Thus, the wet weight of the stomach contents, an indicator of stomach fullness, was not
statistically different in both studies (p>0.05, t-test; data subjected to a natural logarithm
transformation to comply with normality and homoscedasticity of variances), the same being
true for the number of prey items and prey taxa in each stomach (p>0.05, t-test). In
addition, the categories of prey types (fish, cephalopod, crustacean) were also present in
similar proportions (p>0.05, chi-square test) in the two studies. Four out of the six most
important prey (M. undulatus, C. arenarius, B. chrysoura, and L. brevis), numerically and in
terms of frequency of occurrence, were the same in 1986-87 and 1990.
Although there are differences in the two data sets (samples analyzed in Barros and
Odell (1990) were collected during a 2-year period (1986-87), and only from dolphins
stranded in the vicinity of Galveston; samples from the present study were collected from
dolphins stranded along the entire coast of Texas during early 1990), the results obtained
in the present study show that bottlenose dolphins stranded during the 1990 Gulf of Mexico
mortality event had a similar prey spectrum as in years of no unusual mortality. These
results, although preliminary, suggest that the food habits of these dolphins were not
significantly altered during the mortality event.
30
Table I. Stomach contents of bottlenose dolphins from the 1990 Gulf of Mexico mortality (n = 37).
Field
#C #SE Sex' Length Stomach Prey^ Prey Prey
(cm) CtW(g)^ Type Taxa Items
Season^
ccoss
orw
4993
M
216
171
F,C
12
297
W
GA290
C82S
4892
M
(257)
116
F
3
7
W
GA291
C824
4893
M
(213)
5
F,C
8
130
W
GA293
C844
4895
M
(182)
<1
F,C
4
28
w
GA297
rssi
4899
U
(200)
59
F.C
13
101
w
GA298
r8')2
4900
F
(239)
- <1
F.C
4
10
w
GA299
r8S3
4901
U
(184)
44
F.C
14
267
w
GA301
C855
4903
F
(239)
<1
F
6
18
w
GA302
CH'iS
4906
M
262
258
F.CJC
12
402
w
GA304
C860
4904
F
219
214
F.C
6
22
w
GA311
(jjf/e
4913
F
233
39
F.C
10
189
Sp
GAS 12
C877
4914
F
233
645
F.CJC
19
788
Sp
GA313
C878
4915
F
(236)
2
F
9
948
Sp
GA314
OW4
4916
M
260
<1
F.C
3
51
Sp
GAS 15
C885
4917
F
269
<1
F.C
7
183
Sp
GAS34
LV21
5170
M
194
638
F.C
15
730
Sp
GASS5
C9S1
5174
F
245
<1
F
5
8
Sp
GA344
C955
5411
F
206
824
FJC
10
173
Sp
PA18S
C843
4980
F
205
39
F.C
15
315
w
PA189
C881
4986
F
198
3
F.C
9
875
Sp
PA192
C920
5163
F
243
6
F.C
8
63
Sp
PA195
C940
53%
F
240
220
F.CK
24
319
Sp
P0121
C826
4955
M
256
1424
F.C
21
1044
w
P0122
C827
4956
U
238
74
F
5
16
w
P0123
C837
4957
M
229
<1
F.C
9
41
w
P0125
C8S9
4959
F
245
174
F.C
22
327
w
P0127
C846
4%1
M
(179)
<1
F
3
5
Sp
P01S4
C873
4968
F
254
<1
F.C
8
68
Sp
P0135
C874
4969
F
211
3382
F.C
7
74
Sp
P01S6
C875
4970
F
160
42
F.C
5
16
Sp
P0137
r882
4971
U
201
94
F.C
10
135
Sp
P0141
C897
4975
F
247
118
F.CK
9
137
Sp
SPlll
C842
4857
M
254
<1
F
6
31
w
SP112
C856
4858
M
260
16
F.C
10
114
w
SP114
C862
4860
F
251
<1
F
1
3
w
SP115
C870
4861
F
(250)
112
F.C
10
152
Sp
SP12S
C948
5404
M
208
74
F
6
21
Sp
' M = male; U « unlcnown; F " Female
^ Stomach contents weight m g
' F = fish; C • cephalopod; K « cnisiacean
* W = winter, Sp « cpring
31
Table 11. Frequency of occurrence (P.O.) and number of prey (N) taken by botUenose dolphins (n= 37) from
the 1990 Gulf of Mexico mortality.
PREY SPECIES
FAMILY
F.O.
%
N
TELEOSTS
Micropogonias undulatus
Sdaenidae
28
75.7
2152
26.9
Bairdiella cfuysoura
Sciaenidae
26
70.3
1025
12.8
Cynoscion arenarius
Sciaenidae
21
56.8
444
5.6
Unidentified leleost I
-
21
56.8
248
3.1
Stellifer lanceolatus
Sciaenidae
17
45.9
72
0.9
Anchoa sp.
F.ngraulidae
15
40.5
836
10.5
dJ^agodon rhomboides
Sparidae
14
37.8
121
1.5
Menticirrhus sp.
Sciaenidae
12
3Z4
85
1.1
Cynoscion nothus
Sciaenidae
11
29.7
477
6.0
Porichthys plectrodon
Batrachoididae
11
29.7
204
2.6
Urophycis sp.
Gadidae
11
29.7
147
1.8
Leiostomus xanthums
Sciaenidae
11
29.7
44
0.6
Unidentified teleost II
—
9
24.3
105
1.3
Cynoscion nebulosus
Sciaenidae
8
21.6
85
1.1
Unidentified pleuronectifonn I
—
8
21.6
78
1.0
Mugil cf. M. cephalus
Mugilidae
8
21.6
20
0.3
Larimus sp.
Sciaenidae
7
18.9
119
1.5
Unidentified pleuronectiform II
-
6
16.2
387
4.8
lOrthopristis chrysoptera
Sparidae
6
16.2
12
0.2
Trichiurus lepmrus
Trichiuridae
5
13.5
50
0.6
Synodus foetens
Synodontidae
5
13.5
35
0.4
Ariusfelis/Bagremarinus
Ariidae
5
13.5
32
0.4
Unidentified dupeid
-
4
10.8
6
0.1
Pogonias cromis
Sciaenidae
3
8.1
44
0.6
Orlhopristis chrysoptera
Sparidae
3
8.1
9
0.1
Cynoscion sp.
Sciaenidae
3
8.1
8
0.1
IPeprilus sp.
Stromateidae
2
5.4
66
0.8
Unidentified teleost III
—
2
5.4
20
0.3
IBrevoortia sp.
Qupeidae
2
5.4
9
0.1
dSardinella aurita
Engraulidae
2
5.4
4
0.1
lOpsanus sp.
Batrachoididae
2
5.4
2
0.0
Pomatomus saltatrix
Pomatomidae
2
5.4
2
0.0
Unidentified engraulid
Engraulidae
Z7
67
0.8
Unidentified teleost IV
—
2.7
39
0.4
Bairdiella IStellifer
Sciaenidae
2.7
17
0.2
Unidentified teleost V
—
2.7
6
0.1
Unidentified teleost VI
_
2.7
4
0.1
Unidentified teleost VII
~
Z7
4
0.1
Unidentified teleost VIII
_
2.7
3
0.1
Unidentified teleost IX
~
2.7
3
0.1
Unidentified teleost X
—
2.7
3
0.0
Opsanus beta
Batrachoididae
2.7
2
0.0
Unidentified teleost XI
-
2.7
2
0.0
32
Table II. Continued
TELEOSTS (ConL)
PREY SPECIES
Unidentified teleost XII
Unidentified teleost XIII
Unidentified clupeiform
ICynoscion sp.
Sciaenops ocellata
Unidentified teleost XTV
Unidentified teleost XV
Unidentified teleost XVI
Unidentified teleost XVII
Unidentified teleost XVIII
Unidentified teleost XDC
FAME.Y
Sciaenidae
Sciaenidae
P.O.
%
N %
1 2.7
I 0.0
1 2.7
1 0.0
1 2.1
I 0.0
1 2.7
I 0.0
1 2.7 ]
I 0.0
1 2.7 ]
I 0.0
1 2.7 ]
[ 0.0
1 2.7 ]
[ 0.0
1 2.7 ]
1 0.0
1 2.7 ]
0.0
1 2.7 ]
0.0
CEPHALOPODS
LoUiguncula brevis
Unidentified loliginid
Doryteuthis sp.
Unidentified ?oaopodid
Loliginidae
Loliginidae
Loliginidae
?Oaopodidae
25 66.7 736 9.2
16 43.2 111 1.4
8 21.6 67 1.8
1 2.7 1 0.0
CRUSTACEANS
Penaeus sp.
Squila empusa
Penaeidae
Stomatopodidae
6
1
16.2
2.7
58
1
0.7
0.0
33
Table HI. Comparison of the food habits of bottlenose dolphins from Barros and Odell (1990) and this study.
Wet weight
Prey items
Prey taxa
Prey type
X'
R'
X
R
SD
n
X
R
SD
n
F
F.C
F.CK
FJC
Barros & Odell (1990)
This study
Statistics
852.67
398.95
N.S.'
25-6550
16-3382
(p>0.05.
1497.01
748 7?
t-test)
18
22
272.83
219.14
N.S.
1-1073
1-1044
(p>0.05.
338.78
287.80
t-test)
23
37
9.48
9.41
N.S.
1-16
1-24
(p>0.05,
4.94
5.49
t-test)
23
37
6(26%)
9(24%)
N.S.
9(39%)
23(62%)
(p>0.05.
8(35%)
4(11%)
chi-
-
1(3%)
square)
u =
X = mean; R ^ range; SD ■= standard deviation; n ^ sample size; N.S. » non-significant; F • Gsb; C • cephalopod; K ~ crusucean
Literature Cited
Barros, N.B., and D.K. Odell. 1990. Food habits of bottlenose dolphins (TUrsiops truncatus)
in the southeastern United States. Pages 309-328 in S. Leatherwood and R.R. Reeves,
eds. The bottlenose dolphin. Academic Press, San Diego, CA.
Geraci, J.R. 1989. Clinical investigation of the 1987-88 mass mortality of bottlenose dolphins
along the U.S. central and south Atlantic coast. Final Report, U.S. Marine Mammal
Commission, Washington, DC, 63 pp.
Geraci, J.R., D.M. Anderson, R.J. Timperi, DJ. St. Aubin, G.A. Early, J.H. Prescott, and
CA. Mayo. 1989. Humpback whales {Megaptera novaeangliae) fatally poisoned by
dinoflagellate toxin. Canadian Journal of Fisheries and Aquatic Sciences 46:1895-
1898.
34
SECTION V
ENVIRONMENTAL FACTORS
Robert A, Blaylock
Southeast Fisheries Science Center
Miami Laboratory
75 Virginia Beach Drive
Miami, FL 33149
Background
The National Marine Fisheries Service monitors marine mammal mortalities in the
Gulf of Mexico with the cooperation of the Southeast Marine Mammal Stranding Network
(SEUS). This is a mainly volunteer network of federal, state, and imiversity researchers
having varying levels of expertise and resources. In January-March 1990, bottlenose dolphin,
Tursiops truncatus, stranding in the northern Gulf of Mexico received an unusually high
degree of pubhc attention. The highest incidence of bottlenose dolphin stranding was
reported in Texas. This may reflect a higher level of detection and reporting effort in Texas
rather than a higher mortality rate; however, the relatively large Texas data base allowed
analysis of bottlenose dolphin stranding in relation to the physical environment. This study
examined environmental variables and the Texas bottlenose dolphin stranding record for
relationships which might account for the
high stranding incidence reported in early
1990.
Methods
Environmental factors
The Texas Gulf of Mexico coast was
stratified for analysis into five statistical
zones of equal lengths of shoreline totahng
approximately 556 km (300 nm) and
extending from north of Galveston to the
U.S.-Mexico border (Areas I-V, Figure 1).
Bottlenose dolphin stranding records listing
the species stranded, date of discovery, and
location of the stranding and other
pertinent information were obtained firom
SEUS and the Smithsonian Institution
TEXAS
GULF
OF
MEXICO
Fignre 1. Locations of environmental dau stations along Texas coaxt.
Filled circles show Elunan transport stations and unOlled circles show
temperature data station locaiioiis.
35
Marine Mammal Stranding Program. Stranding data from the period January 1986 through
June 1990 were selected for analysis because reporting effort in Texas became consistent
begiiming in 1986.
60
200
ISO
100
-
1—1
so
n
-
r— 1
r— 1
CO
50
MEAN
Q. 40
-I
o
o
30 - ^
20 -
o
UJ
Q
Z
<
V) 10 -
S2 U 84 85 as 87 86 09 80
YT>R
Figmc 2. Yearly bottlenose dolphin stranding rqxsrts bom
the Texas coast from 1982 through 1990.
i^ftftFiFiA^i^
ZmKQ:>-Z-lC9Q.H->U
<UJ<Q.<333lijOOLLl
-3ii-2<2-3'9<(nOza
MONTH
Flgiire 3. Monthly mean bottlenoce dolphin stranding along
The Texas Marine Mammal Stranding Texas coast, 1986-1990, with standard deviations.
Network (TMMSN) was established in 1980 and stranding reports increased yearly through
1985 (Figure 2). After 1985, stranding reports remained more or less constant indicating
that marine mammal stranding detection and reporting had become somewhat consistent (G.
Barron, TMMSN, pers. comm., March 1991). Average monthly bottlenose dolphin stranding
rates in Texas were low during July through December (Figure 3) and no data were
available past June 1990.
Daily air and sea surface temperature and salinity data were obtained from
NOAA/NOS, Tidal Datum QuaUty Assurance Section, Rockville, Maryland, for four stations
along the Texas Gulf coast (Figure 1). Air and sea surface temperature and salinity data
were available for only three sections of the Texas Gulf Coast (Table 1). Monthly dolphin
stranding rates were regressed against monthly mean temperature and salinity values from
each section.
Table 1. Locations of Texas/Gulf of Mexico environmental data stations (sea surface and air temperatures, and salinity).
STATION NAME
SECTION
LA'ITI-UDE
LONGrrUDE
GALVESTON PIER21
1
29° 18.6'N
94° 47.6'W
GALVESTON PLEASURE PIER
I
29° 17.2-N
94° 47.4'W
FREEPORT HARBOR
II
28° 56.8-N
95° ISJ-W
PORT MANSFIELD
V
2<°333*N
97°25.8'W
36
Offshore transport data (Mt/sec/100 km) were obtained from the NOAA/NMFS,
Pacific Environmental Laboratory, Monterey, California, for locations corresponding to the
mid-point of each statistical area at the 10 fm contour (Figure 1). A negative value
indicated a net shoreward movement. These data were available for all five sections (Table
2) and stranding data from each section was analyzed by regression with offshore transport.
Table 2. Appnmmate
kxations
of Tons/Gulf of Monca
ElcDun
transpon
dau ttaiioDS.
^
SECTION
UilTlUDE
LONOrrUDE
I
28°5TN
94°22'W
U
28°44*N
95° IS-W
III
28°14*N
9<S» 26W 1
rv
27° 24'N
vrii-w 1
v
26»29'W
97°05'W 11
Statistical Analyses
Single factor analysis of variance (ANOVA) was used to examine differences in
bottlenose dolphin stranding rate among months and years. ANOVA was used to examine
environmental variables for significant differences among months and years. Possible
relationships between monthly stranding rates and environmental variables were evaluated
using linear regression and ANOVA. Pairwise comparisons were performed using the
Student-Newman-Keuls test (SNK test) if ANOVA determined significant differences.
Significance was determined with o = 0.05.
Results
Stranding rates
Bottlenose dolphin stranding in the Texas Gulf Coast peaked annually in March
during the period January 1986 through June 1990 (Figure 4). Stranding rates did not differ
significantly among the months January-June except for during March when the stranding
rate was significantly higher (P < 0.01, N = 30). Comparison of monthly stranding rates for
January through May, 1986-1990 and December 1985-1989, using ANOVA detected no
significant difference in stranding rates among years (P = 0.46, N = 30); however, the
relatively low power of the ANOVA (1 - B - 0.55) suggested that there might be significant,
but undetected, differences between years. Using the upper 95% confidence interval on the
1986-1990 monthly stranding means as a boundary criteria for detecting significant
differences, the number of strandings during January-March 1990 appeared significantly
greater than during the preceding four years (Figure 4). Table 3 lists bottlenose dolphin
strandings by month for the period January-June, 1986-1990, along the Texas coast.
37
Tabic 1 BotUcnose dolphin strandings from Texas coait January-June, 1986-1990. Mean a 1986-1990; S£. - standard erron
upper 95% confidence level (UCL) calculated with t(..ao}jif.4)-2.132.
MONTH
1986
1987
1988
1989
1990
MEAN
S£.
UCL
JAN
10
5
8
11
42
15
6.1
28
FEB
10
19
19
8
40
19
23
30
MAR
42
50
37
32
59
44
3.0
53
APR
29
36
28
19
16
26
2.7
32
MAY
0
10
3
0
4
3
1.7
7
JUN
4
4
3
0
4
3
0.7
4
JAN-JUN
95
124
98
72
163
110
8.2
128
Sea and air temperature
Monthly and annual mean sea surface
and air temperatures varied significantly among
months (Table 4), among years (Table 5), and
among months within years (Tables 6 and 7).
The regression slope of stranding against sea
surface temperature averaged for the entire
Texas coast was significant; however, low
correlation coefficients indicated that there was
no simple linear relationship between bottlenose
dolphin stranding and temperature variables
(Table 8). No significant relationships were
detected between dolphin stranding and
temperature variables for each section treated
individually.
60
50
5 40
30
20
10
JAN
V77> 1986
ES 1987
^1988
^^ 1989
■1 1990
I 1 95X C.I
FEB MAP APR
MONTH
MAY JUN
Figure 4. Bottlenose dolphin tlrandings on Tescas coast
by month, 1986 through 1990.
Table 4. Monthly mean sea surface temperature (SST °C), air temperature (AIR °C), salinity (SAL °lg^ averaged from three
stations on the Texas Gulf Coast for January-June. 1986-1990 and monthly mean bottlenose dolphin strandings from the Texas
coast for the years 1986-1990. Values withm a column having the same letter were not tigniflcantly different (SNK test, a =
0.05, A = highest value, F « lowest value, N • number of data observations).
JAN
13.5 F
14.2 F
24.0 B
407
«
FEB
15.2 E
15.2 E
25.0 AB
379
19
MAR
183 D
18.7 D
25.2 AB
439
44
APR
21.8 C
22.6 C
24.8 AB
402
26
MAY
26.0 B
26.7 B
24.2 B
437
3
JUN
28.9 A
28.1 A
26.1 A
423
3
38
Table 5. Yearly mean ica lurfacc temperatures (SST "C), air temperatures (AIR "Q, and lalmity (SAL °/^) for the ta month
period January-June averaged from three stauons along the Teas Gulf CoasL Values within a cnlumn having the same letter
were not signifjcantly different (ShfK test, a « 0.05, A = highest value, E " lowest value, N « number of data observations)
Strandmgs are for the tame six month period from the entire coast
YEAR
SST
AIR
SAL
N
STRANDINGS
1986
21,2 A
21,8 AB
21J E
soo
95
1987
19,8 B
19.9 D
2i8 D
514
124
1988
20,5 B
203 CD
29.0 A
464
98
1989
20.9 A
21.1 EC
26,2 B
559
72
1990
21.4 A
22.4 A
25,2 C
450
163
Tabic 6. Monthly mean tea surface temperatures (°C) by year along Teacas Gulf Coast. Values with the tame letters were not
tignincantty different (SNK test, a « 0.05, A = highest value, D « lowest value).
YEAR
JAN
FEB
MAR
APR
MAY
JUN
DEC
1985
-
-
-
-
-
-
14.2B
1986
13.7B
16,2A
19,4A
23.2A
25.7BC
29,08
13,88
1987
12.9C
15,08
17.48
20JOC
25,8BC
28.4C
ISSA 1
1988
10.7D
13^C
17,88
21.68
25.4C
28.78C
16.1A
1989
16.0 A
13.9C
17.98
2238
27.0A
28,88c
10.6C
1990
12.7C
16.8A
19.4A
21,88
263B
29.7A
Table 7. Monthly mean air temperatures (°C) by year along Texas Gulf Coast. Values with the tame letters were not
significantly different (SNK test, a ° 0.05, A = highest value, D — lowest value).
YEAR
JAN
FEB
MAR
APR
MAY
JUN
DEC
1985
-
-
-
-
-
-
13JB
1986
14.4BC
15.98
21.0A
23.9A
26.08
28.9AB
13.18
1987
13.0C
14.9BC
17,28
20.68
26.18
28.28
15.5A
1988
10,9D
13.9BC
18.6B
23.0A
27.0A8
23.1 C
16.0A
1989
16,5A
133C
17.68
22,9A
27JA
29.2AB
10.2C
1 1990
15.4AB
17.8A
19.1B
22.6A
26.7A8
31 OA
-
Table & Results of linear regression of monthly botllenoce dolphin ttrandinp on monthly mean tea turface temperature (SST),
air temperature (AIR), salinity (N - 30) and offshore transport (OFFSHORE).
VARIABLE
SLOPE
INTERCEPT
RJ
P
SST
-1.15
41.59
0.14
0.04
AIR
-1.06
39,82
0.11
ojn
SALINITY
0.08
1.56
0.01
0.49
OFFSHORE
-0.004
2.23
0.00
OJO
39
There was a negative exponential
relationship between first semester stranding rates
and the preceding December-January mean sea
surface temperature. The lowest January-
December mean sea surface temperatures
preceded the highest January-June stranding
incidence, 124 strandings in 1987, and 189 in 1990
(Table 6). Regression of log-transformed
January-June stranding totals (logS) against the
corresponding log- transformed December-January
mean sea surface temperatures (logT) resulted in
the following statistically significant (P = 0.02, N
= 5) relationship: logS = 5.4 - 2.951ogT, r^ =
0.89 (Figure 5). A weak (r^ = 0.65) relationship
between dolphin strandings and air temperature
for the same periods was not significant (P =
0.10).
S
V
2.3
1990 \
•
\
\
2.2
\
\
\
\
2.1
-
V '^
\ 'k
^^vl988\ ^^-,
2.0
\19B\
1.9
'\ \ 1989
\ \
1.8
\
\
1.00
1.05
1.10 1.15
LOG TCMPERATURE
1.20
1.25
Figure 5. Relationship between Dec -Jan tea surface
temperature and Jan-Jun strandings on Tocas coast.
1986-1990. LogY - 539 - 2.95 log X r^ - 0.89, P -
0.02.
Sea surface temperature anomaly data from NOAA's Oceanographic Monthly
Summary for December 1989-January 1990 ranged from -0.4 to -2.0 °C. The monthly
temperature means upon which these anomalies are based are the data from the Robinson,
Bauer and Schroeder (1979) climatologies. Because of the historical nature of the
climatology, these data should be considered qualitative; however, the persistence of the
negative anomaly throughout the winter of 1989-90 suggests that sea surface temperatures
were abnormaUy low.
Salinity
Mean salinity varied significantly among months (Table 4), among years (Table 5),
and among months within years (Table 9); however, there was no significant relationship
between bottlenose dolphin stranding and sahnity (r^ < 0.01, P = 0.99).
Table 9. Monthly mean salinity (ppt) by year along Texas Gulf Coast. Values with the same letters were not signiPicantly
different (SNK test, • - 0.05, A - highest value, E - lowest value).
YEAR
JAN
FEB
MAR
APR
MAY
JUN
DEC
1985
-
-
-
-
-
-
15.0E
1986
19.8B
21 3B
23.6B
213C
18.6C
24 .2C
17.6D
1987
I7.8B
21JB
20.8D
25 .28
28.9A
27. 3C
25JC
1988
27 JA
283A
26.8AB
29.2A
293A
32.2A
31. 6A
1989
283A
26.4A
28.5A
25.1B
24.2B
24.2C
29.1B
1990
28.0A
28.2A
26.08
21 .IC
19,5C
27.4B
-
40
Offshore transport
Monthly mean offshore transport varied significantly among years (Table 10), but
there was no significant relationship between overall monthly mean ofishore transport and
monthly mean bottlenose dolphin stranding (r^ = 0.02, P = 0.50, N = 35, Table 6).
Examination oi seasonal stranding and ofishore transport suggested a weak (r^= 0.08), but
significant (P = 0.01, N=41), inverse relationship during the spring (Figure 6).
Tabk 10. Monthly oQshore transpon (MT/100km*ec) by yor averaged from five tutions along Toas Gulf CoaxL Negative
values indicate onshore transport. Values with the tame letten were not significantly diaereni (SNK test, a. ~ 0.05, A « highest
value, C = lowest value).
YEAR
DEC
JAN
FEB
MAR
APR
1985
-56.6B
-
-
-
-
1986
-53.7B
■nsc
-10.4B
19,2B
41^AB
1987
1.2A
-15,2B
-17,7B
-15.0C
28.9B
1988
2,2a
-34,58
-S,8AB
41,7AB
21.18
1989
-84,8C
-7,8B
-5.7AB
26,28
38.4AB
1990
-
183A
21.1A
54.4A
5S.9A
Discussion
Although standard statistical analyses (ANOVA) failed to estabhsh a significantly
higher Texas Gulf Coast bottlenose dolphin monthly mortality rate during the first semester
of 1990 than for the same period during the previous four years, it is clear that there were
a high number of dolphin mortaUties on the Texas coast during January-March 1990. The
low power of the significance test may explain the inability to reject the null hypothesis of
no significant difference between years. Further evidence that 1990 was unusual was given
by the occurrence of 12 bottlenose dolphin strandings in Texas in November 1990 as
compared to a mean of about five strandings for Novemeber 1985-89.
Colder than normal water temperatures may directly affect bottlenose dolphin health
by increasing energy expenditure to maintain body temperature, or may indirectly affect it
by reducing local food supplies. Low sahnity, resulting from increased fresh water runoff,
may similarly affect prey distribution. Alternatively, unusual current patterns may result in
a higher than usual number of stranded dolphin carcasses with no actual increase in
mortahties.
Other than an inverse relationship between winter sea surface temperatures and
spring dolphin mortality rate, these analyses detected no strong significant relationships
between bottlenose dolphin mortahties and other environmental variables. In all of the
relationships between environmental variables and dolphin stranding rates which were
examined, the power of the significance tests (1 - B) exceeded 0.99. The association of low
winter sea surface temperatures with an increased spring dolphin mortality rate suggests the
41
possibility of thermally-induced stress, perhaps
lowering resistance to opportunistic infection;
however, clinical evidence is lacking due to the
small sample size of fresh carcasses. Although
data are available for other species of marine
mammals (see reviews in Gaskin, 1982, and
Whittow, 1987) httle has been published on
thermoregulatory response of Tursiops tnincatus
in spite of its relatively long history of captivity.
Unpublished data suggest that the thermoneutral
Tninimnm for TuTsiops truncatus is approximately
4-5 °C, but may vary with acclimation (S. H.
Ridgway, pers. comm., December 1991).
o
a
X
s
o
o
150
OFFSHORE TOANSPORT
FigOTB 6: Monthly bottlanosc dolphin ■trmndingi a* m
function of oSihorc trmnsport along Texaa coast, iphng
1986- 19M. (Only months with mora than on* reported
stranding w«r« used.)
Alternatively, the observed association
between sea surface temperature and bottlenose
dolphin stranding may be less direct. Gunter
(1941, 1952) noted increased fish mortahties in
the Texas Gulf Coast region associated with cold
weather. Decreased food availabihty could affect dolphin health. An estimated 2.7 million
fish, of which approximately 2.6 milHon were striped muDet (Mugil cephalus), died in East
Matagorda Bay after a severe cold spell in December 1989 and smaller fish kills occurred
in Texas Bays fi-om Sabine to Lagima Madre Bay; however, there was no evidence of a
similar fish kill in the Gulf of Mexico (pers. comm., Larry McEachron, Fish Resources
Program, Texas Parks and Wildlife Dept., Rockport, TX, March 1991).
Striped mullet are reported to be a prey item of bottlenose dolphins in the northern
Gulf of Mexico (Gunter, 1942; Leatherwood, 1975); however, these reports are contradicted
by more recent studies (Barros and OdeU 1990, and Barros, Section FV, this report). It is
possible that, because of unusual weather-related fish migration patterns or fish mortalities,
bottlenose dolphins were forced to switch to alternative prey items which may have been
nutritionally inadequate. Data regarding fish migration during December 1989-March 1990,
were unavailable. All but one of the 38 stranded bottlenose dolphins examined had food
in their stomachs (Barros, Section IV, this report), and comparison of prey items among
Texas-stranded dolphins from earher dates showed no significant differences in prey species.
An inverse relationship between spring bottlenose dolphin stranding rale and offshore
currents (Ekman transport) suggests the possibility of an apparent spring mortahty increase.
An increase in beach-cast dolphin mortalities may merely reflect an increased probability of
washing ashore due to onshore Ekman transport during the spring. Alternative^, an
apparent increase in mortality rate could occur with a seasonal increase in the nearshore
bottlenose dolphin population; however, this did not appear to be the case (see Hansen,
Section III, this report).
42
As a first attempt at establishing a basis upon which to evaluate bottlenose dolphin
mortalities in relation to environment, these results emphasize the general inadequacy of the
Gulf of Mexico marine TnamTnal stranding data base. Texas was the only state for which
there existed a sufficiently accurate time series stranding record for quantitative analysis.
Consistent stranding data for the other states bordering the Gulf of Mexico, with the possible
exception of southwest Florida, were lacking.
Uteratnre Cited
Barros, N. B. and D. K. OdeH 199a Food habits of bottlenose dolphins in the
southeastern United States. Pages 309-328, in S. Leatherwood and R.R. Reeves
(cds.), The Bottlenose Dolphin. Academic Press, New York.
Gaskin,D. E. 1982. The ecology of whales and dolphins. Heinemann Educational Books,
Ltd^ London. 459 pp.
Gunter, G. 1941. Death of fishes due to cold on the Texas Coast, January 1940. Ecology
22(2): 203-208.
Gunter, G. 1942. Contnbutions to the natural history of the bottle-nosed dolphin, Tursiops
tnmcams (Montague), on the Texas coast, with particular reference to food habits.
J. Mammal. 23: 267-276.
Gunter, G. 1952. The importance of catastrophic mortalities for marine fisheries along the
Texas coast J. Wildl. Manage. 16(1): 63-69.
Leatherwood, S. 1975. Some observations of feeding behavior of bottle-nosed dolphins
(Tursiops mmcatus) in the norther Gulf of Mexico and {Tumops cf. T. gUIi) off
southern California, Baja California, and Nayarit, Mexico. Mar. Fish. Rev. 37(9): 10-
16.
Robinson, M., R. Bauer, and £. Schroeder. 1979. Atlas of North Atlantic-Indian Ocean
monthly mean temperatures and mean salinities of the surface layer. Nav. Ocean.
Off. Ref. Pub. No. 18.
Scott, G.P., D. M. Bom, and L. J. Hansen. 1988. The dolphin die-off: Long-term effects
and recoveiy of the populatioiL Proc Oceans '88 Conference, Baltimore, Maryland,
October 31-November 2, 1988.
Whittow, G. C 1987. Thermoregulatory adaptations in marine Tnammak- Interacting
effects of exercise and body mass. Marine Manunal Science 3(3): 220-241.
43
SECTION VI
PHYTOPLANKTON DISTRIBUTION
Patricia A. Tester
Southeast Fisheries Science Center
Beaufort Laboratory
Beaufort, NC 28516
Gymnodinium breve (Steidinger 1990; fonnerly Ptychodiscus brevis) is a toxic
dinoflagellate species generally restricted to the Gulf of Mexico where it is responsible for
red tides, particularly off the west coast of Florida (Baden et al. 1984). This species blooms
sporadically (bloom = 5 x ICP cells 1"^) and can reach concentrations of 2-5 x 10'' cells 1"'
(Woodcock 1948; Steidinger and Ingle 1972). G. breve blooms are thought to be initiated
in offshore coastal waters, primarily in late summer-fall months and transported inshore
(Steidinger, 1975). Dinoflagellates are phototactically positive organisms and are frequently
more abundant in surface waters than at depth. Because of this, winds, currents, and tides
are important factors in the transport (concentration or dispersal) of red tide cells.
Consequently, areas of high cell counts may be patchy even during blooms. Bloom
conditions though, are associated with a stratified water colimin or a defined water mass and
the integrity of this water mass is believed to affect the durations and extent of a bloom
(Steidinger and Haddad 1981).
The more or less armual red tide blooms along the west Florida coast and sporadic
blooms elsewhere in the Gulf cause fish kills, neurotoxic shellfish poisoning, and respiratory
irritation in humans (Gunter et al. 1947; Woodcock 1948; Pierce 1986). Prior to 1987, the
knowledge of the effect of these toxins (collectively known as brevetoxins) on marine
mammals was little more than anecdotal (Gunter et al. 1948). Since the implication of
dinoflagellate brevetoxins in the dolphin deaths of 1987 (Geraci 1989), examination of
phytoplankton samples from areas of high dolphin mortality (especially in the Gulf) is
indicated.
Methods
Water samples for phytoplankton analysis were collected 22-25 March 1990 in the
primary study area between Galveston Bay and the Mississippi delta region (Figure 1). Four
samples were collected at each station: one at the surface, one near the bottom, and the
other two equidistant from each other and the surface and bottom samples. Since no basin-
wide (Gulf) data exist on background G. breve cell concentrations, and little is kiown of its
seasonal occurrences or natural variation except in Florida waters, we obtained a number
of water samples from the northern Gulf for comparative purposes.
44
OriginaUy, all 145 water samples from the primary study area were examined for
presence of G. breve cells. A 2 ml aliquot of the Utermohl preserved material, settled at the
bottom of one liter sample jars, was observed using an inverted microscope. Of these, some
from the deepest bottles from the near-bottom sampling contained too much sediment to
allow reliable observations. Initial observations indicated that more than 78% of 123
remaining samples contained G. breve cells (Appendix V).
Results and Discussion
Seventy-two of the original samples (from the upper half of the water column) were
reexamined in detail and quantitative counts of G. breve cells were made. More than 91%
(66 of 72) of these samples contained G. breve cells and 35% (25 of 72) had cell numbers
>50 cells 1"^ (Appendix V). Areas of highest G. breve concentration at the surface had a
broad seaward distribution with a tongue of higher cell counts onshore near Station 25
(Figure 1). Information or evidence of an offshore bloom outside the primary study area
at the time of sampling was lacking.
The discolored water areas noted during our aerial observations of the primary study
area were caused by high concentrations (9 x 10^ cells 1'^) oiNoctUuca (scintUlans = miliaris),
a phagotrophic dinoflagellate not generally thought to be toxic. Note the distribution of the
Noctiluca bloom (Figure 1, open circles) parallels the western edge of the high G. breve
concentrations.
Since the numbers of G. breve cells in samples from the primary study area were not
high enough to be considered a bloom, we needed some information on "normal" or
background concentrations of G. breve to put our results in context. Surface water samples
were collected from the NOAA Ships OREGON II and FERREL during their routine work
in the Gulf. Examination and quantitative counts of these "comparative samples" allow the
following observations: 1. The incidence of G. breve in the primary study area was high
(89%) but compared well with other nearshore areas of the northern Gulf (Table 1, Figs.
1. 2 (box) and 3).
2. The proportion of samples in the primary study area during March 1990 with G. breve
concentrations >50 cells 1'^ is 2 to 3 times greater than in other nearshore areas of the
northern Gulf or from the same area in September 1990 (Table 1, Figs. 1, 2 and 3),
3. Both the incidence and concentration of G. breve cells is lower in offshore (open water)
areas than on the shelf/nearshore areas (Table 1, Figure 4).
During the examination of "comparative samples" we noted, on two occasions, high
concentrations (5 x 10^ cells T') of the toxic dinoflagellate Gonyaulax monilata (Connell and
Cross 1950). These samples were taken on two different cruises during Sept. and Oct. 1990
from stations very near the Mississippi delta region in the northern Gulf. No G. monilata
were seen in the primary study area.
45
Summary
One hundred and twenty-three phytoplankton samples from the primary study area
were examined for the presence of the toxic dinoflagellate Gymnodinium breve. Eighty
percent of these samples contained G. breve cells. Seventy samples from the upper half of
the water column were examined in detail, and quantitative counts confirmed that 94%
contained some G. breve cells while 65% contained >50 cells r\ These concentrations are
far below those considered a 'TDloom" (>5 x 10^ cells 1'^). Comparative samples from other
areas in the Gulf suggest that G. breve concentrations in the primary study during the March
1990 sampling period were within "normal backgroimd levels" but consistently higher than
quantitative counts of samples from similar areas or from the primary study area later in the
sunamer.
The discolored water patches noted during aerial observations of the primary study
area were blooms (approx. 1 x 10^ cells 1"^) of the dinoflagellate NoctUuca spp. This genera
is not normally known to be toxic. However, it should be noted that a toxic dinoflagellate
species, Gonyaulax monalata, was found in elevated concentrations near the Mississippi delta
in late summer 1990.
Literature Cited
Baden, D.G., T.J. Mende, M.A. Poli and R.E. Block. 1984. Toxins from Florida's red tide
dinoflagellate Ptychodiscus brevis. Pages 359-367 in E.P. Ragelis (ed.), Seafood toxins.
American Chemical Society, Washington, D.C.
Connell, C.H. and J.B. Cross. 1950. Mass mortality of fish associated with the protozoan
Gonyaulax in the Gulf of Mexico. Science 112:359.
Geraci, J.R. 1989. Qinical investigations of the 1987-1988 mass mortality of bottlenose
dolphins along the U.S. central and south Atlantic coast. Final report to National
Marine Fisheries Service and U.S. Navy, Office of Naval Research and Marine
Mammal Commission, April 1989. 63 pp.
Gunter, G., G.F. Walton Smith, and R.H. Williams. 1947. Mass mortality of marine animals
on the lower west coast of Florida, November 1946-January 1947. Science 105:256-257.
Gunter, G., R.H. Williams, C.C. Davis and F.G. Smith. 1948. Catastrophic mass mortalities
of marine animals and coincidental phytoplankton bloom on the west coast of
Florida, November 1946 - August 1947. Ecological Monographs 18:309-324.
Pierce, R.H. 1986. Red tide {Ptychodiscus brevis) toxin aerosols: a review. Toxicon 24:955-
965.
46
Steidinger, K-A. 1975. Basic factors influencing red tides. Pages 153-162 in V.R. LoCicero
(ed.), Proceedings of the first international conference on toxic dinoflagellate blooms.
Massachusetts Science and Technology Foundation, Wakefield, MA.
Steidinger, K-A. 1990. Species of the tamerensislcatenella group of Gonyaulax and the
fucoxanthin derivative-containing Gymnodiinoids. Pages 11-16 m E. Graneli et al. (eds),
Toxic marine phytoplankton. Elsevier.
Steidinger, YLA. and IC Haddad. 1981. Biological and hydrographic aspects of red tides.
BioScience 31:814-819.
Steidinger, ICA. and R.M. Ingle. 1972. Observations of the 1971 summer red tide in Tampa
Bay, FL. Environmental Letters 3:271-278.
Woodcock, A-H. 1948. Notes concerning human respiratory irritant associated with high
concentrations of plankton and mass mortalities of marine organisms. Journal of
Marine Research 7:56-62.
47
Table 1. Comparisons of Gymnodinium breve concentrations in the Gulf of Mexico. Comparison are for
surface samples only.
Gulf of Medoo
location
Number of
samples
% of samples
with C. breve
% of samples
with >50 cells l'
Nortbwcst-
Primary Study Area
GalvestoD Bay to
Mississippi Delta
RV PELICAN
March 1990 Fig. 1
35
»
29
Norttawcsi -
Comparative Area
Entire Teacas coast
FRV FERREIX, Sept-
OcL 1990 Fig. 2
3S
S2
3
Subset - of data above
Stations which cor-
respond to those in
primary study area.
Fig. 2 (box)
9
»
11
Northern Golf -
Comparative Area
Northern Florida
to Galveston Bay
FRV OREGON II
ScpL 1990 Fig. 3
<3
M
11
Subset - of data above
Sutions which cor-
respond to those in
primary study area.
Fig. 3 (box)
6
33
17
North Ccntn] -
Comparative Area
Offshore
FRV OREGON II, April-
May 1990 Fig. 4
66
15
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SECTION vn
Sununaiy of Brevetoxin Analysis
Staff
Southeast Fisheries Science Center
75 Virginia Beach Drive
Miami, FL 33149
The mass mortality of bottlenose dolphins along the U.S. east coast during 1987-88
was believed to possibly have been caused by a naturally-occurring neurotoxin (Geraci, 1989;
Anderson and White, 1989). The suspected neurotoxin, brevetoxin, is produced by a toxic
dinoflagellate, G. breve. Because of the previous implication of brevetoxin as a cause of mass
mortalities of bottlenose dolphins, a total of 50 bottlenose dolphin liver samples were
analyzed for individual brevetoxins. Forty of the samples were from January-June, 1990, Gulf
of Mexico strandings; 10 were control samples. The control samples were included to
determine if the assay methods could accurately detect samples spiked with brevetoxin. The
brevetoxin analysis was conducted under contract; the contract report is presented in
Appendix VI. TTie results of the analysis are summarized and discussed below.
Summary and Discussion
Toxicity was determined by several methods: 1) fish bioassay - Gambusia affinis, fish
death at a fixed interval indicates toxin present but does not necessarily indicate brevetoxin;
2) HPLC separation of toxin fractions - HPLC separation provides a means to confirm or
deny the presence of brevetoxins in comparison to valid PbTx-standards; 3)
Radioimmunoassay provides a means to positively identify brevetoxin-like materials and is
sensitive to authentic PbTx-3.
Following the first thin-layer chromatography (TLC) plate, 33 of the 50 samples were
found non-toxic in the fish bioassay and were not tested further. Of the remaining 17
samples that tested positive in at least one fraction of the first TLC plate, nine had multiple
toxic fractions. Of the 17 samples, 12 tested negative by fish bioassay foUowing the second
TLC plate. Of the five fractions found toxic after the second TLC separation, three were
judged to be in such limited quantity to preclude further TLC separation. The other two
retained toxicity after the third TLC separation.
The three toxic fi"actions of limited quantity were judged to contain less than 5ug
toxin/total original sample by HPLC; this was presumed to be a negative result. The other
53
two fractions, purified through the third TLC, appeared to contain PbTx-2 by HPLC
separation and co-elution. Radioimmunoassay was performed on these five fractions, using
tritiated PbTx-3 as the internal displacement standard. Based on this assay, the three
fractions purified through 2 TLC steps contained 10.2, 12.2, and 9.33 ng toxin/g liver; the two
fractions purified through 3 TLC steps contained 17 and 240ng toxin/g liver.
The process of extraction, purification, chromatographic separation, and
radioimmunoassay conducted on the 50 samples led to the conclusion that five of the
samples contained brevetoxin or some very similar toxin. Reported concentrations in original
samples were calculated by proportion of sub-sampling at the various steps and were based
on "PbTx-3 equivalents" in the radioimmunoassay.
Of the five toxin-spiked control samples only one was detected as containing
brevetoxin; this sample was spiked with the largest amoimt of PbTx-3, 25ug. Two other
samples were spiked with 20 and 15ug of PbTx-3 respectively, but were not identified as
containing brevetoxin. PbTx-1 and PbTx-2 were also added to several of the samples; PbTx-1
is known to hydrolyze quite quickly. The fact that purified toxins "stick" to glass- and plastic-
ware may expleiin the low level of apparent spike of the liver samples. It is quite possible
that neither the PbTx-1 or PbTx-2 spikes were effective, or it is possible that they do not
effectively displace radio-labeled PbTx-3 in the radioiiim[iunoassay.
Of the five carrier-spiked control samples (treated with MeOH only), three were
identified by the radioimmunoassay as containing brevetoxin. It is difficult to explain this
finding. The other two carrier-spiked samples were found to be negative when purified to
the second TLC step. It is possible that an interfering substance was removed in the early
cleanup phases of some of the controls and not in others.
The sample reported to contain the largest cmiount of brevetoxin, as determined by
radioimmunoassay, was one of the non-toxin (MeOH only) spiked control samples. The only
dolphin liver sample from the strandings that was identified as containing brevetoxin at all
stages contained 10.2 ng toxin/g liver. This level of toxin is considered to be very low.
The problems encountered in properly identifying the spiked and non-spiked control
samples raised serious questions concerning the efficacy of this assay method for detecting
brevetoxin in bottlenose dolphin liver samples. The assay of the control samples resulted in
both false-positives and false-negatives. Certainly, the results of this brevetoxin analysis
caimot be considered conclusive. That is, based on the incorrect assay results of the control
samples, brevetoxin poisoning cannot be ruled out as a proximate cause or factor in the 1990
bottlenose dolphin strandings. These results also indicate that other studies (e.g., Geraci
1989) of brevetoxin poisoning in bottlenose dolphins which have employed similar assay
methods without adequate controls (both known positives and known negatives) should also
be considered inconclusive.
54
Xhe author of the contract report (Appendix VI) suggests that samples should
continue to be coUected so that assays for brevetoxin detection may be refined. His research
group will be conducting collaborative research on the assay of brevetoxins in marine animal
tissues- this process should assist in the further development and verification of the assays.
A major difficulty in establishing an assay of this type is obtaining a true "control liver"
sample, known to be free of toxins or other substances that interfere with the assay.
Literature
Cited
Anderson, DJ^^ and A.W. White. 1989. Toxic dinoflagellates and marine mammal
mortalities: proceedings of and expert consultation held at the Woods Hole
Oceanographic Institution. Woods Hole Oceanog. Inst Tech. Rept., WHOI-89-36
(CRC-89-6). 65 pp.
Geraci, J.R. 1989. Clinical investigations of the 1987-1988 mass mortality of bottlenose
dolphins along the U.S. central and south Atlantic coast. Final report to National
Marine Fisheries Service and U.S. Navy, Office of Naval Research and Marine
Mammal Commission, April 1989. 63 pp.
55
SECTION vra
Chemical Contaminants in Bottlenose Dolphins
Stranded along the Gulf of Mexico during IS^O
Usha Varanasi, Karen L. Tilbury,
Donald W. Brown, Margaret M. Krahn, Catherine A. Wigren,
Robert C. Qark, Sin-Lam Chan
Northwest Fisheries Center
Environmental Conservation Division
2725 Montlake Boulevard East
Seattle, WA 98112
56
The author of the contract report (Appendix VI) suggests that samples should
continue to be collected so that assays for brevetoxin detection may be refined. His research
group will be conducting collaborative research on the assay of brevetoxins in marine animal
tissues; this process should assist in the further development and verification of the assays.
A major difficulty in establishing an assay of this type is obtaining a true "control liver"
sample, known to be free of toxins or other substances that interfere with the assay.
Literature Cited
Anderson, D^., and A-W. White. 1989. Toxic dinoflageUates and marine mammal
mortalities: proceedings of and expert consultation held at the Woods Hole
Oceanographic Institution. Woods Hole Oceanog. Inst Tech. Rept., WHOI-89-36
(CRC-89-6). 65 pp.
Geraci, J.R. 1989. Clinical investigations of the 1987-1988 mass mortality of bottlenose
dolphins along the U.S. central and south Atlantic coast. Final report to National
Marine Fisheries Service and U.S. Navy, Office of Naval Research and Marine
Mammal Commission, April 1989. 63 pp.
55
CHEMICAL CONTAMINANTS IN BOTTLENOSE DOLPHINS
STRANDED ALONG THE GULF OF MEXICO DURING 1990
report to
Dr. Nancy Foster
Director, Office of Protected Resources
National Marine Fisheries Service
NOAA
by
Usha Varanasi, Karen L. Tilbury,
Donald W. Brown, Margaret M. Krahn, Catherine A. Wigren,
Robert C. Clark, Sin-Lam Chan
Environmental Conservation Division
Northwest Fisheries Center
National Marine Fisheries Service
National Oceanic and Atmospheric Administration
2725 Montlake Boulevard East
Seattle, Washington 98112
September 20, 1991
57
TABLE OF CONTENTS
Introduaion 59
Methods 61
Sample Collection 61
Analyses for Metals 61
Analyses for Chlorinated Hydrocarbons 62
Quality Assurance Measures for Metals and Chlorinated
Hydrocarbons 62
Analyses for Percent Lipid 63
Analyses for DNA-xenobiotic Adducts 63
Results 64
Metals in Liver and Kidney 64
Chlorinated Hydrocarbons in Blubber and Liver 64
Quality Assurance Measures for Metals and Chlorinated
Hydrocarbons 65
DNA-xenobiotics Adducts in Livers 65
Discussion 66
Metals in Liver and Kidney 66
Chlorinated Hydrocarbons in Blubber and Liver 67
DNA-xenobiotics Adducts in Livers 69
Summary and Recommendations 70
Acknowledgments 71
Bibliography 72
Figures 76
Tables 80
58
INTRODUCTION
In recent years there have been several notable strandings of bottlenose
dolphins (Tursiops truncatus) along the U.S. Coast Approximately 740 dolphins
were stranded along the East Coast in 1987-1988 and another 350 along the Gulf
Coast in 1990. One of the reports on the earlier dolphin strandings (Geraci
1989) suggests that environmental contaminants found in the dolphin tissues were
of possible health concem even though the direct cause of death was possibly due
to brevetoxin. After the 1990 stranding, there was renewed concem that
environmental pollutants might be instrumental in Ae in[q)aired health of these
animals.
Elevated levels of chlorinated hydrocarbons (CHs) — including PCBs,
DDTs and other CHs (e.g. pesticides) — and certain metals have been reported in
a number of marine mammal species. For example, CHs and heavy metal
contaminants have been reported in white-beaked dolphins (Lagenorhynchus
albirostris) and pilot whales (Globicephala melaena) from Newfoundland, Canada
(Muir, et al. 1988), striped dolphins (Stenella coendeoalba) from Japan (Honda,
et al. 1983), short-finned pilot whales (Globicephala macrorhyncha) and long-
snouted dolphins (Stenella longirostris) from the Caribbean (Gaskin, et al. 1974),
and a bottlenose dolphin {Tursiops gephyreus), franciscana dolphins {Pontoporia
blainvillei) and a pigmy speim whale (Kogia breviceps) from the Southwest
Atlantic beaches in Argentina (Marcovecchio, et al. 1990).
Although the literature on bottlenose dolphins is more limited than for
certain other marine manunal species, environmental contaminants have also been
identified in this species of dolphins by several research groups. O'Shea, et al.
(1980) reported high concentrations of PCBs (- 440 jig/g, wet weight) and DDTs
(~ 2200 fig/g, wet weight) in blubber of two bottlenose dolphins from California.
Similarly, the concentration of total PCBs in blubber of one bottlenose dolphin
calf from Cardigan Bay, England (Morris, et. al. 1989) was also elevated
(~ 290 ^g/g, wet weight). Two bottlenose dolphins sampled near the British Isles
had somewhat elevated concentrations of mercury (- 22 ^g/g, wet weight) in
liver (Law, er a/. 1991). In addition, Geraci (1989) reported the mean
concentration of PCBs in blubber of a subset of dolphins (n = 56) from the 1987-
59
1988 strandings was - 142 ± 1 10 ^g/g (wet weight). Kuehl, et al. (1991 ) also
analyzed for PCBs, and other halogenated compounds in a different subset of
dolphins (n = 7) from the 1987-1988 stranding; the results for CHs were similar
to those found by Geraci.
In this study, we analyzed samples of blubber, liver and kidney from 20
bottlenose dolphins from the 1990 Gulf Coast stranding for a broad spectrum of
chemical contaminants such as (THs and certain toxic metals. Chlorinated
hydrocarbons are among the most widespread and persistent chemical
contaminants in the near coastal environment and can accumulate in the lipid-rich
blubber tissue as well as in the liver of marine mammals. Several metals were
analyzed because of their toxicological significance and their possible
accumulation in the liver, as well as in the kidney — an extrahepatic organ with a
specific affmity to certain toxic metals (e.g. mercury).
In additon to CHs and toxic metals, polycyclic aromatic hydrocarbons
(PAHs) are ubiquitous pollutants in urban areas. However, because of extensive
metabolism by marine mammals and fish, PAHs are not detected in their original
fomi. The presence of PAH metabolites can be determined by analysis of bile, a
biological fluid which accumulates PAH metabolites, and has been effective in
demonstrating PAH-exposure in harbor seals and sea lions (Varanasi et al. 1991).
However, dolphins do not have gall bladders, thus precluding the use of this
technique. Levels of DNA-xenobiotic iddncts (Varanasi et al. 1989A) which
represent the binding of contaminants such as carcinogenic PAHs to DNA may be
a useful indicator of exposure to PAHs, and hence we attempted to analyze liver
samples for DNA-adducts.
The results to date showed that several of these stranded dolphins contained
elevated concentrations of PCB, DDTs and certain metals. DNA analyses showed
that samples with better integrity need to be tested before drawing any firm
conclusion with regard to PAH-exposure of the manunals.
60
METHODS
Sample Collection
Samples of blubber, liver and kidney, collected from a subset (n = 20) of
about 350 bottlenose dolphins that were beached along the Gulf of Mexico from
February to June, 1990 (Figure 1, Table 1), were provided to us by Larry
Hansen (Southeast Fisheries Science Onter, NMFS, NOAA). The samples were
placed in aluminum foil, frozen and shipped to Northwest Fisheries Science
Center.
The 20 bottlenose dolphins analyzed in tiiis study were stranded over a
wide area in the Gulf of Mexico (Figure 1, Table 1). Two do^hins were
stranded in areas south of Galveston, Texas, 10 in the immediate vicinity of
Galveston and 2 in areas north of GalvestoiL Additionally, one dolphin was
stranded at a site in Lousiana, one in Mississippi, one in Alabama, and three
animals in the Florida area. Ten of flie dolphins were males and 10 females; each
sex was represented by various ages. The age of 8 dolphins was estimated from
sectioned teeth, and the age of the other 12 do^hins was estimated using a
length/age chart (Table 1). Eight of the animals were immamre (< 1 year, 3
females and 5 males), six animals were maturing (1-8 years; 3 females and 3
males) and six animals were mature (> 8 years; 4 females and 2 males) [Table 1].
One dolphin was alive (condition 1) when fi«t sQ-anded. The others were in
various stages of decomposition: eight animals had been dead < 24 hours
(condition 2) and 1 1 dolphins were moderately decomposed being dead from one
day to one week (condition 3).
Analyses for Metals
The analytical methodologies and quality assurance procedures used here
were modified from those used in NOAA's National Benthic Surveillance Project
(NBSP). Thawed tissue (1.0-1.8 g) of liver and kidney were digested with 10 mL
of concentrated ultrapure nitric acid for 2 hours at room temperature in a sealed
Teflon bomb. Then the bomb was heated in a microwave oven at 650 watts for 6
min. The digestate was further treated to destroy organic matter by digestion
with 4 mL hydrogen peroxide and again heated in the microwave oven. The
61
digestates were diluted with deionized water to a final volume of 25 mL.
Selected metals were determined by atomic absoqjtion spectrophotometry: Cold
vapor hydride generation was used for determining Hg; acetylene/air flame was
used for Fe and Zn; graphite furnace was used for Al, Mn, Ni, Cu, Cr, Sn, and
Sb; and Zeeman-corrected graphite fiimace was used for Ag, As, Se, Cd, and Pb.
The results for the metal analyses are all discussed on a wet weight basis for this
report.
Analyses for Chlorinated Hydrocarbons
As with the metals analyses, the analytical methodologies and quality
assurance procedures were those used in NOAA's NBSP, with flic procedure for
CH analyses modified for the lipid-rich blubber tissue.
Samples of Aawed liver and blubber tissue from bottlenose dolphins were
extracted for CHs modified from procedures of Kiahn et al. (1988). Tissue
(1 g) was extracted with methylene chloride following mixing with sodium
sulfate, and the mixture was macerated. The extract was filtered through a
column of silica gel and alumina, and the extract concentrated for further
cleanup. Size exclusion chromatography with HPLC (flow rate of 5 ml/min) was
used and a fraction containing the CHs was collected. The dichloromethane
solvent in the HPLC fraction was exchanged into hexane as the volume was
reduced by evaporation to app^oxi^late^y 1 mL. The extracts were analyzed by
capillary column gas chromatography (GC) with an electron capture detector
(MacLeod et al. 1985). GC peak identifications were confirmed on selected
samples using GC-MS. The results for the CH analyses are aU discussed on a wet
weight basis for this report.
Quality Assurance Measures for Metals and Chlorinated Hydrocarbons
Metals. Quality assurance included the use of Certified Reference
Materials (CRMs), method blanks, solvent blanks, and Certified Calibration
Standards. The CRMs used included National Instimte of Standards and
Technology (NIST) Standard Reference Materials #1566a Oyster Tissue and
#1577a Bovine Liver, and the National Research Council of Canada's (TRM
DORM-1 Dogfish Muscle Tissue, DOLT-1 Dogfish Liver, LUTS-1 Non Defatted
62
Lobster Hepatopancrcas, and TORT-1 Lobster Hepatopancreas. NIST Standard
Reference Solutions were used for instrument calibration. Five to 29 tissue
replicate CRM analyses were done for the various metals.
Chlorinated Hydrocarbons. Quality assurance measures for CHs included
the analyses of method blanks, replicate analyses of a frozen wet tissue standard
reference material (NIST SRM 1974) and a duplicate sample. The SRM is
certified for selected PAHs by NIST and reported along with non-ccrtified values
for selected CHs. Analyte concentrations were reported on the basis of the
surrogate standard dibromooctafluorobiphenyl added at the beginning of the
sample extraction. Graduated concentrations of GC-calibration-check standards
were used for multilevel response-factor determinations. The criteria for
instrument stability was that the response for each analyte or surrogate in a GC
calibration standard be reproducible within ± 10 %. A method blank and one
sample of SRM 1974 were analyzed with each sample set of 10 samples. When
the recovery of any surrogate standard for a sample was < 50 %, corrective
action was taken, including instrument repair, inlet cleaning, column
replacement, and/or reanalysis.
Analyses for Percent Lipid
An aliquot of tissue extract was evaporated from a measured Auction of die
total tissue extract of each sample to dcf^miine the extractable lipids. Evaluation
of the results showed that this procedure gave results for lipids in marine
mammal blubber and liver comparable to using the method of Hanson and Olley
(1963).
Analyses for DNA-xenobiotic adducts
The levels of hepatic DNA-xenobiotic adducts were detemnined by the
3^P-postlabeling assay modified from Randerath et al. (1984), as described in
Varansie/a/. (1989B).
63
RESULTS
Metals in Liver and Kidney
Liver and kidney samples of bottlenose dolphins were analyzed for 15
metals and the results arc reported on a wet weight basis with the percent dry
weight included for each sample (Table 2, Appendix Al, A2). The range of
concentrations of metals among individual animals was quite wide, often varying
over two orders of magnitude in both liver samples (e.g. mercury, 0.18-117
p.g/g; selenium, 0.70-34.9 fig/g) and kidney samples (e.g. mercury, 0.10-8.70
p.g/g; selenium, 0.77-2.01 M-g/g). Generally, the concentratioiis of mercury in
liver samples were approximately 10 times higher than in kidney samples (Rgurc
2, Table 2) with the concentrations of mercury in the two tissue types being
significantly correlated (r = 0.80, P < 0.0001). As with mercury, the
concentrations of selenium in dolphin livers were much higher than in respeaive
kidney tissue — by a factor of five (Hgure 2, Table 2), with no definite
correlation between the two types of tissue (r = 0.58. P ^ 0.(X)1).
There was no significant correlation between the concentrations of
mercury and age in these dolphins. For example, the two females with the
highest levels of mercury in their livers were PO 095 (5-8 years) and GA 311
(27 years).
Chlorinated Hydrocarbons in Blubber and Liver
The results of the analyses for CHs (Table 3) of individual samples of
blubber (Table 4) and liver (Table 5) tissues are reported on a wet weight basis;
the percent dry weight and percent lipid weight are also included for each sample
with a summary in Table 2. As with the metals, there was a large variability
(wide range) of concentrations of total CHs in blubber (3.0-190 p.g/g) and liver
(0.5-58 ^ig/g) of individual animals (Figure 3). Concentrations of the analytes
were approximately 10 times higher in blubber than in the corresponding liver
sample. However, calculating PCB concentrations on a lipid weight basis resulted
in similar concentrations of PCBs in the blubber and liver of each animal (Table
6). The concentrations of 17 of the 209 PCB congeners are reported in Appendix
A3.
64
The concentrations of total PCBs were higher than the concentrations of
DDTs or other CHs in 18 of the 19 blubber samples; the same pattern was
observed for liver samples (Figure 3). However, one animal (MS 018) from the
Mississippi coast had DDT concentrations higher than PCBs in blubber (Figure 3,
Table 4). In addition, the liver of a dolphin (SCHM 077) from the Alabama coast
had considerably higher levels of DDTs than PCBs — no blubber sample was
available from this animal.
The ratio of the concentrations of p,p'-DDT to p,p'-DDE ranged from 0.01
to 0.62 in blubber tissue from these 20 dolphins (Table 7) with three dolphins
having a ratio of p,p'-DDT to p,p'-DDE that was greater than one-tenth (e.g. PO
095, 0.62; GA 344, 0.12; MS 018, 0.11) indicating a presence of an unusually
high proportion of unmetabolized DDT.
Quality Assurance Measures for Metals and Chlorinated Hydrocarbons
Mean recovery of metals from CRMs was 104 ± 3^ % and the analyses of
replicates agreed within ± 6 %. The grand mean recovery (120 ± 19 %) was
calculated from the mean recoveries for ceitain CH analytes in SRM 1974 by
calculating the ratio of the concentrations of analytes from this series (n = 4) to
those of previous analyses (n = 9). Variabilty increased as the concentrations of
analytes approached trace levels (Horwitz et al. 1980). Replicate analyses (n = 2)
agreed within ± 12 %. The mean recovery for the surrogate standards (n = 48)
was 84 % with a relative standard deviation (RSD) of 17 %.
DNA'Xenobiotic adducts in Liver
Liver samples were analyzed for levels of DNA-adducts. The fmdings
suggest that the length of time between death and sampling of liver may
compromise the quality of the data. For DNA-adducts, the results showed that, in
dolphins sampled at times > 1 day after death, the level of DNA-adducts were
lower than the levels in dolphins sampled at < 1 day after death.
65
DISCUSSION
The stranding of approximately 350 bottlenose dolphins along the
Gulf Coast raised concerns about the health and survival of this species of marine
mammals as well as the quality of the environment in which they live. These top
predators in the marine food chain can accumulate high concentrations of
contaminants in their tissues and organs. Of the metals analyzed, only mercury
and selenium appeared to have concentrations high enough to be of possible
concern. In addition, elevated concentrations of CHs in some of the dolphins may
be of concern.
Metals in Liver and Kidney
The suite of metals (Appendix Al, A2) were chosen for analysis to allow
monitoring and evaluation of their synergistic and antagonistic characterictics
with respect to some of the known toxic contaminants. The range of
concentrations of metals found in ttitsc Gulf Coast dolphins, especially in livers,
was wide (e.g. mercury, 0.18-117 jig/g and selenium, 0.70-34.9 ^g/g). The
differences in concentrations are probably associated with several factors —
including diet, exposure to anthropogenic and natural sources, as weU as age, sex
and reproductive cycle.
Elevated l«»vels of mercury and s'^hnium in two of ^h^-se 'dolphins may be
of concem. The limit of tolerance for mercury in mammalian liver tissues has
been suggested to be approximately l(X)-400 jig/g (Wagemann and Muir, 1984).
Accordingly, the elevated levels of mercury found in livers from two of these
dolphins (> 1(X) ^g/g) are of concem because of potential biological effects.
Additionally, two more dolphins had mercury levels (~ 40 P-g/g) that were
considerably higher than the remaining animals. Geraci (1989) also reported
elevated levels of mercury in dolphins from the previous stranding (range, 0-110
p.g/g; n = 59). Interestingly, the concentrations of mercury were approximately
ten times higher in liver than in kidney (Figure 2) — the reverse of what is
normally found in terrestrial mammalian species (Doull, 1980). This anomoly
may be of sigiiiflcance because the predominant form of mercury found in the
liver (methyl mercury) may add to the burden of organic pollutants that can
accumulate in this organ.
66
Marine mammals tend to have much higher mercury concentrations than
other marine organisms, with particularly high concentrations being found in the
hver (Law et al. 1991). As a general rule, mercury accumulation in marine
mammals increases with age, although there was no significant correlation
between age and mercury concentrations in these dolphins, the six animals with
the highest levels of mercury were all at least five years old. Mercury is a highly
toxic, nonessential metal, (Thompson, 1990), particularly in one of its organic
forms (methyl mercury) and is believed to affect the central nervous system.
Methylation of mercury, due to the action of aquatic microorganisms could be
followed by bioaccummulation up the food chain through the diet of marine
mammals. In addition, mercury may exhibit toxicity by combining with
sulfhydryl groups inhibiting enzyme systems (Doull, 1980).
Selenium is an essential metal within a narrow range — above that range it
is quite toxic (Cooper, 1967). Selenium concentrations were elevated in die two
animals which also had high mercury levels. This finding is not unexpected
because, as in other species, selenium generally covaries with mercury (Muir et
al. 1988). The selenium concentrations in liver samples of stranded dolphins
were similar to the concentrations reported earlier for this species (Geraci 1989).
Selenium, like mercury, may have an inhibiting effect on activities of many
sulfhydryl enzymes, but is also believed to have an important protective action
against the toxic effects of mercury, by readily complexing with methyl mercury.
To what extent selenium may have a protective effert against mercury in these
dolphins remains to be studied.
Chlorinated Hydrocarbons in Blubber and Liver
Although the levels of CHs were relatively low in most of these dolphins,
some of the animals had concentrations of these contaminants at levels of possible
toxicological concern. In general, the concentrations of contaminants in these
stranded dolphins were consistent with previously published data of stranded
dolphins. Three of the dolphins have concentrations of PCBs (77, 78 and 120
^g/g) in their blubber tissue higher than a level of toxicological concern (SO
^g/g) for marine mammals suggested by Wagemann and Muir (1984); three
additional animals had concentrations of PCBs > 40 p.g/g in blubber tissue.
67
However, the differences in analytical methods and quality assurance measures
make it difficult to rigorously compare contaminant concentrations among data
from various researchers.
Even dolphins stranded in the same area had a wide variablity of the
concentrations of CHs (Tables 4, 5), indicating the source of contaminants was
not related to their stranding sites. For example, among the 10 Galveston area
dolphins, 7 were among those with the lowest PCB and DDT concentrations in
blubber while the other 3 were among the highest (Figure 3). A knowledge of
migratory and feeding patterns of these animals, together with data on age. sex,
and reproductive status, would be essential to help explain the observed
contaminant variability. Also, analyses of stomach contents of these animals may
shed some light on immediate sources of contaminants.
Of the 209 PCB congeners, only a few of fliese are demonstrably or
potentially toxic and of these few, the planar (non-ortho substituted) congeners
may account for most of the toxicity exerted by PCTBs in the environment
(McFarland and Clarke, 1989 and Safe, 1984). Several recent studies report the
presence of low levels of these planar PCBs in a variety of marine mammals
(McFarland and Clarke, 1989 and Tanabe, et al. 1987). Preliminary results
from the analysis of blubber (Figure 4) of these dolphins showed the presence of
low concentrations of a number of planar PCBs (Krahn et al. unpublished data).
Funlicr analyses arc needed to evaluate toxicological implications of these initial
analyses.
Two dolphins had higher concentrations of DDTs than of PCBs in the
blubber samples, similar to results reported by O'Shea, et al. (1980) for two
California bottlenose dolphins with elevated concentrations of DDTs. These
anomalies are interesting as most researchers report PCB concentrations to be
higher than DDTs in tissues of marine mammals and fish. The profile of CHs
(i.e. PCBs vs. DDTs) in California bottlenose dolphins reported by O'Shea et al.
(1980) is similar to patterns observed from sites in southem California where
fish, invertebrates and sediment sampled in our field surveys for the National
Benthic Surveillance Project (NBSP) of NOAA's Status and Trends Program
(NS&T) show relatively high proportions of DDTs. Most other U.S. sites
sampled for the NBSP show contaminant profiles in which concentrations of
68
PCBs are higher than DDTs in sediment and biota (Varanasi et al. 1989C). A
substantial amount of DDT was directly discharged into Southern California
waters over several years especially prior to 1972, contributing to the
contaminant exposure of marine mammals from that area (O'Shea, et al. 1980).
The Gulf coast dolphins in this present study showing high proportions of DDTs
may reflect concentrations in the environment where they may have foraged, but
little is known about their migratory habits or sources of DDTs in the area. It is
obvious therefore that more inforaiation on profiles of PCBs, DDTs and other
CH concentrations is needed for the habitat and food organisms of these dolphins
as well as for incidentally caught animals to better assess the importance of
relative levels and distribution of these compounds found in the stranded animals.
Detailed evaluation of profiles of CUs revealed another interesting finding
showing that three of the dolphins had a higher ratio of p,p'-DDT to one of its
breakdown products p,p'-DDE than has previously been found in various species
of dolphins from U.S. waters (O'Shea, et al. 1980). In fact, the DDT:DDE ratio
of one of these three Gulf Coast bottlenose dolphins (PO 095) better compares
with cetaceans from Asia where DDT may still be used (O'Shea, et al. 1980).
The higher ratio of DDT:DDE in the bottlenose dolphin could indicate exposure
to a relatively recent source of the pesticide. The use of DDT has been highly
restricted in the U.S. since 1972. Since DDT breaks down in the environment
into several products, including DDE and DDD, the latter are the predominant
foims fnnnd now in U.S. coastal waters. Onoe again, stomach content analyses
would be helpful to shed light on the relative recent source of contaminants that
these animals may have encountered. Both tissue and stomach content
contaminant levels could aid in assessing the short and long term effects of toxic
levels of pollutants.
DNA-xenobiotic adducts in Livers
The initial analyses of the livers for levels of DN A-adducts showed that tissue
integrity appeared to be an important factor in the ability to use these biochemical
indicators with tissues from stranded dolphins. In light of these findings, a
comprehensive study is underway to assess the effect of tissue quality on the use of
hepatic DNA-adducts as bioindicators of exposure to contaminants such as aromatic
hydrocarbons.
69
SUMMARY AND RECOMMENDATIONS
Although the concentrations of CHs and metals were relatively low in most
of the bottlenose dolphins some of these animals had concentrations of
contaminants at levels of possible toxicological concem. The concentrations of
mercury in the liver samples of two dolphins were elevated. Three animals had
elevated concentrations of CHs. Additionally, two dolphins had concentrations of
DDTs higher than concentrations of PCBs and die ratio of DDT to DDE, one of
its breakdown products, may indicate a possible exposure to recently released
DDT in three dolphins.
Because of the concem and awareness that chemical contaminants may act
directly or indirectly to bring about consequences deleterious to the health of
these dolphins, we need to better understand the extent of contamination and
effects of these pollutants on marine mammals that frequently suffer from mass
strandings. Various endogenous factors (age, sex, lipid content of tissues,
reproductive cycle) and enviromnental factors (sources and types of
contaminants) need to be systematically investigated widi good quality samples
from a significantly larger number of stranded animals. If possible, samples such
as small portions of blubber from wild populations (non-stranded) would be
useful in evaluating contamination levels in apparently healthy animals. It is
essential to continue to generate a scieiitlfically credible and comprehensive data
base on types and concentrations of contaminants and possible biological effects in
marine manunals using state-of-the-art procedures with quality assurance
measures.
70
ACKNOWLEDGEMENTS
We are grateful to our colleagues in the Office of Protected Resources,
Dean Wilkinson and Ted Lillestolcn, NMFS, NOAA, who provided valuable
assistance and funding support in organizing this project We appreciate the
additional assistance of Larry Hansen from NMFS/SEC with this project in the
collection of samples. Finally, a number of EC Division scientists and technicians
ably assisted this study in the sample analyses and the data management. In
alphabetical order they are, Kristin Blair, Richard Boyer, Katherine Dana, Don
Ernest, Tara Felix-Slinn, Barbara French, Rebecca Hastings, Dr. John Landahl,
Ron Modjeski, John Shields, Dr. John Stein, Dave Rees, Susan Pierce, Dr.
William Reichert, Paul Robisch, Dana Whimey and Gladys Yanagida.
71
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75
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81
Table 3. Chlorinated hydrocarbon analytes measured in samples
from bottlenose dolphins stranded along the Gulf of
Mexico, 1990. The analytes within each group were
summed for tabulation.
DDTs
H2l
other CHs
o,p'-DDE
o.p'-DDD
o.p'-DDT
p.p'-DDT
p.p'-DDE
p.p'-DDD
trichlorobiphenyis
tetrachlorobiphenyls
pentachlorobiphenyls
hexachlorobiphenyls
heptachlorobiphenyls
octachlorobiphenyls
nonachlorobiphenyis
decdol ilorobiphenyl
hexachlorobenzene
lindane
heptachlor
aldrin
heptachlor epoxide
alpha-chlordane
trans-nonachlor
dieidrin
mirex
82
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84
Table 6. Concentrations {^g/g) on a lipid weight basis of PCBs in blubber and liver of
bottlenose dolphins collected from the Gulf of Mexico, 1990.
Specimen #
PA 183
PO095
GA311
GA332
GA319
GA330
GA304
GA333
GA334
GA336
GA342
GA344
SP112
SP114
LA 001
MS 018
SHCM 077
MM 9013
MM 9012
MM 9008
Blubber Liver
74
82
240
220
28
17
20
24
4.4
3.9
14
11
30
21
20
43
67
56
11
11
77
65
100
57
270
130
44
43
70
23
130
110
•
1000
74
54
24
14
70
77
85
Table 7. Ratios of p.p'-DDT to p,p'-DDE in blubber of bottlenose dolphins from the
GulfofMexico,1990.
p.p'-DDT
Specimen # p.p'-DDE
PA 183
PO 095
GA311
GA332
GA319
GA330
GA304
GA333
GA334
GA336
GA342
GA344
SP112
SP114
LA 001
MS 018
SHCf^ 077
MM 9013
MM 9012
MM 9008
0.05
0.62
0.03
0.03
0.04
0.02
0.01
0.02
0.04
0.02
0.03
0.12
0.01
0.04
0.01
0.11
0.02
0.02
0.02
86
This page intentionally left blank.
87
SECTION rx
Summaiy of Available Pathology Reports
Staff
Southeast Fisheries Science Center
Miami Laboratory
75 Virginia Beach Drive
Miami, FL 33149
ainical necropsy reports were available for 13 of the 367 bottlenose dolphin
strandings that were investigated during the 1990 Gulf of Mexico stranding event. This was
due, in large part, to the state of decomposition of the stranded animals; only 32 (about 9%)
of the stranded animals were in a condition suitable for a clinical necropsy. Another
contributing factor to the paucity of clinical necropsies was the limited availability of
personnel skilled in post-mortem examination of dolphins. Of the 13 clinical necropsy
reports, four are brief pathological summaries and nine are lengthy reports. The reports are
appended in Appendix VIII.
General necropsies were performed on all of the 13 bottlenose dolphins.
Histopathological analyses were done for five animals (samples were collected for two other
animals, but were not examined), microbiological analyses for six and hematological analysis
for one.
The following direct causes of death were indicated: possible mastitis (1), a fisheries
interaction mortality (1), bacterial pneumonia (gram-negative) (4) and septicemia (3) (three
with lung congestion and edema which were thought to be agonal), and severe pancreatic
fibrosis and nodular pneumonia with peripheral skin abscesses (1). In three animals the
cause of death was not indicated, but was thought in one animal to be related to the fact
that the most significant lesions were pulmonary animal; another was severely emaciated.
There was moderate to severe post-mortem autolysis (PMA), preventing microscopic
examination and determination of cause of death in three animals. Of these three, one was
indicated as a possible calving associated mortality and another as pulmonary edema as
cause of death or possibly drowning.
Skin lesions (moderate to severe) were observed in nine animals; one was non-
specific, five were listed as punctate, one as an ulceration, two were caused by septicemia,
88
one was related to severe local ischemic necrosis, one had a large nodular grey lesion on an
auricula, and one was active but non-erupted.
Multi-focal circulations with evidence of dermzil involvement were spread over the
entire body of one animal, one other animal had numerous light grey irregular spots on its
ventral abdomen and a third animal had white circular punctate scars distributed over its
body as well as a few healed parallel scars (probably healed con-specific tooth-rake marks).
An endometrial cyst and a Monorygma cyst were observed in one animal (the possible
mastitis diagnosis), and a tan mass was observed on the ovary of another animal.
Serosanguinous fluid was found in the abdominal cavity of one animal and in the
thoracic and peritoneal cavities of another.
A full-term fetus was found in two animals. One of these animals was the possible
calving-associated mortality and the other was a non-indicated mortality.
Adhesions were observed between the limg to the thoracic wall, and in the intestinal
loops to each other, to the pseudopancreas and to the mesentery, in one animal, and
between the intestinal loops and the liver and diaphragm (preventing diaphragmal reflection)
in another.
Mild to severe fibrosis was observed in the lungs, liver, lymph nodes, pancreas and
pseudopancreas of six animals. Lymphoid atrophy was noted in one animal, and hyperplasia
and lymphoid depletion were found in three animals.
The following bacterial isolates were reported: enterics, Salmonella and gram-negative
bacilli from skin samples; enterics, Clostridium, gram-negative bacilli, Pseudomonas and
Aeromonas from lungs; enterics and Aeromonas from intestines; gram-negative bacilli and
ciliated protozoans from the spleen; and an acute E.coli infection in a lymph node
(suggesting immunocompromise).
No viral isolates were found.
Worn teeth (moderate to extreme), indicating an older animal, were observed in five
animals. Minimal teeth wear was observed in two animals, one of which had immature
testes, all indicating a younger animal.
89
SECTION X
UMTTATIONS AND RECOMMENDATIONS
Lany J. Hansen
Southeast Fisheries Science Center
Miami Laboratory
75 Virginia Beach Drive
Miami, FL 33149
Limitations
As with all investigations of unpredictable events of this nature, the scope of the
investigation, and therefore it's ultimate usefulness, was limited to a degree due to available
resources; including financial, human, and the existing baseline information. Some difiiculties
in executing both the data collection and analytical phases of the investigation were
encountered, which limited to some degree the scope of the investigation. The inability to
recognize the anomalous mortality event, and the lack of a rapid response plan and a
dedicated response team also hampered the investigation.
Because of the timing and locations of stranded animals relative to available
persormel, data collection was generally limited to areas where stranding networks were in
place and had sufficient resources to deal with the increased workload. Because of this, after
the mortality event was perceived as anomalous, the Texas network received considerable
assistance from SEFC, but other areas did not. Lack of pre-existing standard and emergency
tissue collection protocols was also a problem. After the event was recognized, SEUS
network participants were asked to collect a specific set of tissues for the investigation. This
lag in recognition of the event resulted in the protocol being established after about half of
what has been defined as the anomalous mortality event occurred. Before the mortality
event occurred, some data were lost from those animals investigated but for which no
standard tissue sampling protocol was in place. The condition of the stranded animals also
had a limiting effect on data collection. As is common in most investigations of stranded
animals, few of the strandings were fi-eshly dead, and thus few provided tissues useful for
histopathology and other pathological examinations (the available clinical necropsy and
histopathology reports are presented in Appendix VIII). The inability to examine numerous
live healthy and live "affected" dolphins limited the possibility of identifying disease agents
or other potential causes of mortality. Few of the voluntary network participants have the
skills required to conduct an adequate gross pathologic examination on marine mammals.
The scope of the anomalous mortality event was not evident until about the end of
February, 1990. At first, the event was somewhat obscured by the mass stranding in
90
Matagorda Bay during January, 1990. The delay in stranding recovery reporting (required
within 30 days after recovery) resulted in a lag in recognizing the event. Although contacts
with the SEUS network coordinator were in place before the event, SEFC contacts with the
network were limited, and most were established during the mortality event. The lack of
contacts had a impact on recognizing the event, and thus determining the scope of the event.
The pre-existing database had a direct impact on the type and extent of analyses that
could be done with the 1990 database. For example, the lack and/or inconsistency of life
history tissue collection (teeth, reproductive tracts) limited the breadth of the age structure
analysis and eliminated any examination of reproductive history. The inconsistency in
stranding recovery efforts, and/or the lack of a measure of stranding reporting and recovery
efforts, reduced the statistical strength of inter-aimual and other comparisons of stranding
rates. Obviously, the lack of information on the effects and levels of contaminants and
biotoxins in healthy bottlenose dolphins, and the lack of information on levels in the pre-
1990 sample constrained the extent of the analyses on contaminants and biotoxins.
The SEFC implemented a bottlenose dolphin monitoring program in 1987 (Scott and
Hansen 1989). This program is designed to detect a halving or doubling in abundance. The
monitoring program did not detect such a decline in abimdance after the mortality event.
However, smaller scale variations in abundance are much more difficult to detect. In
addition, the scope of the aerial surveys conducted during 1990 was limited, and thus
weakened any conclusions relative to 1990 and prior abundance levels. But it is not clear
that "unlimited" surveys would have strengthened any conclusions.
Although the investigation was not successful in conclusively identifying a single factor
(or multiple factors) as a cause of the anomalous stranding event, several suspicious factors
were identified. In addition, the investigation identified weaknesses, explored new methods
and resulted in the implementation of several efforts which will improve future
investigations. These include: a more thorough statistical treatment of stranding fi-equencies,
correlations with envirormiental variables, and comparisons of two methods of assessing
stranding frequency. Stranding protocols have been refined and further standardized, and
the stranding networks have been augmented. The baseline data collected during this
investigation will be invaluable in the evaluation of future episodes of mortality among
bottlenose dolphins. The process of this investigation identified critical areas where the
required information to understand patterns of bottlenose dolphin was lacking, and has
resulted in the development of a plan of action which is described below.
Recommendations and SEFC Program
The 1990 bottlenose dolphin anomalous mortality event in the Gulf of Mexico
illustrated that the SEUS network was not well prepared to mount an adequate response
to increased strandings. Furthermore, gross inconsistencies in regular data collection and
reporting among Network participants have resulted in a lack of consistent baseline
information and the inability to monitor the stranding rate in a timely fashion. These
91
problems are primarily the result of relying on a partially organized, mostly ill-trained, and
generally poorly equipped, volunteer Network. The SEFC has taken steps to improve the
capabilities of the Network by assuming some responsibilities for reporting, by establishing
collection protocols and providing collection materials, by providing training and
arrangements for clinical necropsy of suitable specimens, by providing for analyses of tissue
samples, and by informing Network participants on the results of their reponing and data
collection efforts.
The SEFC stranding response activities center on three areas: monitoring stranding
rate, specimen necropsy, collection and analyses, and dissemination of results. The stranding
rate is monitored by a system for rapid reporting of basic data on stranded animals.
Consistent specimen collection is being accomplished by providing manuals, collection kits
and training to Network participants. The SEFC is establishing pathways for ensuring clinical
necropsy and tissue analyses of suitable specimens. Results will be disseminated to Network
participants in a quarterly report produced in conjimction with the Network Coordinator.
The most important component of all these activities is the development and maintenance
of communication between the SEFC and the Network participants.
The Network already had a system for reporting strandings, some data collection
protocol, and for dissemination of results. However, the level of these activities was not
sufficient to meet the SEFC information requirements. It should be clear that the SEFC is
not attempting to takeover the Network, but that the SEFC is trying to supplement the
Network by providing assistance for particular activities.
Although the Network has been improved and efforts to further improve the Network
continue, the system is not perfect. There will likely be difficulties, both anticipated and
unanticipated, should another anomalous event occur. It is not possible to assure that
adequate staff and funding will always be available to respond to anomalous events. The
development of contingency plans and funding mechanisms at the national level for
anomalous events was recommended by Wilkinson (1991). The establishment of a stranding
emergency response team, much like the national oil spill response team, would eliminate
many of the types of problems encountered during this investigation.
Monitoring Stranding Rate
The SEFC has estabhshed a system to receive stranding reports from the Network
volunteers for near real-time monitoring of the stranding rate. Appropriate staff at each of
the SEFC laboratories and the Regional Office have been identified as SEFC area
representatives and have established contacts in their area with Network participants. The
Network participants are required to report basic data (what, when, where and condition)
to the SEFC area representative within 48 hours of a stranding event The area
representative reports the basic data within 48 hours of receipt to the Miami Laboratory.
A computer bulletin board system has been established for receiving basic data reports. The
92
Miami Laboratory area representative is responsible for reviewing the basic data reports and
for weekly monitoring of the stranding rates throughout the southeast. This allows for rapid
identification of anomalous stranding events and the transfer of this information to NMFS
Headquarters and others in a timely maimer.
Specimen Necropsy Collection and Analyses
The Charleston Laboratory has developed necropsy protocols, specimen collection
protocols and collection kit specifications. The protocols and kits have been distributed to
the appropriate Network participants.
The Miami Laboratory is presently identifying necropsy persoimel and necropsy
facilities in the southeast. The SEFC area representatives will ensure that appropriate
specimens are delivered to necropsy facilities.
The SEFC area representatives receive, track, store and transfer collected samples.
Arrangements have been made with the Armed Forces Institute of Pathology (AFIP) to
conduct histopathological studies on appropriate specimuis. Other collected specimens are
transferred for analyses when suitable investigators are identified (e.g., for genetic, food
habits, aging, stock studies; some funding may be required and faculty appointments used
to bring investigators onboard). These activities will ensure that adequate information is
available to begin evaluating causes and potential effects of both normal and anomalous
mortality events.
Dissemination of Results
A quarterly newsletter which provides stranding summaries, information on analyses
underway or planned, and any noteworthy events or tips, is being prepared and will be
distnljuted to each Network participant. The newsletter is being produced by the SEFC and
the Network coordinator. Although this is a minor activity in terms of funding, it is critical
for maintaining communication and cooperation between the SEFC and the Network
participants. The primary purpose of this activity is to let the Network participants know that
their efforts made to provide the SEFC with information and specimens are worthwhile.
A biennial Stranding Network meeting should be held, sponsored by the SEFC and
the Network coordinator. The meeting will provide a forum for reviewing the Network
activities, providing training in necropsy and specimen collection, reporting related research
findings, and for establishing and maintaining contacts between the Network participants and
the SEFC.
The proposed activities require varying amounts of staff time from each SEFC area
representative. Initially, each area representative will spend a significant (probably 20 hrs or
more per week for two to four weeks) amount of time identifying and contacting area
93
participants and clinical necropsy facilities and personnel. Subsequently, less time, probably
one to five hours per week, will be required for reporting, delivering or transferring
specimens, and maintaining contacts. Some area representatives may also participate in
recovering stranded animals. The Miami Laboratory area representative was responsible for
development and implementation of the computer bulletin board system. This required
about one person-month. Approximately one-half of the Miami Laboratory area
representative's time is spent on reviewing and analyzing reports, distributing specimens,
reviewing results, maintaining and developing contacts, and preparing stranding program
reports.
Appendices DC and X provide more information on specific responsibilities and the
planned implementation schedule for the improvement to the SEUS network.
Literature Cited
Geraci, J.R. 1989. Clinical investigation of the 1987-88 mass mortality of bottlenose dolphins
along the U.S. central and south Atlantic coasL Final Report to NMFS, ONR, and
MMC.
Wilkinson, D.M. 1991. Report to: Assistant Administrator of Fisheries. Program review of
the marine mammal stranding networks. National Marine Fisheries Service, Office of
Protected Resources. 171 pp.
94
Appendix I. StraraM bottlanoM dolphirw docta^ttad during j««jiry-Jin>, 1990, aleno the U.S. Cutf of Mexico coMt.
SEUS COLLECTION i C LEM 6LSS X C NO/DA ST LAT LOM TN BL MS KD LV M ST GO V SK EX CI BT CT FN HE
4762 CC083
4851
SE4851
«767 MNL-9001
4850 SE4850
4891
CA289
4768 »«L-9002F
47*9
11-0011
4793 90-11-0025-CH
47M
SHCM-033
4763
CC084
4792
90-11-031
4892
aA290
4765
SHCM-a34
4766
SHCM-035
4981
PA184
4984
PA187
4982
PA1B5
4771
SHCM-036
4781
HS2-90
4947
P0113
4948
P01U
4949
P0115
4950
P0116
49S1
P0117
4952
P0118
4953
P0119
4934
PO100
4932
P0098
4941
PO107
4940
P0106
4945
P0111
4939
P0105
4936
P0102
4943
P0109
4935
P0101
4942
P0108
4931
PO097
4933
P0099
4946
P0112
4944
PO110
4938
P0104
4937
PO103
4849
SE4849
4782
MS3-90
4930
P0096
4929 PO095
492B
P0094
4807 C90-11-0046CI(
4796
C90-11-0045
4893
CA291
4978
PA 181
4772
SHW-037
4769 90-04-0093FW>
4885
NAP-00a3
4954
PO120
4960
P0126
4753
MS190
4773
SHCM-038
4959
P0125
4958
P0124
4774
SHCK-039
5216 C90-01-0051CM
4783
MS4-90
4856
SP110
4775
SHCM-040
T 191
■ U 5
T 201
■ U 5
T 258
T 249
T 194
T 253
Tf 84
T 196
T 251
T 191 J
T? 2S4
T 257
T 236 •
T 270
T 2U
T 164 •
T 213
T 240 •
T 229
T 191
T 282 •
T 207 •
T 216 ■
T 269 ■
T 261
T 254
T 262 •
T 288
T 216 ■
T 225 ■
T 256 •
T 7 ■
T 220
T 258
T 218
T 254
T 176
T 270
T 262 ■
T 256
T 275
T 245
T 188
T 247
T 240
T 235
T 196
T 137
T7 180
T 213
T 7
T 110
T 249
T 99
T 7
T 236 :
T 235
T 164
T 245 4.9
T 163 0.9
T 154 ••-
T 206 —
T 234 ---
T 240 15
T 99
...
01/02
01/07
01/08
01/08
01/08
01/09
01/11
01/13
01/14
01/15
01/16
01/16
01/17
01/18
01/18
01/18
01/18
01/19
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/20
01/21
01/21
01/21
01/21
01/21
01/22
01/22
01/22
01/22
01/23
01/23
01/23
01/23
01/24
01/25
01/27
01/27
01/27
01/28
01/28
01/28
01/28
01/29
TX
U
FL
U
TO
FL
FL
FL
AL
TO
FL
TO
AL
AL
TO
TO
TO
FL
MS
TO
TO
TO
TO
TO
TO
TO
TO
TO
TO
TO
TO
TO
TO
TO
TO
TO
TO
TO
TO
TO
TO
TO
LA
NS
TO
TO
TO
FL
FL
TO
TO
AL
FL
NS
TO
TO
MS
AL
TO
TO
AL
FL
NS
TO
AL
27^.0
29*47.0
27*24.5
29*47.0
2r47.8
27*38.0
29*43.0
30*24.1
30^7.5
27T2.5
30*24.1
29rS2.1
30*18.5
30*14.0
28*08.
27*42.
28*08.
30*17.5
30*12.1
28*38.6
28*38.9
28T9.
28^.3
28*39.6
2r41.
28*42.8
2r40.7
28*39.9
28*42.2
28*42.1
28*38.3
2r41.8
28*40.9
28*43.4
2r40.8
28*42.3
28*38.6
2r40.1
28*38.4
2ru.3
2r41.7
28*41.2
29*43.0
30*13.5
2r43.5
28*37.7
28*37.
30*25.7
30*20.9
28*53.9
27*50.1
30-13.5
27*31.2
•
28*21.3
28*31.2
30*21.0
30*25.5
28*32.2
28*32.7
30*14 J
29*49.7
30*12.1
29*37.4
30-14.0
97^8.0
93*10.0
8r39.5
93*10.0
95nK.7
82^.6
84*45.0
86*30.0
87T2.5
97*12.5
86*30.0
94*25.
87*31.0
88W.0
96*55.
87*28.0
88*28.1
95*53.8
95*53.4
95^2.6
95*52J
95*51.7'
95*49.
95*47.2
95*56^
95<56J
95*54.7
95*55.2
95*55.4
95*55.9
95*56.7
95^1.5
95*56.8
95*54.6
95*57.1
95*56.2
95*54.1
95*49.4
95*56.1
95*56.6
9r47.0
88*54.3
95*41.8
95*53.9
95*55.6
86*30.0
87*24.7
95*21.
97*03.2
87*49.0
82*38.1
•
96^54.
96*09.5
88*30.5
87*54.5
96*07.2
96*06.3
88*14.0
8518.7
8r40.0
94*11.8
87*54.0
♦ ♦ ♦ ♦ •
♦ ♦ ♦ ♦ ♦
T •
-♦♦♦♦♦
♦ -♦♦♦♦
- . . -. . «
♦ ♦
♦ ♦
♦ ♦
♦ ♦
♦ ♦
♦ ♦
♦ ♦
♦ ♦
♦ ♦
♦ ♦
♦ ♦
♦ ♦
♦ ♦
♦ ♦
♦ ♦
♦ ♦
♦ ♦ ♦ •
♦ ♦ ♦ .
♦ ♦ ♦ •
♦ ♦ ♦ ♦
♦ ♦ ♦ .
♦ ♦ ♦ .
♦ ♦ ♦ •
♦ ♦ ♦ -
♦ ♦ ♦ •
♦ ♦ ♦ •
♦ ♦ ♦ •
♦ ♦ ♦ •
♦ ♦ ♦ •
♦ ♦ ♦ .
♦ ♦ ♦ .
♦ ♦ ♦ •
♦ ♦ ♦ •
♦ ♦ ♦ •
♦ ♦ ♦ •
♦ ♦ ♦ •
♦ ♦
♦ ♦
♦
T Y T
♦ « ♦ ♦
♦ ♦ ♦ ♦
♦ ♦ «
♦ ♦ ♦
♦ ♦♦♦♦♦
♦ ♦♦♦♦♦
♦ ♦♦♦♦•
T T T
♦ ♦♦♦♦♦♦♦
95
A|]p«idix I. (canttnuad)
SEUS aJLLECTIOW # G LEM CLCS X C W/OA ST UT
4957 P0123 1
r 229 2.5
M :
I 01/29
TX
4784 W5-90 1
r 111
....
N .
I 01/30
NS
4786 M7-90 1
r 246
....
f i
I 01/31
NS
4785 »tS6-90 1
r 205
....
M i
I 01/31
MS
4894 SA292 1
r 302
16
M i
I 01/31
TX
4964 PO130 1
r 216
....
U i
i 02/01
TX
4857 a>^^^ i
r 254
12.5 M :
( 02/01
TX
4980 PA1B3 1
r 205 3
F ;
I 02/02
TX
4895 CA293
182 0.9
M *
• 02/03
TX
4896 CA294 1
r ?
13
u :
S 02/03
TX
4809 9001 1
r 97
U i
> 02/04
FL
4991 CC086 1
r 7
F i
I 02/04
TX
4778 MK-1-90 1
r 108
M 2
S 02/05
FL
4996 PI035 1
r 241
F 3
t 02/05
TX
4805 SHCM-042 1
r 165
U !
> 02/07
AL
4791 90-2-1 1
r 7
F i
> 02/07
U
4801 m900^ 1
r 220
F ]
i 02/08
FL
4993 CC08a 1
r 216 4
M i
. 02/08
TX
4961 P0127 1
r 179 2
M i
> 02/08
TX
4813 90007 1
r 186
M :
> 02/09
NS
4956 P0122 1
r 239 20
U 4
I 02/09
TX
4955 P0121 1
r 261
16
M :
( 02/09
TX
4977 P0143 1
r 241
9
M ]
( 02A)9
TX
4898 CA296 1
r 241
0
M :
( 02/10
TX
4897 GA295 1
r 268
N 2
( 02/10
TX
5224 90-01-0070 1
r 137
N "
f 02/11
FL
4983 PA186 ^
r 237
....
M i
. 02/11
TX
4963 P0129 1
r 111
0
F 3
5 02/11
TX
4962 P012B 1
r 152
....
U !
> 02/11
TX
4994 CC089 1
r 183
M i
. 02/12
TX
4900 CA298 1
r 239
15
F i
. 02/12
TX
4899 CA297 1
r 200 5
U i
. 02/12
TX
4803 SHCM-044 1
r 270
H :
» 02/13
AL
4806 SHCM-046 1
r 7
U 2
S 02/13
AL
4802 SHCM-045 1
r ?
U 2
S 02/13
AL
4808 9002 1
r 180
F 2
\ 02/13
FL
4873 MM-9003 1
r 205
F i
. 02/13
FL
4872 MM9002 1
r 240
M I
. 02/13
FL
4841 MS011-90 1
r ?
U 2
i 02/13
NS
4842 MS012-90 1
r ?
U 2
( 02/13
NS
4901 CA299 1
r 184
1.5
U i
. 02/13
TX
4902 CA300 1
r 7
U t
. 02/13
TX
4903 GA301 1
r 239
17
F i
. 02/13
TX
4815 90009 1
r 110
U 2
\ 02/14
NS
4858 SP112 1
r 260
M :
» 02/14
TX
4995 CC090 1
r 234
F i
, 02/14
TX
4874 HM-9004 1
r 212
U i
. 02/15
FL
4905 CA303 1
r 140
U i
. 02/15
TX
4904 CA304 1
219 30
T 2
1 02/15
TX
4859 SP113 1
241
M 2
1 02/15
TX
4906 CA302 1
r 262
17
M i
. 02/15
TX
4875 MM-9005 1
r 190
F 2
( 02/16
FL
4907 CA305 1
r 240
F :
i 02/16
TX
5151 PI036 1
7
U 2
1 02/19
TX
5226 90-01-0082 1
183
F 1
f 02/20
FL
4839 MS015-90 1
2S2
M i
■ 02/22
NS
4860 SP1U 1
251
F 2
I <a/22
TX
4965 P0131 1
200
M 2
I 02/22
TX
4966 P0132 1
7
13
U !
i 02/22
TX
4908 CA306 1
211
....
F i
> 02/22
TX
4967 P0133 1
260
ir
U 2
1 02/22
TX
4876 MH-9006 1
260
—
M i
> 02/24
FL
4974 PO140 1
97
—
M 2
1 02/26
TX
5176 CC095 1
7
—
U 1
02/26
TX
4910 CA308 1
121
0.8
H t
. 02/27
TX
LON TMILMSDLVWSTGOBKEXCirrCTFHME
2r38.3
' 95*52.4'
30^2.5
' 88*59.0'
30^4.5
8r53.2'
30M4.0
88*53.5'
2r58.3
9515.5'
2rJ7.2
«6*05.S'
29^.2
94*06.5 •
2r47.
97*06. •
29^4.
94*53. '
29^9.3
94*U.6'
3019.5
8718.3'
zrzT.
9718. •
zera.2
8r48.3'
26*32.8
9716. '
3013.8
87^.8'
29rS5.0
90*04.5'
2r42.1
82*28.4'
2719.
97*20. '
28'39.
95*50.6'
3013.0
88*55.8'
2813.2
96*38.2'
2814.
96*36.7'
2r46.3
95*36.2'
29T3.7
94'43. '
29*28.
94*37.1'
3010. '
85*48. '
27-50.
97*03. •
28T7.4
96*37.5'
28*27.8
96*24.6'
27*25.
9717. '
2915.4
94*50.5'
29*00.8
9512.2'
30*15.0
87*39.5'
3016.2
87*34.8'
3017.0
87*32.0'
3018.2
87*23.5'
27T7,2
82*42.8'
2r49.7
82*31.0'
3012.0
88*26.0'
3012,0
88*29.0'
29*03.4
95*08.5'
29*04.1
95*08.2'
29*03.9
95*07.2'
3013.5
88*57.7'
29*39.0
94*07.5'
27*30.
9717. •
2r44.1
82*38.1*
29*32.
94*25.6'
29*27.8
94*36.2'
29*39.4
94*06.6'
29*28.4
94*35. '
27*52.7
82*35.1'
29*22.2
94*49.6'
26*30.2
9715.1'
3015.
85*40. •
3312.7
88*57.7'
29*35.
9417.4'
28*49.1
95*30.9'
28*08.
96*45.7'
28^9.2
95*22.2'
28T)4.4
96*50.2'
27*54.7
82*31.8'
27*24.
96*30.2'
zra.t
9712.1'
29*09.7
95*00.3'
♦ ♦♦♦♦♦♦♦
Y T T. -
♦ ♦♦♦♦♦♦
- T - T
Y T T t
- - - Y
♦ •♦♦♦♦♦
♦ . •
♦ ♦ ♦
♦ •♦♦♦♦
♦ •♦♦♦♦
♦ ♦ ♦ ♦
♦ ♦♦♦«♦
T T Y Y C
♦ ♦ ♦ ♦ ♦
♦ ♦ ♦ ♦ ♦
Y T - Y
- Y - Y
Y Y • Y C
96
• THE REPORTED LENGTH FOR SPECIMEN GA304 NAY BE INCORRECT. THE REPORTED LENGTH IS ABOUT 20CN LESS THAN WHAT
WOULD BE EXPECTED FOR A FEMALE OF THIS AGE (30 YRS). THE ORIGINAL DATA SHEET FOR THIS SPECIMEN WAS LOST AND THE
LENGTH CANNOT BE VERIFIED.
Appendix I. (continued)
SEUS COLLECTION « G LEW CLCS X C MO/OA ST LAT
LON
4877 MM -9007
T 265 ---
- M
02/28
FL
2r45.3
■ 82*45.7'
4909 CA307
T 97 -•-
- M
02/28
TX
29°14.5
' 94*52.3'
4852 MML-9004
T 215 ---
- F
03/01
FL
27°38.0
• 82*33.6'
4888 MAP -0006
T 97 --■
- F
03/01
MS
e
1 ■ 1
4881 MS020-90
T 190 --■
- U
03/01
MS
3ff23.5
' 89°5«,2'
4887 MAP -0005
I 196 ---
- F
03/01
MS
4838 MS013-90
I 198 ---
■ M
03/01
MS
30*14.6
' 88*46.4'
4861 SP115
T 250 26
F
03/01
TX
29°37.1
94*12. '
4880 MS019-90
r 91 ---
• U
03/02
MS
30°16.7
89*22.4'
4970 P0136
r 160 1
F
03/02
TX
2831.1
96*09.6'
4969 P0135
r 211 7.9
F
03/02
TX
28T3.1
96*05.3'
4968 P0134
r 254 18
F
03/02
TX
28°33.1
96*05.2'
4878 MM-9008
r 137 ---
■ F
03/03
FL
2r42.2
82*40.2'
4840 HS17-90
r 7 --•
■ U
03/03
MS
30°18.0
88rS5.8'
4911 GA309
r 95 ---
• M
03/03
TX
29^5.6
95*06.4'
48U SHCM-047
r 95 ---
M
03/04
AL
30'14.7
88*12.0'
4845 SHCM-048
r 240 ---
• M
03/04
AL
30*17.2
87*31.2'
5147 DRC-90-1
r 257 ---
M
03/04
FL
24*30 .
81*49. '
5010 MS027-90
r 112 ---
U
03/04
MS
30*12.3
88*26.6'
4843 MS014-90
r 7 ---
U
03/04
MS
30*12.0
88*28.0'
4913 GA311 1
r 233 27
F
03/04
TX
29*06.
95*07. •
5177 CC096 1
r 7 ---
U
03/04
TX
26*54.
97*28.5'
4837 MS016-90 1
r 180 ---
F
03/05
MS
30*25.
88*57. '
4914 GA312 1
r 233 12
F
03/05
TX
29*00.7
95*12.5'
5203 SHCM-068 1
r 241 ---
F
03/06
AL
30*14.
88*20. '
5202 SHCM-067 1
r 279 ---
M
03/06
AL
30*14.
88*20. '
4853 MML-9005-B 1
r 66 ---
U
03/06
FL
2ri7.8
82*34.0'
4883 MS022-90 1
r 109 ---
U
03/06
MS
30*22.6
89T)2.6'
4882 MS021-90 1
r 102 ---
u
03/06
MS
30*22.4
89*02.6'
4985 PA 188 1
r 95 --
F
03/07
TX
2r47.3
97TK. '
4915 GA313 1
r 236 21
F
03/07
TX
29*25.
94*41.3'
4916 GA314 1
r 260 31
M
03/07
TX
29*13.9
94*52.9'
4972 PCI 38 1
r 274 20
F
03/08
TX
28*25.8
96*18. '
4917 CA315 1
r 269 29
F
03/08
TX
29*25.7
94*40.7'
5152 PI037 1
r 235 ---■
U
03/08
TX
26*07.
97*09.9'
4918 GA316 1
104
U
03/08
TX
29*17.8
95*46.2'
4971 P0137 1
201 ---■
U
03/08
TX
28*37.7
95*54. '
4986 PA189 1
198 ---■
F
03/08
TX
28*01.
98*10.8'
5221 90-01 -0109CM 1
114 ---■
F
03/09
FL
30*15.
85*57. '
4973 P0139 1
7 28
U
03/09
TX
28*15.8
96*33.9'
4919 GA317 1
100 ••-•
M
03/09
TX
29*29.6
94*32.2'
4846 SHCM-049 1
219 ----
F
03/10
AL
30*13.5
87*59.5'
4889 MAP -0007 1
122 •■•-
F
03/10
MS
•
• 1
4921 GA319 1
109 0
M
03/10
TX
29*00.7
95*12.5'
4920 GA318 1
231 34
U
03/10
TX
29*28.
94*35.8'
5011 MS028-90 1
244 ---•
U
03/11
MS
30*12.3
88*26.6'
5144 CC085 1
177 0.9
M
03/11
TX
27*29.3'
9ri7.3'
4862 SP116 1
7
U
03/11
TX
29*34.8'
94*18.2'
5130 11-90-0157 1
86 ----
M
03/12
FL
30*27. '
86*35. '
5216 90-01 •0122CM 1
137 •--■
U
03/12
FL
30*20. '
86*15. •
4890 MAP -0008 1
241 •---
M
03/12
MS
• 1
• 1
4863 SP117 1
253 - •
U
03/12
TX
29*40. '
94*04.4'
4864 SP118 1
237 23
u
03/12
TX
29*40.2'
94*04.2'
4922 GA320 1
249 9.3
u
03/12
TX
28*59.5'
9514.2'
4975 P0141 1
247 27
F
03/12
TX
28*34.1'
96*02.1'
4976 P0142 1
191 ---■
F
03/13
TX
• 1
• 1
4927 GA325 1
220
U
03/13
TX
29*26.2'
94*54.5'
4923 GA321 1
162 --•-
F
03/13
TX
29*29.2'
94*33.4'
4879 MS018-90 1
161
M
03/14
MS
30*22.9'
88*33.9'
4924 GA322 1
237 12
F
03/14
TX
29*16.1'
94*49.3'
4925 GA323 T
247 ----
F
03/14
TX
29*29.3'
94*32.4'
5370 SHCM-082 T
190
U
03/15
AL
30*16.5'
88*06.9'
4926 GA324 T
251 13
M
03/15
TX
29*27.9'
94*36. '
5149 CC091 T
289 ■--•
U
03/16
TX
27*31.1'
97*15.9'
5150 GA326 T
89 --■•
U
03/16
TX
29*23. '
94*43.3'
TH BL MS KD LV BM ST GO BO SK EX CI 8T CT FH NE
4.. ..«.«. ..«.Y---
♦ ---♦
♦ ♦♦♦♦♦♦ Y-
♦ --♦♦♦♦♦ Y-
♦ -♦♦♦♦♦■♦■ Y-
♦ -♦♦♦♦♦♦♦- -Y-Y-
■♦-♦♦ YY---
- ♦♦.
♦ ♦♦♦■♦•♦♦♦• - -YYYYC
♦ ♦♦♦♦♦♦ Y-
♦ ♦♦♦♦♦♦♦♦♦•♦•
*■*****■* Y-Y-
♦ •♦•♦♦♦♦♦ YYY-
♦ - ♦
♦ ♦♦♦♦♦-♦----Y---
♦
♦ ♦--♦-
♦ ♦♦♦♦♦♦- - Y-
♦ ♦♦♦♦♦♦♦♦- - -YY-
♦ •---•♦
♦ ♦•-♦
****** YY-..
♦ -
♦ ♦♦♦♦♦• -♦■••-Y--
♦ ♦♦••♦
♦ •♦♦♦
♦ ♦♦♦♦♦♦-♦
♦ - + -♦♦♦
♦ ♦ + ♦♦♦♦♦ Y-
♦ ♦♦♦♦♦
♦ --•-♦
♦ ♦♦♦♦♦♦♦■ -YY-C
- ♦♦♦♦♦♦♦- -♦YY-
♦ ♦♦♦♦♦♦♦♦
♦ -♦♦♦♦♦-♦
♦ ♦♦♦♦♦♦•♦
•♦• -
*......
97
Appendix I. (continued)
SEUS COLLECTION « G LEN CLGS X C MO/OA ST LAT
LON
4987 PA 190
T 213
...
- M
4
03/16
TX
27°50.5
' 97*02. 7-
A988 PA191
T 73
...
- M
4
03/16
TX
27^0.5
' 97*02. 7'
5254 SE52W
T 312
...
- U
4
03/17
U
29°45.
' 93T^. '
5255 SE5255
T 167
...
- U
4
03/17
LA
29°46.
' 93*43. '
5012 MS029-90
T 239
...
- M
3
03/17
HS
30*16.
' 88*41. •
4884 MM-9009
T 125
...
- H
4
03/18
FL
27^2.7
• 82*35.0'
5020 MS031-90
T 94
...
- U
4
03/18
LA
30'14.6
' 89*46.4 •
5029 SHCM-054
f 163
...
- F
3
03/19
AL
30*14.9
88*04.9'
5025 SHCM-050
r 255
...
- F
3
03/19
AL
30*17.
8r45. '
5027 SHCH-052
r 239
—
- M
3
03/19
FL
30*22.
8ri4. '
4997 MS023-90
r 7
...
- U
3
03/19
HS
30*19.
88*30. '
5026 SHCM-051
r 86
—
- M
4
03/20
AL
30*14.2
87^3.3'
5153 GA327
r 254
...
■ U
4
03/20
TX
29*55.1
95*19.3'
5041 41650-1
r 183
...
- H
3
03/21
FL
29*40.
85*11. '
5155 CC092
r 146
...
• U
?
03/21
TX
27*31.
9ri5.9'
5154 GA328
r 7
...
■ U
4
03/21
TX
29*06.8
95*04.8'
5030 SHCH-055
r 160
..."
• F
3
03/22
AL
30*15.
87*49. ■
5028 SHCM-053
r 266
...
■ M
3
03/22
AL
30*18.6
88T)8.2'
5233 SE5233
r 232
...
■ M
3
03/22
LA
29*46.5
93*35. '
4998 MS024-90
r 109
—
■ U
2
03/22
MS
30*12.
88*25. '
5164 PA193
r ?
13
U
5
03/22
TX
2n8.2
96*47.6'
5159 P0146
r 235
...
F
4
03/22
TX
28*16.8
96*37.2'
5160 P0147 1
r 7
...
U
5
03/22
TX
2n4.3
96*37.6'
5157 P0144 1
r 239 23
H
4
03/22
TX
2r46.9
95*35.1'
5156 SP119 1
r 114
0
H
3
03/22
TX
29*36.7
94*13.3'
5158 P0145 1
r 219
25
F
4
03/22
TX
28*48.6
95*31. 8'
5007 90-11-186 1
r 94
—
M
3
03/23
FL
30*27.
86*30. '
SOU LA-TT-03 1
r 247
...
F
3
03/23
LA
29*03.
90*38. '
5163 PA192 1
r 243
15
F
3
03/23
TX
27*57.
96*59.7'
5178 P0151 1
r ?
—
U
3
03/23
TX
28*25
96*22.7'
5162 GA329 1
r 260
...
U
4
03/23
TX
29*17.3
94*47.2'
5230 LA3 1
r 257
...
H
3
03/24
LA
29*45.
93*38.6'
5231 LA2 1
r 177
...
M
4
03/24
LA
29*46.
93*27. •
5232 LAI 1
173
...
F
3
03/24
LA
29*46.4
93*27.3'
5165 GA330 1
r 106
0
M
3
03/24
TX
29*26.2
94*36.6'
5132 90-11-0199-CM 1
168
...
U
7
03/25
FL
30*20.
87*20. '
5042 LA-TT-01 1
251
...
F
3
03/25
LA
29*03.
90*23. '
5043 LA-TT-02 1
265
...
N
4
03/25
LA
29*03.
90*28. •
5013 MS030-90 1
244
...
F
3
03/25
HS
30*13.8
88*40. '
5008 MS025-90 1
229
...
F
3
03/25
HS
30*20.4
88*31.5'
5166 GA331 1
229
40
F
3
03/25
TX
29*20.1
94*43.7'
5179 P0152 1
?
...
U
3
03/25
TX
28*25
96*27.8'
5031 SHCM-056 1
240
...
F
3
03/26
AL
30*17.2
8r44.1'
5045 LA-TT-04 1
228
F
4
03/26
LA
29*03.
90*38. '
5143 MS042-90 1
109
U
3
03/26
HS
30*18.
88*35. •
5009 MS026-90 1
272
H
3
03/27
HS
30*25.
88*51. '
5022 MS033-90 1
234
U
3
03/28
MS
30*13.7
88*37.0'
5021 MS032-90 1
145
H
3
03/28
HS
30*14.6'
88*46.4'
5167 GA332 1
106
0
F
3
03/28
TX
29*13. 7-
94*53.7'
5168 PI038 1
247
10
F
3
03/28
TX
26*16.1'
9ri1.1'
5372 SE5372 1
7 7
U
?
03/29
LA
29*16.9'
91*19.1'
5371 SE5371 1
7 ?
U
7
03/29
LA
29*13.2'
91*12.0'
5170 GA334 1
194
M
3
03/29
TX
29*11.5'
94*57.3'
5174 GA335 1
245
15
F
3
03/29
TX
29*10.2'
94*59.5'
5173 P0149 1
145
1
U
5
03/29
TX
28*38.2'
95*52.7'
5169 GA333 1
118
0
M
3
03/29
TX
29*15.8'
94*50. '
5175 GA336 1
118
0
N
3
03/30
TX
29*26.4'
94*39.2'
5032 SHCM-057 1
130
H
4
04/01
AL
30*18.5'
87*30.8'
4989 CC094 1
158
N
2
04/01
TX
27*32. •
9ri3. '
5023 MS034-90 1
91
U
4
04/02
HS
30*12.1'
88*28.0'
5024 MS035-90 1
254
M
4
04/02
HS
30*12.1'
88*28.0'
5148 PA194 T
110
F
3
04/03
TX
2r43.7'
97*07.8'
5249 SE5249 T
241
U
3
04/04
LA
29*34.4'
92*29.0'
5250 SE5250 T
232
U
4
04/04
LA
29*35.2'
9ri0.1'
5251 SE5251 7
326
u
3
04/04
LA
29*47.6'
93*22.5'
TH BL HS KD LV BN ST GO BO SK EX CI BT CT FH NE
♦ -•--♦
♦
♦ ♦♦♦♦♦-♦•♦
♦ ----■♦
♦ ♦•♦♦-♦
♦ --••♦
♦ --•-•♦•
♦ ♦♦•«••♦ Y--
♦ ----♦
♦ ♦♦♦♦♦♦ Y-Y-
♦ ----♦
♦ ♦ + + ♦♦ YY---
♦ ♦♦♦♦•» YY---
*********
♦ ♦♦♦♦♦ YY-
♦♦♦♦♦♦♦♦- ---YYYC
♦ ♦♦♦♦♦♦-♦- -YYYY-
♦ ♦♦♦♦♦♦♦♦♦♦•Y-Y-
♦ ♦♦♦♦♦♦- - - -YYYY-
♦ ♦♦♦♦♦ YYY--
♦ ♦♦♦♦♦ YY-C
98
Appendix I. (continued)
SEUS COLLECTION # G LEN GLCS X C MO/DA ST LAT
5247 SE5247 1
r 238 ---
• u
04/04
U
29'41.0
92*51 .7'
5253 SE5253 1
r 250 -•-
■ M
04/04
LA
29°45.2
93*36.2'
5248 SE5248 1
r 226 ---
• u
04/04
U
29T6.6
92*40.7'
5252 SE5252 1
r 246 ---
u
04/04
LA
29°48.4
93*27.1'
5040 MML-9006F 1
r 173 ■•-
F
04/07
FL
27°28.5
82*43.2'
5396 PA195 1
r 240 25
F
04/07
TX
27°43.7
97•V6.^'
5243 SE5243 1
r 151 ■•-
F
04/08
U
29°47.
93*31. '
5244 SE52U 1
r 245 -•-
M
04/08
LA
29°46.
9r42. '
5397 PA196 1
r 235 ---
H
04/08
TX
27°46.6
97*05.9'
5398 GA338 1
r 105 0
H
04/08
TX
29°09.5
95*00.6'
5399 SP120 1
r 101 0
F
04/09
TX
29°39.8
94*04.9'
5401 SP122 1
r 213 --■
F
04/09
TX
29°35.7
94*15,5'
5400 SP121 1
r 117 0
U
04/09
TX
29°39.7
94*05.0'
5193 SHCM-058 1
r 269 ---
H
04/11
AL
30°23.1
88*17.5'
5242 SE5242 1
r 249 --•
H
04/12
U
29°46.2
93*24.2'
5194 SHCM-059 1
r 221 -•-
F
04/13
AL
30^.
87*50. '
5385 SE5385 1
r? 7 --•
U
04/13
U
29°19.0
89*48.4'
5379 SE5379 1
r? 7 ■--
U
04/13
LA
29°14.3
89*59.3'
5381 SE5381 1
r? ? • - -
U
04/13
LA
29°16.7
89*56.2'
5384 SE5384 1
r? ? ---
U
04/13
LA
29°18.8
89°51.6'
5382 SE5382 1
r? ? ---
U
04/13
LA
29-18.4
89*56.2'
5377 SE5377 1
r? ? -•-
U
04/13
LA
29'08.5
90*07.5'
5376 SE5376 1
r? 7 •--
U
04/13
LA
29°05.2
90*13.5'
5374 SE5374 1
r? 7 ---
U
04/13
LA
29°34.4
92*92.2'
5383 SE53a3 1
r? ? - - -
U
04/13
LA
29*18.4
89*56.2'
5373 SE5373 1
r? ? - - •
u
04/13
LA
29»16.6
91*18.8'
5378 SE5378 1
r? ? - - -
u
04/13
LA
29°09.6
90*05.9'
5380 SE5380 1
r? ? - - -
u
04/13
LA
29°14.6
89*58.7'
5375 SE5375 1
r? ? ---
u
04/13
LA
29T)2.6
90*45.8'
5137 MS036-90 1
267 ■--
M
04/13
MS
30'15.2
88*42.9'
5402 PA 197 1
r ? ---
U
04/14
TX
28*D4.4
97*02.1'
5196 SHCM-060 1
208 -•-
F
04/15
AL
30*18.5
87^1. '
5197 SHCM-062 1
93 ---
F
04/15
AL
30*15.1
88*08.8'
5191 HS039-90 1
94 ...
U
04/15
MS
30*13.2
88*52.4'
5403 GA339 1
244 32
F
04/15
TX
29*23.
94*43.4'
5196 SHCM-061 1
112 -■■
F
04/16
AL
30*16.2
87*33.8'
5186 FLGM41690-3 1
90 -••
H
04/16
FL
30*27.
86*30. '
5240 SE5240 1
? —
M
04/18
LA
29*49.0
93*43.2'
5198 SHCM-063 1
171 -•-
F
04/19
AL
30*18.2
8r44. '
5404 SP123 1
208 4
H
04/19
TX
29*48.1
93*55.7'
5405 GA340 1
256 ---
F
04/19
TX
29*21.4
94*43.3'
5199 SHCH-064 1
104 --■
F
04/21
FL
30*18.2
87*24. '
5239 SE5239 1
r 113 •--
U
04/21
LA
29*47.
93*09. '
5192 MS040-90 1
r 257 ---
U
04/21
MS
30*11.6
88*59.3'
5406 GA341 1
r 97 0
F
04/21
TX
29*17.6
94*47. '
5407 PA198 1
r 240 ---
H
04/22
TX
28*04.7
97T)2.7'
5200 SHCH-065 1
7 ---
H
04/23
AL
30*19.1
88*11. '
5140 DSU90-01 1
250 ---
F
04/23
FL
26*06.5
81*48.2'
5183 MS037-90 1
185 •--
F
04/24
AL
30*13.6
88*18.6'
5184 MS038-90 1
223 •--
F
04/24
MS
30*20.8
88*31.3'
5409 PA199 1
? —
F
04/25
TX
28*01.5
96*57.8'
5408 GA342 1
r 220 7
H
04/25
TX
29*30.8
95*10.8'
5201 SHCM-066 1
r 140 ■--
U
04/26
AL
30*
sr
5138 HM9010 1
215 ---
H
04/26
FL
27*53.2
82*28.2'
5234 SE5234 1
220 ---
U
04/28
LA
29*45.
93*43. '
5238 SE5238 1
r 240 --■
U
04/28
LA
29*U.2
93*41.5'
5209 SHCH-074 1
168 ---
F
04/30
AL
30*14.
88*00.5'
5139 MM9011 1
r 7 •-•
U
04/30
FL
2r4a.o
82*48.4'
5279 MS049-90 1
r 163 ---
U
04/30
MS
30*13.3
88*30.4'
5210 SHCH-075 1
r 218 ---
U
05/02
AL
30*14.5
87*54. '
5211 SHCH-076 1
168 ---
U
05/03
AL
30*14.4
87*53.7'
5204 SHCM-069 1
r 118 -•-
H
05/03
AL
30*14.8
8r41.4'
5205 SE5205 1
r ? ---
M
05/04
AL
30*18.7
88*08.2'
5206 SHCH-071 1
7 ---
U
05/07
AL
30*13.9
87*54.2'
5142 MS041-90 1
r 150 •--
U
05/07
MS
30*13.
88*31. •
LON TH BL MS KD LV BN ST GO BO SK EX CI BT CT FH NE
♦ ♦♦♦♦♦•♦•♦♦- -YYYC
♦ ♦♦♦♦♦•♦♦♦♦♦
♦ ♦♦♦♦♦■•♦
♦ •-••♦
♦ •♦•♦♦♦♦♦-■♦■
♦ •---♦
♦ •♦•♦♦♦♦♦♦♦♦*
♦ ♦♦♦♦♦♦♦♦-♦-Y-
♦ --••♦♦ Y-
♦ ♦♦♦♦♦♦♦- •♦•Y-
4.4.4.. ...... ......
♦ --
♦ + ♦♦♦•♦
♦ ♦♦♦♦♦
♦ ♦♦♦♦♦--♦-. YYY-C
99
Af]pendix I. (continued)
SEUS COLLECTION # G LEN GLGS X C MO/DA ST LAT
LON
5207 SHCH-072
r 286 --■
- M
2
05/09
AL
30*13.6
87*49, 9-
5235 SE5235
r 179 --■
- F
4
05/10
LA
29°47.
93*30. ■
5236 SE5236
r 261 -■■
■ U
5
05/10
LA
29"47.
93*25. '
5237 SE5237
r 202 ---
• M
4
05/10
LA
29°47.
93*23. '
5262 MS045-90
r 267 -•-
• U
3
05/10
MS
30*19.
88*29. '
5260 MS0«3-90
r ? ■•-
• U
4
05/10
HS
30*19.
88*30. '
5261 MS044-90
r 102 ---
• U
4
05/10
MS
30*19.
SBTSO. '
5410 GA343
r 93 0
H
3
05/10
TX
29*19.4
94*U.5*
5263 MS046-90
r 168 --•
U
4
05/13
MS
30*21.
88*24. •
5208 SHCH-073
r 215 ---
• M
3
05/15
AL
30*32.
88*04, '
5264 MS047-90
r 231 ---
U
4
05/15
MS
30*20.
88*31. '
5265 MS048-90
r 229 -•-
U
4
05/15
MS
30*20.
88*31. '
5282 MHL-9007F
r 207 ■•-
F
3
05/17
FL
26*54.7
82*21.2'
5271 MM9012
r 96 ■■-
F
3
05/20
FL
27*43.8
82*44.6-
5571 PA
r 7 ---
U
5
05/21
TX
zeroz.5
96'51.5'
5U6 DRC-90-2 1
r 244 ---
U
1
05/22
FL
24*44.1
81*00.4'
5283 SHCM-077 1
r 261 ---
H
2
05/23
AL
30*27.5
87*55. '
5431 MCSM-90-r0009 1
r 251 ---
F
4
05/24
MS
30*24.
88*54.5'
5272 MM9013 1
r 118 ---
H
3
05/27
FL
27*53.2
82*28.2'
5267 CK-01-90 1
r 254 ---
M
4
06/02
FL
27*59.0
8r48.5'
5617 90/11/466 1
r? 240 ---
F
7
06/02
FL
30*23.
87*27. '
5280 DSW-90-02 1
r 252 ---
M
3
06/05
FL
26*38.3
82T)4.2'
5356 90-11-0491 1
r 229 ---
H
7
06/07
FL
30*23.
86*30. '
5281 FLGM60790-4 1
r 231 - -
F
3
06/07
FL
30*23.
86*30. '
5288 MS050-90 1
r 47 ---
U
3
06/08
MS
30*13.5
88*54.3'
5411 GA3U 1
r 206 ---
F
2
06/08
TX
e
• 1
5412 P0153 1
r 248 16
H
3
06/09
TX
28*08.
96*45.8'
5357 MML-9008F 1
r 203 ---
H
3
06/10
FL
26*52.2
82*19.5'
5421 LA001-90 1
r 262 ---
U
5
06/11
LA
30*07.7
89*25,8'
5289 MS051-90 1
r 206 ■--
H
3
06/11
MS
30*13.5
88*54.3'
5432 MCSM-90-F0010 1
r 126 ---•
F
2
06/12
MS
30*20.
88*07. '
5424 MS052-90 1
r 102 ---■
U
5
06/18
MS
30*22.1
88*50.2'
5133 MM9014 1
r 118 --•■
H
4
06/23
FL
27*55.7
82*32.0'
5366 SHCM-078 1
232 ---•
M
4
06/25
AL
30*14.9
88*10,6'
5363 41650-26 1
r 167 ■--■
M
3
06/25
FL
29*40.5
8512. '
5358 MML-9009F 1
r 89 -■-■
M
4
06/25
FL
26*58.5
82*23.1'
5433 MCSN-90-M0011 1
r 146 ---•
M
3
06/26
MS
30*20.
89T)4. ■
5413 CC097 1
280 ---■
H
4
06/26
TX
2ri2.
97*26. '
5415 P0155 1
209 ---
U
5
06/26
TX
28*07.7
96*46.2'
5367 SHCH-079 1
127 ----
H
3
06/28
AL
30*14.9
88*10.7-
5368 SHCM-080 1
250 ----
H
4
06/29
AL
30*14.2
88*16. '
5369 SHCM-081 1
249 ---•
F
3
06/30
AL
30*38.
88*01.7'
TH BL MS KD LV BN ST GO BO SK EX CI BT CT FH NE
- ♦-♦♦ ♦- -Y-C
Y Y
♦ ♦♦•♦♦
♦ Y Y
Y Y
Y Y Y Y C
COOES: SEUS=archive #; COLLECTION it. Field Collection #; G, genus, T=Tursiops: LEM= length, cm; GLGS=age; X=8ex; C=condition;
MO/DA=month/day; ST=state; LAT^latitude; LON=longitude; TH=teeth; BL«blubber; MS=»nuscle; KD»kidney LV«l iver; BN=booe; ST=stoniach;
G0=gon8ds; BD=blood; SIC=skull; EX=other; CI, Y=tissues sent to MWFC; BT, Y=brevetOKin done; CT, Y«tissues sent to EPA; FH, Y=food
habits done; NE, C=clinical necropsy
100
Aspendix II: Bottlertose dolphin strandings in the northern Gulf of Mexico. 1982-90. For Texas, the proportion listed is the
proportion of Gulf strandings from Texas.
YEAR
STATE
82
83
84
85
86
87
88
89
90
82-9
TOTAL
TX
29
.56
32
.51
84
.73
50
.59
116
.73
111
.57
100
.52
90
34
201
.42
612
.60
813
LA
0
0
6
0
6
17
14
0
49
43
92
MS
1
14
3
12
14
19
36
22
84
121
205
AL
1
0
4
1
0
7
9
11
58
33
91
FL
21
16
18
22
23
41
34
42
86
217
303
TOTAL
52
62
115
85
159
195
193
165
478
102
6
1504
101
Appendix HI: Overall sex ratios and sex ratios for animal <140cni by year for January-June Texas
bottlenose dolphins strandings.
YEAR
I FNGTH
TOTAL
I FNGTH
<140cin
MAIF
TOTAL
MAf F
<140cm
FEMAIF
TOTAL
FEMAIf.
<140cni
M:F
MF
<140cin
1984
54
18
33
31
10
32
14
4
1.00:
0.45
1.00:
0.40
1985
31
4
.13
13
2
.15
12
1
.08
1.00:
0.92
1.00;
0.50
1986
82
29
35
37
15
.40
23
7
30
1.00:
0.62
1.00:
0.46
1987
114
35
31
52
15
.29
38
5
.13
1.00:
0.73
1.00:
033
1988
91
28
31
42
17
.40
22
3
.14
1.00:
0.52
1.00:
0.18
1989
59
15
.25
23
9
39
27
5
.18
0.85:
1.00
1.00:
0J5
1984-89
431
129
30
198
68
34
136
25
.18
1.00:
0.69
1.00:
037
1990
142
22
.15
58
13
.22
57
6
.10
1.00:
0.98
1.00:
0.46
102
Appendix FV. Report on aerial surveys of bottlenose dolphin abundance conducted in near- and offshore
waters off the Texas coast during 1990.
103
Aerial Surveys
Keith D. Mullin
Southeast Fisheries Science Center
Mississippi Laboratory
Pascagoula Facility
3209 Frederic Street
Pascagoula, MS 39567
Methods
Survey Blocks
Aerial surveys were conducted in response to two events: the 1990 bottlenose dolphin
mortality event and the oil spill from the oil tanker MEGABORG. The surveys completed
in response to the mortality event were conducted in block 154 during March, 1990. The
surveys associated with the oil spill were conducted during Jime, 1990 in the vicinity of the
MEGABORG and with one exception (block "B") duplicated survey blocks studied by Scott
et al. (1989) (Figure 1): 152 - 1,296 km^, 153 - 1,588 km^, 053 - 16,292 km^ and 154 - 11,040
km^. Block "B", a 5,850 km^ area in the immediate vicinity of the MEGABORG was also
surveyed (see Section II, Figure 1). Sampling methods for all surveys were similar.
Sampling
Aerial surveys using line transect methods (Bumham et al. 1980) were used to sample
the survey blocks. The sampling strategy was similar to that presented by Scott et al. (1989).
Transects were selected randomly and were placed perpendicular to water depth isobaths.
Samples were designed to sample 7.5% of the surface area of blocks 152, 153, and 154, and
5% of 053.
Survey flights were conducted during daylight hours from 22-24 March 1990 and 14-18
June 1990 when the weather was clear to partly cloudy and the Beaufort Sea State was 3 or
less. The survey platform was a DeHavilland (DHC-6) Twin-Otter aircraft maintained and
operated by NOAA's Aircraft Operations Center. The aircraft had a large plexiglas bubble
window on each side which allowed for an unobstructed view of the transect line.
Transects were surveyed from an altitude of 230 m (750 feet) at an airspeed of 204
km/hour (110 knots). The flight crew consisted of a NOAA pilot and copilot, and 3
experienced NMFS observers. While surveying transects, one observer was stationed at each
bubble-window. The third observer entered data on a laptop computer. The computer was
104
interfaced with an aircraft LORAN system. A data acquisition program downloaded the
time and date, and the latitude, longitude, speed and heading of the aircraft whenever
sighting data was entered. The observers rotated positions about every 30 minutes.
Observers and the flight crew commimicated through headsets via the aircraft
intercom system. Observers searched for marine mammals, sea turtles and other marine life
at the surface of the water from directly beneath the aircraft out to a perpendicular distance
of 629 m. Whenever a sighting was made, the distance of the sighting from the transect hne
was measured using calibrated marks delineating 7 perpendicular distance categories on each
bubble window (40, 83, 132, 192, 273, 397, 629 m). When necessary, the aircraft was diverted
from the transect line to make species identifications and to estimate marine mammal herd
sizes.
Density Estimation
Bumham et al. (1980) recommended that sighting functions should be based on a
minimum of 40 sightings, but stated 60-80 sightings were preferable. However, White et al.
(1989) suggest that over 200 sightings may be required. Because only 94 cetacean herds
were sighted (91 were bottlenose dolphin herds) within 629 m of the transect line during
both surveys, the perpendicular distance sighting data were pooled with 1,523 bottlenose
dolphin herd sightings coUected by the same survey team in the northern Gulf of Mexico
from the same aircraft. These pooled data were used to construct a sighting histogram. To
estimate !(0), the value of the probability density function evaluated at the transect line, a
hazard-rate model (Buckland 1985) was fit to the histogram. The hazard-rate model was
selected for two reasons: (1) the number of parameters in the model is fixed (there was no
subjective decision making regarding the number of parameters), and (2) the model always
has a shoulder near the transect line (distance zero).
Bottlenose dolphin density for each survey block was estimated as the product of a herd
density estimate and a estimate of mean herd size. Herd density was estimated separately
for each survey block. In order to increase sample sizes and reduce variability of mean herd
size estimates, all herds sighted in inshore blocks (152, 153, 154) were pooled as were all
herds sighted in offshore blocks (054, B). [Bottlenose dolphin herds in the Gulf of Mexico
may increase in size in deeper water.] Of the bottlenose dolphin herds sighted during both
surveys, 89% were of 10 dolphins or less. However, three herds were sighted that were
greater than 40. Because of relatively small sample sizes, these large herds had a
tremendous influence on the means and substantially increased variability. Therefore data
were trimmed from each end of the herd size distributions until the means stabilized. This
generally occurred after a total of 15% of the data were excluded. The "trimmed" mean
herd size for offshore and inshore blocks was estimated as the arithmetic mean.
105
The herd density, t)^, for each replicate transect, i, was estimated as
where 1^ was the transect length and n was the number of herds sighted. To insure that no
herds were coimted more than once (during each replicate), each transect was considered
a replicate. The herd density, 6^, for each survey block was estimated from R replicate
transects by
and the variance of this estimate was approximated as
Dolphin density, t)^, was calculated as
^a'^h
the product of the mean herd size (fl) and estimated herd density. The variance of t)^ was
estimated using Goodman's (1960) method of estimating the variance of a product
v^r (Z?d) -^s% (4) 2+i5^s% (/f) ==-s% (i/) ^sfe (4) 2
where n was the number of herds used to estimate the mean. The standard error was
estimated as
The approximate 95% confidence interval, assuming lognormal error, was estimated as
where
1.96 ./ln(i*(-^)*)
C-e V ^
106
Dolphin abundance was estimated as the product of sampling block surface area and the
associated density estimate.
Results
Transects surveyed during the mortality-event-related surveys totaled 1,845 km. Over
2,200 transect kilometers were surveyed during the MEGABORG-related surveys. Forty-nine
cetacean herds, all bottlenose dolphins, were sighted during the mortality event investigation
surveys. A total of 42 herds of bottlenose dolphins were sighted during the MEGABORG
surveys. Two herds of Atlantic spotted dolphins {SteneUa frontalis) and a dolphin that was
probably a Risso's dolphin {Grampus griseus) were also sighted.
Table L Estimates of bottlenose dolphio density and related parameien.
Block
R
le(H)
n
t>.
«e(l5h)
R
t>.
•c(l5a)
km'
f^LSS
fi
«U«
22-24 March 1990
154
33
036
41
0.042
0.007
27
0.14
0.027
11,040
1,063
1,564
2048
14-18 June 1990
054
6S
1.67
8
0.033
0.010
4
0.21
0.083
1632
1,621
3,421
7,218
-B"
6.5
1.67
5
0.014
0.006
15
0.09
0.044
5350
213
527
1305
152
52
0.95
0
0
-
8
0
-
1,296
-
0
-
153
52
0.95
3
0.035
0.020
6
0.18
0.108
1,588
96
286
847
IS4
5.2
0.95
26
0.048
0.010
13
0.25
0.069
11.040
1.623
2.760
4.694
H - mean bottlenose dolphin herd size
n - number of herds sighted
C-b-
bottlenose
dolphin
herd density (herds/km')
R - number of
replicate
transects
t>,-
bottlenose
dolphin
density
(dolphins/km^)
km'.
survey block surface area
«L95.
f^. I^U95 -
bottlenose dolph
lin abundance estimates.
lower 95%
interval bound, point estimate, and upper 95% interval bound,
respectively
An f(0) of 3.08 km"^ (se = 0.11 km"^) was estimated using the hazard-rate model.
The estimated average bottlenose dolphin herd size used for density and abundance
estimation ranged from 3.3 to 6.5 dolphins/herd (Table I). The largest herd sighted was of
52 bottlenose dolphins. Sighting conditions were less than optimal in block 152 and no
bottlenose dolphin herds were sighted. In survey blocks where bottlenose dolphin herds
were sighted, estimated dolphin densities ranged from 0.09 dolphins/km^ in the vicinity of
the MEGABORG (block "B") to 0.25 dolphins/km^ in block 154 (Table I).
107
Literature Cited
Buckland, S.T. 1985. Perpendicular distance models for line transect sampling. Biometrics
41:177-95.
Bumham, ICP., D.R. Anderson and J.L. Laake. 1980. Estimation of density from line
transect sampling of biological populations. Wildlife Monographs 72:1-202.
Goodman, L~A- 1960. On the exact variance of products. Journal of the American Statistical
Association 55:708-713.
Scott, G.P., D.M. Bum, LJ. Hansen, and R.E. Owen. 1989. Estimates of bottlenose dolphin
abundance in the Gulf of Mexico from regional surveys. National Marine Fisheries
Service, Miami Laboratory, 75 Virginia Beach Drive, Miami, Fl 33149.
White, G.C., R.M. Bartmami, L.H. Carpenter and RA. GarrotL 1989. Evjiluation of aerial
line transects for estimating mule deer densities. Journal of Wildlife Management
53:625-635.
108
Appendix V. Gymnodinsum brtvt presence/absence' and quantitative cell counts^
SutioD BottoiD Sample Oymnodijuum breve
Number Depth(m) Dcpth(m) Pre»cnce/Ab«cncc Quantitative
+ ± cellar'
CaauDoils
10
11
13
24
30
34
36
32
32
30
28
24
26
0
3^
8.9
12.7
0
+
10.4
+
16.5
+
22.8
0
+
14.5
+
20.5
+
273
+
0
+
16.6
+
23.1
+
32J
+
0
+
15.8
+
24.6
+
33.5
+
0
+ +
13.4
19J
+
29.9
0
16.0
+
243
+
293
0
++
8.2
+ +
13.7
++
28
0
11.2
17.2
27.1
0
11.1
+
183
23.4
+ +
0
+
8.7
+
14.1
+
243
20
7
21
3
80
22
35
48
18
21
11
38
160/40
51
22
14
180^1
75
Sediment'
Sediment
Sediment
Sediment
109
Appendix V. Continued.
Station Bottom Sample
Number Dcptli(m) DcptJi(m)
Gynviodmjwn breve
Prcscnce/AbscDce Quantitative
+ ± - cells r'
Comments
12
13
14
15
16
17
18
19
20
21
24
22
17
17
12
14
17
10
11
0
+
8.4
+
15.8
+
23.4
+
0
+
IZl
17.6
+
23.1
+
0
+
8.2
+
12.2
+
21.9
0
+
S2
+
95
+
15.8
+
0
5.2
93
+
15.5
0
7.0
9.2
12.0
0
+
3.2
+
7.6
++
n.i
0
5S
+
82
16
0
Zl
+
45
+
8.7
+
0
4.6
73
95
88
66
46
24
440/413
122
66
71
8
14
0
40/2
86
21
104
71
0/32
80/38
Sediment
Sediment
Sediment
Sediment
Sediment
110
Appendix V. Continued.
SutioD Bottom Sample
Number Deptli(m) Dcpili(m)
Gymnodmhan breve
Prcsence/Abceoce Quantitative
+ ± cells r'
ConuDcnu
21A
22
24
25
12
14
20
16
13
0
0/19
3.4
+
240/45
7.9
+
11.2
+
0
38
53
32
10.2
^
1Z9
-
0
+
180/170
5.7
80
15.1
±
193
±
0
+
«9
5.7
+
58
10.4
+
14.7
+
0
.
176
3.4
+
133
8.6
+
11.6
+
25A
Surface water discolored NoaUuca
appnxL 900,000 ceUs r>
25B
26
27
28
29
16
20
21
20
0
±
8,000 r
3.2
+
2-4,000 r'
6J
±
9.2
+
0
on
4.1
5
9.9
-
14.8
+
0
45
3J
56
5.8
+
19.6
+
0
+
38
43
+
80/19
7.9
+
20J
-
0
±
10
6.7
+
51
143
±
19.6
±
Noaihtca
NoaHuca
111
Appendix V. Conltoued.
SutiOD Bottom Sample OymFiodinaon brevt
Number Depth(m) £>epth(m) Presence/Absence Oiuntiutive
+ ± cdl$r'
Coauiiails
30
31
32
33
34
18
18
14
0
+
S.0
+
10.6
+
17^
+
0
+
5^
lOJ
+
17.0
0
+
S2
+
92
\Z2
+
0
+
\Ji
+
$2
8.4
0
A2
6.6
26
30
3
14
7010
5
0
7
Sediment
Sediment
Sediment
Sediment
'initially, all samples were checked for presence/absence of Oymnodinium breve cells. This was a range finding pnxxdure to evaluate the
techniques needed to do quantitative counts. These cunofy observations do not always agree with the quantitative observatioiu.
Quantitative counts. See text for details on method for quantitative counts.
^Samples marked "sediment* had too much sediment to allow reliable ccamination.
112
Appendix VI. Contract report on brevetoxin analysis. Analysis and report prepared by the Chiral
Corporation.
113
(Cover Sheet)
Reference Order #: 40WCNF002505
Issuing Office:
National Oceanic and Atmospheric Administration
CASC Procurement, CC33
601 R 12th Street
Kansas Oty MO 64106
Description:
Analysis of bottlenose dolphin liver samples and fish samples for
presence of brevetoxins as part of NMFS Emergency Investigation of
Mass Mortality of Bottlenose Dolphins in the Gulf of Mexico.
Issuing Date:
April 19, 1990
Work Commencement Date:
September 19, 1990
Work Completion Date:
March 18, 1991
Report Date:
March 18, 1991
U4
TABLE OF CONTENTS
INTRODUCTION AND BACKGROUND 116
STATEMENT OF WORK 116
PRE-AWARD CONDITIONS 116
AWARD CONDITIONS H*^
PROTOCOL FOR LIVER ANALYSES 118
RATIONALE FOR METHODS 121
EXPERIENCE, EXPERTISE, AND PROHCENCY OF PERSONNEL 121
SUBMITTED SAMPLES 122
FEE SCHEDULE FOR BREVETOXIN ANALYSES 123
GENERAL OBSERVATIONS ON SAMPLES RECEIVED . 124
BIOASSAY DURING PURIHCATION 124
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 124
RADIOIMMUNOASSAY 125
POTENTL\L SOURCES OF ERROR 125
RECOMMENDATIONS 126
RADIOIMMUNOASSAY FIGURES AND CALCULATIONS OF
BREVETOXIN EQUIVALENTS 127
115
ISTRODUCnON AND BACKGROUND
The SEFC has reqxsnsibility for an Emergency Invesdgnion inio the eoent, cause and effea of the
mass mortality of bottknose dolphins in the US Gulf of Mexico. Bottlenoce do^hins have been stranding at
about 3 times the normal rate along the Gulf of Mexico from north central Florida to south Texas smce the
fim of January (over 300 stranded dolphins have been observed). The majority of animals (circa 140) have
stranded along the Texas coast. A biotoxin was identiSed as a possible cause of the 1987-88 east coast
bottlenoce do^hin dieoS, and it is imperative that samples from the current dieoCT be analyzed for presence
of biotcodns. Chiral Corporation was selected to cany out the assays for brevetoxins, based on expertise in
the isolation and purification of these potent biotoxins.
STATEMENT OF WORK
The contractor shall complete analyses of bottlenose do^hin bver saiiq>les for brevetoxins for $2S0iX)
per sample NTE S12400 in a term of 6 months. The contractor shaD analyze available marine mammal liver
samples and fish samples selected by the NMFS OOTR, in consultation with the oontraaor and other
researchers. The contractor agrees to conduct analyses of fifty (SO) san^)les for individual brevetoxins. A
detailed schedule of activities wiD be provided by the contractor and agreed to by the OOTR before award.
The contraaor also agrees to the following award conditions. Award of this contract is contingent upon
acceptance by the CX)TR of the schedule and aU items identified by the OOTR below as award conditions
before contract items 1 through S. Rnal paymem is oontingem vpoa acceptance by the OOTR of aD items
identified below as Award Conditions Fioa] Report items 1 through 5.
The contractor may submit biweekly invoices for saiiq>les analyzed, or may submit an invoice after all
tissues are analyzed. The final invoice shaD consist of a draft final iqxm which nieets the specifications of
aD items identified below as Award Conditions Final Rqiort items 1 throu^ 5. The draft final repon shaD
be received by Dr. T. Siekicki of the Southeast Fisheries Science Center. Comments on the draft need to be
incorporated into the final fqx>rt before final paymem can be made.
NOTE: Subsequent to Award Notification, Dr. T. Siewicdd was replaced as OOTR by Dr. Sylvia
Galloway. We operate under the assumption that Dr. Galloway shaO receive both the Draft and Revised Final
Report
PRE- AWARD CONDITIONS
1. Provide and agree to detaliled protocols for aD steps of sample preparation, extraction, purification,
bioassay, and chromatography. Details should be provided to allow any competent researcher to repeat aD
steps, (attached herein as ITEM #1]
2. Provide rationale for methods that sippon the effectiveness of the methods for detection and
quantification of brevetoxins in marine mammal tissues, (attached herein as ITEM #2]
3. Identifywhat samples to analyze based on proximity of impacted area, confirmed bloom, freshness of
carcass, case history (ind. pertinent Uological dau), proper handling and norage of the sample. AD samples
wiD be coded and provided Mind to the contractor. A total of ten suitable control and qiiked-oontrol samples
wiD be included in the total (attached as ITEM #3].
4. Documentation of experience, expertise, and profiden^ of of brevetoxin analyses of tissue samples
for aD persoiuiel must he provided. Similarly drtailrd documentation must be provided for any subcontractors
to be used. Approval of subcontractois by the OOTR is required before award, (attached as ITEM #4]
5. Spediy amount of tissue needed for each analysis and any tpedai collection, handling or storage
procedures required.
U6
AWARD CONDITIONS
1 Provide quimiutive bioassay results for each cnidc extract and each preparation resuhing from major
puriScation steps.
2. Include aS raw data for all procedures employed.
3. Provide quantitative results on each brevetaxin found in each sample analyzed (per unit sampkr
weight).
4. Provide qualitative and quantitative resuhs from all procedures employed.
5. Identify any problems encountered and thoroughly describe any concerns or Umitations to the use.
interpretations, or inferences made from the results.
INTRODUCTION AND BACKGROUND. SOW, AND AWARD CONDITIONS were communicated by FAX,
with follow-up hard-copy, on 7 June 1990.
U7
Innovative Probes for Molecular Research
PROTOCOL FOR LIVER ANALYSES
BREVETOXINS IN DOLPHIN LIVER
Spprif"" kems related to tamfde mhwhtioe are otmibered:
(1) RECEIPT: Sampbi chaD be received Toesday-Tbursday (shipped Monday-Wedoetday) ai the
Chira] CorporatioD Research and Developoeal Uboratory. The proper ^bippiag address is:
Chira) Corporaiiao R&D Laboratory
Uaivenity of Miami Rosenstid School of Marine and Atmoqiheric Scsence
4600 Rickeabadcer Causeway
Miami norida 33149
ATTN: Daniel G. Bades
The shipper should make every effort to retain portions of each sample shipped m case of Iocs of sh^nnent,
unavoidable loss of sample, or for eonfirmatioe by additioB labocatories.
(2) SIZE: Livers shaD be of "«"«i«"'"" size 45-60 f, and iwfividual samples shaD be padcaged in separate
swirl packs or other DOs-water-solnUe p»'*^E™£ Other types of sample should be of similar size.
(3) SHIPPING: Samples shaD be padced in dry ice, and shipping eootainers shaD be marked as
appropriate for infectious agents, as idrntiftcd by the lATA. Appnyriate labels may include those pertaining to
toxins, dry ice, and infectious agents.
(4) CODING: All samples should be eoded in a random £ashiofi, with copies of the coding retained
by the shipper.
(5) SAMPLE QUALITY: Liver integrity is of principal importance in assays for brevetonns. No
guarantee can be made for analyses resulting froin decomposed, improperly p«'*ig^'<. or otherwise non-pristine
materials.
(6) SAMPI ^. AGE All hough not qnantififid, cge may have Sipme bearing on the coocentratioas of
toads remaining. We do not set a time limit oe sam|rie storage for data is incompleie, but draw anentioo to it
as a possible complicating faaor in certain analyses.
(7) CONTROLS: Appropriate eoded control amplrs should be submitted, mixed with authentic samples.
Two coofirmed cootrcds should be marked as CONTROL, and shipped with the first set of samples, for batft'T
confumation.
Specific Protocol Steps are Numbered:
(1) Upon receipt, fiver samples are nnparkfd, logged, weighed, and placed m a -20^ freezer. We shall
analyze 10 samples per week, and based on shipments of this number, no sampk shaD be in our freezer for more
than one week. AD samples are treated as iafectioos agenu as defined by HHS publication No. (NIH) 88-8395.
(2) AD protocols ntifize pristine ^assware which has been nordtased medficaDvfor the proien md h».; not
been used previousK. This is of predominant importance to prevent background lewds of tonn in re-used
glassware. AU soh«Dts are of rea^at grade or better.
(3) Individual livers an placed m SOO mL gUkt beakers, 300 mL acetone is added, and each is covered with
a glass watchglass. Livers soak owemight at room temperature b acetone to dehydrate. The fiver suspension is
homogenized using a double blade Vtrtis hoaogenizer operated at 2S00 RPM m an ice bath. The acetone/bver
bomogeaate is powed off and is fihered throii^ a bochner funnel using Whatman #1 fiherpaper and eoltected
118
■Bto SOO bL nde am fiasks aader vaaniiB atpiraiMCL Arrfopc tfttuiiotu arc truuicrrco u> mojviau^ loag
■ed SOO mL ranad boooB fiaikt and eadi ■ fiasb ewiponted oo a rotary evaporator operating at nduccd
flrtsuxre. Reclaimed ioKeal ■ placed is five pBoe Bed drmas for appropriate rtnpnaal.
(4) Hooogenate fiber caken, togetber with the fiber papert, are placed back iato their mdivuluil SOO mL
beakerv muI ^OO mL chtorofarm it added. Hooogeaizaticxi and fibratioc it rcpeaiti
(5) The cfalorofonn fihrates are added to the appropriate acetooe residues from ctep (3) and the chlorofons
■ remowed by rotary flasb-evaporation. [Note 1].
(6) Ib the SOO mL flasks «^n«ifm^£ the fiver residues, add 20 g dry silica gel (ICH chromatograpby grade
or better) and follow with 100 mL acetone. Swirl each flask nitQ the silica gel scours the residue from the flask
liie. and is uniformly tan to brown in color. Flash evaporate acetooe and apply the dried brown silica gel/liver
enraa to the top of a 120 mL silica gel flash chromatography column (Baker Chemical). Add 2S mL acetone
to the original round bottom flask to rinse any remaining material from the sides and pipette this toUuion oo
top of the dried sibca gel in the column. R^ieat once.
(7) Assemble the flash chromatography column resevoir and add Z7D oiL acetooe. Apply 4 psi nitrogen
(99.999%) pressure to the column qiparatus and ooDect aD ehoed niateiial into dean SOO mL loqg neck
round bonom flasks. Flash evaporate loivenl in each case. [Note 1].
(8) Repeat step (6). Substitute 20 mLehlorofonnimethanoLacetic add (100:10:1) flask rinse for the acetone
rinse in step (6).
(9) AssemUe the flash chromatogra|^ column resevoir and add 310 mL cfaloroformaaethanokacetic acid
(100:10:1). Apply 4 pu nitrogen pressure to the column qiparatns and collect aD ehaed material imo a dean
SOO mL loog neck round bottom flasL Flash evaporate. [Nou 1, Note 2].
(10) The residue is redissolved m L0-2J) mL solvent (either acetooe or cUoroform is fine) and is appfied to
a preparative fluorescent silica gel presorbant thin-layer chrooiatqgraphy plate (20 x 20 cm., 1000 » **«i<*»»^».
Whatman PK6F or equivalent) using an Applied ScicBcrs TLC breaking system, nates are chromatogn^ihed
using 100 mL of mobile phase ***«i«*"£ ctf acetooedight prtrolrmn (30:70). [Note 1, Note 3).
(11) Toxic fractions frt» the TLC plate are scrqied m a safety hood, groimd to a fine powder using a
porcelis mortar and pestle [Note 4), and ehtted from the siEca gd in 30 mL sintered ^ass fibers using 2S mL
afnot>f or methanol Eluted tooon is placed in individual SO mL round bottom flasks and flash evaporated.
(12) Toadc frsefioBS tre redisstrfved m methanol, ud applied to semi-preparative fhioreseeat nlica gd thin-
layer chromatography plates C^ x 20 bO., SOO a thirknr.w, Ar^hr^i Uoiplate or equivalenl) and
chromatographed using 100 mL ethyl acetaieJight petrofeum (SChSO) as molnle phase. [Note 1, Note 3).
(U) Repeal txep (11) for toobc fractions.
(14) TooDC fractions are redissolved in methanol, and applied to semi-preparative fluorescent silica gel thin-
layer chromatograiAy plates (20 x 20 cm., SOO a riiifknrti, Anahedi Uniplate at equivalettt) and
chromatographed using 100 mL ethyl acetateJighi petroleinn (70-30) as mobile phue. [Note 1. Note 3J.
(15) Repeat st^ (11) for tooac fraaioos.
(16) Toobc fraetioes are £ssoh«d from SO mL round bottom flasks nsiag '"i"^*"*' (03-10 mL ^lic grade
methanoL High performance liquid chromatography is performed in ana^ticd mode using a Ranun C-18
analytical reverse phase column (4j6 mm diameter x 2S ^ long) using isocratic 8S% methanol in water, 1.4
mL/minute flow rate. Effliimt is moohored at 215 am [Note 1). f^itmin are caEbrated with known
cooceatrationt of analytical grade purified brevetoxins FbTx-L PbTx-2, and FbTx-3, the &ree predominant toxins
m natural or cultured systems. Excepting note below and ""'**>^">£ steps, we «^ coofirm the identity of the
|^)lc peaks by mixing equal proportioos of anthentv brevetooon and suspect brevttooun from samples. Mixed
samples would then be sAjeaed to oo-migratioo in hplc analysis. Quantification of the amount of btevctoaan
m the origina] unknown samples can be calculated from hplc data.
119
NOTE: Protocols beyood this aep are BOt withxa the eofltnaed S2S0X10 sunple. We will leek the achue of the
Coetraaing Officer prior to imdaiakiqg apy mrrrrrirug a^(, and woold reqoetl approval prior to aaalyus
Thuc procedum iDclude brevetosis radiobnaimoasuy, Fourier iraosforD infrared aaalyus, NMR, or bxmss
tpeOTcmtuy.
NOTES
|1] Bioauay: Gombiuia it^intf, or moiqmto fish, are ittcd as bioasuy ^Kdmea for aD tte|&. Assays
■re cooduaed is 20 mL leawaier of 15% salinity uiiig 1 fish per 50 mL beaker. For lohitioas, cuspea toxxo
■ a<V*^^ in OJOl mL methaDoI at ooocemratioos equtvakst to 5% of the ongjnal sasi|^ extract. For thts-laycr
dirooatograpby plates, a 03 em wide oohuBD is ca b devdoped plates fron ongtn to soKeai from, and 1 cxs
taD fractioits are cut, crushed, and added to dni^icate iadividua] beakers mntaining fish. Lethahty is assessed
24 hr (or 48 hr) later aod toaddty (death '4* or bo death '-*) soled. Sensttivity (LDjd approximates 7D «tg/kg.
Assay at steps (5) and (7) are alinoct uniformly taadc, and may be dispensed with in favor of sample conservation.
Only '* ' samples proceed to the subsequent step.
[2] Aberrant Samples: If any single sample is very oily at this stage, it is partitioned between aqueous
methane^ and li^t petroleum by dissolvii^ the lamf^ in 100 mL 95% wrthannl and nnrarting with 3- 100 mL
portions of light pe&oleum, m a 500 mL separatory htnneL the three ii^ pctrolmm fractions are combined
in a fresh 500 mL separatory funnel and are back-extracted once with 100 mL 95% methanol/water. Both
nethano fraoions are combixked in a 500 mL round bonon flask and the sfdvent is removed by flasb-evqiaration.
The light petroleum fradioos are discarded in the solvenl fcayvery (
[i] TLC Plate Preparation for Toodcity Testing: Developed thin-layer plates are air-dried, followed
by observation under short wave ohraviolet Ught. Using pencil, enorde in>>-absarbing bands. Remove lie plate
from nv Ughi and cot two grooves from origin to solvent front, placed 03 cm qaait, qiprooomately 10 cm in from
the sides of the plate. From origin to solvent fatrnt, cut 1 cm laments fatn the developed plate betwuji the
two parallel score marks, and cnuh each 03 cm x 1 cm silica gel rmanglr into a powder to be added tobioassay
beakers.
f4] CAUTION: Brevetoxins are potent re^nratory irritants, especially when adsorbed oo siEca gel
particles. Take aO precautions necessary for respiratary irritants &at are riwrififd as Class I poisanous
ffuKtt"":* NIOSH certified panicle filtration masks may be worn in qiplicableiitnatiaas where expossre could
occur. Al 1 scraping operations and grinding of silica gel powders tbould be canied out in an approved
ventilation hood.
120
Rationale for Methods
Hreveiniim Tc maieriils which becnae eoaceatraied in nmae titniet throngfa annwl feeAng trtjuit^t
jf PP«f fwnittr<< rrfmniant Thc tonu, lynthrtTTT/l by thc marine faofUgellate ft>cfcadbaa b»rw «rc aorm*l)>
fcKf^^n^wnuUte/i b fiher-feedtog orguisnu, but recent evidence hxfirnci that biomagnificatioc through the food
T*i«iti by ahenaie roates oiay ako occur.
The »<^»*«f« and parificatoB procedure and iafividna] protocols for brevetoiis from dolphio bvcrs is
both jr^**^^ and laborious. Liver, bung the prindpa] drinrififatinn organ of «"»"""■>■■" ipcocs (and more
ao in dolphins which lack a gaD bladder), is the organ we nprrt to find breveiams in their largest conccntratiom
■hould they east. Liver by its nature contains high coBceatratioBS of the eazynes necessary to detoxify organic
ppH^ftwn isduding toodns. Also by its funetional nature is the ttor^ge depot for er»n«wim«tm to which the bviag
doti^iin has been exposed.
Iffitial aeps in the pnrificatioo seek to first denature any enzymatic machinery which may exist in th;
frazen qiedsens using a modified acetOBe predpitatioo stq>. TUs ttep also tends to dehydrate the specimen
and make e&raetioo of Iqiid soluble materiids easier (tlep 3 of protocols). Following dehydratioo, a noo-polar
solvent (chloroform) is used to extract the brevetoxins from the tissue samfde. Brevetooons are freely sofa^ in
the chloroform (step 4). Steps 5-16 are steps utthzed routinely and daily by Chira) Corporation in our protoctds
for extradioo and purificatioo of brevetoiins from laboratory cultures of the **^***^^g'"**^. and as sudi are the
results of 16 years of optimizatioa and progressioo from anal3^tical separation to large scale quantitative recovery.
Flash chramatogra|diy (steps 6-9) is a routine and powerful way to separate toodc materials from
fPfffftnmatmg oOs and pigments, the former of which is a massive probefan in marine mammal tissues. These
initial steps are utlized in the order — and on the scale that thqr art 'to isolate miotigram quantities of toadn
from gram quantities of interfering snlnianrrt
Three sfqumtial thin-layer chromatograpfay oqs are utiEzed to isolate brevetoxins from one another,
and are the exact trfhniqnrt that were used in our laboratory to first determine the individual nature of PbTi-
1, PbTx-2, and PbTz-3. When compared to the known migration of standardized brevetoaons, preliminary identity
(tf suspect toxins can be made.
The final step (tf high performance Equid riiTnmatogra|Ay is an analytical technique, «4iidi when ^ipiied
in the parallel presence of standard brevetoasas, can ^ve a further jmjjiigj basis for ooofirming or denying
brevetoxio identity.
The Cambusia ajfinis fish bioassay is the most sensitive bioassay kimown for the brevetoxins, and
individual brevetoiins exhibit lethal dose ranges in the nanooolar ooBcentratioB ranges (Le. aanomoles of toxio
per liter of test water, fish are placed in 20 mL water «4iich yields a calculated sensitivity of 17.9 ^assay
volume. We bebeve it is accessary to emphasize that '*' bioassay means the fish dies witUn the time period
of observation. For most sensitive applications, observatioos are oiade over 48 hr, but positives fi«quently are
exhibiied within hours of initial laqxKure. Those fraaions which test *•', Le. do death, are not pursued futher.
Thus, a positive value does ajt necjsiarily iadi^tte br. noapn, bat a amative by these criteria does not contain
brevet axis.
Eadi step of the purification and analysis which tests positive lends increasiag support to the indications
that brevetoxis are present in the sample. By back<alculatioa (knowing the amounts ot ataterial tested in a
destructive manner at each step) one can arrive at a value for toxin concentration in the origiaa] sample. The
value is calculated based on liplc traces against known concentrations of standards.
For those samf^ which test positive throughout the entire assay protocol to step (16), we would suggest
further analysis to further confirm identity. The method requiring the least amount of material is brevetoada
radioimmunoassay, a technique pioneered at the University of Miami, and whi^ is part of the product line of
Chiral Corporation. Recent work confirms the polyether Vevetooon-Iike* sensitivity of the assays and a complete
insensitjvity to okadaic acid-type polyether toxins. Non-destructive tesu which could be employed included
Fourier transform infrared qiectrometry, or mass spectnnetry or FT nuclear magn^tir resonance qwctrometry
(the laner two being sub-contracted out). Short of X-ray crystallography, ao sia^e technique wiU unequivocally
identify brevetoxins.
In summary, the progression of the steps m the protocol (i) ^'^™»»» potentiaOy imerferiiv f^Kr^ftuf^;
(a) have bees optimized over many years of hands-on fjjgiicDce; (m) are »>^4»««M2ii>f ased routinefy by the
Corporation to purify brevetoaias; and (W) progressively affirm or deay the ideatity of toobc «i««tiTi«K as
wevetoiins
E)q>erience, Expertise, and Proficiency of Personnel
The cnnicnhun vitae for Daniel G. Baden. President of Qiirs] Corporation, and Lloyd S. Schuhnaa, pan-
121
tne tr'*"^^-" for Chiral Carparatiae. art iadiided with tbete doamnrtt Dr. Bades has 17 ycMn qpciieace
«i^ Uitiiuiim. aod Uoyd SchnlmaB has ewer four yean inrtmiral rrptiriwr wilb puriCcatioo aod
aysuSijxMacm el hrwcuaaai.
Submitted San^les (dupHratr^l oo Pratoco] iheeu)
(1) RECEIPT: Saaplck ^aD be received Tuesday-Tbanday (chipped Moaday-WcdBctday) at the
Chin] Con»oratjoo Reieardi and Dewelopncal Labocaioiy. The proper shippiqg addmt it
CUral Corporatioo R&D Laboratoiy
Usfvenity of Miami RoKsstiel Scbool of Marine and Atmospheric Sdeoce
4600 Rickeabuker Causeway
Miami Florida 33149
ATTN: Daaiel C. Bades
The chipper should make eveiy effort to retain portioes of each campk sh^iped in case of Iocs of shipnenl,
Bnavoidable lou of campk, or for eonfirmatioo by additios laboratoriec
(2) jgTTF- Liven chaD be of miaimnm cize 45^ f. and iwfividua] camples dull be pack«ged in separate
twirl packs or other noo-water-cohible packaging Other types of cample cbonld be of cimilar cize.
(3) SHIPPING: Samples duD be packed in dry iee, and chipring oootainen chaD be marked as
appropriate for infectious agents, as identified by the lATA. Appropriate labels may indude those pmaining to
toodns, dry ice, and infectious agents.
(4) CODING: All camples choold be coded in a landom fashioB, with copies of the oodiqg retuned
by the chipper.
(5) SAMPLE QUALITY: Liver imegrity is of princ^ importance in assays for brevetoadns. No
guarantee can be made for analyses resulting froa decomposed, improperly parkagrd, or otherwise noe-pristine
materials.
(6) SAMPLE AGE Ahhou^ not quantified, ^e may have come beariqg on the concrnrrations of
tooDo remaining. We do not set a time bnit on cample ctorage for data is incomplete, bat draw anentioo to it
as a posuble coofdicating factor m certain analyses.
(7) CONTROLS: >^>propriate coded rootrol camples chould be cidmutted, miad with authentic camples.
Two confirmed controls slxwld be marked as CONTROL, and chipped with the firtt cet of camples, for baseline
(8) RIGHT OF REFUSAL: Chiral Corporatioo leaves biolopcal oceanqgr^diic questions of
appropriateness of each cam|^ to the '■**'*«^^««g organizatioo(c). Our tuk is to affirm or deny the presence
of brcnctooDns in the supplied camplft Chiral Corporation retains the right to refuse any roned or purifying
camples, old or obviously decayed materials, bven of a less-than*pristine nature, or aaaterials whidi may pose
a safety risk to our personnel Refused camples wfll be returned to the oootratxii^ agency.
122
FAXNombcr. (305)661-0140
Fee Schedule for BrevetoxiB Aiulyses
Per Sample Costs
(1) HomogenlMtlon, extractloii, and Cambusia fish
bioassay of trade samples *l25.w
(2) Solvent Partitioning, Silicic acid flash
^^ chromatography, and bioassay of crade extract $ 35.00
(3) -nun-layer Plate Silica Gel Chromatography of
fractions from (2) * ^^
(4) High Performance Uqnid Chromatography of fra^ns , ,_ .^
from (3), compared with brevetoxin standards L2LBI
Total Cost Per Assay $250.00
Additional Analytical Protocols Available hot not Included in above assay price
(5)
(6)
(7)
Radioimmnnoassay for Brevetoxins $ 20.00
Sodium Channel Receptor Assays for Brevetoxins $ 35.00
Fourier Transform Infc ared Analysis of purified materials $ 35.00
123
GENERAL OBSERVATIONS ON SAMPLES RECEIVED
AQ samples ippeared to be of bver, with « fibrous constitution consistent with dolphin bver examined
in the past. Samples ranged in size from a low mass of 12.411 g to a high mass of 110.614 g. Twenty-eight
of the samples weighed below the ^jcdfied 45-60 g. Twenty-dght samples were judged to be of good condmon
when received with no evidence of decomposition, eight were slightly freezer-burned, an additionaJ four were
severely freezer-burned, and ten were received in homogenized condition, except as noted for sac. no sample
was deemed unsuitable for testing. Samples were numbered in the laboratory as #]-#50 based on leoeipt and
were not re-correlated with NMFS identification untS completion of assays.
BJOASSAY DURING PURIFICATION
Based on the size of a substantial number of the sanq>les, bioassay was not performed at neps (1)
through (9) of the "Protocol For Liver Analyses: Brevetoxins in Dolphin Liver" [labeled ITEM #1J. Further,
based on the consistent quality of the supplied samples, note [2] 'Aberrant Saiiq>les" did not tppYf.
At step (10) in the protocols, the first preparative thin-layer silica gel diromaiogr^hic step, thirty-five
(35) of the samples were non-taodc by Gambtaia affitiis fish bioassay and analyses were terminated in these
cases. Negative sao^les at this stage were:
GA342
SP112
MM9012
MM9008
SHCM0T7
PO095
MM9013
GA334
GA336
GA311
GA344
MS018-90
GA319
lAOOl
GA332
PA1S3
GA321
SPlll
GA314
P0123
GA335
PA192
P0134
SPllO
F0125
GA313
GA315
GA339
GA302
P0135
MM-9007
2505-7
2505-8
The remaining sao^les tested positive by fish bioassay in at least one fraction of the panected thin-
layer dut>matography plate. Nine of the samples were tone in multiple sections of the plate indicating a
multiplicity of toxic materials. Of the samples which tested positive at the first TLC plate, twelve tested
negative following thin-byer duomatography on the second plate (stq> 12). The samples «iuch tested negative
at this step were:
GA333
GA304
GAllO
SP114
2505-1
2505-2
2505-3
2505-4
PA195
PIQ38
P0121
GA340
Following this diromatogn^hic step, five (5) san^les remained for further analysis. Of these, OO094,
2505-5, and 2505-6 were judged to be of limited quantity and were not subjected to the third thin-layer step
(Rq> 14). Samples 2505-9 and 2505-10 possessed toxic fractions following cfaromatognqihy according to Step
#14_
HIGH PERFORMANCE UQUID CHROMATOGRAPHY
Based on migration of standard brevetoxins, san^les CX3)94, 2SQ5-S, and 2505-6 were negative by
HPLC That is to say, they contain < 5 Mg toxinAotal original sample. Samples 2505-9 and 2505-10, when
liPLCd, indicated the presence of PbTx-2 and this was confirmed by mixing with authentic PbTx-2 and re-
injecting. Co-migration of the mixed samples further suggested authenticity.
124
RADIOIMhfUNQASSAY
This procedure wis not within the oontncted SOW, and wis bsted in the Fee Schedule as ar
Additional Ana)ytical Protocol. However, based on the hmhed number of samples reaching HPLC
immunoassay of leveral samples wis undertaken to confirm identity, at our expense. At present, only
brevetoxins and dguatoxin are known to cross-react in this radioimmunoassay. The %amp)es tested were:
CC094 (podtivt through two TLC pises, negative by HPLQ
2505-5 (positive through two TLC plates— two fractions, negative by HPLQ
2505-6 (positive through two TLC ptates, negative ly HPLQ
2505-9 (positive through three TLC plates— four fractions, positive Yry HPLQ
2505-10 (positive through three TLC plates, positive by HPLQ
T^ resuhs of RL\ are flhistrated in Rgures 1-5. An intemal standard displacement carve for
unlabeled PbTx-3 as compcihor (against fixed trttiated PbTx-3) is used in each experiment. Based on these
resuhs, and we feel these are unequivocal, each of the five samples indicated above was oontammated with
brevetoxin to one degree or another. Based on RIA, the amooirt of brevttoxin contammatmg the ongmaJ
samples was calculated to be as follows:
cam 0390 Mg per 38i)36 g, or 102 Bg^ liver
2505-5 0.460 Mg per 41.255 g, or 12.1 ng^ bver
2505-6 0385 Atg per 43.482 g, or 933 ag^ liver
2505-9 im Mg per 46.639 g. or 240 Bg^ liver
2505-10 0.815 Mg per 45332 g, or 17jO ng/g bver
POTENTIAL SOURCES OF ERROR
We antic^>ated and were informed that random brevetoxin-spiked samples were induded amongst the
50 samples submitted for analysis, and that^amples fiom the previous Atlantic do^hin die-off of 1987-1988
might also be induded. In the former instance, that of ^iked samples, we expea to have little dif&uhy in
identifying PbTx-2 and PbTx-3 (fairiy stable materials) and greater difBcuhy with Pbli-l or aiy of its oogeners
(an unstable polyether backbone structure). We in fact expntsed our uncertainty with respeo to toxins with
the PbTx-1 backbone prior to contract issuance. With reqject to samples from the previous Atlantic bottlenose
die-off, we expressed a concern for the length of time in storage, whidi if it was not at -80*C might not be
sufficiently cold to inhibh aO enzymatic activity. Our previous experience with a progressive reduction in
dguatoxicity in shark bver is our basis for this concern. In these latter two cases, we might eipcfl to tee less
or no brevetoxin in samples, whether q>iked or authentic
Trace brevetoxin contamination within our R & D laboratories is a potential problem, there being
brevetoxin purification progressing at any time. To alleviate the potential for oontamiiuaion, pristine glassware
was purchased for these analyses, and was used only once. Thin-layer chromatography plates were newly
unopened boxes and soWents were freshly opened prior to use. HPLC syringes were sequentially rinsed in an
duotrophic series of soWents as were aniJytical HPLC cohimns prior to use. Fixed baseline at the absorbance
sensitivity used for analyses were a pre-requisite. Ehition times which match authentic brevetoxin retention
times are circumstantial and not ifieatiBactioa means in an authentic sense. Nor is oo-migration of authentic
and unknown in the same sample. However, co-migration lends an iitcreased measure of certainty to the
conclusions. Sensitivity of deteaion is prinqpaOy a function of ultraviolet absorbance, and this decreases from
PbTx-2 to PbTx-3 to PbTx-9, and from PbTx-1 to PbTx-7 to PbTx-10. Thus, h wiD be more difficuh to detect
PbTx-9 and PbTx-10, less difficuh to see PbTx-7 and PbTx-3, and relatively easy to see PbTx-1 and PbTx-2.
125
RadioimmuBoassay. again is not an analytical tool in the cxhct lense of the word, although posnive
resihs in this test would tend to indicate a simDamy in stniauie more finely ascertained than TLC or HPLC
migration. Being that brevetojms and dguatoxin are the only materials known to inhibit specific binding of
brevetoxin to its ^ledBc antibody, one can condude with tome certainty that the five samples identified above
contained brevetoodns or a very similar toodn filce dgiutonn.
Concentrations in origina] samples are based on aliquot and cub-samples at cadi step, and are
ultimately based on "PbTx-S^qtiivalents* in brevetoxin immunoassay. Efficacy of di^laoement of tritiated
brevetoxin by unlabeled brevetoxin oogeners varies about 15-20 %. This obviously is a potential source of
error.
We can make no conclusions about the presence of toxin in any particular sample relative to ecologiuJ
constderations, bloom conditions, proximity to contaminated fish sources or the like. All samples were received
in a coded fashion, make-up and origin unknown to us.
RECOMMENDATIONS
(1) Continue collection of data and refine assaiys for detection.
(2) Notify Chiral Corporation of identity of aD ^riked oontit^ and unknowns for our record-
keeping and to aid us in refining our techniques.
(3) Maintain a groi^ on alert to properly coDea and marie tissues (preferably fieeze-damped with
dry ice to preserve).
(4) Establish r^id nspoast protocols to aid in detection.
(5) Establish protocols for handling of tissues (We treat aD samples as if they were infectious
agents as defined by DHHS puUication No. (NIH) 88-8395.)
126
0.1 1.0 10.0 100.0
COMPETITOR CONCENTRATION (units)
1000.0
Figure 1. RIAof (•) 2505-9 fraction 4^ [plate 3], (^ 2505-9 fnctioii 73 \pi»te 3). (▲) 2505-9 HPLCpeak.
(^) PbTx-3 intenia] nandard curve.
01 1.0 10.0 100.0
COMPETITOR CONCENTRATION (units)
1000.0
Figure 2. RIA of (•) 2S05-6 fraction 3 {plate 2), (^) FbTz-3 inteiiul standard curve.
127
I looi
m
u
t2 80-
u
u
Qu
OQ
D
60--
40--
20--
0-
Z
u
u
£ -20
a. l.OE-2
0.1 1.0 10.0 100.0
COMPETITOR CONCENTRATION (units)
1000.0
Figure 3. RIA of (•) 2505-10 fraction 7 Iplate 31, (♦) PbTx-3 intenul fUndard anve.
0.1 1.0 10.0 100.0
COMPETITOR CONCENTRATION (iiniU)
1000.0
Figure 4. RIA of (•) OO094 fraakn 5 [plate 2), (#) PbTx-3 mtenu] stnadaid cnrve.
128
^ 100
0.1 1.0 10.0 100.0
COMPETITOR CONCENTRATION (units)
1000.0
Figure 5. RIA of (•) 2505-5 fraaion 3 [plate 2], (T) 2505-5 fraction 5 [plate 2], PbTx-3 internal standard
curve.
129
CALCULATIONS FOR RADIOIMMUNQASSAY
PbTx-3
Unh concentrations ^ OJOl nMoies/bter to 100 nMoles/lher, or OJOl pMol/mL to 100 pMoI/mL
50% displacement (EDjg) of tmiated toxin occurs at IjO pMol/mL (0.895 pgrams)
Unknowns
Unit concentration « 125 fiL of iOOfd, original san^le (125%)« 100 Units
1 Unit » 0JQ12S% of sample
Figure 1
( #) 2505-9 fraction 4^ [plate 3] adjudged incondusive
Iy) 2505-9 fraction 73 [plate 3] adjudged incondusive
(A) 2505-9 HPLC peak 50% di^lacement at 11 Units or 0.1375 % at ehited peak (11 z 0i)125%)
Therefore, 0.1375% * 895 pgram equivalents in HPLC peak fay oonqurison with standard curve for PbTx-3.
100% / 0.1375% - 72727. Yielding (895 x 727)- 650,655 pg. or 650 ng in total peak.
The peak assayed was 50 mL/IOOO^L total, wfaidi « 650 ngx (1000/50)- 10 ug PbTx-3 equivalents in sample
prior to HPLC
Back calculations to original amount:
three plates, using 42% or each plate for bioassay - 87i^ toodn remaining at HPLC stq>.
(87.9% X 10 Mg equivalents calculated against standards) - apppiujumately 11 ^g brevetoxin in sample 2505-9.
'Based on 46.639 g of liver supplied, appioximately 024 fig toodn per g liver was presem in the sanq>le.*
Figure 2
( • ) 2505-6 fraction 3 [plate 2] 50% displacement at 20 units or 0253% of sample at this stage (20 x
0i)125%).
Therefore, 0253% - 895 pgram equivalents in fraction 3 from the TLC plate by comparison with standard
curves for PbTx-3.
100%/D253% - 39526, Yielding (895 x 39526) - 353,757 pg. or 353 ng in total sample.
That 353 ng is equivalent to 91.8% of the total sanqile (100% z 0i^8 z 0.958 - 91 J%).
Therefore, the sample was calculated to contain 385 ng brevetoxin (353 x 100%A)1.8%).
'Based on a liver we^t of 41255 g, appfoiimately 933 ng taadn per g fiver was present*
130
fisurej
(•) 2505-10 fraction 7 [plate 3] 50% di^laoement at 10 unhs or 0.125% of sampie at this stage of puriScation
(10 X 0.0125%).
Therefore, 0.125% * 895 pg eqtiivaients in fraaion 7 from the TLC plate by comparison with standard curves
for PbTx-3.
100%A3.125% - 800, Yielding (895 x 800) « 716,000 pg. or 716 ng in total sample.
That 716 ng is equivalent to 87.9% of the total sample (100% x 0.958 x 0.958 x 0.958 = 87.9%)
Therefore, the sample was calculated to contain 815 ng brevetoxin (716 x 100%/87.9%).
•Based on 45^33 g of liver supplied, approximately 17 ng of brevetoxin per g Bver was present.*
Figure 4
(•) CCD94 fraaion 5 [plate 2] 50% displacement at 20 unit or 025% of sample at this stage (20 x 0.0125%).
Therefore, 025% « 895 pgram equivalents in fraction 5 from the tic plate l^ comparison with standard curves
for PbTx-3.
100%/025% » 400, Yielding (895 x 400) ■ 358,000 pg, or 358 ng in total sample.
That 358 ng is equivalent to 91 J% of the total sample (100% x 0.958 x 0.958 > 91.8%).
Therefore, the sample was calculated to contain 390 ng brevetoxin (358 x 100%^1.8%).
* Based on a weight of 38.036 g liver supplied, approximately 102 ng toxin per g liver.*
Figure 5
( •) 2505-5 fraction 3 [plate 2] 50% displacement at 100 units or 125% of sample at this stage (100 x
0.0125%).
Therefore, 1 25% > 895 pg equivalents in fraction 3 from the TLC plate by comparison with PbTx-3 standard.
100%/125% > 80. Yielding (895 x 80) - 71600 pg. or 71 ng in sample.
That 71 ng is equivalent to 91 J% of the total sample (100% x 0.958 x 0.958 - 91.8%).
Therefore, the sample was catailatrd to contain 77 ng brevetoxin (71 x 100%^1.8%).
'Based on a liver mass of 412S5 g. approximately 19 ng brevetaxin ▼« present in the sample.*
131
(9) 2505-5 fraction 5 [pUte 2] 50% drqiUcrment u 20 or 025% of sample at this Rage (20 x Oi)I259b).
Therefore, 025% ■ 895 pgram equivlaents in fraction 5 from the TLC plate by oompasion with standard
curves.
100%yD25% - 400. Tielding (895 x 400) - 358.000 pg. or 358 ng in total sample.
That 358 ng is equivalent to 91.8% of the total sample (100% x 0.958 x 0.958 * 91 J%).
Therefore, the sample was calculated to contain 390 ng birvetoxin (358 x 100%^1.8%).
* Based on a weight of 38i)36 g bver supplied, appimlmately 102 ng toxin per g bver.*
132
Appendix VII. Results of analyses for metals and chlorinated hydrocarbons, including quality assurance.
133
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Explanatory Notes for Tables A-3 through A-12.
nd - indicates that the analyte was not detected above the limit of detection which
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156
Appendix VIII. Available clinical necropsy and histopathology reports of bottlenose dolphins stranded in
the U.S. Gulf of Mexico during Januaiy-June, 1990.
157
This page intentionally left blank.
158
TEXAS MARINE MAMMAL STRANDING NETWORK
NECROPSY EXAMINATIONS
SPRING 1990
Eight animals (all Tursiops truncatus) have recently been
examined. Three (GA-321, PI-38, CC-94) were necropsied at the
Texas Veterinary Medical Diagnostic Laboratory (TVMDL) in College
Station, Texas and five (GA-287, PI-35, SP-114, PA-195, GA-342)
were examined at the Galveston Marine Research Laboratory (GMRL)
in Galveston, Texas. Veterinary affiliations of investigators:
TAMO - College of Veterinary Medicine, Texas A&M University
TVMDL - Texas Veterinary Medical Diagnostic Laboratory
AFIP - Armed Forces Institute of Pathology
NOSC - Naval Ocean Systems Center
Sea World, Inc.
In order of stranding date:
GA-287
Date stranded: 12/11/89
Sex: Female (adult)
Length: 242.7 cm
Weight: 168.0 kg
Pathology notes: Held in freezer prior to necropsy at GMRL.
Necropsy by Dr. Linnehan (NOSC, San Diego) and Dr. Tarpley
(TAMO) . Older animal - teeth fairly worn. Parallel rake
marks over body -some fresh. Several punctate lesions on
skin. Viscous yellowish exudate from central lacteal of
right maimnary gland. SevereJ. nematodes in lung - not
extensive. Several Nasitrema in left pterygoid sinus; one
in right. Monorygma cyst at apex of urinary bladder.
Corpora on both ovaries. Endometrial cyst in one uterine
horn. Histopath tissues collected but not yet examined.
Pathology summary: Possible mastitis.
PI-35
Date stranded: 2/5/90
Sex: Female
Length: 231.0 cm
Weight:
Pathology notes: Held in freezer prior to necropsy at GMRL.
Necropsy by Dr. Magee (NOSC, Hawaii) and Dr. Tarpley (TAMD) .
Moderate PMA and no remarkable gross lesions. Evidence of
coyote scavenging around head. Lead pellet embedded in
dorsal fin base but no sign of entry - probably ante-mortem.
Pathology summary: No diagnostic conclusions.
159
SP-114
Date stranded: 2/22/90
Sex: Female
Length: 234.5 cm
Weight: 138.6 kg
Pathology notes: Held in freezer prior to necropsy at GNRL.
Necropsy by Dr. Tarpley (TAMD) . Many wraps of monofilament
fishing line around left tailfluke blade. Blubber thickness
1.5 - 2.4 cm. Several punctate skin lesions over body.
Minimal tooth wear. Essentially no parallel rake marks.
Adhesion (17 X 15 cm) of cranial portion of right lung to
thoracic wall. Extensive adhesions of loops of intestine
with each other and with pseudopancreas and mesentery. No
gross tissue reaction appeared to be associated with these
adhesions.
Pathology summary: Fisheries interaction mortality.
6A-321
Date stranded: 3/13/90
Sex: Female (juvenile)
Length: 161.5 cm
Weight: 40.5 kg
Pathology notes: Held on ice and necropsied without freezing at
TVMDL. This was a live stranding but received no treatment
(died before TMMSN personnel arrived). Necropsy by Dr.
Robinson (TVMDL) and Dr. Teirpley (TAMO). Microbiology by
Dr. Whitford (TVMDL). Several rake marks (some fresh) and
severe skin lesions randomly distributed. Skin lesions
resulted from local ischemic necrosis caused by blockage of
vessels by bacteria. Lymphoid hyperplasia in lymph node.
Focal necrosis and inflaiOJiu'jory infiltration of lung tissue.
Bacterial isolates: Enterics from skin and Aeromonas lung.
No viruses isolated.
Pathology summary: - Bacterial pneumonia and septicemia.
PI-38
Date stranded: 3/28/90
Sex: Female (adult)
Length: 246.5 cm
Weight: Not weighed
Pathology notes: Held on ice and necropsied without freezing at
TVMDL. Considerable PMA. Necropsy by Dr. Fiske (TVMDL) and
Dr. Tarpley (TVMDL). Histopath by Dr. Fiske. Microbiology
by Dr. Whitford (TVMDL). Term fetus wrapped in amnion found
free in abdominal cavity. A tear in the uterine wall may
have been caused by knife cut when abdomen was punctured by
unknotm person on beach. Bacterial isolates: Enterics and
Aeromonas from intestine (no Salmonella) j Enterics and
Clostri'dlum perf ringens) from lung.
Pathology sunmiaryl Possible calving-associated mortality.
l60
CC-94
Date stranded: 4/1/90
Sex: Male (juvenile)
Length: 160.0 cm
Weight: Not weighed . ^ .^w ^ *
Pathology notes: Held on ice and necropsied without freezing at
TVMDL. Necropsy by Dr. Jones (TVMDL) and Dr. Tarpley
(TAMU) . Histopath by Dr. Robinson (TVMDL). Microbiology by
Dr. Whitford (TVMDL). Several rake Barks and skin
ulcerations with bacteria present in dermal vasculature.
Pancreas very fibrotic with enlarged and granulated
hepatopancreatic duct. Lymph node hyperplasia. Some portal
fibrosis in liver. Lung contained fibrotic nodules and
fibrosed terminal bronchioles. Salmonella isolated from
skin lesion.
Pathology summary: Severe pancreatic fibrosis. Nodular
pneumonia with peripheral skin abscesses.
PA-195
Date stranded: 4/7/90
Sex: Female (adult)
Length: 240.0 cm
Weight: 113.6 kg
Pathology notes: This was a live stranding from Port Aransas
region but died before TMMSN personnel arrived. Put in
freezer initially but removed before freezing and
transported to GMRL and held in cooler prior to necropsy.
Necropsy by Dr. Tarpley (TAMD) and Dr. Schimeca (UTMB) .
Histopath by Dr. Robinson (TVMDL). Microbiology by Dr.
Whitford (TVMDL) . Extensive parallel rake marks (mostly
fresh) and soiae dkin lesions with vasculitis and necrosis of
epidermis. Older animal — considerable tooth wear.
Generally emaciated. Congested lungs with regions of
emphysema and -interstiticd fibrosis. Lymph node fibrotic
and depleted of lymphoid cells. Spleen also has lymphoid
depletion. Considerable portal fibrosis in liver.
Pseudomonas and Enterobacter isolated from lung.
Pathology summary: Possible bacterial septicemia associated with
pneumonia.
161
GA-342
Date stranded: 4/25/90
Sex: Male (subadult)
Length: 220.0 cm
Weight: 94.5 kg
Pathology notes: Live stranding from Galveston region. Therapy
was limited to steroids (Solu-delta>corte£ 100 mg iv) on
beach, fluids (water via stomach tube), vitamins (A&D
combination, B12 and B complex - 5 cc of each IM) and Valium
(100 mg IM) . Several blood samples were taken for clinical
evaluation and culture. RBC and NBC counts low with no band
neutrophils.
Laboratory Submissions:
Clinical Hematology — • St. Mary's Bospiteil, Galveston, TX
Blood taken for CBC and chemistries (two samples)
Clinical Toxicology ~ 3 cc serum to be sent to Dr. Ridgway
(NOSC, San Diego) for heavy metal analysis.
Microbiology
Bacteriology and Mycology
1. St. Mary's HospitcJ. — blowhole swabs (2) ,
blood for culture (bacteria and fungi) , lung
tissues (necrotic and hemorrhagic) , lung
swabs, lymph node tissues.
2. The University of Texas Medical Branch in
Galveston <~ blowhole swabs, heparinized
blood sample for media inoculation.
3. TVMDL (Dr. Whitford) — lung and lymph node
tissues for culture.
Virology — Dr. Al Smith (Oregon State University),
viral swabs taken from blowhole and anus and a
seel- ion of active skin lesion and placed in media
— saiu^les frozen at - 80 C at DTMfl to await
shipment.
Histopathology
1. APIP (Dr. Lipscomb) - set of tissues
2. TVMDL (Dr. Robinson and Dr. Jones) - set of
tissues
Necropsy performed immediately after death at GMRL by
Dr. Lipscomb (AFIP) , Dr. Dover (Sea World, San Diego), Dr.
Magee (NOSC, Hawaii) and Dr. Tarpley (TAMO). A young animal
- minimal tooth wear and immature testes. Several punctate
skin lesions. Several regular shark bites (not cookie-
cutter) . One active lesion (not erupted to surface but
swelling during the clinical course) with fibrin thrombi and
neutrophilic vasculitis. Evidence of chronic
bronchopneumonia (may be associated with lungworms or
infectious organism) and acute septicemic pneumonia (short
rod bacteria found in lung tissue and in pulmonary
arteries) . Pulmonary lymph nodes and pseudopancreas contain
162
areas of necrosis (granulomatous inflammation) with
multinucleated giant cells. Both lymph nodes and
pseudopancreas appear depleted of lymphoid cells, other
lymph nodes and spleen do not appear depleted. Congestion
and portal fibrosis with mild fatty change in liver and
lymphoid aggregates in portal areas and beneath liver
capsule. Approximately 10 ulcers in mucosa of distal
esophagus, a bleeding ulcer in fundic chamber near junction
with forestomach and several longitudinal mucosal erosions
in pyloric chamber.
Lung and lymph node isolates from by both St. Mary's
and TVMDL were E. coli (said by TVMDL to be hemolytic) . No
bacterial isolates on blood culture (including no acid fast
bacilli or anaerobes) — fungal culture still in progress.
TVMDL commented that immunocompromise must be considered for
a relatively non-pathogenic organism such as E. coli to have
caused an acute effect.
Pathology suomary: Hematogenous bacterial pneumonia.
163
Tursiops truncatus Fenale
This dclDhin stranded in Karch 1990 and was held frozen in
Galveston unt'il necropsy on 5 June 1990.
GROSS NECROPSY
EXTERNAL
This appears to be an older dolphin with moderate tooth wear.
However, there is ? remarkable absence of teeth, especially In three
arcades (all but left upper). Crown wear in the teeth which are present
does not appear to justify the complete loss of so many teeth. The
alveolar sockets are healed, indicating that this loss was not extremely
recent. The abdomen is rotund and suggests pregnancy. The genital
groove is somewhat dilated in support of this. There are numerous light
gray irregular spots in the otherwise unpigmented ventral abdomen. This
is apparently normal coloration for this animal. There are several
severe and recent rake marks fairly widely distributed but very evident
around the genital area. Rake marks are present as well over the fluke
blades, some on the flippers and dorsal fins and over the dorsum
(especially in front of the dorsal fin caudal to the blowhole) and over
the tail stock. The left eyelids are scarred and the dorsal eyelid has
fibrotically adhered to tiie cornea. A few punctate skin lesions are
randomly distributed. A few Xenobalanus are attached to the trailing
edge of the left fluke blade near the notch. A tumor or papllloma-like
growth extends from the left lateral of the tongue about midway along
its length. The dorsal fin Is deeply notched (healed^ approximately
midway along the caudal edge. A few Isolated lonoituctinal scars are
present on the ventrum in front of the genital groove caudal to the
umbilicus. There is a lengthy cut with some healing penetratina the
epidermis at points along its course, running from the anterior
insertion of the left flipper up and onto the lateral thoracic wall. A
shorter, longitudinal scar runs parallel to this one at a distance of
about 6 cm. Venipuncture site on ventral keel between fluke blades 3 cm
deep and lies between two tendons..
ABDOMINAL
An apparently full term fetus was found free In the abdominal
cavity wrapped in amnion. The rostrum was oriented toward, and produced
an indention upon, the caudal surface of the liver. The tailstock
curved at the caudal limits of the abdominal cavity to make "U" so that
the tailflukes pointed anteriorly. The body o* the uterus was ruptured
along an anterior line which measured approximately 22 cm. The right
uterine horn was slightly larger than the left and the supporting CL was
on the right ovary. Both ovaries containec" many corpora. The right
mammary gland measured approximately 1.3 an in height and 9.5 cm in
width; however, there was nc mtcroscopic evidence of lactation. The
abdominal cavity contained approximately 2-3 L of a dark reddish fluid.
There were no stomach contents but the stomach was 11 gated and frozen
for later formalin injection and use In rreparatlon of plastination
164
section. Fish remains were present In the esophagus.
THORACIC
The base of the left lung was adhered to the thoracic cavity near
the pericardial sac.
HEAD
HISTOPATHOLOGY
Not conducted due to frozen specimen.
MICROBIOLOGY
Not conducted due to frozen specimen.
TOXICOLOGY
Specimens of blubber (), muscle (), liver (). kidney () and bone ()
were collected and frozen for contaminant analysis. Liver (2 sections
^rom right lobe) were collected for brevetoxin assay. Stomach contents
were frozen in situ.
AGING
Teeth were collected from the following arcades In formalin for
aging:
LL — 13th from rear
LR — 10th, 12th. 13th, ISth from rear
FEEDING HABITS
Stomach was empty.
SPECIMENS COLLECTED
TMMSN archives
Li qui pa k Drum
' Reproductive tract (entire Including external genitalia
and ovaries)
Brain with pituitary Intact
Left mammary gland section
Left tailfluke blade
Left fluke blade swelling
165
Right kidney (entire)
JdM imprint on left dorsum
Longitudinal scarring from anterior flipper Insertion
Jars
Right mammary gland section
Renlcular lesion from left kidney
Adrenals (right and left)
Venipuncture vessels In midkeel
Left eye
Teeth for aging
Frozen
Tissue sections for toxicology
Blubber sections for G. Worthy
Dorsal fin
Stomach (entire, empty)
MEASUREMENTS
LENGTHS
Total body length 234.0 cm
Intestinal length 2990.0 cm
Uterine body rupture 22.0 cm
Fetal length 92.5 en
TMMSN, Navy and G. Worthy blubber depth measurements taken
WEIGHTS
Total body weight 16B.3 kg
Brain (with pituitary) 1228.4 g
Kidney, right 639.4 g
Kidney, left 516.9 g
Adrenal, right 11.6 g
Adrenal, left 11.8 g
Stomach (with duodenal ampulla and contents) 3422.8 g
Spleen 50.1 g
Liver • 3635.0 g
Heart (flushed) 997.7 g
Lung, right 2787.5 g
Lung, left 3116.9 g
Lymph node (pulmonary at base of lung near diaphragm) .. 2.1 g
Fetal weight 9446.3 g
RATIOS
Body length to body weight {)
Brain weight to body weight (kg/kg) 0.007
Brain weight to body length (g/cm) 5.25
Intestinal length to body length (cn/cm) 12.78
166
NECROPSY NOTES SP nH
General comments
The most remarkable external finding Is the entanglement of
many wraps of monofilament line around the left fluke blade.
Severe lacerations are on the leading and trailing edges of the
left fluke blade (see photos prints and slides). Possible single
net marks are at the leading base of dorsal fin.
There is minimal tooth wear. The two posterior crowns on the
upper and lower arcades are moderately damaged. All teeth are
present. Small lingual papillae which are fairly regressed are
found on the lateral edges of thp tongue.
A few white circular punctate scars are scattered over the
entire body approximately Mmm In diameter (the appearance is
similar to protozoal lesions). There Is a punctate scar about 8
mm in diameter that is approximately 8 cm dorsal to the cranial
insertion of the left flipper. Another punctate scar Is present
on the left tailstook and several punctate scars are present on
the right side (see diagram for exact locations). A few well-
healed parallel scars approximately 3 to 15 cm in length and 2 to
li mm apart are noted in the right scapular region and on the dorsum
above the scapular region are, but no reoent lacerations are
detected.
The thoracic cavity contains some serosangulnous fluid - the
majority is frozen (ice formation). There is an area of adhesion
of the cranial portion of the right lung to the thoracic wall about
170mffl by 150mm. There are 12 ribs on the left rib cage and the
last (12th) is free. The first 11 ribs are attached to the
vertebral column. The first four are double headed ribs.
Abundant serosangulnoun fluid is present in the peritoneal
cavity. Left ovary has a large round tan colored mass associated
with the cranial pole. Both ovaries seem otherwise fairly
inactive. Urinary bladder is empty. An oval lobulated spleen and
a small accessory spleen is present. The loops of intestine are
tightly adhered to each other and to the pseudopancreas. The
mesentery is also tightly and almost entirely adhered to the
intestines.
Weights and Measures
Heights (g) — L Kidney (with capsule) s 465.8
L Adrenal s ^t^,^
R Adrenal b 12.1
Spleen s 193.2
Liver = 6252.9
Measures (cm) — Intestine length s 123 x l8om
Area of lung adhesion « 170mm x 150mm
Punctate lesion acar s Smm
#
Anatomy Specimsns
Formalin
Gross — Brain
167
Tongue, larynx, hyoid bones, lungs , thyroids ,
epiglottic spout (in situ)
Entire reproductive tract
Blubber (8)
1 . anal girth
2. lOcD cranial to umbilicus
3> lateral body wall
4. lateral inbetween girth axilla - dorsal fin
5* 15cD caudal to blow hole
6. ventral Inbetween girth axilla - dorsal fin
7. dorsal inbetween girth axilla - dorsal fin
8. anal girth along lateral prooess
F Eye
L Adrenal
R Adrenal
L Ovary ,
H Ovary
Histo — Kidney
Frozen — Stomach
Toxicology tissues
Liver (1 left lobe; 2 right lobe)
Blubber (4)
Kidney (1)
Bone (4)
Muscle (4)
Blubber samples for Graham Worthy
Skeleton
Fluid in peritoneal cavity
Dorsal fin
Pleural fluid
L Eye
168
-sp\^^
HSCJtoPSY rrror.T sp 112
Multiple parallel linear aoars are pr<»sent ranging In aire
from 3 en to 15 ea In leiigth and about 2 to ^ mm apnrt over the
entire body (cicatrization). There ia a remarkable absence of
fr*?. : parallel lacerations. The animal appears to be old and
nutritionally compromised. Ee is emaciated, determined by his
general body condition. Generalized over the entire body are
multifocal circular ulcerations with evidence of dermal involvement
rangin«r in size from 0.5 cm to 3.5 cm (see diagram for exact
locations). No external parasites are present.
The teeth show extensive wear with fractures present on
numerous crowns over the entire mouth. No lingual papillae are'
present. Tooth counts are as follows;
Dpper right arcade: 24 slots with tooth #15* #16 and #23
missing.
Upper left arcadet' 23 slots with tooth #24 missing.
Lower right arcac'c: 23 slots with tooth #13 missing.
Lower left arcadet 22 alots with no teeth missing.
Intestinal adhesions predonlnate In the anterior portion of
the intestines. Mutifocal neorotio areas about 3 om In diameter are
present throughout the entire Intestinal tract. There are
extensive adhesions between the liver and diaphragm so that the
diaphragm cannot be reflected.
The lung is firm, solid and heavy. Both lungs are flbrotlc
with a diffusely mottled pink and white appearance. On out surface
of the lungs white fibrous tissue is presei^t throughout the
parenchyma. There are numeroua thin white nematodes present
bilaterally. There are black as well as white nematodes present
in the left lung, suggesting antemortem hemorrhage ingested by the
worms, but only white nematodes are found in the right lung. There
is a large nodular gray vegetative l^'ion In the left auricle.
Weights and Measures
Weig»-ts (c) — L Kidney (vlth capaulf) « 487.3
L Adrm-.l s 15.7
L Testicle with epidldymus s 534.4
L Testicle without epidldymus s 444.9 *
R Testicle with epidldymus « 488.2
R Testicle without epidldymus s 435.2
R Kidney r *o9.2
R Adrenal « 11."
R Lung = 3702.0
L Lung r 3004.2
Heart « 999.1 •
Spleen s 94.0
Liver « 45?1.3
£pl»en « ?4,0
Eraln = 1260.9
Measures (cm) — L Testicle 25op x 8cm
R T^st?.ele 23o»". r. Se-
Intestines « 2r''4ei"
169
Anatomy Specimens
Formalin
Gross — L Adrenal
R Adrenal
Parathyroid and Thyroid
Spleen (Whole)
Histo — L Testicle
Frozen — R Eye
Toxicology Tissues
Liver (»« left lobe; 2 right lobe)
Blubber (1)
Kidney (4)
Bone (1)
Muscle (4)
Graham Worthy Blubber Samples
Stomach with contents
Blood from Heart
Dorsal fin
Skeleton
170
«E»»1.VT0
ATTEMTIONOF
ARMED FORCES INSTITLrrE OF P/CTHOLOGY
WASHINGTON. DC 20306-6000
September 2B, 1990
Larry Hansen
Southeast Fisheries Center
75 Virginia Beach Drive
Miami, FL 33155
Dear Larry,
Reports on three Texas Gulf Coast dolphins are enclosed. Two
other reports will be sent to you next week. I will then write up a
summary of the findings in the five animals and forward it to you.
. Best wishes.
THOMAS P. LIPSCOMB, DVM
MAJ, VC, USA
Dept. of Veterinary Pathology
171
AHMtO FORCES INSTTTUTk 0»- CATHOLOCY
WASHINGTON. DC 2030»-*0O0
KS'f^] J> ?iffK
.T to
Aiiutnon w
PATIENT IDENTIFICATION
ATIP ACCESSION NUMSEK
3362007-1
n.£ASC USE An^ ACCCSSKM
CNccxoiaiT
01
SEQUENCE
SSAN
ASIUAL. CETACEl DOLPHIS Dr.
00130138 S T
SUWICAL/AUTOWT PATH ACCtSSIOM rS
Tarplcy
PtEASE iwfoWM US or *nr PAtion iOE>fnr»cATX3N ehwows
Dr. EAymond T*rpl«y
TtxA« VAttrinAry Itodic&l C«nt«r
Colltg* of VtttrlnAry lUdlcln*
T«x«s ALU Unlvcrflty
l_£oll«<« StAtion. TX 77843-44B8
n
J
CPU-V
JMP/TPL/Jlp
DATE 11 Jun* 1000
CONSULTATION REPORT ON CONTRIBUTOR MATERIAL
AFIP DIAGNOSIS:
00120126 1. Lun|: Pn«UBonlA» aoutc, b«BorrbA|io, aniltifooAl.
aodtrat*, with oolonlct of fraB-ntgativ* bAOiUl. AtUntlo
bottlcnof* dolpbln (Iy£Elfi21 lllfflfiftlyi) • eatAOtan.
2. Lung: Bponcbopnauaonla, aout* to aubacuta, multlfooal,
Bodarata, witb |pam-na|atlva baallli.
3. Lung: BronobopnauBonla, obronio-aotiva, aoa Inopbilic.
Btultlfooal. Bodarata, witb aataatrongylld nanatodaa.
4. Lung: Pblabltia, acuta, Bultlfoaal, nlld, witb
gram-nagativa bacilli.
5. Livar. bapatocytac: Vacuolar cbanga, ulerovaaicular,
diffuaa, Bodarata.
a. Livar: Hapatitia, lympbocytic. portal, pariportal and
eapaular, Bultifocal, Bild.
7. Livar: Congaation. cantrilobular , diffuaa. Bild.
Livar: Haaoaldaroaia, sultifocal, Bild.
Livar: Extraaadullary baBatopoiaaia, sultifooal, Bild.
Livar: Artaritia, acuta, focal, Bild.
6.
0.
10
11
Lyapbadanitia,
Bild.
Lyapboid daplation, diffuaa.
»aantaric: lacroaia, acuta, Bultifocal,
Lymph nodaa , pulBonary:
nacroauppurativa, Bultifocal,
13. Lyapb nodaa, Baaantaric:
Bodarata.
13. Lymph noda,
Bild.
14. Lyapb noda,
nacroauppurativa
giant oallf.
15. Lymph noda, maaantaric:
diffuaa, Bild.
16. Lymph noda, sadiaatinal;
Bodarata, with hamoaidaroaia,
17. Lymph noda, aadiaatinal:
diffuaa, Bild.
18. Lymph noda, axillary: HaBorrhaga and
arytbropbagocytoaia, multifooAl, Bild.
10. Kidnay; Congaation, Bultifocal, Bodarata.
20. Stomach: aaatritia. aoalnophillo, diffuaa. Bild. with
adama.
Baaantaric:
, Bultifocal
Lympbadanitia,
Bodarata, with aultinuclaatad
Lympbadanitia, aoainopbilic,
HaBorrbaga, diffusa,
Lympbadanitia, aoainopbilic.
AFIPF/L61
15 May 87
172
2282907-1
ANIMAL, CJTTMrEA DCaifHIN Dr. Tarpley
90120128 S T
cro-v
JMP/TPVjlP
11 June 1990
21. Stonach: Dloer, focal, noderate, with
htguuiilizujii (jiiiinn diagnosis.
22. StoDacii, pylorus: Gastritis, lynrhnrytic, focal, mild.
23. Stonach, pylorus: Erosion, aaxte, fnmlly extensive,
moderate.
24. Duodenum: Btteritis, lyntiyrfhl 1 1 ml nr, mil ti focal,
moderate.
25. Spleen, c^sule: Ccngesticn and beonrxhage,
multifocal, mild.
26. Trachea: Tradieitis, iyHY*vifttnir»ii^T- and
eosinophilic, nultifocal, mild.
27. £sc|iiagus: Ulcer, acute, focal, moderate.
28. Skin: Dermatitis, acute to subacute, focally
extensive, moderate, v±^ acute necxotizing vasculitis and
fiisrin thrcnbi.
Ihe cause of deatii is bacterial pneumonia. Ihe bacterial mocphology and staining
characteristics are consistent with Escherichia ooli which was cultured frcm the
Ixng. Ihe pulmonary lesions eure ocoplex. Ihe distribution of the hesmrxhagic
pneumonia is consistent with hematogenous dissemination. Other findings that
support septinpmjn are the presenoe of gram-negative bacilli within pulmonary
veins, zuaite h^iatic arteritis and necxotizing vasculitis in the skin. Ihe
bacterial bronchopneumonia %ras of longer duration than the very acute hemorrhagic
pneumonia. The Ixmgworm a«pyici ntPti pneumonia was chronic and relatively mild. Ihe
conbined effects of the lung lesions %«ere fated. Ihe microvesicular vacuolar
change in hepatocytes is consistent vdth lipidosis. lesions of this type have been
associated with a variety of toodns in other species. Specigd stains failed to
reveal the cause of the necrotizing lesions in pulmonary and mesenteric lynph
nodes. The gastric iiloer probably caused significant pain. The splenic congestion
and raysular hanorxhage probably reflect the sqitioemia. Ihe causes of the other
lesions were not ^parent.
JOHN M. FLETCHER, EMI, MFH
Colcnel, VC, USA
Chairman, Department of Veterineury Pathology
IHOAS P. LIF5GCHB, ZfM
MAT, VC, USA
Department of Veterinary Pathology
173
2297924-9
ANIMAL, CETACEA DOLPHIN
C90101256 SBT
Dr. Raymond J. Tarpley
Dept. of Veterinary Anatomy
Texas A&M University
College Station, TX 77843
CPU-V
jMP/TPL/mab
25 September 1990
C90101256
1. Spleen: Dermatitis, necroulcerative, acute, focal ly
extensive, moderate, with numerous mixed gram-negative bacilli
and ciliated protozoa, Atlantic bottlenose dolphin (Tursiops
truncatus), cetacean.
n Liver: Hepatitis, portal to periportal, lymphoplasmacytic
and eosinophilic, diffuse, mild to moderate.
3. Liver: Congestion, centri lobular, diffuse, moderate.
4. Liver: Extramedullary hematopoiesis, multifocal, mild.
5. Lung, pleura: Fibrosis, focal ly extensive, moderate, with
fibrous adhesions.
6. Lung: Congestion and alveolar edema, diffuse, moderate,
with alveolar histiocytosis.
7. Lymph node: Lymphoid depletion, diffuse, moderate.
8. Lymph node: Lymphadenitis, eosinophilic, diffuse, mild.
9. Lymph node: Congestion, diffuse, moderate, with mild
hemorrhage.
Comment: The skin lesion is suggestive of cutaneous infarction. The presence of
bacteria deep within the lesion raises the possibility that the lesion was caused
by septic thromboembolism. However, bacterial invasion through the skin is also
possible. Ciliated protozoa are found rather commonly in skin lesions of this
type in dolphins. They are generally considered to be secondary invaders. The
hepatic and pulmonary congestion are probably agonal. The pleural fibrosis and
adhesions are probably the result of a resolved pneumonia and pleuritis. The
causes of the other lesions are not apparent.
JOHN M. PLETCHER, DVM, MPH
Colonel, VC, USA
Chairman, Department of Veterinary Pathology
THOMAS P. LIPSCOMB, DVM
MAJ, VC, USA
Department of Veterinary Pathology
174
2297921-5
ANIMAL, CETACEA DOLPHIN
90092184 SBT
Dr. Raymond J. Tarpley
Dept. of Veterinary Anatomy
Texas A&M University
College Station, TX 77843
CPU-V
JMP/TPL/mab
25 September 1990
90092184
1. Lung: Edema, alveolar, diffuse, moderate, with alveolar
histiocytosis, Atlantic bottlenose dolphin (Tursiops
truncatus), cetacean.
T. Lung: Pyogranulomas, multifocal, moderate.
3. Lung: Fibrosis, multifocal, moderate.
4. Pancreas: Fibrosis and acinar cell atrophy, diffuse,
severe, with mild multifocal chronic and chronic -active
pancreatitis.
5. Liver: Vacuolar change, microvesicular, diffuse, moderate.
6. Liver: Sinusoidal dilatation, multifocal, moderate.
7. Lymph nodes: Lymphoid atrophy, diffuse, mild.
8. Thymus: Atrophy, diffuse, mild.
Comment: Postmortem autolysis hindered microscopic evaluation. The pulmonary
edema may have been agonal or caused by drowning. The pulmonary pyogranulomas
were probably caused by metazoan parasites. The fibrosis in the lungs probably
represents areas of resolved pneumonia. The caM«;e of the severe pancreatic
fibrosis and atrophy was not apparent. Lesions of this type have been recognized
previously, but generally in older dolphins. It is likely that both exocrine and
endocrine pancreatic function were impaired. The cause of this lesion is
unknown. Hepatic vacuolar change can be caused by a number of different factors.
Microvesicular fatty change, which this vacuolar change resembles, has been
associated with toxic hepatic injury in some species. The thymic atrophy may
represent a normal physiologic process. The causes of the other lesions were not
apparent.
JOHN M. PLETCHER, DVM, MPH
Colonel, VC, USA
Chairman, Department of Veterinary Pathology
THOMAS P. LIPSCOMB, DVM
MAJ, VC, USA
Department of Veterinary Pathology
175
ARMED POHCES INSTITUTE OF PATHOLOGY
WASHrNGTON. DC 2030&-6000
REPIY TO V.
October 11, 1990
Department of Veterinary Pathology
Larry Hansen
Southeast Fisheries Science Center
National Marine Fisheries Service
75 Virginia Beach Drive
Miami, TL 3J149
Dear Larry,
Enclosed are the last two necropsy reports on Gulf of Mexico
dolphins. I will get a summiury of the pathological findings in the
five animals written up in the next week or so. Let me know if you
need the report in a particular format.
Sincerely,
THOMAS P. LIPSCOMB, DVM
MAJ, VC, USA
Department of Veterinary Pathology
176
2298103-9
ANIMAL, CETACEA DOLPHIN
C90073283 B
Dr. Raymond 0. Tarpley
Department of Veterinary Anatomy
TVMC, Texas A&M University
College Station, TX 77843-4458
CPU-V
JMP/TPL/mab
11 October 1990
C90073283
1. Skin, dermis: Necrosis, coagulative, focally extensive,
severe, with acute dermatitis and gram-negative bacilli,
Atlantic bottlenose dolphin (Tursiops truncatus), cetacean.
2. Skin: Necrosis, coagulative, focally extensive, severe,
with acute dermatitis, ulceration and ciliated protozoa.
3. Skin: Dermatitis, acute, focally extensive, moderate, with
focally extensive dermal and epidermal necrosis, and fibrin
thrombi .
4. Skin: Dermatitis, acute, focal, mild, with gram-negative
bacilli.
5. Skin: Dermatitis, chronic, focally extensive, moderate.
6. Lung: Necrogranuloma, focal, moderate, with mineralization.
7. Lung: Congestion and alveolar edema, diffuse, mild to
moderate, with alveolar histiocytosis.
8. Liver: Hepatitis, portal and periportal,
lymphoplasmacytic, diffuse, mild.
9. Lymph node: Lymphoid hyperplasia, diffuse, moderate.
10. Lymph node: Lymphoid depletion, diffuse, mild.
11. Lymph node: Edema, diffuse, moderate, with mild
hemorrhage.
Comment: Gram-negative septicemia is considered the cause of death. Rarely,
gram-negative bacilli were found in nonulcerated areas of acute dermatitis,
strongly suggesting hematogenous dissemination of the bacteria. Several of the
skin lesions were consistent with cutaneous infarcts. We believe that the
majority of cutaneous lesions were caused by septicemia. Ciliated protozoa were
found in some of the cutaneous lesions. This is a relatively common finding in
dolphins; these protozoa are generally considered to be secondary invaders. The
cause of the pulmonary necrogranuloma was not apparent; this lesion was not likely
to have been clinically significant. The pulmonary congestion and edema were
177
2298103
Animal, Cetacea Dolphin
Page two
probably agonal. Histologically similar hepatitis has been seen previously in
dolphins; the cause is unknown. The causes of the other lesions are also unknown.
JOHN M. PLETCHER. DVM, MPH
Colonel, VC, USA
Chairman, Department of Veterinary Pathology
THOMAS P. LIPSCOMB, DVM
MAJ, VC, USA
Department of Veterinary Pathology
178
2295794-8
ANIMAL. CETACEA DOLPHIN
SA344 T
Dr. Raymond J. Tarpley
Department of Veterinary Anatomy
TVMC, Texas A&« University
College Station, TX 77843-4458
CPU-V
JMP/TPL/maD
n October 1990
6A344, C901 62225
1. Lung: Fibrosis, pleural and interstitial, multifocal,
moderate, with mild multifocal chronic pneumonia, Atlantic
bottlenose dolphin (Tursiops truncatus), cetacean.
2. Lung: Necrogranulomas, chronic, multifocal, moderate, with
mineralization.
3. Lung: Congestion and edema, multifocal, moderate, with
alveolar histiocytosis.
4. Liver: Congestion, centri lobular, diffuse, mild.
5. Liver: Fibrosis, portal and capsular, diffuse, mild.
6. Lymph nodes: Lymphadenitis, eosinophilic, necrotizing,
multifocal, mild, with multinucleated giant cells.
7. Lymph node: Lymphoid hyperplasia, multifocal, moderate.
8. Lymph node: Lymphoid depletion, multifocal, moderate.
Comment: The cause of death is not clear. The most significant lesions were
pulmonary. The pulmonary fibrosis probably represents the resolution phase of a
pneumonia; the cause was not evident. The pulmonary necrogranulomas were probably
caused by parasites. The pulmonary congestion and edema could have agonal or
caused by drowning. The hepatic congestion was probably caused by terminal
cardiovascular collapse. Similar eosinophilic necrotizing lymphadenitis with
multinucleated giant cells has been seen in other dolphins; the cause is unknown.
Both hyperplastic and depleted lymph nodes were present.
JOHN M. FLETCHER, DVM, MPH
Colonel, VC, USA
Chairman, Department of Veterinary Pathology
THOMAS P. LIPSCOMB, DVM
MAj, VC, USA
Department of Veterinary Pathology
179
ARMED FORCES INSTTTUTE OF PATHOLOGY
WASHINGTON. DC 20306-6000
»«EPl.r TO Sr.
AmtfnoN Of ■*•'••». p. "•'
November 2, 1990
Department* -G^ Veterinary Pathology
Larry Hansen
Southeast Fisheries Science Center
National Marine Fisheries Service
75 Virginia Beach Drive
Miami , FT. T?i4Q
Sxunmary of Pathologic Findings in Atlantic Bottlenose
Dolphins from the Giilf of Mexico.
The Department of Veterinary Pathology of the Armed Forces Institute
of Pathology received material from five Atlantic bottlenose dolphins
that died from March through June 1990. All were from Texas waters.
Two of the five had gram-negative septicemia. One of these two also
had gram-negative bacterial pneumonia and £. coli was cultured from
lung. Another of the five dolphins had cutaneous lesions suggestive
of gram-negative septicemia. Gram-negative septicemia has not been
found to be a common cause of death in dolphins, although Pseudomonas
pseudomallei has caused septicemias in an aquarium in Hong Kong.
Generally, grzun-negative septicemia tends to occur in the terminal
stages of chronic diseases and in individuals with some basis for
depressed immunity. The remaining two dolphins had various lesions
but no clear-cut cause of death.
A number of interesting lesions were found in these five
dolphins. Diffuse hepatic microvesicular vacuolar change, consistent
with microvesicular fatty change, vas found in two dolphi.ns.
Although hepatic fatty change can be caused by a variety of
physiologic and pathologic processes, diffuse microvesicular fatty
change has been associated with a variety of toxins in other
species. Lymphocytic portal hepatitis was present in two dolphins.
This lesion has been previously described in dolphins from the Gulf
of Mexico; its cause is unknown. Lymphadenitis, lymphoid hyperplasia
and lymphoid depletion were all seen, occasionally in the same
animal. The significance of these lesions is unknown. Significant
amounts of pulmonary fibrosis were present in three dolphins; these
probably represent areas of resolved pneumonia. One dolphin had
severe pancreatic fibrosis and atrophy. Pancreatic lesions of this
type have been recognized previously, but generally in dolphins that
were older. (This dolphin was sexually immature.)
Thomas P. Lipscomb, DVM
MAJ, VC, USA
Department of Veterinary Pathology
180
Sl-Hyocinthe, lll^lt^ 1
Mr. Lorry Hensen
NMFS Miami Loborolory
75 Virginia Beech Drive
Miami, Florida 33149
U.S.A.
Dear Mr. Hensen,
As requested by Mr. Charles A. Oravetz from the Protected Species
Management Branch in his letter to Mr. Bruce McKay, Greenpeace Montreal,
included with this letter, you will find a copy of the necropsy report of a
male bottlenose dolphin I examined in Mobil, Alabama, in May 1990 (5HCM
072). If you need any information or have any question, don't hesitate to
contact me, I will be glad to help you if i con.
Sincerely yours.
jlvain De Guise, D.M.V
cc: Mr. Bruce McKay, Greenpeace Montreal
C.P. 5000
Swit-Hyacmttw (QuMiac)
J2S7C6
(514) 779-6521 — Mt«: 05-060505
Ttltcopwur (514) 773-2161
181
90-3530
Ey^grpaT examination: This is the fresh carcass of 8 1 15 kg Atlantic
bottlenose dolphin on which there are a few external parasites, mostly on
the tail.
prp$s findings:
-brain: No significant lesion, there are no parasites In the auditive canal.
-mouth: All the teeth are worn, their surface being all even.
-lungs: There is a severe extensive bilateral verminous pneumonia
caracterised by whitish and greenish discoloration of the parenchyma on
the cut section of the whole surface of both lungs associated with a heavy
burden of parasites in the bronchi, with a mild to nrtoderete suppurative
resction. The parasites are 2 to 3 cm long, whitish, with a thin
longitudinal black stripe. There Is a mild brown to reddish exudate In the
trachea and large bronchi.
-heart: No significant lesion.
-thymus: There is a small thymus remnant.
-liver: There are a few slightly depressed lines of whitish discoloration
on the surface of the liver with nothing significant on cut section.
-kidneys: No significant lesion.
-spleen: The spleen is about 5x4x4 cm, and a few daric red blackish foci of
discoloration on the surface. The cut surface Is very regular. There are
two small ectopic spleen of 1 and 0.5 cm In diameter.
-pancreas: No significant lesion.
-thyroid: No significant lesion.
-adrenals: The adrenal cortex Is rather thin, with a rather thick medulla.
-1st gastric compartment: No significant lesion.
-2nd gastric compartment: No significant lesion.
-3rd gastric comportment: There ore 3 foci of small pedonculated nodules
182
Appendix I. (conunueo;
ottoched to the gostric mucosa. Those yellowish nodules ore 3 mm in
diometer ond correspond to porosHes.
-4th gastric compartment (duodenal ampula): No significant lesion.
-intestine: No significant lesion.
-testis- No significant lesion.
-epididymis: No significant lesion.
-seminal vesicles:
-urinary bladder: No significant lesion.
Macroscopic diagnosis: Verminous broncho-pneumonia
Laboratory tests:
Bacteriology: yes
Virology: yes
To><1cology: yes
Parasitology: yes
Histopothology:
-general: All the tissues ore nfilldly to nfioderotely autolysed, and there are
many clumps of bactehas In the lumen of blood vessels that sometimes
occlude completely some small vessels.
-brain: There Is a small to moderate amount of llpofuscin in the cytoplasm
of some neurons of the brain, cerebellum and brainstem.
-tongue: There is a moderate multifocal lymphocytic InftUratlon around
the tongue mucus glands end their ducts under the Malplghlan epithelium.
-lungs: We observe a moderate amount of nematodes In small bronchi, with
a mild to moderate neutrophilic Infiltration around the parasite, often
with a moderate Increase In the number of alveolar macrophages, and a
moderate lympho-plasmacytlc Infiltration In the chorion. The same kind of
inflammation extends in the surrounding alveolar lumen and wall, and In
the wall of bronchi, bronchloll. We also note a mild multifocal
183
Appendix 1. (^conunuca;
eosinophilic inflltrotlon In the well of some eWeoll end bronchi end
sometimes In their lumen, with the neutrophils. Further from the moln
inflammatory foci that contain the parasites, we often note alveolar
edema and a mild infiltration of neutrophils and eosinophils. The muscles
around the bronchioli are often hypertrophied, and we find a moderate
amount of corpora amilacea (small calcified plagues) in the lumen of
bronchi, bronchioli and alveoli, and sometimes incorporated into their
wall, both of these lesions are not always seen in relation with the
inflammatory foci. We also note ocasionnal giant cells in airways, with
very few larvae, and a multifocal mild to moderate accumulation of
Gram-negative bactehae sometimes associated with the lesions.
-mediastinal and cervical lymph nodes: There Is a mild to moderate
eosinophilic Infiltration In the mildly edematous cortex and In the
medullary sinuses, where It Is accompanied by a few macrophages and
some red blood cells. The cortex contains very few follicles.
-heart: We note very few small foci of fibrosis In the myocardium, and a
small to moderate amount of llpofuscln In the cardlomyocytes, around the
nuclei.
-aorta: No significant lesion.
-diaphragm: No significant lesion.
-thymus: Only some very thin thymus remnants are still there, and they
ore poorly cellular.
-liver In addition to a mild atrophy of the centrolobular hepatic cords,
there Is a small amount of brown pigmet In the cytoplasm of the
hepatocytes.
-spleen: The white pulp It made of a large number of follicles that have a
large germinal center with a very thin outer ring of mature lymphocytes.
-pancreas: No significant lesion.
-thyroid: Most of the follicles contain little colloid, but the autolysis is
moderate. Some Intact follicles contain a normal amount of colloid.
-adrenals: The cortex/medulla ratio Is low, and the junction Is Irregular.
184
-1st gostnc comportment: No significant lesion.
-2nd gostnc comportment: There ie o emoll focol euperficiol erosion thot
exudates mucus, while the rest of the mucosa is normal.
-3rd gastric compartment: We observe some trematodes {Brounino
cordjformis) attached to the mucosa by a thin layer of submucosa they
incorporate in themselves, between their Inner and outer body.
-4th gastric compartment (duodenal ampula): No significant lesion.
-intestine: No significant lesion.
-testis: No significant lesion, the spermatogenesis Is normal and active.
-epididymis: No significant lesion, the tubules carry a good amount of
spermatozoids.
-urinary bladder No significant lesion, the epithelium Is preety high (not
compressed).
-muscle: A few muscle fibers are swollen.
Final Diagnosis:
-Severe verminous broncho-pneumonia.
Comment: The moderate amount of bacteria sometimes found to be
associated to the lung lesions suggests that a secondary bacterial
infect/bn complicated the primary verminous pneumonia, a fact that could
have precipitated death.
SQlvain Oe Guise, D.n.V
185
I
jtheast Fisheries Science Center representative system for the Man
Mammal Stranding Network.
Appendix EX. Proposal outline for Southeast Fisheries Science Center representative system for the Marir|
186
1
II
.• •'■••
•i.» C
Marine Mammal Stranding Network
Representative System
Organizational Woricsbop
Miami Laboratoiy
75 VirgiiQa Beach E^ive
Miami, FL 33149
May 7^ 1991
FISHERIES
187
Ma-tional Marine Fisheries Servicse
Southeast Fisheries Science Center
Marine Manual Stranding Metwork Representative System
Organizational Moricshop
Miami lAboratory
75 Virginia Beach Drive
Miami, Florida
May 7-8, 1991
188
Marine NasBal Stranding Network
NMFS Area Representative Systea
Southeast Fisheries Science Center
Organizational Norkshop
Miaai, Florida, Nay 7-8, 1991
ASSSDh
Tuesday, Nay 7.
0900 Introductory remarks and introduction of participants
Brad Bro%m.
0920 Overview of SEDS stranding netirark - Dan Odell.
0940 NMFS area representative authority at stranding site •
Jeff Bro%m.
1000 Stranding of protected species. National perspective •
Dean Wilkinson
1030 Coffee break.
1045 Discussion of NMFS stranding network area
representative responsibilities - Larry Hansen.
1115 Discussion of basic aarine BaBmal stranding data
reporting - Ben Blaylock.
1130 Denonstration of reporting software - Lee Weinberger.
1200 Break for lunch.
1330 Specinen and data collection protocols - Sylvia
Galloway.
1430 Discussion of specinen storage and transfer - Larry
Hansen.
1500 Coffee break.
1530 Ad hoc discussion of aspects of iapleaenting the
reporting systea relative to individual NMFS
laboratories .
1700 Adjourn.
189
Wednesday, May 8.
0900 Reconvene ... continue previous afternoon's discussion
1030 Coffee break.
1100 Ad hoc discussion period.
1200 Adjourn meeting.
190
WORKSHOP PARTTCTPAITPS
Mr. Ben Blaylock
Southeast Fisheries Science Center
Miami Laboratory
75 Virginia Beach Drive
Miami, FL 33149
(305) 361-4299/FTS 350-1299
Mr. Bill Bowen
Southeast Fisheries Science Center
Beaufort Laboratory
Beaufort, NC 28516
(919) 728-3595/FTS 670-9740
Dr. Brad Brown, Center Director
Southeast Fisheries Science Center
75 Virginia Beach Drive
Mieuni, FL 33149
(305) 361-4286/FTS 350-1286
Mr. Jeff Bro%m
Southeast Regional Office
Management Division
9450 Koger Blvd.
St. Petersburg, FL 33702
(813) 893-3366/FTS 826-3366
Dr. Charles Caillouet
Southeast Fisheries Science Center
Galveston Laboratory
4700 Avenue U.
Galveston, TX 77550
(409) 527-6500/FTS 527-6500
Mr. Bill Fable, Jr.
Southeast Fisheries Science Center
Panama City Laboratory
3500 Delwood Beach Road
Panama City, FL 32407
(904) 234-6541
Mr. Larry Hansen
Southeast Fisheries Science Center
Miami Laboratory
75 Virginia Beach Drive
Miami, FL 33149
(305) 361-4264/FTS 350-1264
191
Mr. Wayne Hoggard
Southeast Fisheries Science Center
Mississippi LsUboratories
P.O. Drawer 1207
Pascagoula, MS 39567
(601) 762-4591
Dr. Sylvia Galloway
Southeast Fisheries Science Center
Charleston Laboratory
P.O. Box 12607
Charleston, SC 29412
(803) 762-1200
Mrs. Ann Jennings
Southeast Fisheries Science Center
Charleston Laboratory
P.O. Box 12607
Charleston, SC 29412
(803) 762-1200
Dr. Dan Odell, SEUS Coordinator
Sea World of Florida
7007 Sea World Drive
Orlando, FL 32821
(407) 363-2158
Dr. Joe Powers, Laboratory Director
Southeast Fisheries Science Center
Miami Laboratory
75 Virginia Beach Drive
Miami, FL 33149
(305) 361-4284/FTS 350-1284
Ms. Kathy Prunier
Southeast Fisheries Science Center
Miami Leiboratory
75 Virgina Beach Drive
Miami, FL 33149
(305) 361-4596/FTS 350-1596
Dr. Gerry Sco-tt, Chief
Oceanics and Pelagics Division
Southeast Fisheries Science Center
Miami Labora-tory
75 Virginia Beach Drive
Miami, FL 33149
(305) 361-4530/FTS 350-1530
192
Mr. Lee Weinberger
southeast Fisheries Science Center
Data Management Division
75 Virginia Beach Drive
Miami, FL 33149
(305) 361-4287/FTS 350-1287
Mr. Dean Wilkinson
National Marine Fisheries Service
Division of Protected Species
1335 East West Highway, Room 8259
Silver spring, MD 20910
(301) 427-2322/FTS 427-2322
193
Appendix 1.
PreliMinaxy Proposal for Enhancing
<the Southeast U.S. Marine m»—»i stranding Network
Prepared By
Staff
National Oceanic and Ataospheric Adninistration
National Marine Fisheries Service
Southeast Fisheries Science Center
75 Virginia Beach Drive
Mieu&i, Florida 33149
Contribution: MIA-90/91-55
194
.Tustific^i^ion and Introduction
The 1990 bottlenose dolphin anomalous mortality event in the
Gulf of Mexico illustrated that the Stranding Network was
unprepared to mount an adequate response to increased strandings.
Furthermore, gross inconsistencies in regular data collection and
reporting among Network participants have resulted in a lack of
baseline information and the inability to monitor the stranding
rate in a timely fashion. These problems are primarily the result
of relying on a not-uniformly organized or trained, under-funded,
volunteer Network. The SEFC can improve the capabilities of the
Network by assuming some responsibilities for reporting, by
establishing collection protocols and providing collection
materials, by providing training and arrxmgements for clinical
necropsy of suitable specimens, by providing for analyses of tissue
samples, and by informing Network participants on the results of
their reporting and data collection efforts.
The SEFC stranding response activities will center on three
areas: monitoring stranding rate, specimen necropsy, collection and
analyses, and dissemination of results. The stranding rate will be
monitored by establishing a system for rapid reporting of basic
data on stranded animals. Consistent specimen collection will be
accomplished by providing manuals, collection kits and training to
Network participants. The SEFC will establish pathways for ensxiring
clinical necropsy and tissue analyses of suitable specimens.
Results will be disseminated to Network participants in a quarterly
report produced in conjunction with the Net«rork Coordinator. The
most important component of all these activi-tles is the development
and maintenance of communication between the SEFC and the Network
participants .
The Network already has a system for reporting stramdings,
some data collection protocol, emd for dissemination of results.
However, the level of these activities is not sufficient to meet
the SEFC information requirements. It should be clear that the SEFC
is not attempting to takeover the Hetwork, bnt that the SEFC is
trying to supplement the Hetwork by providing assistance for
particular activities.
Monitoring Stranding Rate
The SEFC will establish a system for near real-time monitoring
of the stranding rate. Appropriate staff at each of the SEFC
laboratories and the Regional Office will be identified as SEFC
area representatives and will establish contacts in their area with
Network participants. The Network participants will be required to
report basic data (vrtiat, when, where and condition) to the SEFC
area representative within 48 hours of a stranding event. The area
representative will then report the basic data within 48 hours of
receipt to the Miami Laboratory. Ultimately, a computer bulletin
board system will be established for receiving basic data reports.
The Miami Laboratory area representative will be responsible for
195
reviewing the basic data reports and for wee)cly monitoring of the
stranding rates throughout the southeast. This will allow for rapid
identification of anomalous stranding events and the transfer of
this information to NMFS Headquarters and others in a timely
manner .
Actions Required
• Center Director
o Request leiboratories to assign staff as SEFC area
representatives
o Coordinate with Regional Director to assign
regional staff as an SEFC area r^resentative
• Regional Director
o Modify letters of Authorization to incliide
reporting reguireBent of basic data to SEFC area
representative within 48 hours
o Assign regional staff as an SEFC area
representative
Specimen Necroosv Collection and Analvses
The Charleston Laboratory is currently developing necropsy
protocols, specimen collection protocols and collection Icit
specifications. The protocols and )cits will be distributed to the
appropriate Network participants.
The Miami Laboratory is presently identifying necropsy
personnel and nacronsy facilities in the southeast. The SEFC area
representatives will ensiire that appropriate specimens are
delivered to necropsy facilities.
The SEFC area representatives will receive, track, store and
transfer collected samples. Arrangements will be made with the
Armed Forces Institute of Pathology (AFIP) to conduct
histopathological studies on appropriate specimens. Other collected
specimens will be trzmsferred for analyses when suiteible
investigators are identified (e.g., for genetic, food habits,
aging, stock studies; some funding may be required and faculty
appointments used to bring investigators onboard) . These activities
will ensure that adequate information is available to begin
evaluating causes and potential effects of both normal and
anomalous mortality events.
Actions Required
• Center Director
196
o Approve fiinding and faculty appointments
nisseminatiion of Results
A quarterly newsletter which provides stranding sumnaries,
information on analyses underway or planned, and any noteworthy
events or tips, will be distributed to each Network participant.
The newsletter will be produced by the SEFC and the Network
coordinator. Although this is a minor activity in terms of funding,
it is critical for maintaining communication and cooperation
between the SEFC and the Network participants. The primary purpose
of this activity is to let the Network participants know that their
efforts made to provide the SEFC with information and specimens are
worthwhile.
A biennial Stranding Network meeting should be held, sponsored
by the SEFC and the Network coordinator. The meeting will provide
a forum for reviewing the Network activities, providing training in
necropsy and specimen collection, reporting related resezurch
findings, emd for estedslishing and maintaining contacts between the
Network participants and the SEFC.
Actions Required
• Regional and Science Directors
o Approval for Newslsttar and Meeting
Estimated Personnel Costs
The proposed activities will require varying amounts of staff
time from each SEFC area representative. Initially, each area
representative will spend a significant (probably 20 hrs or more
per week for two to four weeks) amount of time identifying and
contacting area participants nnd clinical necropsy facilities and
personnel. Subsequently, less time, probably one to five hours per
week, will be required for reporting, delivering or transferring
specimens, emd maintaining contacts. Some area representatives may
also participate in recovering stranded animals. The Miami
Laboratory area representative will be responsible for development
and implementation of the computer bulletin board system. This will
take about one person-month. Approximately one-half of the Mieuni
Laboratory area representative's time will be spent on reviewing
and analyzing reports, distributing specimens, reviewing results,
maintaining and developing contacts, and preparing stranding
program reports.
The following draft proposal outline provides more information
on specific responsibilities and a tentative implementation
schedule.
197
OUTLINE FOR ENHANCING NMFS CETACEAN STRANDING DATA COLLECTION
To foster closer cooperation between local SEUS stranding network
participants and NHFS Marine Maaonal Stranding Area
Representatives .
To improve data collection quality, quantity, and consistency.
To provide near real -tine reporting of cetacean stranding to the
NMFS.
To provide flow of information to network participants from NMFS
representatives and SEUS coordinators.
To foster information exchange and cooperation among network
peurticipants .
UPWARD DATA FLOW
Products
A. COMPLETED SEUS DATA - Sent to SEUS Network Coordinator
within 30 days of collection as specified in the NMFS Letter
of Agreement (LOA) .
B. BASIC STRANDING DATA - Sent to NMFS Miami Laboratory from
NMFS Area Representatives within 48 hours of initial report
from SEUS Nettrork Coordinators (total elapsed time since
initial discovery will not exceed 96 hours);
1. Field Number
2 . Species
3. Sex
4. Length
5. Yeeur, Month, Day of 1st report
6 . State
7. Latitude/Longitude qz zone (to be developed)
8. Condition (1-5)
9. Fishery Interaction (Yes fic No)
10 . Time report 1st received by NMFS staff
11. SEUS nettrork participant reporting to NMFS
representative .
C. SPECIMENS - Collected by SEUS network participants and
delivered to, or picked up by, NMFS area network
representative (numbers in parentheses refer to specimen
condition) :
1. Pathology:
a. Necropsy reports from participating veterinary
clinics (1&2).
b. Histology specimens for AFIP (1&2).
c. Contaminants (1-4).
d. Biotoxins (1-4).
Charleston Laboratory is currently establishing
198
protocols for pathology speciaen collection.
2. Life history specimens:
a. Entire head if at all possible (1'5), otherwise
collect 5 teeth from mid lower jaw of each
stranded specimen (I'S).
b. Gonads and reproductive tract (1-4),
c. Stomachs (1-4).
e. T%ro mid-thoracic vertebrae (1-4).
3. Genetics specimens:
a. Blood (1-4).
b. Liver (1&2).
c. Heart (lfc2).
DOWNWARD INFORMATION FLOW
Products
A. QUARTERLY REPORT - Produced jointly by SEDS/SEFC. Will
include information of general interest to stranding network
participants and quarterly summary of stranding activity in
SEUS. Will not duplicate the Smithsonian quarterly report;
it is intended to be more of an informal newsletter.
B. Biennial SE Stranding Network (or regional subset) meeting.
Should include meurine mammal research activity in SEUS in
addition to stranding network.
RESPONSIBILITIES
WMFS Area Renresentatlve
A. Each NNFS laboratory under the Southeast Fisheries Center
will have a staff me^ober (NMFS Ar^a Representative) with
backup personnel, assigned to receive stranding reports and
specimcms required by the NNFS. A telephone
answering/message recording machine will be used during off-
duty hours.
B. NMFS Area Representative will forward stranding BASIC DATA
to NMFS Miami Lab via coiqniter bulletin board in
standardized format within 48 hours of receiving report from
SEUS regional coordinator.
C. NMFS Area Representative will receive or retrieve biological
sa^>les required by the NMFS and f ortrard to the appropriate
NMFS laboratory specified by Miami Lab.
D. NMFS Area Representative will coordinate with participating
veterinary clinical pathology laboratories for detailed
necropsies of condition 1 & 2 carcasses and will forward
199
results to Miami Lab.
E. NMFS will provide materials for specimen collection to SEUS
Stranding Network and NMFS Area Representatives will accept
collect telephone calls for reporting stranding.
F. NMFS Miami Laboratory will manage NMFS Area Representatives,
arrange for transfer of biological samples to analytical
laboratories, coordinate final disposition of specimens, and
with SEUS Network Director, produce newletter and assist in
maintenance of regional organization. Miami Laboratory will
maintain real-time cetacean stranding database for the NMFS
southeast region.
G. NMFS will not usxirp SEDS stranding network coordination and
r espons ibi 1 it i es .
gffng jgl^randlno Network
A. Nettrork participants will report BASIC DATA (above) to SEDS
Regional Coordinator by telephone within 24 hours of
discovery of stranding as a requirement of the LOA. Voice
confirmation of receipt of information is required. If SEUS
Regional Coordinator cannot be reached on the first try,
then the network participant will immediately telephone the
NMFS Area Representative.
B. SEUS Regional Coordinator will be required in LOA to report
stranding to NMFS Area Representative within 24 hours of
receipt from network participants.
C. Network participants will perform general necropsies on
carcasses to the extent that condition warrants. General
necropsies will include morphological measurements,
photographs, examination for fishery interaction and
external pathology, and collection of specimens for life
history, pathology, and genetics studies as detailed in
necropsy manual and outlined herein. If the carcass is
condition 1 or 2, and size permits, the «fhole carcass will
be transported to a participating veterinary clinic for
detailed necropsy.
D. SEDS Stranding Network participant will deliver or
coordinate delivery of NMFS-required specimens to the NMFS
Area Representative and ensure proper storage of specimens
until delivered to NMFS.
E. SEDS Stranding Network participants will provide stranding
data to SEUS Stranding Network Director as directed in the
LOA.
200
TENTATIVE IMPLEMENTATION SCHEDULE
A. BASIC:
1. NMFS area netirork representative selection...! March 1991.
2. Reporting requirements to SEUS nettrork participants via
LOA 1 April 1991.
3. Collection and transfer of life history
specinens iaaediately .
4. BASIC data reporting fully i]q>leBented 1 April 1991.
B. PATHOLOGY:
1. Draft pathology xsanual 1 ^nril 1991.
2. Schedule training workshops ?
C. GENETICS:
1. Collection of genetics speciaens iaaediately.
D. FACILiry APPOINTMENTS
1. Pathology AFIP.
2 . Genetics ?
3. Life history
a. Stonach content analysis
Tursiops Sea Norld.
Others ?
b. Teeth for ageing ?
c. Reproductive tracts and gonads ?
d. Other ?
4. Other ?
201
SOUTHEAST REGIONAL STRANDING NETWORK DATA FLOW
- SEUS STRANDING NETWORK PARTICIPANTS -
TX LA,MS,AL NW FL. C-SW FL. KEYS-GA GA-SC
BASIC DATA
24 Hoxxr Reporting Li ait
NC
GAL
PAS
SEUS REGIONAL COORDINATORS
BASIC DATA
24 Hour Reporting Limit
i
NNFS AREA REPRESENTATIVES
PAN ST. PETE. MIA CBASTON
BEAD
COMPLETE STRANDING RECORD
30 Day Reporting Limit.
i
SEUS Nettrork Coordinator
T
JOINT QUARTERLY REPORT
TO SEUS AND NNFS
NETWORK PARTICIPANTS
BASIC DATA
48 Hour Reporting Limit
i
NNFS Miami Laboratory
EMERGENCY REPORTS
TO NMFS HQRTRS.
AND MMC.
202
Appendix X. Report on the Southeast Fisheries Science Center Marine Mammal Stranding Network
Representative System Organizational Workshop.
203
This page intentionally left blank.
204
^
OF,
^
•n
*«i«T 0» '
Report on the Southeast Fisheries Science Center
Marine Mammal Stranding Network Representative
System Organizational Workshop
Southeast FisbariBS Scimacm Canter
Miami, Florida
May 7-8, 1991
I.
Prepared by Staff
Southeast Flsberiec Science
Contribution KIA-91/92-24
December 1991
205
Report on the Soirtheast Fisheries Science Center
Marine MaBsal Stranding Network Representative Systea
Organizational Workshop, May 7-8, 1991
I. INTRODDCnOir
The Marine Mammal Stranding Network NMFS Area Representative
System Organizational Workshop was held at the Southeast
Fisheries Science Center in Miami, Florida, May 7-9, 1991. The
following participants attended the workshop:
Mr. Ben Blaylock - Mizuni Laboratory
Mr. Bill Bowen - Beaufort LeUooratory
Dr. Brad Brown, Center Director - SETC
Mr. Jeff Brown - Southeast Regional Office
Dr. Charles Caillouet - Galveston Laboratory
Mr. Bill Feible - Panama City Laboratory
Dr. Sylvia Galloway - Chcurleston Laboratory
Mr. Larry Hansen - Miami Laboratory
Mr. Wayne Hoggeurd - Mississippi Laboratory
Mrs. Ann Jennings - Charleston Laboratory
Dr. Dan Odell, SEUS Coordinator - Sea World
Dr. Joe Powers, Laboratory Director - Miami Laboratory
Ms. Kathy Prunier - Miami Laboratory
Dr. Gerry Scott - Miami Laboratory
Mr. Lee Weinberger - Miami Laboratory
Mr. Dean Wilkinson - Office of Protected Resources
The workshop was convened to discuss methods to enhance the
Southeast D.S. Marine Mammal Stremding Net%rork and to implement
the Southeast Science Center Marine Meunmal Strtmding Network Area
Representative System. It was recognized that the volunteer
Stranding Network is non-uniformly organized and trained, and it
is under-funded, resulting in two major problems. These are: (1)
the Stranding Network is unprepared to mount an adequate response
to increased stranding, and (2) gross inconsistencies in data
collection and reporting have resulted in a lack of baseline
information and in an inability to monitor the stranding rate in
a timely fashion.
The purpose of the SEPC Area Representative System is to
supplement the Stranding Network by providing assistance to
enhance the efficiency of the net«rork; however, it will not usurp
SEUS Stranding Net«rork coordination and responsibilities. The
system is designed to:
Facilitate near real-time reporting of cetacean
stranding to NMFS.
Improve data collection quality, quantity, and
consistency.
206
Foster information exchange and cooperation among
Network Participants and NMFS Representatives.
The SEFC Stranding Network Representative System will focus
on three areas: (1) monitoring the stranding rate; (2) sp>ecimen
necropsy, collection and analyses; and (3) dissemination of
results .
7ntTT>ductorv l^f^mr^tR- Brad Brown
The following are efforts designed to strengthen the
Southeast Fisheries Science Center (SEFC) involvement in the
activities of the SEUS Stranding Network:
Improve funding for the stremding coordinator.
Enlist and pay for veterinarians to perform necropsies.
Assign responsible people at each laboratory to act as
a major link to enhance collection of stranding
information .
This meeting occurred as a commitment to respond quickly to
major stranding events. Although the SEFC has previously
responded to stranding events analytically, it will become more
active in the SEUS Stranding Nettrork.
SEDS stranding Hetwork! Dan Odell
There has been a lack of coordination in the SEUS Stremding
Network and there is a perceived need for greater organization.
The network began wii-h data cards and a mailing list involving
Sea World and Marine Land (David and Melba Caldwell). The first
Marine Mammal Stranding workshop %ras in Georgia in 1977, emd was
sponsored by the Marine Meunmal Conoaission (MMC). In the first
year of the network, less than 100 stranded mammals were
reported, primarily Tursiops. Data increased greatly from 1978
to 1990.
An individual field number is assigned to each stranded
marine meusmal. It should be the primary number for tracking the
specimen and oxir goal should be to identify a specimen with as
few numbers as possible. There must be a standard developed to
achieve iiniformity throughout the entire area. One suggestion is
a series of numbers %rtiere the first two are symbolic of the state
code, and the rest related to the area. However, it is importemt
to not interfere with any previous long-term systems.
The number of strandings depends on the amount of coastline
in a given eurea and the number of people involved in the Net%rork.
207
There are approximately 250 people in the SEUS Stranding Network
and although many state and local official agencies participate,
there have often been problems in getting sufficient information
from strandings. For instance, better coordination with the
Marine Patrol is needed in parts of Florida. A training video is
being created that will focus on the. data sheet used by the
Stranding Network, with examples using real animals.
Data collection sheets should be carried to each stranding,
and everyone should keep a log book. It would be a good idea to
package a log book and data collection materials for %rorkshop
participants. Area Coordinators mxist collect and organize data.
Reduction of time lags and duplicate information require more
screening and organization of Network Peurticipants at the Area
Coordinator level.
There are two key forms used in the network, but only the
short form is required by the Letter of Authorization (LOA). The
information on the short form is entered into the SEUS Stranding
Network computer information bank.
II. NONirORING THE STRAMDIIIG RATE
The SEFC has newly established a system for near real-time
monitoring of the stremding rate. Appropriate staff at each of
the SEFC laboratories and the Regional Office were identified as
SEFC Area Representatives to esteiblish contact in their area with
SEUS Network Participants. Each NMFS Area Representative must
have back-up personnel assigned to receive stranding reports and
specimens in their eibsence. A telephone emswering/message
recording machine should be used during off-duty hours.
The SEUS Network Participaa^ will report basic data to the
SEUS Area Coordinator by telephone within 24 hours of discovery
of the stremding as a requirement of the LOA. Voice confirmation
of receipt of information is required. If the SEUS Area
Coordinator cemnot be reached on the first try, then the Network
Participant will immediately telephone the NMFS Area
Representative. Completed SEUS data will be sent to the SEUS
Network Coordinator within 30 days of collection as specified in
the NMFS Letter of Agreement (LOA). The SEUS Area Coordinator
will be required in the LOA to report strandings to NMFS Area
Representatives within 24 hours of receipt from the Network
Participants .
A computerized system was established for transferring basic
data reports to the Miami mainframe computer. NMFS Area
Representatives will send stranding data to the SEFC NMFS Miami
Laboratory within 48 hours of initial receipt from SEUS Area
Coordinators (total elapsed time since initial discovery should
not exceed 96 hours) using the computerized system. The basic
208
stranding data transmitted to the Miami Laboratory are:
Field number.
Species.
Sex.
Length.
Year, month, day of first report of stranding.
State.
County .
Condition (1-5).
Fishery interaction (yes or no) .
Time report first received by NMFS staff.
SEUS Network Participant reporting to NMFS.
Incidental remarks.
The Miami Laboratory Area Representative is responsible for
reviewing the basic data reports and for monitoring the stranding
rates throughout the southeast. This system will allow for rapid
identification of anomalous stranding events, the transfer of
this information to NMFS Headquarters and others in a timely
manner, and maintencmce of a real -time cetacean stranding
database for the NMFS southeast region. The "chain of custody"
within the 96 hour reporting system is from Seus Net%rork
Participants to the SEUS Area Coordinator to the NMFS
Representative to Miami. In Florida, there are three Area
Representatives; however, the SEDS State Coordinator provides the
real-time stranding reports directly to the Miami Laboratory.
The NMFS Area Representatives are:
• Bill Bowen
• Ann Jennings
• Ben Blaylock
• Jeff Brotm
• Bill Fable
• Wayne Hoggard
• Charles Caillouet
Beaufort
Charleston
Miami
St. Petersburg
Panama City
Pascagoula
Galveston
North Carolina
So. Carolina, Georgia
South Florida
Central Florida
Florida Panhandle
Miss, Ala, La
Texas
III. SPECIMEN COLLECTION, NECROPSY, AMD ANALYSES
Newly proposed regulations will require the collection of
certain tissues from stranded marine mammals. This could lead to
a decrease in coverage by volunteer participants of the stranding
network. In order to maintain a cooperative atmosphere between
the NMFS and SEUS Stranding Net%rork Participants, NMFS Area
209
Representatives should assist with code 1 and 2 strandings
whenever possible to ensure that appropriate samples are
collected.
The Charleston Laboratory is currently developing necropsy
and specimen collection protocols, and tissue collection kits
which will be distributed to the appropriate personnel. The
Mieuni Laboratory is presently identifying necropsy personnel and
necropsy facilities in the southeast. The NMFS Area
Representatives should enstire that appropriate specimens are
delivered to the necropsy facilities.
The SEFC Area Representatives will receive, track, store,
and transfer collected samples as necesseury. Arremgements have
been made with the AFIP to conduct histopathological studies on
appropriate specimens. Specimens collected for genetic, food
habit, aging, emd stock identification studies will be
transferred for analyses when the appropriate investigators are
identified. Some funding will be made available for faculty
appointments to perform some of these studies. These activities
should ensure that adequate information is available to begin
evaluating causes and potential effects of both normal and
anomalous mortality events.
Official Responsibilities and Authority; Jeff Brown
The three topics discussed were: who is on the network;
non-government people and LOA's; and jurisdiction.
SEDS Stranding Network
There are approximately 250 participants in the network; all
are listed in the Stranding Directory which is currently being
updated. A suggestion was made to put the Area Representatives
higher on the list, emd to alphabetize the list.
Letters of Authorization (LQA)
LOA holder qualifications vary. Potential peurticipants
sxibmit a letter to the Regional Office stating %^y they are
interested in participating in the Network, along with a resume
indicating education and experience. Qualifications for LOA
issuance are subjective and depend on the locality. Issuance of
LOA's and compliance with rules and responsibilities of LOA
holders is the responsibility of the Regional Office. It is
important to keep the rules and the application process simple as
the majority of people in the Stranding Network are volunteers.
They must understand %fhy they are in the Network, that we do not
impose anything on them that we trould not do ourselves, and that
210
proper procedures must be followed.
The large turnover in peurticipants necessitates frequent
review of the Network membership and LOAs are generally renewed
annually. The renewal process consists of a letter from the
Regional Office asking if there is a desire to renew, how many
strandings they were called for, and how many they responded to.
This information is reviewed and the renewal of the LOA is
decided.
A number of questions arose during this disciission. At a
stranding can other people assist the LOA holder? The response
was that legally, if a person touches an emimal they need an LOA.
Obviously, LOAs cannot be issued for each person %rho helps in a
stranding; thus, appropriate subjective judgement must be used
for each situation.
Section 109H (Appendix I) of the Marine Hanmal Protection
Act, about the taking of meurine mammals is importcmt to read and
understand. Section 112C (Appendix IZ) covers LOA holders and the
federal government. Section 109H, concerning stranded or dead
imimals, specifically states that federal, state and city
employees need not have an LOA.
Jurisdiction
Theoretically, the first LOA holder or city or state
official «rtio arrives at the stranding site has control; however,
the stranding is ultimately under Federal jurisdiction and the
NMFS may take control of the situation if necessary. He must
deal tactfully with network members at strandings, but we also
have a responsibility to ensure that the emimals receive humane
treatment, including eutheuiasia, if necesseury. We must employ
diplomacy emd coordinate with people on the scene. In the
Florida Keys, it may be necessary to have a 2-3 person management
team to make contacts; however, decision making must be
restricted to 1 or 2 persons.
Responsibility for disposal of carcasses varies regionally.
For example, burial is not allowed in the Florida Keys. City
agencies usually take care of carcass disposal for public health
reasons «rtien a stranding occiirs near a municipality. A carcass
may sometimes be left to rot in a remote eurea. NMFS Area
Representatives should contact local authorities in advance and
find out about carcass disposal policies and facilities.
NMFS Area Representatives should become acquainted with key
people in local and state agencies and coordinate these efforts.
It is the job of the Area Representatives to ecttablish local
contacts and work with them to facilitate retrieval of data and
specimens and assist with carcass disposal if necessary. Local
211
veterinarians who work with large animals may provide assistance
in carcass disposal.
.qtrandina fi£ A PTPtggted SPCCJeS =. L National Persneetive;
Dean Nilkinson
A couple of regulatory chjmges have occxirred:
The newly enacted requirement of proper registration
and tracking of tissue taken from stranded animals.
A revision of permit regulations concerning the public
display of rehabilitated animals (not applicable to the
NMFS Stranding Network Representative System) .
Regional differences in LOA issuance and requirements
currently exist; however, a national standard will be established
later this year.
An exciting development in the proposed fiscal year 1992
budget is $0.5 million to be split between the Stranding Networks
and the National Marine Mammal Tissue Bank. This will provide
approximately $20-25,000 to each region for basic Stranding
Network support (ie. the prepeuration of guidebooks, training
meetings, for equipment, and for responses to unusual stranding)
if approved by Congress. There may be money funded for the SE
region to hire a person to enhemce Stranding Network activities
in the northern Gulf area. $100,000 will be withheld by OPR for
unusual marine meunmal stranding.
Si-ndi*»s id«»ntlf le*^ for gathering basic information on
Tursiops and the use of stranded animals are: (1) an intra-
tissue contaminant distribution study; (2) a conteuainant
degradation study in 1992, to determine how many hours after
death tissues can be used for the National Tissue Bank; and (3) a
quality assurance program to obtain standard prep tissues for
calibration. A training video by Dan Odell and a general
stranding response manual by Dr. J. Geraci should be available by
the end of 1991 for Stremding Network Members.
ResponsibllltJgs fl£ AZfiA Reprcscntati veg i Larry Hansen
Reporting:
The primary goal of the enhancement project is to move the
stranding information as quickly as possible. Presently, tiie
Area Representative sends information to the state coordinator
(if they're not the same person). The information is then passed
212
on to the Miami Lab. In Miami, Ben Blaylock or Kathy Prunier are
the contacts to call about stranding reports. Lee Neinberger is
in charge of the software organization in Miami. The report to
Mieusi should occur within 48 hours of receipt by the NMFS Area
Representative. The flow of information is from the SEUS state
coordinator to the NMFS Area Representative to the Miami
Laboratory. If the event seems unusual, the information will be
passed to the SE Regional Office and the Office of Protected
Resources to determine an appropriate response (e.g., should an
emergency investigation be initiated?).
Communication:
It is importemt to commxinicate with area Nettrork
Peurticipants the need for a better response to stranding and to
give moral support. Jeff Bro%m will distribute a memo concerning
legalities emd goals to the entire network. The memo will
include the responsibilities of the Area Representatives, and the
purpose of the program. It would be a good idea to include a
copy of the 1987 Stranding Workshop Report with these letters.
This will provide both information and feedback to participants.
Approximately one week after the memo is sent, all SEDS Net%rork
Participants should be personally contacted by the State
Coordinators about the new protocol.
Necropsies :
Tissue collection from anything beyond Code 2 strandings
should be collected as the situation warrants. It is a
responsibility of the NMFS Area Representative to assist the SEUS
Network Participants in tissue and data collection from Code 1 &
2 animals. Area Representatives are responsible for contacting
qualified pai'hologists and v^terinnrian^ to see that quality
necropsies take place.
There are two methods to handle this. One is through a
formal . contract with local veterinarians. There may also be
local veterinarians who already SEUS Network Participants. The
NMFS Area Rep. should make contact with these resources. Another
is to set up a coordinated progreua through nearby veterinary
schools. Small animal veterinarians may be helpful, but there is
a big difference between small animals and large marine mammals.
Hopefully, some formal veterinary necropsy arrangements will be
made later this yeeur.
There will be paid faculty appointments for basic life
history studies with cost varying according to the event.
Faculty appointees may be taxonomists, specimen analysts, emd/or
veterinary pathologists. This will be easier if the appointee is
affiliated with a university. In some instances faculty are
allowed to consult but may not be allotted to use university
facilities; however, there may be other facilities available.
213
NMFS Area Representatives should identify local available
prospects for faculty appointments in advance. Expertise
available in each area will also be investigated by the Miami
LaOjoratory in consultation with the SEDS Network Coordinator.
Our primary interest in each area should be to find people to do
clinical necropsies and histopathological collection, and
secondarily to do the other analyses.
Area Representatives must ensure that Network Participants
remain aware of the importance of data collection. Care must be
taken in the collection of all data, as some participants are
prone to collect only ceirtain data. The protocol being developed
by Sylvia Galloway will clarify data collection requirements and
procedxires .
Discussion fi£ Specimen Storage aiul TmilBferL Larry Hansen
Specimen Storage:
It is important to know what facilities eure available.
Ultra-cold freezers are necessary for storing tissues for toxin,
biotoxin, and metal analyses. One cubic foot per animal is
sufficient. If a regular chest freezer is used, place the
specimens as far towards the back as possible before they are
shipped to a facility which has an ultra-cold freezer.
Sei^ile Care:
A standard set of tissue will be collected. Life history
seunples include teeth, stomachs, and reproductive organs.
Contaminant analysis samples are organs such as the liver,
kidney, and blubber. Detailed collection procedures will be in
the protocol. Tissues will be sent to Miami or Charleston for
distribution to the appropriate laboratory. Each facility must
have temporary storage capability for both frozen and formalin-
stored samples.
Basic M*ii-i nm M»i—»»i Stranding I^ta Reporting; Ben Blaylock
A primary purpose of this meeting was to establish the real-
time marine mtunmal stranding reporting system. Prom data
obtained within 96 hours of the discovery of a marine mammal
strzmding, the Miami Laboratory will produce a summary of
regional stremding activities, listing data such as species,
year, state, sex, county, condition, and fishery interactions.
To implement the system. Area Representatives received a set of
computer disks %ihich allow automated reporting of level A data
directly to the Mieuai Laboratory's mainframe computer, and a
memual describing the reporting system. The data collected in
214
the progreuB were designed to facilitate the immediate reporting
of data. The Miami Lab will produce a 7 and a 30 day report.
Specimen and Cfi^ Collection Protocols: Sylvia Gallmfay
Why do dolphins die? We would like to collect samples to
explain how they die, and for metal and contaminimt analyses. A
group convened by the NMFS reviewed the investigation of the 1990
Gulf of Mexico die-off and reached the consensus that a multi-
disciplinary approach was necessary to gain adequate data from
strandings. As a result, a data inventory list was prepared in
advance of another large die-off.
In addition, a kit was prepared in order to deal with
unusual stranding events. These kits will go to a few trained
individuals to begin building a database. The kits were designed
by working with both experienced and inexperienced people in a
triple necropsy. Peirticipants discussed each specimen and the
requirements of each protocol, identified conflicts, and resolved
most of the conflicts in a round-table discussion.
The integrated protocol included a sample collection
checklist. It was determined that a team is needed to perform a
good necropsy on a code 2 animal, and a very fresh animal is
necessary for microbiological analysis. Large pieces of tissue
are needed for hi stopatho logical work. A veterinarian responding
to a Code 2 stranding can obtain most of these data.
It was decided to collect complete stomachs for pathology
examination along with stomach contents. Locations and methods
for obtaining blubber measurements needed to be specified. A
compromise was to identify three points on the luiimal: one
varieQ}le and dependent upon the species; one a standard point;
and for the third, more information is needed before a
determination can be made.
Disposable supplies from the kits will be replenished by the
user. These are not intended as field necropsy kits. Large
tissues should be taken back to the lab for clean sub-sampling.
There will be instructions for the appropriate use of the kits.
Each Area Representative will maintain a fully-stocked kit. The
kits are to be used to respond to an unxisual stranding event (as
defined by a designated peuiel of experts), or Code 2 strandings.
They should not be used for other strandings. Federal and army
surplus stores cure good soxirces for general field necropsy
supplies.
i)ata Collection
The NMFS will provide materials to the SEDS Stranding
215
Network for collecting samples specified by the NMFS (ie., life
history siunples, tissues for contaminant analyses, histopathology
samples, etc.) and NMFS Area Representatives should accept
collect telephone calls for rep>orting strandings. SEUS Network
Participants will F>erform general necropsies on carcasses to the
extent that conditions weurxant. General gross necropsies include
morphological measurements, photographs, examination for fishery
interaction, external pathology. Specimens must be collected for
life history, pathology, and genetics studies as detailed in the
necropsy manual and outlined in this report.
If the carcass is Code 2, emd size permits, the entire
carcass should be tremsported to a participating veterinary
clinic for detailed necropsy. The following specimens (condition
code in parentheses) should be collected by SEUS Nettrork
Participants emd delivered to, or picked up by, the NMFS Area
network Representative.
For Pathology:
Necropsy reports from participating veterinary clinics
(2).
Histology specimens for AFIP (2).
• Contaminants ( 2-4 ) .
• Biotoxins (2-4).
The Charleston Laboratory is currently establishing
protocols for pathology specimen collection.
For Life history specimens:
Entire head if at all possible (2-5), otherwise collect
lower jaw or 5 teeth from mid-lower jaw of each
stranded specimen.
• Gonads and reproductive tract (2-4).
Stomachs ( 2-4 ) .
Two mid-thoracic vertebrae ( 2-4 ) .
For Genetics specimens:
Blood (2-4).
Liver (2).
Heart (2).
The SEUS Stranding Nettrork Participant will deliver or
arrange delivery of NMFS-reguired specimens to the NMFS Area
Representative and ensure proper storage of specimens until
delivered to NMFS. The NMFS Area Representative will receive or
retrieve biological samples required by the NMFS and forward them
to the appropriate laboratory as specified by the Miami
ledsoratory. The NMFS Area Representative will coordinate with
participating veterinary clinical pathology laboratories for
216
detailed necropsies of Code 2 carcasses, and will forward tJie
results to the Miami laboratory. The NMFS Miami Laboratory will
manage NHFS Area Representatives, arrange for transfer of
biological samples to analytical laboratories, and coordinate
final disposition of specimens.
The protocols in the manuals are very detailed and the
check-off sheets are important to the procedures. Successful
necropsies take time, practice, patience, and care. Each sample
must be labeled. The SE Fisheries Science Center is willing to
train, emd workshops are being planned. Acquiring contacts who
will respond in the time of need is essential and NMFS Area
Representatives are responsible for decreasing the response time
to stranding. The MMC was pleased and has been complimentary
about recent stranding data collection activities in the
Southeast Region.
Data Flow
State Coordinators are a point source who feed information
to the Area Representatives; howevar, the link between public
authorities and the NMFS and the Marine Mammal Stranding Network
needs to be strengthened, especially in particular areas. In
some areas, using the marine patrol is advantageous and in others
cooperation may be difficult to obtain. Posters advertising the
Network are helpful in increasing public awareness. Often, whole
fresh dolphin carcasses may be kept in local fish houses until
arrangements cem be made for necropsy. The NMFS Area
Representative should contact local and state authorities to
increase their awareness of the system and enlist their help.
General Discussion: Workshop Participants
The following topics were brought up in a general round
tzible discussion:
As there are not many Code 2 necropsies, we should be
able to get our necropsies performed using our protocol from
local veterinary schools at their cost. Present contractual
agreements allot $850.00 for each carcass. Me must determine how
to appropriate veterinary expertise for fresh stremdings. The
Miami Laboratory will contact veterinary schools and report to
Area Representatives.
Are there rehabilitation facilities in each state? There
are no primarily rehabitative facilities in the southeast, but
zoological park -type and oceanarium facilities have participated
in the Net%rark.
217
How suitable are animals for the protocols when they have
been reheibilitated for a F>6riod of time? The microbiological and
viral information is lost but other information is still gained.
Is there a protocol for the first 24 hours that a live
animal is being treated? It would be important to get general
clinical information. Pre-treatment protocol would include
routine blood samples. An experienced veterineurian would likely
do this; however, we need to specify our needs.
Is it possible to determine whether a marine mammal has
drown or suffocated? There eure both wet and dry dro%ming.
Strontium in blood, liver, and kidney, and diatom levels in the
lungs can be used to determine the type of death. These
varieibles are used in human autopsies.
Appropriate seunples to be collected from a stranded
animal are outlined on page 24 of the necropsy protocol, in
general it is as follows: (1) "clean" tissue collection protocol
for code 1 and 2 animals; (2) stomachs and life history samples
collected for all animals; and (3) for code 3-5 animals,
protocols are being developed.
If a rare animal has stranded. Network Participants
should call their Area Representatives immediately. There will
be specific protocols. The Area Representative should be
prepared for this with appropriate information and specific
questions to ask the network people. In this regard, the only
normal stranding is single coastal bottlenose dolphins.
IV. DISSEKXHATIOir OF RESULTS
A quaurterly report will be produced jointly by SEUS/SEFC.
It will include information of general interest to Stranding
Network Participants, a quarterly summary of stranding activity
in SEUS, information on analyses underway or planned, and any
noteworthy events or tips. It will not duplicate the Smithsonian
quarterly report; it is intended to be more of an informal
newsletter. Although this is a minor activity in terms of
funding, it is critical for maintaining communication and
cooperation between the SEFC and SEUS Nettrark Participants. The
primary purpose of this activity is to let Nettrark Participants
know that their effozrts made to provide the NMFS with information
and specimens are worthwhile. The NMFS Miami laboratory, in
coordination with the SEUS Network Director, will both produce
the newsletter and assist in the maintenance of regional
organization.
It was suggested that a biennial Stranding Network meeting
should be held, sponsored by the SEFC and the SEUS Net«fork
Coordinator. The purpose of the meeting would be to monitor the
218
Network activities, provide a forvun for reviewing Network
activities, provide training in necropsy and specimen collection,
report related research findings, and establish and maintain
contacts between the SEUS Network Participemts and the NMFS. It
should not be limited to stranding related research.
V. AD HOC ITEMS
Area Representatives should check into freezer storage
and contact the Miami lab with this information. The Miami lab
can then make the required arrangements.
What communication is there between state agencies, NMFS,
and the Network? Some states apparently have no regulations
dealing with dead mzurine mammals, and in Louisiana and Florida,
for example, permits are required to transport live animals.
Hill there be a defined split in area boundaries in
Louisiana? This question is under consideration.
Network Participant responsibilities must be clear and
well defined in the LOA. The LOA will be modified to include the
48 hour reporting requirement. Area Representative
responsibilities will be detailed. Requirements unique to each
area will also be included.
There was a proposal that the SE region meet bi-amnually
during the marine meunmal meetings, and that other stranding
related persons and cetaceem reseeurchers from the SE region are
invited. Regional gatherings every other year were also
suggested.
219
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