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Full text of "Report on investigation of 1990 Gulf of Mexico bottlenose dolphin strandings"

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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 

















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 





11 


9 


9 


7 


Galveston 


6 





8 





10 


1 


Bryan Beach 


9 





8 





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 





14 





22 


4 


South Padre Island 


20 





21 





1 





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 





472 


0.0177 


508 


0.0142 


491 


GaKeston 





518 





415 


0.0022 


449 


Bryan Beach 





45 





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 





341 





367 


0.0054 


735 


South Padre Island 





600 





600 





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 





16 


9 


32 


7 


Galveston 
Bryan Beach 


45 






11 

1 






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 













13 


4 


South Padre Island 


A 





3 





4 





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 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 



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 - 










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 7 . 


/ / — «i 


s "■•■ 


/ ; MXD SMWJE 

/ t 


5 04. 


^^y^ J 


\ "-'■ 


— _^ 
y y OM . 0J4S0 


O.J • 
0.1 • 


/ ,-'' » < OJ»l 



IJO. 






o.«  






\ 0.0  




'</ mo 


1 0.7 . 




/ — *"• 


S "^ ' 




A — *ca> SM»i£ 


!"■ 




yZt 


3 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 - 


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 





10 


3 





4 


3 


1.7 


7 


JUN 


4 


4 


3 





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 
te mpera ture 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 






48 




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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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Wagemann, R., and D. C. G. Muir, 1984. Concentrations of heavy metals and 
organochlorines in marine mammals of northem waters: Overview and 
evaluation. Can. Tech. Rep. Hsh. Aquat. Sci. 1279: 100 pp. 



75 




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Figure 2. Concentrations of mercury and seienium in liver and kidney of 
dolphin collected from the Gulf of Mexico, 1990. 



77 



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dolphin collected from the Gulf of Mexico, 1990. 



78 



BOTTLENOSE DOLPHIN 
blubber extract after 
isolation of planar PCBs 

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60 70 80 90 

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Retention time (minutes) 



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«ndatef(A)HPLCction ialo graphyon2K11>ywny<)ettiy>(ime^^ TbePCBsm 

identified by lUPAC lUTter vd m «so iderttted in (A) by tte toadiir gra^«^ 

congeners (pure 3-m8Diylctioi<m8wane-lype inducers: 169 not present 

AbbreviatianK: NONA.os4iaracMor.Oia-deidrin:op-.pp4)DT-2.4'-or4,4'-OOT;op-.p|^^ Z4- or 4^-000: op-. nM)DE- Z4'-a4. 

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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 

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aldrin 

heptachlor epoxide 

alpha-chlordane 

trans-nonachlor 

dieidrin 

mirex 



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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 





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 





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 


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 





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 





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 





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 





M 


3 


03/29 


TX 


29*15.8' 


94*50. ' 


5175 GA336 1 


118 





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 


H 




04/08 


TX 


29°09.5 


95*00.6' 


5399 SP120 1 


r 101 


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 


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 


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 


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 








6 





6 


17 


14 





49 


43 


92 


MS 


1 


14 


3 


12 


14 


19 


36 


22 


84 


121 


205 


AL 


1 





4 


1 





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 








- 


8 





- 


1,296 


- 





- 


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 








3^ 




8.9 




12.7 







+ 


10.4 


+ 


16.5 


+ 


22.8 







+ 


14.5 


+ 


20.5 


+ 


273 


+ 





+ 


16.6 


+ 


23.1 


+ 


32J 


+ 





+ 


15.8 


+ 


24.6 


+ 


33.5 


+ 





+ + 


13.4 




19J 


+ 


29.9 









16.0 


+ 


243 


+ 


293 







++ 


8.2 


+ + 


13.7 


++ 


28 









11.2 




17.2 




27.1 









11.1 


+ 


183 




23.4 


+ + 





+ 


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 






+ 


8.4 


+ 


15.8 


+ 


23.4 


+ 





+ 


IZl 




17.6 


+ 


23.1 


+ 





+ 


8.2 


+ 


12.2 


+ 


21.9 







+ 


S2 


+ 


95 


+ 


15.8 


+ 







5.2 




93 


+ 


15.5 









7.0 




9.2 




12.0 







+ 


3.2 


+ 


7.6 


++ 


n.i 









5S 


+ 


82 




16 









Zl 


+ 


45 


+ 


8.7 


+ 







4.6 




73 




95 





88 
66 



46 
24 



440/413 
122 



66 

71 



8 
14 





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 










0/19 


3.4 


+ 




240/45 


7.9 


+ 






11.2 


+ 













38 


53 






32 


10.2 




^ 




1Z9 






- 





+ 




180/170 


5.7 






80 


15.1 




± 




193 




± 







+ 




«9 


5.7 


+ 




58 


10.4 


+ 






14.7 


+ 









. 




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 








± 


8,000 r 


3.2 


+ 




2-4,000 r' 


6J 




± 




9.2 


+ 













on 


4.1 






5 


9.9 






- 


14.8 


+ 













45 


3J 






56 


5.8 


+ 






19.6 


+ 









+ 




38 


43 


+ 




80/19 


7.9 


+ 






20J 






- 







± 


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 






+ 


S.0 


+ 


10.6 


+ 


17^ 


+ 





+ 


5^ 




lOJ 


+ 


17.0 







+ 


S2 


+ 


92 

\Z2 


+ 





+ 


\Ji 


+ 


$2 




8.4 









A2 




6.6 





26 
30 



3 
14 



7010 
5 





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 p ro fiden^ 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 



Sp pri f"" 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. A p p n yr i ate 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 Pr o tocol 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 
homo gen ized 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 Chemic a l ). 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 perfo r m ance 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 br e v tt oo un from samples. Mixed 
samples would then be sAjeaed to oo-migratioo in hplc analysis. Quantification of the amount of bt e v ct oa an 
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 f ca yv er y ( 



[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 b et w uj i 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 precau t i o ns 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 powd ers tbould be canied out in an ap pr o v ed 
ventilation hood. 



120 



Rationale for Methods 

Hreveiniim Tc maieriils which becnae eoaceatraied in nmae titniet throngfa annwl feeAng trtjuit^t 
jf PP«f fwn ittr<< rrfmniant Thc tonu, lynthrtTTT/l by thc marine faofUgellate ft>cfcadbaa b»rw «rc aorm*l)> 
fcKf^^n^wn uUte/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 «"»"""■>■■" i pcoc s (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. 

I ffitial 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) Corp or ati o n 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 
fP fffftnmatmg 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 ' t o 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 perf or m ance 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 supp o r t 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 fjjgiic D ce ; (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 d oamnrtt Dr. Bades has 17 ycMn qp ci ieace 
«i^ Uitiiuiim. aod Uoyd SchnlmaB has ewer four yean inrtmiral rrpt i riwr wilb puriCcatioo aod 
aysuSijxMacm el hrwcuaaai. 

Submitted San^les ( dupH ra tr ^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 
approp r i ate 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 
a ppr op ri ateness 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 ei p c fl 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 
b r ev e toxin purification progr e ssin g 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 ab so rb an ce, 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) - apppiujum ately 11 ^g brevetoxin in sample 2505-9. 

'Based on 46.639 g of liver supplied, appi o xim ately 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, appfoiim a tel y 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. ap p roxim ately 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, appimlm ately 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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135 



Chlorinated Hydrocarbon (CH) Analyses for 

Bottlenose Dolphins 

Explanatory Notes for Tables A-3 through A-12. 



nd - indicates that the analyte was not detected above the limit of detection which 
ranged from 0.2 to 7.0 ng/g (ppb) wet weight 

Results were detennined by GC/ECD (electron captmc detection). 

The letter "a" indicates that the identification of analytes was confirmed by 
GC/MS. 

Percent recoveries for the internal standard 4,4-dibromooctafluorobiphenyl 
(surrogate standard) averaged 84 %, CV = 17 %, n = 48. 



136 



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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. 

TO XICOLOGY 

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 
h tguuiili z uj i i ( ji i i i nn diagnosis. 

22. StoDacii, pylorus: Gastritis, lynrhnrytic, focal, mild. 

23. Stonach, pylorus: Erosion, aaxte, f nmlly 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 

Tt l tcopwur (514) 773-2161 



181 



90-3530 

E y^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. 

pr p$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 Scie n ce Center 

Marine Manual Stranding M e tw o r k R ep r esen tative 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 Di rect or 

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 

nissemin atiion 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 ap pr o p riate 
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 . 

7 ntTT>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 



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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 



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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. 



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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 



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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 



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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