: C ^n ftTWOSp, v/ m - I 4)1 73/ H3^ ' '■^e\-^ o>- REPORT ON INVESTIGATION OF 1990 GULF OF MEXICO BOTTLENOSE DOLPHIN STRANDINGS Edited by ^^^ u- ^^= CO o ru S=o -===D 2=ir^ ^^m a s^ ^ \ V V r QL 737 .C432 H36 V Larry J. Hansen, Investigation Coordinator National Oceanic and Atmospheric Administration National Marine Fisheries Service Southeast Fisheries Science Center 75 Virginia Beach Drive Miami, FL 33149 November 1992 Contribution: MIA-92/93-21 REPORT ON INVESTIGATION OF 1990 GULF OF MEXICO BOTTLENOSE DOLPHIN STRANDINGS Edited by Lany J. Hansen, Investigation Coordinator National Oceanic and Atmospheric Administration National Marine Fisheries Service Southeast Fisheries Science Center 75 Virginia Beach Drive Miami, FL 33149 November 1992 Contribution: MIA-92/93-21 Table of Contents Overview 1 Introduction 10 Section I: Hansen, LJ. - Stranding Rate and Trends 15 Section n: Hansen, LJ. - Age Structure 21 Section ni: Hansen, LJ. - Population Abundance and Strandings 24 Section IV: Barros, N3. - Food Habits 29 Section V: Blaylock, RA • Environmental Factors 35 Section VI: Tester, PA. - Pfaytoplankton Distribution 44 Section VII: Staff - Sunnnary of Brevnoodn Analysis 53 Section VIE: Varanasi, U^ ILL. Tilbuiy, D.W. Brown, MM- Krahn, CA. Wigren, R.C Clark, S. Chan • Chemical Contaminants 56 Section DC: Staff - Summary of Available Pathology Reports 88 Section X: Hansen, LJ. • Limitations and Recommendations 90 Appendices: I: Stranded bottienose dolphins documented during January-Jime, 1990, along the U^. Gulf of Mexico coast 95 11: Bottlenose dolphin strandings fay state in the northern Gulf of Mexico, 1982-90 101 ni: Overall sex ratios and sex ratios for animal <140cm by year for Januaiy-June Texas bottlenose dolphins strandings 102 IV: Report on aerial surveys of bottlenose dolphin abundance conducted in near-and ofEshore waters off the Texas coast during 1990 103 V: Oymnodinium breve presence/absence^ and quantitative cell coimts^ 109 VL Contract report on brevetoxin analysis 113 VII: Results of analyses for metals and chlorinated hydrocarbons, including quality assurance 133 Vni: Available rijpirnl necropsy and histopathology reports of bottlenose dolphins stranded in the U^ Gulf of Mexico during Januaiy-June, 1990 157 DC: Proposal outline for Southeast Fisheries Science Center representative system for the Marine Mammal Stranding Network . 186 X: Report on the Southeast Hsheries Science Center Marine Manunal Stranding Network Representative System Organizational Workshop 203 PREFACE This document was prepared as a source for complete information on the National Marine Fisheries Service's (NMFS) investigation of an apparent anomalous mortality event involving bottlenose dolphins during January-June, 1990, in the coastal northern and western U.S. Gulf of Mexico. As such, it contains numerous tables, appendices, and other information that might not be included in a paper prepared for pubhcation. The report was reviewed by the NMFS Task Force on Unusual Marine Mammal Mortalities; the Service wishes to thank them for taking the time to review such a large document and for providing many useful comments and suggestions for improving the report. OVERVIEW The Southeast Fisheries Science Center (SEFC) coordinated a multi-disciplinary investigation into the cause, extent, and potential effects of higher than previously reported numbers of bottlenose dolphin {Tursiops truncams) strandings which occurred during 1990 in the northern Gulf of Mexico. The majority of the funding for this investigation was provided by the National Marine Fisheries Service Office of Protected Resources. The investigation of the Gulf strandings was organized into seven main themes. Four of the themes were concerned with population biology and included: 1) stranding rate and trends; 2) population abundance and strandings; 3) age structure of stranded animals; and 4) food habits. The other themes related to extent and causes of mortality, and included studies of: 5) environmental factors; 6) biotoxins; and 7) contaminants. Summaries of each theme are provided in the overview; the complete reports on each theme are presented as separate sections. The available pathology reports are summarized in Section DC The SEFC took several steps to assist the Southeastern U.S. Stranding Network (SEUS) in stranding reporting and recovery operations during the mortality investigation. Communications were established with Network participants throughout the Gulf to ascertain stranding rates, to alert participants to the ongoing event, and to request collection of a standard set of tissue samples. SEFC personnel participated in stranding recovery operations in Texas and Mississippi. Strandings along the Louisiana coast were documented, and occasionally examined, by SEFC personnel onboard routine U.S. Coast Guard helicopter flights. Strandings on the barrier islands of Mississippi were documented by SEFC pei^onnel conducting offshore surveys for marine mammals. Small boat surveys, funded or conducted by the SEFC, were conducted along the Texas and Alabama coasts to visually examine bottlenose dolphin herds for the presence of "affected" animals. Authorization was obtained to collect, hold, and examine afff-f red animals. However, no affected animals were observed during the surveys. The Texas Marine Mammal Stranding Network (TMMSN) was provided with equipment and supplies to deal with the large numbers of animals stranding along the Texas coast. Travel expenses were provided to veterinary pathologists and others assisting the TMMSN. The SEFC also provided funds to cover costs for shipping specimens. The Beaufort, Charleston, Galveston, Miami and Mississippi Laboratories of the SEFC and the National Marine Fisheries Service Office of Protected Resources provided support for the investigation. Additional support was provided by a variety of agencies, institutions and other groups and individuals, including: the Armed Forces Institute of Pathology; the University of Miami's Cooperative Institute for Marine and Atmospheric Sciences; Environmental Protection Agency; Florida Department of Natural Resources; Greenpeace; Kansas State University; Louisiana University Marine Consortium; Marine Mammal Commission; National Oceanic and Atmospheric Administration Aircraft Operations Center; Oak Ridge National Laboratories; Sea World Orlando; Smithsonian Institution; Southeast U.S. Marine Mammal Stranding Network; Spring Hill College; Texas A&M University; Texas Marine Mammal Stranding Network; US Coast Guard; and others. This report provides a background on bottlenose dolphin strandings in the Gulf and the various research activities pursued during the investigation. Reports on several research activities are included as separate sections. The rationale for the research directions taken are discussed, as are the difficulties encountered in executing the investigation. The results of each research activity are summarized and discussed, and, when appropriate, integrated with other research results for further discussion. Reconmiendations are given for improving the SEFC's stranding investigation capabihties, along with steps taken to implement these recommendations. Stranding Rate and Trends The mortality event investigated occurred during January through May, 1990. Strandings from January through June, 1986-89, were compared to the January through June, 1990, strandings. Strandings during 1986-89 for these months ranged from 122-174; strandings totaled 367 during the same months in 1990. Compared to the 1986-89 average, during 1990 the largest increase in sfrandings was observed in Alabama (9.68 fold) and the lowest in Texas (1.80 fold). For the U.S. Gulf as a whole, January-Jime 1990 strandings were 2.62 times the 1986-89 average and 2.1 times the prior maximum recorded. The 1990 strandings generally followed the same seasonal pattern previously observed, with a peak in sfrandings during March. The 1990 strandings during January- March were from about 3 to 4 times greater than the 1986-89 averages. April 1990 strandings were about twice the prior 4-year average, and May and June 1990 strandings were also about double, but were relatively low in number, 25 and 23, respectively. Strandings throughout the Gulf during 1990 were the highest on record for all months except August. The sex ratio of the Texas, January- June 1990 sfrandings, was compared to that of 1984-89 Texas strandings. The sex was recorded for 400 animals that stranded during 1984- 89, and the sex ratio was 1.00:0.67, males to females. The sex ratio for 1990 was 1.00:0.98, which still falls within the yearly ranges observed (1.00:0.34 to 0.85:1.00). The length-frequency distribution of Texas, January-Jime 1990 strandings, was compared to that of the Texas, January-June 1984-89, combined length frequency distribution. Contingency table analysis indicated that the differences between the 1990 and 1984-89 distributions were statistically significant. The largest differences occurred in the numbers of animals < 140cm, with proportionally half as many stranding during 1990 as compared to 1984-89. About 30% of the January- June 1984-89 strandings were < 140cm; during 1990 for these months only 15% were < 140cm. This is the lowest proportion observed, except for 1985 (only 35 strandings were recovered during 1985, and only 4 were < 140cm). The proportion of stranded animals measuring < 140cm has been decreasing since 1986. Population Abundance and Trends Estimates of bottlenose dolphin abundance from large-scale aerial surveys of the Gulf of Mexico (Scott et al. 1989) were compared with historical stranding data. The available population abundance estimates for the northwestern Gulf of Mexico indicate that abundance is lowest in the winter, increases during the spring, and remains at about the spring level through the summer and fall. Strandings in the northwestern Gulf peak from February-April; it appears that strandings in the northwestern Gulf peak during a period when abundance is increasing. However, if only the nearshore and inshore £ireas (waters < 18.3m) are considered, strandings peak when abundance is at intermediate levels and declining. Available bottlenose dolphin abundance estimates for the northeastern Gulf of Mexico indicate that the lowest abundance occurs during the summer, with the highest abundance during the winter. Strandings for the northeastern Gulf peak diuing March and April, which appears to be when abundance is declining. When only the nearshore and inshore areas are considered, strandings peak when abundance appears to be declining. Aerial surveys of portions of the northwestern Gulf were conducted during March and June, 1990. The results of these surveys indicate that there may have been more animals present in the inshore areas (waters to 18.3m) and offshore areas (waters 18.3m to 183m) during 1990 than the previous sampling period (1983-84). These results are similar to the findings of nearshore surveys conducted prior to the 1987-88 east coast dolphin dieoff, but differ from the results of offshore surveys conducted during the 1987-88 dieoff which indicated there was a 60% chance of a decline in the offshore stock abundance (Scott and Bum 1987). Age Structure of Stranded Animals The age structure of the bottlenose dolphins stranded along the Texas coast was examined to determine if was different during 1990 as compared to previous years. The available sample of teeth from animals stranded during January- June during 1983-90 were aged using standard techniques for bottlenose dolphins (e.g., see Hohn et al 1989). The available sample consisted of 195 animals, of which 70 were obtained in 1990. Comparison of the 1983-89 and 1990 aged samples indicated that the differences observed were not statistically significant (Kolmogorov-Smimov two-sample test, DN=0.19, p>0.10). However, further examination of the samples indicated that the 1983-89 aged sampled was most likely biased, and that this likely bias resulted in under-representation of young animals in the sample. For this reason, comparisons of the available age samples were considered inconclusive. The length structure of the stranded animals, although only a gross approximation of age, was not considered biased and indicated that proportionally fewer younger animals (<1 year old) stranded during 1990 than during previous years. Food Habits Due to the implication of feeding habits in the recent mortalities of bottlenose dolphins (Geraci 1989) and humpback whales (Geraci et al. 1989) along the eastern U.S. coast, there was concern of a sirnilar occurrence in the Gulf of Mexico during 1990. This study examined the food habits of bottlenose dolphins stranded along the coast of Texas during January-April 1990 and compared the results with a previous study of bottlenose dolphin stranded during 1986-87 in the same area (Barros and Odell 1990), Entire stomachs were collected from 38 stranded bottlenose dolphins and frozen for analysis. A total of 15,950 otohths, 1,681 squid beaks and remains of 59 crustaceans were found, representing 7,109 fish, 915 squid and 59 crustaceans. Prey items averaged 219.1 and prey taxa 9.4 per stomach. Altogether, 46 species of fish (from 11 families), 3 species of cephalopods and 2 species of crustaceans were identified. Six prey species occurred in more than 50% of the stomachs and accoimted for 57% of all prey. These species were: the Atlantic croaker (Micropogonias undulatus), silver perch (Bairdiella chrysoura), brief squid {Lolliquncula brevis), sand seatrout {Cynoscion arenarius) and an unidentified telelost. Wet weight of the stomach contents (an indicator of stomach fullness), the numbers of prey items and prey taxa in each stomach, and the categories of prey types (fish, cephalopod, crustacean) were not significantly different from those reported in Barros and, Odell (1990). The four most commonly and numerically important prey (M. undulatus, C. arenarius, B. chrysoura, and L. brevis) were represented in both 1986-87 and 1990. The results obtained in the present study show that bottlenose dolphins stranded during the 1990 Gulf of Mexico mortality event had a similar prey spectrum as dolphins stranded in previous years, and suggest that the food habits of the dolphins stranded during 1990 were not significantly different from 1986-87. Environmental Factors The Texas marine mammal stranding data base allowed analysis of bottlenose dolphin stranding in relation to certain physical factors of the environment. Bottlenose dolphin stranding records for the period January 1986-June 1990, were analyzed by linear regression with monthly mean sea surface and air temperatures, salinity, and offshore transport. Bottlenose dolphin stranding on the Texas Gulf Coast peaked significantly in March; otherwise, stranding rates did not differ significantly among the months January-June. No significant difference in stranding rates was detected among years; however, the relatively low power of the ANOVA test suggested some undetected differences among years. Using the upper 95% confidence interval on the 1986-1990 monthly stranding means as a criterion for detecting significant differences, the number of strandings during January-March 1990 was significantly greater than during the preceding four years. The slope of monthly stranding rates regressed against Texas coastal monthly mean sea surface temperature was significant; however, a low correlation coefficient suggested that there was no simple linear relationship. A negative exponential relationship between first semester stranding rates and the preceding December-January mean sea surface temperature was detected; the lowest January-December mean sea surface temperatures preceded the highest January- June stranding incidence. A weak relationship between dolphin strandings and air temperature for the same period was not significant. Sea surface temperature anomaly data from NOAA's Oceanographic Monthly Summary for December 1989- January 1990 ranged from -04 to -2.0 °C The persistence of the negative anomaly throughout the winter of 1989-90 suggested abnormally low sea surface temperatures. Mean salinity varied significantly among months, among years, and among months within years; however, there was no significant relationship between bottlenose dolphin stranding and salinity. Monthly mean offshore transport varied significantly among years, but there was no apparent relationship between monthly mean offishore transport and monthly mean bottlenose dolphin stranding. Examination of seasonal stranding and offishore transport suggested a weak, but significant, inverse relationship during the spring. Other than an inverse relationship between winter sea surface temperatures and spring dolphin mortality rate, these analyses detected no strong significant relationships between bottlenose dolphin strandings and other enviromnental variables. The association of low winter sea surface temperatures with an increased spring-time, dolphin stranding rate suggests t ■- possibility of thermaUy-induced stress. Alternatively, the observed association between v, .er sea surface temperature and spring bottlenose dolphin stranding rates may be less dirt -. An estimated 2.7 million fish, of which approximately 2.6 million were striped mullet . Jugil cephalus), died in East Matagorda Bay after a severe cold spell in December 1989. ^nd smaller kills occurred in Texas Bays from Sabine to Laguna Madre Bay. It is possible that bottlenose dolphins were forced to switch to inferior prey items because of unusual weather-related fish migration patterns or fish mortalities. However, the available food habits study results do not indicate M. cephalus as a major prey item in stranded dolphin stomachs. The inverse relationship between spring bottlenose dolphin stranding and offshore currents (Ekman transport), although not strong, may contribute to an increase in beach-cast mortalities in the spring and thus, an apparent increase in mortality rate. An increased nearshore occurrence of bottlenose dolphins, with normal mortality rates, during the spring season could also contribute to an apparent increase in mortality. Phytoplankton Distribution Toxins produced by the dinoflagellate Gymnodinium breve were implicated as a proximate cause of the mass mortality of bottlenose dolphins along the U.S. east coast during 1987-88 (Geraci, 1989). For this reason, 123 phytoplankton samples from the Texas- Louisiana offshore area were examined for the presence of the G. breve. Eighty percent of these samples contained G. breve cells. Seventy samples from the upper half of the water column were examined in detail, and quantitative counts confirmed that 94% contained some G. breve cells while 65% contained >50 cells 1'^ (see Section IV, Figure 1). These concentrations are far below those considered a "bloom" (>5 x 10^ cells 1'^). Comparative samples fi-om other areas in the Gulf of Mexico suggest that G. breve concentrations in the primary study area during the March 1990 sampling period were within normal background levels but consistently higher than quantitative counts of samples from similar areas in the northern Gulf of Mexico of from the primary study area later in the summer. Discolored water patches noted during aerial observations of the primary study area were blooms (approx. 1 x 10^ cells 1'^) of the dinoflagellate Noctihica spp. This genus is not known to normally be toxic. However, it should be noted that a toxic dinoflagellate species, Gonyaulax monalata, was found in elevated concentration near the Mississippi delta in late summer 1990. Brevetoxin Analysis Brevetoxin is a neurotoxin produced by a toxic dinoflagellate, G. breve. Poisoning by this toxin was believed to have caused the 1987-88 mass mortality of bottlenose dolphins along the U.S. east coast. Because of the previous implication of brevetoxin as a cause of mass mortalities of bottlenose dolphins, a total of 50 bottlenose dolphin liver samples were analyzed for individual brevetoxins (40 samples from 1990 Gulf of Mexico samples, and 10 control samples). Toxicity was determined by several methods: 1) fish bioassay - Gambusia affinis, fish death at a fixed interval indicates toxin present but does not necessarily indicate brevetoxin; 2) HPLC separation of toxin fractions - HPLC separation provides a means to confirm or deny the presence of brevetoxins in comparison to valid Pb Tx-standards; 3) Radioimmunoassay provides a means to positively identify brevetoxin-like materials and is sensitive to authentic Pb Tx-3. Following the first thin-layer chromatography (TLC) plate, 33 of the 50 samples were found non-toxic in the fish bioassay and were not tested further. Of the remaining 17 samples that tested positive in at least one fraction of the first TLC plate, nine had multiple toxic fractions. Of the 17 samples, 12 tested negative by fish bioassay following the second TLC plate. Of the five fi-actions found toxic after the second TLC separation, three were judged to be in such limited quantity to preclude further TLC separation. The other two retained toxicity after the third TLC separation. The three toxic fractions of limited quantity were judged to contain less than 5ug toxin/total original sample by HPLC; this was presimied to be a negative result. The other two fractions, purified through the third TLC, appeared to contain Pb Tx-2 by HPLC separation and co-elution. Radioimmunoassay was performed on these five fractions, using tritiated Pb Tx-3 as the internal displacement standard. Based on this assay, the three fractions purified through 2 TLC steps contained 10.2, 12.2, and 9.33 ng toxin/g liver; the two fractions purified through 3 TLC steps contained 17 and 240ng toxin/g liver. The process of extraction, purification, chromatographic separation, and radioimmunoassay conducted on the 50 samples led to the conclusion that five of the samples contained brevetoxin or some very similar toxin. Reported concenfrations in original samples were calculated by proportion of sub-sampling at the various steps and were based on "Pb Tx-3 equivalents" in the radioimmimoassay. Of the five toxin-spiked control samples only one was detected as containing brevetoxin; this sample was spiked with the largest amount of Pb Tx-3, 25ug. Two other samples were spiked with 20 and 15ug of Pb Tx-3 respectively, but were not identified as containing brevetoxin. PbTx-1 and PbTx-2 were also added to several of the samples; PbTx-1 is known to hydrolyze quite quickly. The fact that purified toxins "stick" to glass- and plastic- ware may explain the low level of apparent spike of the liver samples. It is quite possible that neither the PbTx-1 or PbTx-2 spikes were effective, or it is possible that they do not effectively displace radio-labeled PbTx-3 in the radioimmimoassay. Of the five carrier-spiked control samples (treated with MeOH only), three were identified by the radioimmunoassay as containing brevetoxin. It is difficult to explain this finding. The other two carrier-spiked samples were found to be negative when purified to the second TLC step. It is possible that an interfering substance was removed in the early cleanup phases of some of the controls and not in others. The sample reported to contain the largest amount of brevetoxin, as determined by radioimmunoassay, was one of the non-toxin (MeOH only) spiked control samples. The only dolphin liver sample from the strandings that was identified as containing brevetoxin at all stages contained 10.2 ng toxin/g liver. This level of toxin is considered to be very low. The problems encountered in properly identifying the spiked and non-spiked control samples raised serious questions concerning the efficacy of this assay method for detecting brevetoxin in bottlenose dolphin liver samples. Certainly, the results of this brevetoxin analysis and other studies which used the same assay methods (e.g. Geraci 1989) cannot be considered conclusive. That is, based on the incorrect assay results of the control samples, brevetoxin poisoning cannot be ruled out as a proximate cause or factor in the 1990 bottlenose dolphin strandings. The author of the report suggests that samples should continue to be collected so that assays for brevetoxin detection may be refined. His research group will be conducting collaborative research on the assay of brevetoxins in marine animal tissues; this process should assist in the further development and verification of the assays. A major difficulty in establishing an assay of this type is obtaining a true "control liver" sample, known to be free of toxins or other substances that interfere with the assay. Contaminants Tissues from a sub-set of the stranded bottlenose dolphins were examined to investigate the possibility that environmental contaminants may have caused or contributed to the observed strandings. Blubber and liver samples from 20 of the stranded bottlenose dolphins were analyzed for chlorinated hydrocarbons (CHs). In addition, liver and kidney samples of these dolphins were analyzed for certain metals. The concentrations of mercury in the livers of 2 dolphins (114 and 117 jig/g or ppm based on wet weight) were notably elevated and may be of toxicological concern. The concentrations of total CHs in the 20 animals sampled varied widely; 3.0-190 ppm in blubber and 0.5-58 ppm in liver with eight dolphins having levels of total CHs in blubber that were greater than 50 ppm. Interestingly, relative levels of DDT, compared to the levels of DDE (a metabolite of DDT), in three of the dolphins may indicate an exposure to relatively recently released DDT. The concentrations of CHs and certain metals, especially mercury and selenium, in some of the dolphins were sufficiently high to warrant a more systematic study of contaminant exposure of this species and of potential health effects due to this exposure. In addition, special efforts are needed to investigate possible sources of certain toxic chemicals; including CHs and aromatic hydrocarbons, by measuring parent compounds and their metabolites in tissues and stomach contents of these animals. Summary The investigation did not provide any conclusive evidence of a single causal agent, or multiple causal agents, for the increase in strandings. Available abundance estimates indicate that bottlenose dolphin abundance may have been higher in the nearshore northwestern Gulf during the spring and summer of 1990 than during 1984; however, the 1990 estimates are limited in geographic scope and may not reflect the overall abundance patterns. No statistically significant differences were found between the 1990 and the 1984-89 age structure of the stranded animals. But, possible biases in the 1984-89 age sample were identified and the age structure analysis must be considered inconclusive. The results of the food habits analysis indicate that the bottlenose dolphins stranded during 1990 had a similar prey spectrum as in previous years. The analysis of environmental factors (sea surface and air temperatures, salinity, and offshore transport) detected a statistically significant inverse 8 relationship between the winter sea surface temperature and spring bottlenose dolphin strandings. The winter 1989-90 sea surface temperatures were considered abnormally low and suggest the possibility of thermally-induced stress. However, the stranding database covers only 5 years and was insufficient to determine if the inverse relationship between winter temperatures and spring strandings is consistent. The results of the brevetoxin analysis were questionable and must be considered inconclusive, and although the phytoplankton study determined that the brevetoxin producing organism was present, there is no information available on the "normal" occurrence patterns of the organism within the phytoplankton study area. The contaminant analyses indicated that although a few dolphins had concentrations of contaminants at levels of possible toxicological concern, contaminant concentrations in most of the dolphins were relatively low. Fewer than 3% of the stranded dolphins received thorough pathological exams. As a result, essentially almost no pathological information was available. Overall, none of the studies conducted provided conclusive evidence of circumstances or agents which caused the observt.d increase in strandings. INTRODUCTION The Southeast U.S. Marine Mammal Stranding Network (SEUS) was organized in 1977 to document and salvage marine mammal strandings along the U.S. coast from Texas to Virginia (for a review of the network, see Odell, 1991). Most of the Network participants are volunteers, and stranding reporting and salvage efforts vary considerably. For example, salvage efforts range from nearly none along Louisiana to nearly 100% along Texas. Although the salvage efforts are not consistent, stranding records may provide an index of mortality rates and have been used to estimate the potential effects of anomalous mortality events (Scott et al., 1988). Nearly all of the stranding reports and specimens used in the investigation of 1990 strandings were provided by the participants of the SEUS. A list of specimens collected during the mortality event imder investigation is provided in Appendix I. The specimens are listed by stranding date, and information is provided on location, sex, length, tissues collected, and analyses conducted. The investigation was initiated due to reports that higher than normal numbers of bottlenose dolphins were stranding along the U.S. Gulf coast. Begiiming in early 1990, a number of stranding and mortality observations of bottlenose dolphins were made. There was a mass dieoff of 23 dolphins in January in Matagorda Bay, Texas, that may have been caused by unusually cold weather (Miller, 1991). A higher level of strandings than the prior 4-year average was observed in the Gulf along the coasts of Rorida, Alabama, Mississippi, and Texas beginning in January. Floating dolphin carcasses were consistently observed during January and February while NMFS observers were on transit to deep water aerial survey study areas in the central northern Gulf of Mexico. As a result of all of these observations, the SEFC notified the NMFS Office of Protected Resources and the MMC of the observations and began intensified monitoring of the strandings and initiated an investigation of the cause and extent of the strandings. Higher munbers of dolphins than the prior 4-year average continued to strand from the Florida panhandle to Texas during February and March, 1990. Strandings decreased to average or below average along the Texas coast after March, 1990, but continued to occur sporadically at higher than average levels along the Mississippi, Alabama, and Florida coasts from February through May, TaMc L Northern Gulf of Mexico 1990 bottlenose dolphin strandings by slate and month. MONTH STATE J F M A M J J A S O N D ALL FL 10 12 12 6 4 9 7 3 3 6 3 11 86 AL 7 4 12 11 8 4 1 1 1 1 2 6 58 MS 8 5 24 7 8 5 9 4 7 2 3 2 84 LA 3 1 13 27 3 1 0 0 0 0 0 1 49 TX 42 40 59 16 2 4 2 2 4 9 13 8 201 All. 70 62 120 67 25 23 19 10 15 18 21 28 478 10 1990. It appeared that the mortality was within the prior 4-year average by the end of May, 1990. A total of 344 stranded bottlenose dolphins were recovered along the U.S. Gulf coast during January-May, 1990. Investigators from Texas recovered 159 strandings during this period, while strandings recovered in the other Gulf states ranged from 42-52. Strandings by state and month are shown in Table I. Overall, the number of reported strandings for January-May, 1990, in the U.S. Gulf of Mexico was about 2.5 times the 1986-89 average for those months. By comparison, during the 1987-88 U.S. Atlantic coast dieoff over 700 bottlenose dolphins were reported stranded over a 10-month period and represented a 10- fold increase over the prior 4-year average in strandings. Between Januaiy-May, 1990, most of the dolphins recovered on a state by state basis during the investigation stranded along the Texas coast. Approximately 166 dolphins were reported stranded there from January- June, and 163 were recovered. Within January-March and all states, the greatest number of recovered animals were from the Texas coast during March (59), with January and February totals (also from Texas) of 42 and 40, respectively. The totals for these months were the highest on record. Texas strandings for January were 4.4 times the 1986-89 Texas monthly average, February 2.3 times average, and March 1.5 times average. The cumulative Texas total for January-March, 1990, was 2.1 times average. However, the Texas stranding recovery rate decreased after March, 1990, and Texas strandings for April, 1990, were about 50% of average and the lowest total for April since 1986. A total of 204 animals were recovered from January-June along the coasts of the other Gulf states. These represented about 55% of the recovered strandings for that period. A total of 57 dolphins were recovered in Mississippi, 52 in Florida, 48 in Louisiana, and 46 in Alabama. By month, most (24) of the Mississippi strandings were recovered during March, most (12 each) of the Florida strandings during February and March, most (27) of the, Louisiana strandings during April, and most (12) of the Alabama strandings during March. Recovery effort in 1990 in Mississippi and Florida was relatively constant, but the stranding recovery effort along Louisiana was not, because of USCG supported helicopter beach- surveys conducted during March and April, 1990. Stranding recovery effort along the Alabama coast was consistent January-March. Alabama stranding recovery efforts increased significantly during the last half of April, primarily due to Greenpeace efforts to increase stranding reporting and recovery along the Alabama coast. It is likely that the effort expended on reporting and recovering of strandings was not consistent between 1986-89 and 1990. The mass stranding of bottlenose dolphins in Matagorda Bay, Texas during January 1990 was well publicized in Texas. The 1987-88 mass- mortality of bottlenose dolphins on the east coast likely sensitized stranding networks and various groups to strandings. The observed increase in stranding reports could thus be an artifact of the influence of these events on reporting and recovery efforts. The results of regular, controlled effort surveys for marine mammal and sea turle strandings along the Texas coast provided a basis for obtaining an independent measure of 11 stranding rates along the Texas coast. These surveys were conducted by the SEFSC during 1988-90 along portions of the Texas coast. Bottlenose dolphin stranding data collected during the beach surveys were examined to determine if the surveys detected an increase in strandings between 1990 and 1988-89 during the months of January-April. The beach surveys for 8 areas were examined; Table 2 lists the areas surveyed and the number of dolphin strandings recorded. TbUc 1. Texas beach surveys conduaed from 1988-90 (Januaiy-May) by area with reported bottlenose dolphin strandings. 1988 1989 1990 AREA SURVEY STRAND SURVEY STRAND SURVEY STRAND Bolivar 5 0 11 9 9 7 Galveston 6 0 8 0 10 1 Bryan Beach 9 0 8 0 11 1 Sargent's Beach 10 1 8 5 11 2 East Matagorda Peninsula 10 4 8 4 11 3 Matagorda Island 10 4 4 2 10 9 Mustang Island 16 0 14 0 22 4 South Padre Island 20 0 21 0 1 0 TOTAL 86 9 82 20 85 27 The number of surveys per area varied yearly, but the total number of surveys per year was similar, and ranged from 82-86. The number of miles of beach surveyed per year was also similar, and ranged from 2388-2580 (Table 3). Compared to the 1988-89 average, 1990 strandings increased in 5 areas, decreased in 2 areas, and were unchanged in 1 area. Overall, the number of strandings recorded during 1990 were about 1.9 times the 1988-89 average. The beach surveys provided a measure of the stranding rate; the 1990 stranding rate (stranded dolphins per mile surveyed) was approximately 1.8 times the 1988-89 average.- The number of bottlenose dolphin strandings reported by the Texas Marine Mammal Stranding Network (TMMSN) for the beach survey areas showed an increase during 1990 of about 2.5 times the 1988-89 average (Table 4). Potential biases were apparent in comparisons of trends in strandings between the beach survey data and the TMMSN data. First, the yearly number of beach surveys by area was not consistent. For instance, during the first five months of 1988 and 1989 about 20 surveys were conducted each year at South Padre Island, while during 1990 only one survey was conducted (Table 3). Second, surveys were not conducted at an adequate frequency. Surveys averaged about 2 per month (range, 1-4), but during peak stranding periods bottlenose dolphins stranded about once every 3 days. Third, discrepancies between the data sets indicated that under-reporting was occurring in both systems. The beach survey data should be a temporal subset of the Network data, but in fact some of the beach survey areas reported more strandings during 1988 and 1989 than the Network reported for those areas (Table 4). The Network reported 45 dolphins stranded in the Galveston area during 1988 while the Galveston beach survey reported none that year. These discrepancies may indicate under-reporting within each data set, and/or variations in geographic definitions. Overall, the 12 sample size of dolphin strandings by area from the beach surveys was too low to derive any reliable inferences as to monthly trends or differences between areas. However, the beach surveys did show an overaD increase during 1990 in the total number of stranded bottlenose dolphins and in their stranding rate (1.8 times the 1988-89 average). The beach surveys provided an independent measure of the number of strandings and the stranding rate and confirmed the observations of the TMMSN. Tabic 2 Toos beach turveys conducted from 198S-90 (Januaiy-May) by area reporting number of miles surveyed and number ef stranded botilenose dolphins per mile surveyed. 1988 1989 1990 AREA STRAND/ MILE MILES STRAND/ MILE Mn RS STRAND/ MILE MILES Bolivar 0 472 0.0177 508 0.0142 491 GaKeston 0 518 0 415 0.0022 449 Bryan Beach 0 45 0 51 0.0161 62 Sargent's Beach 0.0112 89 0.0532 94 0.0180 111 East Matagorda Peninsula 0.0160 250 0.0196 204 0.0107 280 Matagorda Island 0.0151 265 0.0134 149 0.0275 327 Mustang Island 0 341 0 367 0.0054 735 South Padre Island 0 600 0 600 0 30 TOTAL 00035 2580 0.0084 2388 0.0109 2485 Table 3. Numbers of bottlenose dolphins reponed stranded by the Tecas beach surveys and by the Marine Mammal Stranding Network (MMSN) during January-Arril, 1988-90. 1988 1989 1990 AREA MMSN BEACH MMSN BEACH MMSN BEACH Bolivar G 0 16 9 32 7 Galveston Bryan Beach 45 0 0 11 1 0 0 29 1 1 1 Sargent's Beach 1 3 5 4 2 East Matagorda Peninsula 4 7 4 32 3 Matagorda Island 4 6 2 11 9 Mustang Island 0 0 0 13 4 South Padre Island A 0 3 0 4 0 TOTAL •kj 9 57 20 126 27 13 Literature Cited Miller, W.G. 1991. An investigation of dolphin (Tursiops tmncatus) deaths in East Matagorda Bay, Texas, January 1990. Naval Ocean Systems Center, Code 5107, San Diego, CA 92152. Odell, D.K. 1991. A review of the Southeastern United States Marine Mammal Stranding Network: 1978-1987. Pages 19-24 in J.E. Reynolds III and D.K. Odell (eds.). Marine mammal strandings in the United States. NOAA Tech. Rep. NMFS 98. 157 pp. Scott, G.P., D.M. Bum and LJ. Hansen. 1988. The dolphin dieoff: long-term effects and recovery of the population. Pages 819-823 in Proceedings of the Oceans '88 Conference, Baltimore, MD. IEEE Catalog No. 88-CH2585-8. 14 SECnON I STRANDING RATE AND TRENDS Lany J. Hansen Southeast Fisheries Science Center Miami Laboratory 75 Virginia Beach Drive Miami, FL 33149 Methods Since the mortality event under investigation appeared to have occurred between January through May, 1990, the 1990 stranding records for January through June were compared to those from prior years for the same months. Unless otherwise noted, references to aimual or yearly strandings refer to strandings that occurred during January through June. In those cases where the number of strandings were compared, strandings from 1986-89 were used for comparison (except when comparing the highest number stranded, then records from 1984 on were used). This was done since stranding recovery effort in several areas of the Gulf was probably relatively consistent during those years, although this effort cannot be measured. Prior to 1986, stranding recovery effort was generally sporadic but was assumed to have provided a random sample of the strandings. For analytical purposes, in most cases the strandings were divided into two main groups, Texas strandings and non-Texas strandings. This was done primarily because a consistent stranding network has been operating on the Texas coast beginning in about 1986, and the yearly sample size from Texas was adequate for analysis. The stranding networks in the remainder of the Gulf vary in consistency, and none have adequate yearly sample sizes. For these reasons, additional analyses (sex and length structure) were conducted on the Texas sample. 400 in MO o z i 230 I I TEXAS ?77i LOUIStMM Jga MtSSSSPPI m ALABAMA R^ FUJRIOA 200 Results and Discussion The number of stranded bottlenose dolphins examined between 1984-89 varied from 63 to 174 animals. During 1990, 361 stranded bottlenose dolphins were examined. The number of animals ISO o m too SO Figiirc 1: Bottlenose dolphin strandings nonbern Gulf of Medcx). 1984-90. in the 15 stranded for 1984-90 by state is illustrated in Figure 1 and listed in Appendix II. From 1984- 89, more animals were reported stranded aimually along the Texas coast than in the rest of the U.S. Gulf. However, during 1990, the number of animals reported stranded on the Texas coast was less than that reported in the remainder of the U.S. Gulf (161 vs 200). The rate of increase in strandings during 1990, expressed as January through June 1990 strandings/prior 4-year average, varied by state (Appendix II). The highest increase was observed in Alabama (9.68 times), and the lowest was observed in Texas (1.80 times). For the U.S. Gulf as a whole, strandings during 1990 were 2.62 times the 1986-89 average. Historically, strandings of bottlenose dolphins in the U.S. Gulf of Mexico have shown a seasonal trend. The greatest number of dolphins strand during March, with February and April also showing an increase over the rest of the year (Figure 2). Overall, the 1990 strandings by month showed a pattern similar to that previously observed. Some deviation from this pattern was observed in Alabama, Lx)uisiana and Texas. However, the stranding reporting and recovery effort in Alabama and Louisiana was considerably greater during 1990 than in prior years, and the degree and duration of increases observed in those states may, in part, be an artifact of increased effort. The Texas strandings deviated from the 1984-89 trend during January, with more strandings occurring during that month than during February (Figure 3). However, if the anomalous mortality that occurred in Matagorda Bay, Texas, during January is excluded, the pattern is similar to that previously observed. Although it may be reasonable to exclude the Matagorda Bay strandings, it should be noted that the 1990 pattern for the U.S. Gulf exclusive of Texas shows the same pattern of a higher proportion of strandings during January (Figure 4). With the exception of August, strandings were higher in all months during 1990, and were outside of the upper 95% confidence interval about the mean for the period 1986-89 (Figure 2), and were generally greater than the maximum monthly reported for that period. Strandings in Texas were above the 95% confidence interval for January-March and October- November (Figure 3). Strandings in the remainder of the Gulf were above the 95% confidence interval for all months except August and November (Figure 4). 1*0 120 100 BO 60 40 20 CULF OF MEXICO • 1990 V 1984-89 HIGH ERROR BARS 9SS un^ J F M A M J A S 0 N D Figure 2 Comparison of northern Gulf of Mocioo 1990 monthly bottlenose dolphin strandings with 1986-89 mean, with 1986-89 95% conndence interval High monthly from 1984-89 data. 70 60 50 40 - 30 - 20 10 JFMAMJJASONO Figure 3: Comparison of Texas 1990 monthly bottlenose dolphin strandings with 1986^ mean, showing 1986-89 95% confidence interval High monthly from 1984-89 data. During January-June 1990, Texas TEXAS • 1990 • ^ 19B4-89 HIGH • ^ ERROR BARS 95X .•7' 1 i 7 \ f \ \ yC ■y W^jf^S^ 1 16 reported strandings were >1.5 times the previous high for only January and February, whereas reported strandings in the remainder of the Gulf were >U times the previous high for January-May. Thus it appears that most of the anomalous mortality occurred outside of Texas, both in terms of duration and numbers. The increases in reported stranding rates (strandings/month) in the Gulf were compared to the increases observed during the 1987-89 bottlenose dolphin dieoff along the U.S. east coast. Compared to the prior 3-year average, the east coast dieoff represented a 10 fold increase (Geraci 1989; Scott et al. 1988) while in the Gulf a 2.6 times increase was observed. On a state and month basis, increases along 70 60 50 40 30 20 10 - NON-TEXAS • 1990 ^ 19B4-89 HIGH ERROR BARS 95k JFMAUJJASOND Figure 4: Compahsoo of non-Texas 1990 monthly bottlenoae dolphin strandings with 1986-89 mean, tbowing 1986-89 95% confidence intervals. High monthly from 1984-89 data. the east coast were up to >40 times the average as compared to a maximum of about 12 times the average in the Gulf. Figure 5 illustrates the range of relative increases observed in the Gulf and along the east coast by state and month in reference to prior 4-year averages for each region. The total number of animals stranded during the east coast dieoff was about twice that observed in the Gulf, but the east coast relative strandings were dramatically higher. so X 2 20 ■ • ATUNTC 1987-08 A GULF tmo The 1990 sex ratio of Texas strandings was compared to that observed during 1984-89 for the months January-June. Information on sex was used only for specimens with length data. This was done to avoid the possible bias that may exist in sexing animals (i.e., it may be easier to sex males, even when more than moderately decomposed). It was assumed that specimens with length data would be reliably sexed. During January-June 1984-89, sex and length was recorded for a total of 334 animals, and the resulting sex ratio was 1.00:0.69, males to females (range: 1.00:0.45 to 0.85:1.00; see Appendix III). Except for 1989, the available data suggest that there were more strandings of males than females. The sex ratio for 1990 for the same months was 1.00:0.98, which falls within the previously observed range. A z-test, corrected for continuity (Snedecor and Cochran 1973), was used to test the null hypothesis that there was no significant difference between the proportions of males stranded during 1984-89 and during 1990. The results of this analysis indicated that the proportions of observed during 1984-89 and during 1990 were not significantly different (z = 1.545, p > 0.12). P t , ^ S 0 D J F IIONTH Figiire 5: Bottlenose dolphin strandings by state and month during the 1987-88 easi coast dieoff and the 1990 nonhern Gulf of Modco mortality event compared to prior 4-year averages. 17 During 1984-89, lengths were recorded for 431 Texas strandings during the months January-June. The 1984-89 length-frequency distribution (by 10cm intervals) is shown in Figure 6 and the 1990 January- June length-frequency distribution is shown in Figure 7. Based on the 1984-89 distribution pattern, the length data was separated into three groups: < 140cm, > 139cm to <230cm, >229cm. Contingency table analysis indicated that the differences between the 1984-89 and the 1990 distributions were significant (X^= 13.66, p<0.01). The largest differences are in the first two groups, with proportionally half as many < 140cm during 1990, and proportionally about 30% fewer of > 139cm to <230cm. The proportion in the last group, those > 229cm was nearly the same (1984- 89, 45%; 1990, 50%). 130- 180- 230- 280- 138 1BS 239 l£NCTM CM 289 Figmc 6: Length frequency distribution, based on lOcm intervals, of 1984-89 January-June Texas bottlenose dolphin strandings. Historically, 88% of the yearly strandings of animals < 140cm occurred during January- June; during 1990 over 95% stranded during those months. About 30% of the January-June 1984-89 strandings were < 140cm. During 1990 for the same months, 15% of the strandings were < 140cm. This is the lowest proportion observed, except for 1985. However, only 35 strandings were recovered during 1985, and only 31 were measured (4 were < 140cm). The proportion of stranded animals measuring < 140cm has been decreasing since 1986 (Figure 8). 130- 180- 230- 280- 13S 189 239 LENGTH CM 289 Figure 7: Length frequency dislribution, based on lOcm intervals, of Tecas 1990 January-June bottlenoce dolphin strandings. Figure & Comparison of proportions by length (< 140cm and >- 140cm) and year of Tescas January-June 1984-90 bottlenose dolphin strandings. Males accounted for 58% of the Texas strandings with sex and length information during January-June, 1984-89. During this same period, males accounted for 73% of the strandings < 140cm. Overall, during this period, the ratio of males to females has ranged from 1.00:0.45 to 0.85:1.00, while the ratio of males to females for animals < 140cm has ranged from 1.00:0.18 to 1.00:0.55 (Appendix III). During January-June 1990, males accounted for 68% of the strandings < 140cm. The ratio of males to females during this period was 1.00:0.98 while the ratio of males to females for animals < 140cm was 1.00:0.46. Although the percentage of females < 140cm stranding during 1990 was somewhat less than the 1984-89 percentage (5% vs 7%), the 18 percentage of males < 140cm stranding during 1990 was about half of those stranding during 1984-89 (11% vs 20%). It appears that the apparent decrease in the proportion of strandings < 140cm was accompanied by a decrease in strandings of males < 140cm. This result could reflect a decrease in natality rate, a decrease in mortality rates of animals (primarily males) < 140cm, or some other factor, such as a change in distribution patterns of animals with dependent calves or a differential bias between periods of study. A z-test, corrected for continuity (Snedecor and Cochran 1973), was used to test the null hypothesis of no difference between the proportions of males < 140cm stranded during 1984-89 and during 1990 (where the proportion is equal to (# males < 140cm)/(total males plus females)). The results of this test indicated that 1984-89 and the 1990 proportions were significantly different (z = 2.037, p < 0.04). A z-test was also applied to the proportions of females < 140cm stranded during 1984-89 and during 1990, and the results indicated that the difference in the proportions was not significant (z = 0.614, p > 0.51). However, the 1990 sample size for animals < 140cm was small (n = 19) and therefore the results of the z-test analyses should be regarded with caution. The sex ratio of animals stranding along the U.S. Gulf of Mexico coast outside of Texas during January-June 1984-89 ranged from 1.00:0.50 to 0.87:100, males to females. The overall sex ratio for this period was 1.00:0.91, males to females. The 1990 sex ratio for the same months was 1.00:0.67, males to females. These ratios are opposite those observed for Texas; the 1984-89 Texas ratio was 1.00:0.69 and the 1990 Texas ratio was 1.00:0.98, males to females. For the Gulf outside of Texas during January-June 1984-89, the overall proportion of animals < 140cm was 0.30 and ranged yearly from 0.15 to 0.49. During 1990 for the same area and months the proportion of animals < 140cm was 0.27, which is essentially the same as the proportion during 1984-89. During January-June 1984-89, the proportion of males < 140cm was 0.29, ranging yearly from 0.14 to 0.61, and the 1990 proportion was similar at 0.27. The proportion of females < 140cm for these months during 1984-89 was about 0.25, ranging yearly from 0.00 to 0.37, while the 1990 proportion for the same months was about 0.19. TTiis contrasts with the changes observed for the Texas coast, where the proportion of males < 140cm decreased while the proportion of females remained about the same. However, as with the Texas strandings, the 1990 < 140cm sample was small and the changes in the sex ratio may not be significant. The stranding rate and trends could reflect a change or changes in factors which cause carcasses to reach the beach, rather than a change in the mortality rate. The bottlenose dolphin population in the northern Gulf of Mexico is conservatively estimated to consist of 35,000 to 45,000 animals (Scott et al., 1989). The annual natural mortality rates of bottlenose dolphins are beheved to range from 4% to 14% (Hersh, 1987, Wells and Scott, 1988). Using the estimated range of population size and the estimated range of natural mortality, approximately 1,400 to 5,300 bottlenose dolphins deaths per year would be expected in the northern Gulf of Mexico. If the these estimates are correct, only 2.8% to 12.7% of carcasses available to strand are observed. It is obvious that only a marginal 19 increase in the rate of beachings could result in a doubling to more than five times increase in numbers recovered. It is generally assumed that the stranding recovery rate is an index of the mortality rate. However, in a large complex system with a large population of animals available to strand, the variability in the stranding rate may be more sensitive to factors which cause carcasses to drift to the beach than to changes in the mortality rate. Changes in the pattern and causes of mortality may be a more reliable indicator of anomalous mortality events than increases (which do not deviate from the seasonal pattern) in the number of stranded animals. Both of these conditions were evident during the 1987-88 east coast bottlenose dolphin dieoff (Geraci, 1989; Scott et al., 1988). The occurrence of the 1987-88 strandings deviated from the normal pattern, and the pathology associated with the strandings was unusual. Neither of these conditions were evident in the 1990 Gulf of Mexico bottlenose dolphin strandings. Literature Cited Geraci, J.R. 1989. Clinical investigation of the 1987-88 mass mortality of bottlenose dolphins along the U.S. central and south Atlantic coast Final Report to NMFS, ONR, and MMC. Hersh, S.L. 1987. Mortality, natahty, migration and organismic growth rates of bottlenose dolphins (Genus Tursiops): a review and management considerations. NMFS/SEFC Miami, Contract Report No. 40-GENF-700715. Scott, G.P., D.M. Bum and L.J. Hansen. 1988. The dolphin dieoff: long-term effects and recovery of the population. Pages 819-823 in Proceedings of the Oceans '88 Conference, Baltimore, MD. IEEE Catalog No. 88-CH2585-8. Scott, G.P., D.M. Bum, L.J. Hansen and R.E. Owen. 1989. Estimates of bottlenose dolphin abundance in the Gulf of Mexico from regional aerial surveys. NMFS/SEFC Miami Contribution No. CRD-88/89-07. Snedecor, G.W. and W.G. Cochran. 1973, Statistical methods, 6'*' ed. Iowa State University Press, Ames, Iowa. 593 pp. Wells, R.S. and M.D. Scott. 1988. Estimating bottlenose dolphin population parameters from individual identification and capture-release techniques. NMFS/SEFC Miami Contract Report No. 50-WCNF-7-06083. 20 SECTION n AGE STRUCTURE Lany J. Hansen Southeast Fisheries Science Center Miami Laboratory 75 Virginia Beach Drive Miami, FL 33149 Hersh (1988) found that the age structiire of bottlenose dolphins stranded during the 1987-88 U.S. east coast dieoff differed significantly from the available pre-dieoff composite sample. Specifically, a significantly larger proportion of 5 to 9 year olds stranded during the dieoff. This may have reflected that the hypothesized disease epidemic (Geraci, 1989) caused proportionally higher than normal rates of mortality among an age group which usually exhibits a relatively low mortality rate (Hersh, 1988). Methods An age analysis was conducted on the available sample of teeth from animals that stranded along the Texas coast to evaluate the age structure of the 1990 strandings as compared to the composite sample of previous years. The teeth were examined for growth layer groups (GLG or GLGS; see Perrin and Myrick, 1980) by Ms. S. Fernandez, with assistance from Dr. A. Hohn, a leading expert on techniques for aging odontocete cetaceans. The methods used followed those detailed in Myrick et al (1983) as modified for bottlenose dolphins (Hohn et aL 1989). The sample included animals that stranded from 1983-1990. Of this sample, 195 stranded during January- June, of which 70 were from 1990. Results and Discussion The cumulative distributions of ages of the two samples (animals from 1983-89 and animals from 1990) are shown in Figure 1. These distributions were compared using a Kolmogorov-Smimov two-sample test. There were proportionally more young animals in the 1990 sample, but the results of this test indicate that the cumulative distributions were not significantly different (DN=0.186, p>0.10). The length data form a larger and probably more representative sample of the stranded animals. A comparison of the cumulative distributions of the 1990 zmd 1983-89 length samples (Figure 2) indicates that proportionally more short (i.e., young) animals stranded during 1983-89. The distributions were significantly different (DN=0.0987, p<0.04). 21 This trend was the opposite of that of the aged sample (This trend is also discussed in Section II). IJO • ,.^ ' o.t • .'-^ y^ Oi ■ /'" ^^ 1 0.7 • / jT" / ItM IM3-W OJ. r / 0.5. 1/ 0.4 - 0 J ■ ,^ ^^ f r' ■^ ON • o.ias7 0.1 - / t > 0.104 0.1 - 1 r > I 20 13 CLBS Figmc 1: Cumulative distributions of ages (id GLGS) of Texas January-June 1983-89 and 1990 bottlenose dolphin strandings. 1.0 - o.t - /^ S a* ■ f 1 0.7 . f — itoo 1 0.4. go.. 0.3- 0.1 . /^ ON . 0.0M7 > < 0.040 M no IM laa it* im 2i« zm LEKTTM Ol 290 ]7s no 3ia Fignre 2 Cumulative distributions of lengths of Tens Januaiy-June 1983-89 and 1990 bottlenose dolphin unndings. The aged samples are a sub-sample of the stranded animals with length data. If the aged sample is representative of the length sample, the length structure of the aged samples should reflect that of the overall length samples. To examine this, the cumulative distributions of the lengths of the 1983-89 and 1990 aged samples were compared to the 1983-89 and 1990 length samples, respectively (Figure 3-4). The results of the Kohnogorov- Smimov two-sample tests of these distributions indicate that the while the 1990 aged sample was not significantly different from the 1990 length sample (DN=0.105, p>0.59), the 1983-89 aged sample was significantly different from the 1983-89 length sample (DN=0.245, p<0.01). 1.0 ■ o.s - /T" 1 0.S • / ,' loai-M i 0 7 . / / — «i s "■•■ / ; MXD SMWJE / t 5 04. ^^y^ J \ "-'■ — _^ y y OM . 0J4S0 O.J • 0.1 • / ,-'' » < OJ»l IJO. o.« ■ \ 0.0 ■ ' SM»i£ !"■ yZt 3 0 4 . ^l-'' g OJ- ^„^—' OM - O.I04« o.i. ^^- --=*^' » > 03M 0.1 ■ *S IIS ISO ISO IT! IM 111 130 230 270 l*a III IINCTM Ol Figure 3: Cumulative distributions all lengths and lengths of aged sample ofToas 1983-89 January-June bottlenose dolphin strandings. M no ISO ISO 170 Ita 211 230 290 171 2M 3l« UNCTM CH Figiiic 4: Cumulative distributions lengths of aged sample of Texas bottlenose dolphin strandings. of all lengths and 1990 January-June These results indicate that the 1983-89 aged sample was biased in some way. It appears that shorter (< 140cm) animals were under-represented in the 1983-89 aged sample. The teeth of most of these shorter animals probably were not collected during 1983-89 because the teeth had not yet erupted. 22 Because the 1983-89 aged sample was probably not an unbiased sample, the age structure of this sample most likely does not accurately reflect that of the animals stranded during 1983-89. Therefore, the results of the age structure comparison between 1983-89 and 1990 should be considered inconclusive. Since there is no indication that the length samples were biased, they can be used for a gross comparison of age structure. The 1983-89 and 1990 length samples were significantly different, and indicate that proportionally fewer younger animals and more older animals stranded during 1990 than during 1983-89. A yearly comparison of lengths demonstrates that the proportion of stranded animals < 140cm has been decreasing since 1986 (see Section II, Figure 8). Literature Cited Geraci, J.R. 1989. Qinical investigation of the 1987-88 mass mortality of bottlenose dolphins along the U.S. central and south Atlantic coast. Final Report to NMFS, ONR, and MMC. Hersh, S.L. 1988. Age class distribution of bottlenose dolphins stranded during the east coast die-off of 1987/1988. NMFS/SEFC Miami, Contract Report No. 45-WCNfF-800633. Hohn, A.A., M.D. Scott, R.S. Wells, J.C. Sweeney and A.B. Irvine. 1989. Growth layers in teeth from known-age, free-ranging bottlenose dolphins. Marine Mammal Science, 5(4):315-342. Myrick, A.C., Jr., A.A. Hohn, TA.. Sloan, M. Kimura and D.D. Stanley. 1983. Estimating age of spotted and spinner dolphins {Stenella attenuata and Stenella longirostris) from teeth. NOAA Tech. Rep. NMFS 30. 17 pp. Perrin, W.F., and A.C. Myrick, Jr., eds. 1980. Age determination of toothed whales and sirenians. Reports of the International Whaling Commission, Special Issue 3. 229 pp. 23 SECTION m POPULATION ABUNDANCE AND STRANDINGS Lany J. Hansen Southeast Fisheries Science Center Miami Laboratory 75 Virginia Beach Drive Miami, FL 33149 Methods Estimates of bottlenose dolphin abundance from large-scale aerial surveys of the Gulf of Mexico (Scott et al. 1989) were compared with historical stranding data. Seasonal estimates were available for fall (September-October 1983), winter (January-February 1984), spring (April-May 1984), and summer (July-August 1984) for the northwestern Gulf. For the northeastern Gulf, seasonal estimates were available for fall (September-October 1985), winter (January-February 1986), and simimer (June-August 1985). The estimates were stratified by the following zones: bay (embayments and inshore of barrier islands), inshore (seaward of the coast or embayment boundaries to the 18.3m isobath), and offshore (18.3m isobath to 9.3km seaward of the 182.9m isobath). The area surveyed is shown in Figure 1. M JOT< •• 1 Kw tr* 10^ * ■w •»■» *rm »rw — t ■ ir« ^ ^ .--' ^ ^ ^^ *% f /• 054 c^rcr^^co \ ^^ J jrii -1- v ^^ ■7 * Vs'.-^i An aerial survey of a portion (Block 154) of the northwestern Gulf inshore zone along Texas and Louisiana was conducted during March, 1990, in response to the 1990 anomalous mortality event (see Appendix IV for details). This area, and another adjacent portion of the inshore zone (Blocks 152 and 153) and an adjacent portion of the offshore zone (Block 054 and B) were surveyed during June, 1990, in response to the MEGABORG oil spill (see Appendix IV). The estimated abundance for these areas sampled in 1990 was compared to the survey results from during 1984. Fignrc 1: Aerial »urvcy blocks of the northern Gulf of Mexico used during 1983-86 NMFS regional surveys for bottlenose dolphins (Scott et al^ 1989), and blocks surveyed dunng 1990 (152, 153. 154, 054, B). 24 Results and Discussion The 1983-84 abundance estimates of all zones combined for the northwestern Gulf were lowest in the winter (January- February), increased during the spring (April-May), and remained at that level through the summer and fall (Figure 2). The Texas strandings provide the best sample for comparison with the northwestern Gulf abundance estimates. The peak in strandings occurs from February-April, with monthly levels approximately equal throughout the rest of the year (Section 1, Figure 2). Thus, if the abundance estimates represent the normeil, seasonal trends, strandings occur during a period when abundance is apparently increasing. The trend in 1985-86 abundance estimates of all zones combined for the northeastern Gulf of Mexico differed from that of the northwestern Gulf, with the lowest seasonal abundance in the summer rather than the winter (Figure 3). In fact, the highest seasonal abundance in the northeastern Gulf occurred during the winter. Strandings for the northeastern Gulf peak during March and April (Section I, Figure 4), which would correspond to spring, however a spring abundance estimate is not available. Based on the winter and summer abundance estimates for all zones, it appears that the peak in strandings occurs while there is an apparent declining trend in abundance. This is the opposite of the trend in the northwestern Gulf. UJ o i z r> CD < 20 18 16 14 - 12 - 10 - 8 - 6 - 4- O BAY V INSHORE D OFFSHORE A ALL ZONES ff f V 4 O^r ^ -9-r w S S F -I — I — I I I — I — i—T — I — I — \ — r I I WSSF WSSF ZONE AND SEASON WSSF Fignne 2 Estimates o( seasonal bottlenose cSolphin abundance in the northwestern Gulf of Mexico (Scott et al., 1989). Error bais represent 95% confldenoe intervals. o I z CD < . 1 50 - 45 - 40 - O BAY V INSHORE D OFFSHORE £, ALL ZONES 1 35 - i i 30 - A 25 - I 1 20 - ■ 1 i 1 15 - 1 1 I- 10 - 5 - 0 o6 ^ 1 1 1 1 1 1 1 1 1 1 1 — I— 1 1 1 1 — r— WSSF WSSF WSSF ZONE AND SEASON WSSF Fignre 3: Estimates of seasonal bottlenose dolphin abundance in the nonheastetn Gulf of Mexico (Soott et aL, 1989). Error bart represent 95% confidence intervals. Another aspect of the abundance estimates to consider is the likelihood of whether or not animals which die in the different zones will eventually strand. Mead (1979) discussed factors which affect the discovery of cetacean strandings. He stated that "virtually all cetaceans" are negatively buoyant. Dolphins 25 which die and sink in waters deep enough to keep decomposition gases in solution should not strand. He also suggested that, because of increased exposure to scavengers and predators, the greater the distance from shore at which an animal dies or is debilitated, the less likely the animal will strand. Because of these factors, it is probably reasonable to assume that most of the animals which strand, died or became debilitated within the bay and inshore zones. In the northwestern Gulf, the trends in the bay and inshore zones abundance estimates have a different pattern than that of all zones combined, which essentially reflects the trend of the offshore zone. In the inshore zone, fall had the highest estimated abundance, summer the lowest, and winter and spring had intermediate levels. Using a 95% c.i. criterion, the inshore summer estimated abundance was significantly different from the other seasons, but the other seasons were not significantly different from each other. In the bay zone, winter and fall had the lowest estimated abimdance, with spring and summer having approximately equal, but higher levels. The bay winter estimate was significantly lower than the other seasons, but the other seasons were not significantly different from each other (95% c.i. criterion). If the bay and inshore zones are combined, the apparent trend follows that of the inshore zone. In any case, if the trend in the estimated seasonal abundance does reflect the normal pattern, strandings in the northwestern Gulf peak when abundance in the bay and nearshore zones combined is at intermediate levels and is declining. The trends in estimated seasonal abundance in the northeastern Gulf for all zones combined also reflect that of the offshore zone. However, the trend in the inshore zone is similar, with the lowest estimated abundance occurring during the summer and winter. Estimated abundance in the bay zone seems to be constant. Keeping in mind that no spring abundance estimate is available and that seasonal estimates of abundance may not be statistically significant, strandings peak in the northeastern Gulf while the abundance in the bay and nearshore zones combined appears to be declining, which was similar to the pattern in the northwestern Gulf. The 1990 point abundance estimates for Block 154 were higher than the 1984 point estimates for winter, spring, and fall (Figure 4). Based on the 95% c.i. criterion, the 1990 March estimate was significantly different from only the 1984 spring estimate, while the 1990 June estimate was iiW - 450- O 1984 400 - V 1990 ^ 350 - o i^ 300- UJ o 250- \ r z 200 - " 150 - T y f 100 - { ^ I 50 - 1 6 1 1 ? JAN MAR MAY JUN SURVEY MONTH AUG Figure 4: Estimates of bottlenose dolphin abundance for aerial survey Block 154 (sec Figure 1 for location) (Scolt et al., 1989, and Mullin, this volume). Error bars represent 95% confidence intervals. 26 significantly different from the 1984 spring and summer estimates. Based on these surveys, it appears that there may have been more animals in this area during spring and summer of 1990 than during the 1984 sampling period. The estimated abundance for Block 153 was also higher for June, 1990, than for the spring and summer 1984 estimates (Figure 5). This is consistent with the pattern observed in Block 154. The estimated abundance for the offshore Block 054 for June, 1990, was significantly higher than the spring 1984 estimate (95% c.i. criterion), and was higher but not significantly different from the the summer 1984 estimate (Figure 6). In fact, the June, 1990, estimate was about 20 times higher than the spring 1984 estimate. Similarly, the summer 1984 estimate was about 11 times higher than the spring 1984 estimate. This could indicate that the pattern observed in 1984 (higher summer abundance in Block 054) was evident again in 1990. The 1990 estimates for these adjacent blocks (153, 154, and 054) were consistently higher than the estimates from 1983-84. It is important to note that the boundaries between the inshore and offshore blocks were not based on any known distribution patterns of the Gulf bottlenose dolphins. The available survey results indicate that the distribution of these dolphins was more or less continuous from the shore out to at least the seaward boundary of the offshore zones. 90 - 80 - O 1984 V 1990 TO- GO • 50 - 40 - 30 - ' r 20- 10 - 6 1 1 o MAY JUN JUL SURVEY MONTH Figmc S: Estimaies of boKlenose dolphin abundance for aerial The results of these aerial surveys were similar to those conducted prior to the 1987-88 east coast bottlenose dolphin dieoff. Keinath and Musick (1988) reported that the results of nearshore aerial surveys along the Virginia coast suggested bottlenose dolphin densities were higher during the dieoff than previous years (1980- 86). However, Scott and Bum (1987) reported that their analysis of surveys conducted during the dieoff along New Jersey and Virginia out to the 1000 fathom isobath indicated there was a 60% chance of decline in the offshore stratum abundance ind'jx, as compared to the 1980- 81 abundance index. Data were insufficient to evaluate the nearshore stratum (Scott survey MuUin, intervals Blodc 153 (see Figure 1 for location) (Scott et al., 1989; this volume). Error ban represent 95% confidence z 3 70 - O 1984 V 1990 60 - 50 H 40 - ' r X - ■ 20 - < ) 10 - ■^ . 9 , JUN SURVEY MONTH JUL Figure 6: Estimates of bottlenoae dolphin abundance for aerial survey Block 054 (see Figure 1 for location) (Scott cl aL, 1989; Mullin, this volume). Error bar* represent 95% confidence intervals. 27 and Bum, 1987). Although the information is limited, it appears that anomalous monalities of bottlenose dolphins during 1987-88 and during 1990 may have occurred when apparent densities in nearshore areas were higher than previously observed, but the pattern in offshore densities was not similar. Literature Cited Mead, J.G. 1979. An analysis of cetacean strandings along the eastern coast of the United States. Pages 54-71 in J.B. Geraci and DJ. StAubin (eds.). Biology of marine mammals: insights through strandings. NTIS No. PB-293 890. 343 pp. Keinath, J.A., and J.A. Musick. 1988. Population trends of the bottlenose dolphin {Tursiops tmncatus) in Virginia, 1980-87. NfMFS/SEFC Miami, Contract Report No. 40-GENF- 800564. Scott, G.P., and D.M. Bum. 1987. The potential impact of the 1987 mass mortahty on the Mid- Atlantic offshore stock of bottlenose dolphins. NMFS/SEFC Miami, Contribution No. ML-CRD-87/88-10. Scott, G.P., D.M. Bum, LJ. Hansen and R.E. Owen. 1989. Estimates of bottlenose dolphin abundance in the Gulf of Mexico from regional aerial surveys. NMFS/SEFC Miami Contribution No. CRD-88/89-07. 28 SECTION IV FOOD HABITS Nelio B. Barros Division of Biology and Living Resources Rosensnel School of Marine and Atmospheric Science University of Miami Miami, FL 33149 An unusual mortality of bottlenose dolphins in the Gulf of Mexico (primarily along the Texas coast) took place during early 1990. Due to the implication of food habits in the recent mortalities of bottlenose dolphins (Geraci 1989) and humpback whales (Geraci et al. 1989) in the eastern coast of the United States, there was concern of a similar occurrence in the Gulf of Mexico during 1990. This study analyzes the food habits of bottlenose dolphins stranded along the coast of Texas during January- April 1990 and compares the results with a previous study in the same area (Barros and Odell 1990). Samples obtained from 26 dolphins stranded in Matagorda Bay in late January 1990 were not available for examination and were not included in this study. Methods Samples (entire stomachs) were collected from 38 stranded dolphins and frozen for analysis (see Appendix I for details on stranding date, location, sex, age, etc.). This sample was comprised of all the whole stomachs that were coUected and frozen; it was not possible to determine if the sample was representative of the animals that died during January-April 1990. One stomach was empty and the remaining 37 had food matter (Table 1). In this sample, dolphins ranged in size (total length) from 160 to 269 cm. Fish otoliths, squid beaks and shrimp rostra and/or telsons were the structures utilized in prey identification, following methodology outlined in Barros and Odell (1990). Results and Discussion Wet weight of the 37 stomach contents ranged from 1 to 3382 g (Table 1), with a mean of 237.95 g per stomach. When only stomachs with contents weighing 10 g or more are considered (see Barros and Odell 1990) this figure increases to 398.95 g (n= 22). The number of prey items per stomach averaged 219.14 (SD= 287.80, n= 37) and number of prey taxa 9.41 (SD= 5.49, n= 37) (Table 1). A total of 15,950 fish otoUths (13,816 sagittae, 2,124 lapilli, 10 asterisci), 1,681 (890 upper and 791 lower) squid beaks, and remains (rostra/telsons) of 59 crustaceans were found in all stomachs, representing 7,109 fish, 915 squid and 59 crustaceans. The categories of prey type were: fish only (F): 9 (24%); fish and 29 cephalopod (F,C): 23 (62%); fish, cephalopod, crustacean (F,C;K): 4 (11%), and fish and crustacean (F,K): 1 (3%) (Table 1). Altogether, 46 species offish (19 of which could not be identified) distributed in 11 families, and 3 species of cephalopods and 2 species of crustaceans were identified (Table 2). Six prey species occurred in more than 50% of the stomachs: the Atlantic croaker (Micropogonias undulatus), silver perch (Bairdiella chrysoura), brief squid (LoUiguncula brevis), sand seatrout (Cynoscion arenarius), and a unidentified teleost, and accounted for 57% of all prey taken. The family Sciaenidae was the most important fish family, accounting for 64% of all fish prey. Cephalopods of the family Loliginidae dominated this category, numbering 914 of the 915 specimens found. Shrimp of the genus Penaeus comprised 58 of the 59 crustaceans identified in all stomachs. A comparison of these results with the study of Barros and Odell (1990) (Table 3) indicates that bottlenose dolphins in this study did not differ significantly in their food habits. Thus, the wet weight of the stomach contents, an indicator of stomach fullness, was not statistically different in both studies (p>0.05, t-test; data subjected to a natural logarithm transformation to comply with normality and homoscedasticity of variances), the same being true for the number of prey items and prey taxa in each stomach (p>0.05, t-test). In addition, the categories of prey types (fish, cephalopod, crustacean) were also present in similar proportions (p>0.05, chi-square test) in the two studies. Four out of the six most important prey (M. undulatus, C. arenarius, B. chrysoura, and L. brevis), numerically and in terms of frequency of occurrence, were the same in 1986-87 and 1990. Although there are differences in the two data sets (samples analyzed in Barros and Odell (1990) were collected during a 2-year period (1986-87), and only from dolphins stranded in the vicinity of Galveston; samples from the present study were collected from dolphins stranded along the entire coast of Texas during early 1990), the results obtained in the present study show that bottlenose dolphins stranded during the 1990 Gulf of Mexico mortality event had a similar prey spectrum as in years of no unusual mortality. These results, although preliminary, suggest that the food habits of these dolphins were not significantly altered during the mortality event. 30 Table I. Stomach contents of bottlenose dolphins from the 1990 Gulf of Mexico mortality (n = 37). Field #C #SE Sex' Length Stomach Prey^ Prey Prey (cm) CtW(g)^ Type Taxa Items Season^ ccoss orw 4993 M 216 171 F,C 12 297 W GA290 C82S 4892 M (257) 116 F 3 7 W GA291 C824 4893 M (213) 5 F,C 8 130 W GA293 C844 4895 M (182) <1 F,C 4 28 w GA297 rssi 4899 U (200) 59 F.C 13 101 w GA298 r8')2 4900 F (239) - <1 F.C 4 10 w GA299 r8S3 4901 U (184) 44 F.C 14 267 w GA301 C855 4903 F (239) <1 F 6 18 w GA302 CH'iS 4906 M 262 258 F.CJC 12 402 w GA304 C860 4904 F 219 214 F.C 6 22 w GA311 (jjf/e 4913 F 233 39 F.C 10 189 Sp GAS 12 C877 4914 F 233 645 F.CJC 19 788 Sp GA313 C878 4915 F (236) 2 F 9 948 Sp GA314 OW4 4916 M 260 <1 F.C 3 51 Sp GAS 15 C885 4917 F 269 <1 F.C 7 183 Sp GAS34 LV21 5170 M 194 638 F.C 15 730 Sp GASS5 C9S1 5174 F 245 <1 F 5 8 Sp GA344 C955 5411 F 206 824 FJC 10 173 Sp PA18S C843 4980 F 205 39 F.C 15 315 w PA189 C881 4986 F 198 3 F.C 9 875 Sp PA192 C920 5163 F 243 6 F.C 8 63 Sp PA195 C940 53% F 240 220 F.CK 24 319 Sp P0121 C826 4955 M 256 1424 F.C 21 1044 w P0122 C827 4956 U 238 74 F 5 16 w P0123 C837 4957 M 229 <1 F.C 9 41 w P0125 C8S9 4959 F 245 174 F.C 22 327 w P0127 C846 4%1 M (179) <1 F 3 5 Sp P01S4 C873 4968 F 254 <1 F.C 8 68 Sp P0135 C874 4969 F 211 3382 F.C 7 74 Sp P01S6 C875 4970 F 160 42 F.C 5 16 Sp P0137 r882 4971 U 201 94 F.C 10 135 Sp P0141 C897 4975 F 247 118 F.CK 9 137 Sp SPlll C842 4857 M 254 <1 F 6 31 w SP112 C856 4858 M 260 16 F.C 10 114 w SP114 C862 4860 F 251 <1 F 1 3 w SP115 C870 4861 F (250) 112 F.C 10 152 Sp SP12S C948 5404 M 208 74 F 6 21 Sp ' M = male; U « unlcnown; F " Female ^ Stomach contents weight m g ' F = fish; C • cephalopod; K « cnisiacean * W = winter, Sp « cpring 31 Table 11. Frequency of occurrence (P.O.) and number of prey (N) taken by botUenose dolphins (n= 37) from the 1990 Gulf of Mexico mortality. PREY SPECIES FAMILY F.O. % N TELEOSTS Micropogonias undulatus Sdaenidae 28 75.7 2152 26.9 Bairdiella cfuysoura Sciaenidae 26 70.3 1025 12.8 Cynoscion arenarius Sciaenidae 21 56.8 444 5.6 Unidentified leleost I - 21 56.8 248 3.1 Stellifer lanceolatus Sciaenidae 17 45.9 72 0.9 Anchoa sp. F.ngraulidae 15 40.5 836 10.5 dJ^agodon rhomboides Sparidae 14 37.8 121 1.5 Menticirrhus sp. Sciaenidae 12 3Z4 85 1.1 Cynoscion nothus Sciaenidae 11 29.7 477 6.0 Porichthys plectrodon Batrachoididae 11 29.7 204 2.6 Urophycis sp. Gadidae 11 29.7 147 1.8 Leiostomus xanthums Sciaenidae 11 29.7 44 0.6 Unidentified teleost II — 9 24.3 105 1.3 Cynoscion nebulosus Sciaenidae 8 21.6 85 1.1 Unidentified pleuronectifonn I — 8 21.6 78 1.0 Mugil cf. M. cephalus Mugilidae 8 21.6 20 0.3 Larimus sp. Sciaenidae 7 18.9 119 1.5 Unidentified pleuronectiform II - 6 16.2 387 4.8 lOrthopristis chrysoptera Sparidae 6 16.2 12 0.2 Trichiurus lepmrus Trichiuridae 5 13.5 50 0.6 Synodus foetens Synodontidae 5 13.5 35 0.4 Ariusfelis/Bagremarinus Ariidae 5 13.5 32 0.4 Unidentified dupeid - 4 10.8 6 0.1 Pogonias cromis Sciaenidae 3 8.1 44 0.6 Orlhopristis chrysoptera Sparidae 3 8.1 9 0.1 Cynoscion sp. Sciaenidae 3 8.1 8 0.1 IPeprilus sp. Stromateidae 2 5.4 66 0.8 Unidentified teleost III — 2 5.4 20 0.3 IBrevoortia sp. Qupeidae 2 5.4 9 0.1 dSardinella aurita Engraulidae 2 5.4 4 0.1 lOpsanus sp. Batrachoididae 2 5.4 2 0.0 Pomatomus saltatrix Pomatomidae 2 5.4 2 0.0 Unidentified engraulid Engraulidae Z7 67 0.8 Unidentified teleost IV — 2.7 39 0.4 Bairdiella IStellifer Sciaenidae 2.7 17 0.2 Unidentified teleost V — 2.7 6 0.1 Unidentified teleost VI _ 2.7 4 0.1 Unidentified teleost VII ~ Z7 4 0.1 Unidentified teleost VIII _ 2.7 3 0.1 Unidentified teleost IX ~ 2.7 3 0.1 Unidentified teleost X — 2.7 3 0.0 Opsanus beta Batrachoididae 2.7 2 0.0 Unidentified teleost XI - 2.7 2 0.0 32 Table II. Continued TELEOSTS (ConL) PREY SPECIES Unidentified teleost XII Unidentified teleost XIII Unidentified clupeiform ICynoscion sp. Sciaenops ocellata Unidentified teleost XTV Unidentified teleost XV Unidentified teleost XVI Unidentified teleost XVII Unidentified teleost XVIII Unidentified teleost XDC FAME.Y Sciaenidae Sciaenidae P.O. % N % 1 2.7 I 0.0 1 2.7 1 0.0 1 2.1 I 0.0 1 2.7 I 0.0 1 2.7 ] I 0.0 1 2.7 ] I 0.0 1 2.7 ] [ 0.0 1 2.7 ] [ 0.0 1 2.7 ] 1 0.0 1 2.7 ] 0.0 1 2.7 ] 0.0 CEPHALOPODS LoUiguncula brevis Unidentified loliginid Doryteuthis sp. Unidentified ?oaopodid Loliginidae Loliginidae Loliginidae ?Oaopodidae 25 66.7 736 9.2 16 43.2 111 1.4 8 21.6 67 1.8 1 2.7 1 0.0 CRUSTACEANS Penaeus sp. Squila empusa Penaeidae Stomatopodidae 6 1 16.2 2.7 58 1 0.7 0.0 33 Table HI. Comparison of the food habits of bottlenose dolphins from Barros and Odell (1990) and this study. Wet weight Prey items Prey taxa Prey type X' R' X R SD n X R SD n F F.C F.CK FJC Barros & Odell (1990) This study Statistics 852.67 398.95 N.S.' 25-6550 16-3382 (p>0.05. 1497.01 748 7? t-test) 18 22 272.83 219.14 N.S. 1-1073 1-1044 (p>0.05. 338.78 287.80 t-test) 23 37 9.48 9.41 N.S. 1-16 1-24 (p>0.05, 4.94 5.49 t-test) 23 37 6(26%) 9(24%) N.S. 9(39%) 23(62%) (p>0.05. 8(35%) 4(11%) chi- - 1(3%) square) u = X = mean; R ^ range; SD ■= standard deviation; n ^ sample size; N.S. » non-significant; F • Gsb; C • cephalopod; K ~ crusucean Literature Cited Barros, N.B., and D.K. Odell. 1990. Food habits of bottlenose dolphins (TUrsiops truncatus) in the southeastern United States. Pages 309-328 in S. Leatherwood and R.R. Reeves, eds. The bottlenose dolphin. Academic Press, San Diego, CA. Geraci, J.R. 1989. Clinical investigation of the 1987-88 mass mortality of bottlenose dolphins along the U.S. central and south Atlantic coast. Final Report, U.S. Marine Mammal Commission, Washington, DC, 63 pp. Geraci, J.R., D.M. Anderson, R.J. Timperi, DJ. St. Aubin, G.A. Early, J.H. Prescott, and CA. Mayo. 1989. Humpback whales {Megaptera novaeangliae) fatally poisoned by dinoflagellate toxin. Canadian Journal of Fisheries and Aquatic Sciences 46:1895- 1898. 34 SECTION V ENVIRONMENTAL FACTORS Robert A, Blaylock Southeast Fisheries Science Center Miami Laboratory 75 Virginia Beach Drive Miami, FL 33149 Background The National Marine Fisheries Service monitors marine mammal mortalities in the Gulf of Mexico with the cooperation of the Southeast Marine Mammal Stranding Network (SEUS). This is a mainly volunteer network of federal, state, and imiversity researchers having varying levels of expertise and resources. In January-March 1990, bottlenose dolphin, Tursiops truncatus, stranding in the northern Gulf of Mexico received an unusually high degree of pubhc attention. The highest incidence of bottlenose dolphin stranding was reported in Texas. This may reflect a higher level of detection and reporting effort in Texas rather than a higher mortality rate; however, the relatively large Texas data base allowed analysis of bottlenose dolphin stranding in relation to the physical environment. This study examined environmental variables and the Texas bottlenose dolphin stranding record for relationships which might account for the high stranding incidence reported in early 1990. Methods Environmental factors The Texas Gulf of Mexico coast was stratified for analysis into five statistical zones of equal lengths of shoreline totahng approximately 556 km (300 nm) and extending from north of Galveston to the U.S.-Mexico border (Areas I-V, Figure 1). Bottlenose dolphin stranding records listing the species stranded, date of discovery, and location of the stranding and other pertinent information were obtained firom SEUS and the Smithsonian Institution TEXAS GULF OF MEXICO Fignre 1. Locations of environmental dau stations along Texas coaxt. Filled circles show Elunan transport stations and unOlled circles show temperature data station locaiioiis. 35 Marine Mammal Stranding Program. Stranding data from the period January 1986 through June 1990 were selected for analysis because reporting effort in Texas became consistent begiiming in 1986. 60 200 ISO 100 - 1—1 so n - r— 1 r— 1 CO 50 MEAN Q. 40 -I o o 30 - ^ 20 - o UJ Q Z < V) 10 - S2 U 84 85 as 87 86 09 80 YT>R Figmc 2. Yearly bottlenose dolphin stranding rqxsrts bom the Texas coast from 1982 through 1990. i^ftftFiFiA^i^ ZmKQ:>-Z-lC9Q.H->U 1986 ES 1987 ^1988 ^^ 1989 ■1 1990 I 1 95X C.I FEB MAP APR MONTH MAY JUN Figure 4. Bottlenose dolphin tlrandings on Tescas coast by month, 1986 through 1990. Table 4. Monthly mean sea surface temperature (SST °C), air temperature (AIR °C), salinity (SAL °lg^ averaged from three stations on the Texas Gulf Coast for January-June. 1986-1990 and monthly mean bottlenose dolphin strandings from the Texas coast for the years 1986-1990. Values withm a column having the same letter were not tigniflcantly different (SNK test, a = 0.05, A = highest value, F « lowest value, N • number of data observations). JAN 13.5 F 14.2 F 24.0 B 407 « FEB 15.2 E 15.2 E 25.0 AB 379 19 MAR 183 D 18.7 D 25.2 AB 439 44 APR 21.8 C 22.6 C 24.8 AB 402 26 MAY 26.0 B 26.7 B 24.2 B 437 3 JUN 28.9 A 28.1 A 26.1 A 423 3 38 Table 5. Yearly mean ica lurfacc temperatures (SST "C), air temperatures (AIR "Q, and lalmity (SAL °/^) for the ta month period January-June averaged from three stauons along the Teas Gulf CoasL Values within a cnlumn having the same letter were not signifjcantly different (ShfK test, a « 0.05, A = highest value, E " lowest value, N « number of data observations) Strandmgs are for the tame six month period from the entire coast YEAR SST AIR SAL N STRANDINGS 1986 21,2 A 21,8 AB 21J E soo 95 1987 19,8 B 19.9 D 2i8 D 514 124 1988 20,5 B 203 CD 29.0 A 464 98 1989 20.9 A 21.1 EC 26,2 B 559 72 1990 21.4 A 22.4 A 25,2 C 450 163 Tabic 6. Monthly mean tea surface temperatures (°C) by year along Teacas Gulf Coast. Values with the tame letters were not tignincantty different (SNK test, a « 0.05, A = highest value, D « lowest value). YEAR JAN FEB MAR APR MAY JUN DEC 1985 - - - - - - 14.2B 1986 13.7B 16,2A 19,4A 23.2A 25.7BC 29,08 13,88 1987 12.9C 15,08 17.48 20JOC 25,8BC 28.4C ISSA 1 1988 10.7D 13^C 17,88 21.68 25.4C 28.78C 16.1A 1989 16.0 A 13.9C 17.98 2238 27.0A 28,88c 10.6C 1990 12.7C 16.8A 19.4A 21,88 263B 29.7A Table 7. Monthly mean air temperatures (°C) by year along Texas Gulf Coast. Values with the tame letters were not significantly different (SNK test, a ° 0.05, A = highest value, D — lowest value). YEAR JAN FEB MAR APR MAY JUN DEC 1985 - - - - - - 13JB 1986 14.4BC 15.98 21.0A 23.9A 26.08 28.9AB 13.18 1987 13.0C 14.9BC 17,28 20.68 26.18 28.28 15.5A 1988 10,9D 13.9BC 18.6B 23.0A 27.0A8 23.1 C 16.0A 1989 16,5A 133C 17.68 22,9A 27JA 29.2AB 10.2C 1 1990 15.4AB 17.8A 19.1B 22.6A 26.7A8 31 OA - Table & Results of linear regression of monthly botllenoce dolphin ttrandinp on monthly mean tea turface temperature (SST), air temperature (AIR), salinity (N - 30) and offshore transport (OFFSHORE). VARIABLE SLOPE INTERCEPT RJ P SST -1.15 41.59 0.14 0.04 AIR -1.06 39,82 0.11 ojn SALINITY 0.08 1.56 0.01 0.49 OFFSHORE -0.004 2.23 0.00 OJO 39 There was a negative exponential relationship between first semester stranding rates and the preceding December-January mean sea surface temperature. The lowest January- December mean sea surface temperatures preceded the highest January-June stranding incidence, 124 strandings in 1987, and 189 in 1990 (Table 6). Regression of log-transformed January-June stranding totals (logS) against the corresponding log- transformed December-January mean sea surface temperatures (logT) resulted in the following statistically significant (P = 0.02, N = 5) relationship: logS = 5.4 - 2.951ogT, r^ = 0.89 (Figure 5). A weak (r^ = 0.65) relationship between dolphin strandings and air temperature for the same periods was not significant (P = 0.10). S V 2.3 1990 \ • \ \ 2.2 \ \ \ \ 2.1 - V '^ \ 'k ^^vl988\ ^^-, 2.0 \19B\ 1.9 '\ \ 1989 \ \ 1.8 \ \ 1.00 1.05 1.10 1.15 LOG TCMPERATURE 1.20 1.25 Figure 5. Relationship between Dec -Jan tea surface temperature and Jan-Jun strandings on Tocas coast. 1986-1990. LogY - 539 - 2.95 log X r^ - 0.89, P - 0.02. Sea surface temperature anomaly data from NOAA's Oceanographic Monthly Summary for December 1989-January 1990 ranged from -0.4 to -2.0 °C. The monthly temperature means upon which these anomalies are based are the data from the Robinson, Bauer and Schroeder (1979) climatologies. Because of the historical nature of the climatology, these data should be considered qualitative; however, the persistence of the negative anomaly throughout the winter of 1989-90 suggests that sea surface temperatures were abnormaUy low. Salinity Mean salinity varied significantly among months (Table 4), among years (Table 5), and among months within years (Table 9); however, there was no significant relationship between bottlenose dolphin stranding and sahnity (r^ < 0.01, P = 0.99). Table 9. Monthly mean salinity (ppt) by year along Texas Gulf Coast. Values with the same letters were not signiPicantly different (SNK test, • - 0.05, A - highest value, E - lowest value). YEAR JAN FEB MAR APR MAY JUN DEC 1985 - - - - - - 15.0E 1986 19.8B 21 3B 23.6B 213C 18.6C 24 .2C 17.6D 1987 I7.8B 21JB 20.8D 25 .28 28.9A 27. 3C 25JC 1988 27 JA 283A 26.8AB 29.2A 293A 32.2A 31. 6A 1989 283A 26.4A 28.5A 25.1B 24.2B 24.2C 29.1B 1990 28.0A 28.2A 26.08 21 .IC 19,5C 27.4B - 40 Offshore transport Monthly mean offshore transport varied significantly among years (Table 10), but there was no significant relationship between overall monthly mean ofishore transport and monthly mean bottlenose dolphin stranding (r^ = 0.02, P = 0.50, N = 35, Table 6). Examination oi seasonal stranding and ofishore transport suggested a weak (r^= 0.08), but significant (P = 0.01, N=41), inverse relationship during the spring (Figure 6). Tabk 10. Monthly oQshore transpon (MT/100km*ec) by yor averaged from five tutions along Toas Gulf CoaxL Negative values indicate onshore transport. Values with the tame letten were not significantly diaereni (SNK test, a. ~ 0.05, A « highest value, C = lowest value). YEAR DEC JAN FEB MAR APR 1985 -56.6B - - - - 1986 -53.7B ■nsc -10.4B 19,2B 41^AB 1987 1.2A -15,2B -17,7B -15.0C 28.9B 1988 2,2a -34,58 -S,8AB 41,7AB 21.18 1989 -84,8C -7,8B -5.7AB 26,28 38.4AB 1990 - 183A 21.1A 54.4A 5S.9A Discussion Although standard statistical analyses (ANOVA) failed to estabhsh a significantly higher Texas Gulf Coast bottlenose dolphin monthly mortality rate during the first semester of 1990 than for the same period during the previous four years, it is clear that there were a high number of dolphin mortaUties on the Texas coast during January-March 1990. The low power of the significance test may explain the inability to reject the null hypothesis of no significant difference between years. Further evidence that 1990 was unusual was given by the occurrence of 12 bottlenose dolphin strandings in Texas in November 1990 as compared to a mean of about five strandings for Novemeber 1985-89. Colder than normal water temperatures may directly affect bottlenose dolphin health by increasing energy expenditure to maintain body temperature, or may indirectly affect it by reducing local food supplies. Low sahnity, resulting from increased fresh water runoff, may similarly affect prey distribution. Alternatively, unusual current patterns may result in a higher than usual number of stranded dolphin carcasses with no actual increase in mortahties. Other than an inverse relationship between winter sea surface temperatures and spring dolphin mortality rate, these analyses detected no strong significant relationships between bottlenose dolphin mortahties and other environmental variables. In all of the relationships between environmental variables and dolphin stranding rates which were examined, the power of the significance tests (1 - B) exceeded 0.99. The association of low winter sea surface temperatures with an increased spring dolphin mortality rate suggests the 41 possibility of thermally-induced stress, perhaps lowering resistance to opportunistic infection; however, clinical evidence is lacking due to the small sample size of fresh carcasses. Although data are available for other species of marine mammals (see reviews in Gaskin, 1982, and Whittow, 1987) httle has been published on thermoregulatory response of Tursiops tnincatus in spite of its relatively long history of captivity. Unpublished data suggest that the thermoneutral Tninimnm for TuTsiops truncatus is approximately 4-5 °C, but may vary with acclimation (S. H. Ridgway, pers. comm., December 1991). o a X s o o 150 OFFSHORE TOANSPORT FigOTB 6: Monthly bottlanosc dolphin ■trmndingi a* m function of oSihorc trmnsport along Texaa coast, iphng 1986- 19M. (Only months with mora than on* reported stranding w«r« used.) Alternatively, the observed association between sea surface temperature and bottlenose dolphin stranding may be less direct. Gunter (1941, 1952) noted increased fish mortahties in the Texas Gulf Coast region associated with cold weather. Decreased food availabihty could affect dolphin health. An estimated 2.7 million fish, of which approximately 2.6 milHon were striped muDet (Mugil cephalus), died in East Matagorda Bay after a severe cold spell in December 1989 and smaller fish kills occurred in Texas Bays fi-om Sabine to Lagima Madre Bay; however, there was no evidence of a similar fish kill in the Gulf of Mexico (pers. comm., Larry McEachron, Fish Resources Program, Texas Parks and Wildlife Dept., Rockport, TX, March 1991). Striped mullet are reported to be a prey item of bottlenose dolphins in the northern Gulf of Mexico (Gunter, 1942; Leatherwood, 1975); however, these reports are contradicted by more recent studies (Barros and OdeU 1990, and Barros, Section FV, this report). It is possible that, because of unusual weather-related fish migration patterns or fish mortalities, bottlenose dolphins were forced to switch to alternative prey items which may have been nutritionally inadequate. Data regarding fish migration during December 1989-March 1990, were unavailable. All but one of the 38 stranded bottlenose dolphins examined had food in their stomachs (Barros, Section IV, this report), and comparison of prey items among Texas-stranded dolphins from earher dates showed no significant differences in prey species. An inverse relationship between spring bottlenose dolphin stranding rale and offshore currents (Ekman transport) suggests the possibility of an apparent spring mortahty increase. An increase in beach-cast dolphin mortalities may merely reflect an increased probability of washing ashore due to onshore Ekman transport during the spring. Alternative^, an apparent increase in mortality rate could occur with a seasonal increase in the nearshore bottlenose dolphin population; however, this did not appear to be the case (see Hansen, Section III, this report). 42 As a first attempt at establishing a basis upon which to evaluate bottlenose dolphin mortalities in relation to environment, these results emphasize the general inadequacy of the Gulf of Mexico marine TnamTnal stranding data base. Texas was the only state for which there existed a sufficiently accurate time series stranding record for quantitative analysis. Consistent stranding data for the other states bordering the Gulf of Mexico, with the possible exception of southwest Florida, were lacking. Uteratnre Cited Barros, N. B. and D. K. OdeH 199a Food habits of bottlenose dolphins in the southeastern United States. Pages 309-328, in S. Leatherwood and R.R. Reeves (cds.), The Bottlenose Dolphin. Academic Press, New York. Gaskin,D. E. 1982. The ecology of whales and dolphins. Heinemann Educational Books, Ltd^ London. 459 pp. Gunter, G. 1941. Death of fishes due to cold on the Texas Coast, January 1940. Ecology 22(2): 203-208. Gunter, G. 1942. Contnbutions to the natural history of the bottle-nosed dolphin, Tursiops tnmcams (Montague), on the Texas coast, with particular reference to food habits. J. Mammal. 23: 267-276. Gunter, G. 1952. The importance of catastrophic mortalities for marine fisheries along the Texas coast J. Wildl. Manage. 16(1): 63-69. Leatherwood, S. 1975. Some observations of feeding behavior of bottle-nosed dolphins (Tursiops mmcatus) in the norther Gulf of Mexico and {Tumops cf. T. gUIi) off southern California, Baja California, and Nayarit, Mexico. Mar. Fish. Rev. 37(9): 10- 16. Robinson, M., R. Bauer, and £. Schroeder. 1979. Atlas of North Atlantic-Indian Ocean monthly mean temperatures and mean salinities of the surface layer. Nav. Ocean. Off. Ref. Pub. No. 18. Scott, G.P., D. M. Bom, and L. J. Hansen. 1988. The dolphin die-off: Long-term effects and recoveiy of the populatioiL Proc Oceans '88 Conference, Baltimore, Maryland, October 31-November 2, 1988. Whittow, G. C 1987. Thermoregulatory adaptations in marine Tnammak- Interacting effects of exercise and body mass. Marine Manunal Science 3(3): 220-241. 43 SECTION VI PHYTOPLANKTON DISTRIBUTION Patricia A. Tester Southeast Fisheries Science Center Beaufort Laboratory Beaufort, NC 28516 Gymnodinium breve (Steidinger 1990; fonnerly Ptychodiscus brevis) is a toxic dinoflagellate species generally restricted to the Gulf of Mexico where it is responsible for red tides, particularly off the west coast of Florida (Baden et al. 1984). This species blooms sporadically (bloom = 5 x ICP cells 1"^) and can reach concentrations of 2-5 x 10'' cells 1"' (Woodcock 1948; Steidinger and Ingle 1972). G. breve blooms are thought to be initiated in offshore coastal waters, primarily in late summer-fall months and transported inshore (Steidinger, 1975). Dinoflagellates are phototactically positive organisms and are frequently more abundant in surface waters than at depth. Because of this, winds, currents, and tides are important factors in the transport (concentration or dispersal) of red tide cells. Consequently, areas of high cell counts may be patchy even during blooms. Bloom conditions though, are associated with a stratified water colimin or a defined water mass and the integrity of this water mass is believed to affect the durations and extent of a bloom (Steidinger and Haddad 1981). The more or less armual red tide blooms along the west Florida coast and sporadic blooms elsewhere in the Gulf cause fish kills, neurotoxic shellfish poisoning, and respiratory irritation in humans (Gunter et al. 1947; Woodcock 1948; Pierce 1986). Prior to 1987, the knowledge of the effect of these toxins (collectively known as brevetoxins) on marine mammals was little more than anecdotal (Gunter et al. 1948). Since the implication of dinoflagellate brevetoxins in the dolphin deaths of 1987 (Geraci 1989), examination of phytoplankton samples from areas of high dolphin mortality (especially in the Gulf) is indicated. Methods Water samples for phytoplankton analysis were collected 22-25 March 1990 in the primary study area between Galveston Bay and the Mississippi delta region (Figure 1). Four samples were collected at each station: one at the surface, one near the bottom, and the other two equidistant from each other and the surface and bottom samples. Since no basin- wide (Gulf) data exist on background G. breve cell concentrations, and little is kiown of its seasonal occurrences or natural variation except in Florida waters, we obtained a number of water samples from the northern Gulf for comparative purposes. 44 OriginaUy, all 145 water samples from the primary study area were examined for presence of G. breve cells. A 2 ml aliquot of the Utermohl preserved material, settled at the bottom of one liter sample jars, was observed using an inverted microscope. Of these, some from the deepest bottles from the near-bottom sampling contained too much sediment to allow reliable observations. Initial observations indicated that more than 78% of 123 remaining samples contained G. breve cells (Appendix V). Results and Discussion Seventy-two of the original samples (from the upper half of the water column) were reexamined in detail and quantitative counts of G. breve cells were made. More than 91% (66 of 72) of these samples contained G. breve cells and 35% (25 of 72) had cell numbers >50 cells 1"^ (Appendix V). Areas of highest G. breve concentration at the surface had a broad seaward distribution with a tongue of higher cell counts onshore near Station 25 (Figure 1). Information or evidence of an offshore bloom outside the primary study area at the time of sampling was lacking. The discolored water areas noted during our aerial observations of the primary study area were caused by high concentrations (9 x 10^ cells 1'^) oiNoctUuca (scintUlans = miliaris), a phagotrophic dinoflagellate not generally thought to be toxic. Note the distribution of the Noctiluca bloom (Figure 1, open circles) parallels the western edge of the high G. breve concentrations. Since the numbers of G. breve cells in samples from the primary study area were not high enough to be considered a bloom, we needed some information on "normal" or background concentrations of G. breve to put our results in context. Surface water samples were collected from the NOAA Ships OREGON II and FERREL during their routine work in the Gulf. Examination and quantitative counts of these "comparative samples" allow the following observations: 1. The incidence of G. breve in the primary study area was high (89%) but compared well with other nearshore areas of the northern Gulf (Table 1, Figs. 1. 2 (box) and 3). 2. The proportion of samples in the primary study area during March 1990 with G. breve concentrations >50 cells 1'^ is 2 to 3 times greater than in other nearshore areas of the northern Gulf or from the same area in September 1990 (Table 1, Figs. 1, 2 and 3), 3. Both the incidence and concentration of G. breve cells is lower in offshore (open water) areas than on the shelf/nearshore areas (Table 1, Figure 4). During the examination of "comparative samples" we noted, on two occasions, high concentrations (5 x 10^ cells T') of the toxic dinoflagellate Gonyaulax monilata (Connell and Cross 1950). These samples were taken on two different cruises during Sept. and Oct. 1990 from stations very near the Mississippi delta region in the northern Gulf. No G. monilata were seen in the primary study area. 45 Summary One hundred and twenty-three phytoplankton samples from the primary study area were examined for the presence of the toxic dinoflagellate Gymnodinium breve. Eighty percent of these samples contained G. breve cells. Seventy samples from the upper half of the water column were examined in detail, and quantitative counts confirmed that 94% contained some G. breve cells while 65% contained >50 cells r\ These concentrations are far below those considered a 'TDloom" (>5 x 10^ cells 1'^). Comparative samples from other areas in the Gulf suggest that G. breve concentrations in the primary study during the March 1990 sampling period were within "normal backgroimd levels" but consistently higher than quantitative counts of samples from similar areas or from the primary study area later in the sunamer. The discolored water patches noted during aerial observations of the primary study area were blooms (approx. 1 x 10^ cells 1"^) of the dinoflagellate NoctUuca spp. This genera is not normally known to be toxic. However, it should be noted that a toxic dinoflagellate species, Gonyaulax monalata, was found in elevated concentrations near the Mississippi delta in late summer 1990. Literature Cited Baden, D.G., T.J. Mende, M.A. Poli and R.E. Block. 1984. Toxins from Florida's red tide dinoflagellate Ptychodiscus brevis. Pages 359-367 in E.P. Ragelis (ed.), Seafood toxins. American Chemical Society, Washington, D.C. Connell, C.H. and J.B. Cross. 1950. Mass mortality of fish associated with the protozoan Gonyaulax in the Gulf of Mexico. Science 112:359. Geraci, J.R. 1989. Qinical investigations of the 1987-1988 mass mortality of bottlenose dolphins along the U.S. central and south Atlantic coast. Final report to National Marine Fisheries Service and U.S. Navy, Office of Naval Research and Marine Mammal Commission, April 1989. 63 pp. Gunter, G., G.F. Walton Smith, and R.H. Williams. 1947. Mass mortality of marine animals on the lower west coast of Florida, November 1946-January 1947. Science 105:256-257. Gunter, G., R.H. Williams, C.C. Davis and F.G. Smith. 1948. Catastrophic mass mortalities of marine animals and coincidental phytoplankton bloom on the west coast of Florida, November 1946 - August 1947. Ecological Monographs 18:309-324. Pierce, R.H. 1986. Red tide {Ptychodiscus brevis) toxin aerosols: a review. Toxicon 24:955- 965. 46 Steidinger, K-A. 1975. Basic factors influencing red tides. Pages 153-162 in V.R. LoCicero (ed.), Proceedings of the first international conference on toxic dinoflagellate blooms. Massachusetts Science and Technology Foundation, Wakefield, MA. Steidinger, K-A. 1990. Species of the tamerensislcatenella group of Gonyaulax and the fucoxanthin derivative-containing Gymnodiinoids. Pages 11-16 m E. Graneli et al. (eds), Toxic marine phytoplankton. Elsevier. Steidinger, YLA. and IC Haddad. 1981. Biological and hydrographic aspects of red tides. BioScience 31:814-819. Steidinger, ICA. and R.M. Ingle. 1972. Observations of the 1971 summer red tide in Tampa Bay, FL. Environmental Letters 3:271-278. Woodcock, A-H. 1948. Notes concerning human respiratory irritant associated with high concentrations of plankton and mass mortalities of marine organisms. Journal of Marine Research 7:56-62. 47 Table 1. Comparisons of Gymnodinium breve concentrations in the Gulf of Mexico. Comparison are for surface samples only. Gulf of Medoo location Number of samples % of samples with C. breve % of samples with >50 cells l' Nortbwcst- Primary Study Area GalvestoD Bay to Mississippi Delta RV PELICAN March 1990 Fig. 1 35 » 29 Norttawcsi - Comparative Area Entire Teacas coast FRV FERREIX, Sept- OcL 1990 Fig. 2 3S S2 3 Subset - of data above Stations which cor- respond to those in primary study area. Fig. 2 (box) 9 » 11 Northern Golf - Comparative Area Northern Florida to Galveston Bay FRV OREGON II ScpL 1990 Fig. 3 <3 M 11 Subset - of data above Sutions which cor- respond to those in primary study area. Fig. 3 (box) 6 33 17 North Ccntn] - Comparative Area Offshore FRV OREGON II, April- May 1990 Fig. 4 66 15 0 48 I E a O i u >„• ec - f 8 8 II Q e £• A ll 8 c Ov I e i i o m a > s 0 B 3 t 8 ■B O E e 0 ii !«■ |i c « 8 E li en 0 e o IT) M e M o OB a 9 M I M K Q. UJ CO o o UJ o I* M N N N V o 8 I 1 1 I B • z • o o u b: o > 8 IS. E 3 & 8 •5 O E «n 2 o. < O O tt) ei O > t a. e a I 15 E V f C o t o t a. B 3 .1 C S. E 8 o n M M M lb 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 BIBLIOGRAPHY Cooper, W. C, 1967. Selenium Toxicity in Man. Symposium: Selenium in Biomedicine, Avi, Westport, Conn., 185-199. Doull, J., C. D. Klaassen, and M. O. Amdur (ed.), 1980. Casarett and DouU's Toxicology: Macmillan Publishing Co., Inc. New Yoii:, p. 239. Gaskin, D. E., D. J. D. Smith, P. W. Amold, M. V. Louisy. R. Frank, M. Holdrinet and J. W. McWade, 1974. Mercury, DDT, dieldrin and PCB in two species of Odontoceti (Cetacea) from St Lucia, Lesser Antilles. J. Fish. Res. Board Can., 31: 1235-1239. Geraci, J. R., 1989. Clinical investigation of the 1987-88 mass mortality of bottlenose dolphins along the U.S. Central and Soudi Atlantic Coast Rept to NMFS, ONR, and Marine Mammal Conmiission, Guelph, Ontario, Caiuida, 63 pp. Hanson, S. W. F., and J. Olley, 1963. Application of the Bligh and Dyer method of lipid extraction to tissue homogenates. Journal of Biochemistry, 89: 101. Honda, K., R. Tatsukawa, K. itaiio, N. Miyazaki, and T. Fujiyama, 1983. Heavy metal concentrations in muscle, liver and kidney tissue of striped dolphin {Stenella coendeoalba ) and their variations with body length, weight age, and sex. Agric. Biol. Chem., 47: 1219-1228. Horwitz, W., L. R. Kamps, and K. W. Boyer, 1980. Quality assurance in &e analysis of foods for trace constiments. J. Assoc. Off. AnaL Chem., 63: 1344- 1354. 72 Krahn, M. M., C. A. Wigren, R. W. Pearce, L. K. Moore, R. G. Bogar, W. D. MacLeod, Jr., S-L. Chan, and D. W. Brown., 1988. Standard analytical procedures of the NO A A National Analytical Facility, 1988: New HPLC cleanup and revised extraction procedures for organic contaminants. U. S. Dep. Commer. Tech. Memo, NMFS F/NWC-153, 52 p. Kuehl, D. W., R. Haebler, and C. Potter, 1991. Chemical residues in dolphins from the U.S. Atlantic coast including Atlantic bottlenose obtained during the 1987/1988 mass mortality. Chemosphere, 22: 1071-1084. Law, R. J., C. F. Fileman. A. D. Hopkins, J. R. Baker, J. Harwood, D. B. Jackson, S. Kennedy, A. R. Martin, and R. J. Monis, 1991. Concentrations of trace metals in the livers of marine mammals (seals, porpoises and dolphins) from waters around the British Isles. Mar. PolluL BulL, 22: 183-191. MacLeod, W. D.. Jr., D. W. Brown, A. J. Friedman, D. G. Burrows, O. Maynes, R. W. Pearce, C. A. Wigren, and R. G. Bogar, 1985. Standard analytical procedures of the NOAA National Analytical Facility, 1984-1985: Extractable Toxic Organic Compounds, Second Ed., U. S. Dep. Commer. Tech. Memo, NMFS F/NWC-92, 121 p. Marcovecchio, J. E., V. J. Moreno, R. O. Bastida, M. S. Gerpe, and D. H. Rodriguez, 1990. Tissue disuibution of heavy metals in small cetaceans from the South westem Atlantic Ocean. Mar. PolluL Bull.. 21: 299-304. McFarland, V. A. and J. U. Clarke, 1989. Environmental Occurrence, Abundance, and Potential Toxicity of Polychlorinated Biphenyl Congeners: Considerations for a Congener-Specific Analysis. Environ. Health Perspect., 81:225-239. Morris, R. J., R. J. Law, C. R. Allchin, C. A. Kelly, and C. F. Fileman, 1989. Metals and organochlorines in dolphins and porpoises of Cardigan Bay, West Wales. Mar. PolluL Bull., 20: 512-523. 73 Muir, D. C. G., R. Wagemann, N. P. Grift, R. J. Norstrom, M. Simon, and J. Lien, 1988. Organochlorine chemical and heavy metal contaminants in whiic- beaked dolphins (Lagenorhynchus albirosnis) and pilot whales {Globicephala melaena) from the coast of Newfoundland, Canada. Arch. Environ. Contam. Toxicol., 17: 613-629. O'Shea, T. J., R. L. BrowneU Jr., D. R. Clark Jr., W. A. Walker. M. L. Gay, and T. G. Lamont, 1980. Fish, wildlife, and estuaries. Organochlorine pollutants in small cetaceans from the Pacific and South Atlantic Oceans, November 1968- June 1976. Pesticides Monitoring Journal, 14: 35-46. Randerath, K. E., E. Randerath, H. P. Agrawal, and M V. Reddy, 1984. Biochemical (postlabeling) methods for analysis of carcinogen-DNA-adducts. In: A. Berlin, M. Draper, K. Hemminki, and H. Vaninio (Eds.), Monitoring Human Exposure to Carcinogenic and Mutanogenic Agents. lARC Scientific Pubhcations No. 59, International Agency for Research on Cancer, Lyon, pp. 217-231. Safe, S., 1984. Polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs): biochemistry, toxicology, and mechanism of action. CRC Crit Rev. Toxicol.. 13: 319-395. Tanabe, C, N. Kannan, An. Subramanian, S. Watanabc and R. Tatsukawa, 1987. Highly toxic coplanar PCBs: Occurrence, source, persistency and toxic implications to wildlife and humans. Environ. Pollut. 47: 147-163. Thompson, D. R., 1990. Metal levels in marine vertebrates. In Heavy Metals in the Marine Environment, edited by R.W. Fumess and P.S. Rainbow, pp. 143- 182. CRC Press, Boca Raton, FL. Varanasi, U.. J. E. Stein and M. Nishimoto, 1989 A. Bkotransformation and disposition of polycyclic aromatic hydrocarbons in fish. In: Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment U. Varanasi (ed.). CRC Press, Boca Raton, FL, pp. 93-149. 74 Varanasi, U., W. L. Reichert, and J. E. Stein, 1989B. 32p.postiabeling analysis of DNA-adducts in liver of wild English sole {Parophrys vetidus) and winter flounder (Pseudopleuronectes americanus). Cancer Res., 49: 1171-1177. Varanasi, U., S-L. Chan, B. B. McCain, J. T. Landahl, M. H. Schiewe, R. C. Clark, Jr., D. W. Brown, M. S. Myers, M. M. Krahn, W. D. Gronlund and W. D. MacLeod, Jr., 1989C. National Benthic Surveillance Project: Pacific Coast, Part n. Technical Presentation of the Results for Cycles I to m (1984-1986). NOAA Tech. Memo. NMFS/ F/NWC-170. 159p. Varanasi, U., J. E. Stein, W. L. Reichert, K. L. TUbuiy, and S-L. Chan, 1991. Chlorinated and Aromatic Hydrocarbons in Bottom Sediments, Rsh and Marine Mammals in U. S. Coastal Waters: Laboratory and Held Studies of Metabolism and Accumulation. Persistent Pollutants in the Marine Environment C. Walker (ed.) in press. 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 o o a> 3 o c o CO m c O "D O CO o c © o m ffl To O) c C CO CO 3 76 120 a. c o 0> u c o O Uver < O a o. r ^ 0» CM CO * ^ T- CO CO £2 CO CO CO CO to < < < < < CM < 1 I r O) ooooooooooweoj CO o CM CO O O s s s s c o c a> u c o O ^oocoeortco««wcoco^T-o 8 i:: S 5! 5 <0<<<<<<<<< Figure 2. Concentrations of mercury and seienium in liver and kidney of dolphin collected from the Gulf of Mexico, 1990. 77 O) 0) c o 0> o c o U w CO T- < Q. in ^ O CO O < a. o CM CO £2 ^ W O CO CO CO CO CO CO (DCMVC^^T-Or^COOIg o> o> o> (0 >J S O S 2 2 X 2 S S (0 OOOOOOOOOC _0) 20- S. 10- c «> o c o O CO in ^ ca CO o> 1 I I I I i 1 I I r a»o«co«cDcv«oi«^eoKcocM cocococoncococococo^T^ooooo 5 o O. Q. <<<<<<<<<ywny<)ettiy>(ime^^ TbePCBsm identified by lUPAC lUTter vd m «so iderttted in (A) by tte toadiir gra^«^ congeners (pure 3-m8Diylctioi E u evieMeMrjneooeoeow'-eMeMeMrjeueviwoeo "^KvVVWV^VI^AAA^AVVV oeoeoooor'.'-w CMOCOIOIOCDCD^qCO I tl. u. u. u.2Stt2ZZSu.Zu.u.2ZSu. U. '-' -I o o o o o> p S" CO h^ en cj uS o in «- r«. in tn ^ u) O) O) o> O) o> o o o o h> in 8> 0> 0> in at a» o o ot o> CM CM OJ 60 O O O^OCMCM^^-CMCO KcoaaAocDoatooat CMCMCMCMCMCMCMCMCMCMCM X X 0 % e o I O I 0) c & E Tl a c 8 CM CM CM J5 ^ o CM CM CM m : : o ro r<» ^«> O CM CM CM I E S s 1 r> pO ?t^? in ^ CM Oft o 9 CO CM eo OB 0> 0> ^S»^nSgHSSc^?^^::go| o a. X lU lU m o »- Ui < > I < o I »- o z o o ffi o < z « < V) o —I < z LU O H (J UJ UI W CD < >- t- < < ^i fo Z < o °E 5° 1^ UJ 2g I — *- lU O O z < UJ O I 2 c < Ui 2 80 9 3 c JO a. (0 o c « xs CM O 1 e s CM ^ CM ^ CD •5. "S c m ^ £ 0) c o ^^m ^ ? • ^ c 3 g 8 ^M o « c — o -c T5 n» « » 2: 5 ■o c5 •E ^ 5 E C Q. CO Q. ^ " O 2 ^ "S A c o 3 CO ^ S CO s E tt 5 "5. C (0 nconce Kidney CD "O O C 5 « oj 9 •Q CM CO 0» • • • ID r«> o to ^ CD O CD r) CM o • f^ CM U> ^ CO CM r>: CO CM fsT o « « k. CD H> » O o t§ is 81 Table 3. Chlorinated hydrocarbon analytes measured in samples from bottlenose dolphins stranded along the Gulf of Mexico, 1990. The analytes within each group were summed for tabulation. DDTs H2l other CHs o,p'-DDE o.p'-DDD o.p'-DDT p.p'-DDT p.p'-DDE p.p'-DDD trichlorobiphenyis tetrachlorobiphenyls pentachlorobiphenyls hexachlorobiphenyls heptachlorobiphenyls octachlorobiphenyls nonachlorobiphenyis decdol ilorobiphenyl hexachlorobenzene lindane heptachlor aldrin heptachlor epoxide alpha-chlordane trans-nonachlor dieidrin mirex 82 en c 1 8 i = ^ CO 5 1- o c m a a u Q. 0 E Q- (0 o CO £ ^ ;;■ l2 5>i CD M (DtoiflCMoeopoocneviCDincM^u)'^, nqcM (O (D ^ino>!ocoogir>'5r w eg ^ (O (D (D (O iO 5^ CO CO O ^ cs c §1 • o •" "*^ CO CD O- o " i "5. o c « 1st o £ e Ex© Q. O _ ^ o 5 CO CJ ; 2 E 2 C O CO ® ~ ■— ni CO en X o £ o CO O O ^f*.in«Dej«or>o>eoio«>eMU>''>^. lo o> «>{ f*: <«- 00 CO CM U) o u)tnOcqr>:CMr», ^co^oco(DCmn«M^'»-CMu>'»-«D5i > u) r^ CM CM o) a> CO CO o CM (0 ID O 0. o c CM r«- o) rj o a> o o o o CO CO r^ CO CO CM IT) 1^ ui (^ oi CM rs.Seo(DCDOmcD ^SScM'^r^.Q.^'- o o t^ CM CO ^ CD C o E u a> < 0. If) a> o O Q. CO CM m CM 8 o i § § (O CM i CM ^ § CO o o o o < S < < < < s s < < 0. (0 0. (0 5 CO 2 I (0 Z 2 Z 2 CO o o ^ CM CO (O r«> CD o> > ^ t- cc z (0 0> o n cs ■ c c o ^ > 2 5 oc a. (O 83 c c I 8 '^ 2 o. ^ 1 o o « 2 Q. O E •= aS iJisrK.*^ir)r>a>tNie>ic>iu>»rir»^r5C\i^io«r)^o wwcuwwSS-wcNieNieMOieMeMeMWOJcucj'O- S 2 = * CO OC S m a c ^ 8 i5 7^ M ffi e- S "So. Q- S E o c S ^" "S V S> « •§. ? o -5 » o» ^ — o> o ^ o eI o O S o. a. 3 a> «■ O Q c o cS 5 £ « 2 E 2 i 2 - 9 CO X o 9 £ o CO O O to «D d ▼^ -: «SS in cvcM'^eoo^r»»eMO>coin^eo oOodd°°°® CMOOOOCOOOO 5 lO eg

o ■H ^^r^eDO tM CD ^ 0> »^ ui CM CM d d o o CO o en CD o CO ^ 9 E t « oi • <0<<<<<<<<< O 9 *" Q *" £2 o CO rt o O CO Z 2 CO CO 0) N^CM o c c I 5 CO — ' I; 9 cc CO ca.co 84 Table 6. Concentrations {^g/g) on a lipid weight basis of PCBs in blubber and liver of bottlenose dolphins collected from the Gulf of Mexico, 1990. Specimen # PA 183 PO095 GA311 GA332 GA319 GA330 GA304 GA333 GA334 GA336 GA342 GA344 SP112 SP114 LA 001 MS 018 SHCM 077 MM 9013 MM 9012 MM 9008 Blubber Liver 74 82 240 220 28 17 20 24 4.4 3.9 14 11 30 21 20 43 67 56 11 11 77 65 100 57 270 130 44 43 70 23 130 110 • 1000 74 54 24 14 70 77 85 Table 7. Ratios of p.p'-DDT to p,p'-DDE in blubber of bottlenose dolphins from the GulfofMexico,1990. p.p'-DDT Specimen # p.p'-DDE PA 183 PO 095 GA311 GA332 GA319 GA330 GA304 GA333 GA334 GA336 GA342 GA344 SP112 SP114 LA 001 MS 018 SHCf^ 077 MM 9013 MM 9012 MM 9008 0.05 0.62 0.03 0.03 0.04 0.02 0.01 0.02 0.04 0.02 0.03 0.12 0.01 0.04 0.01 0.11 0.02 0.02 0.02 86 This page intentionally left blank. 87 SECTION rx Summaiy of Available Pathology Reports Staff Southeast Fisheries Science Center Miami Laboratory 75 Virginia Beach Drive Miami, FL 33149 ainical necropsy reports were available for 13 of the 367 bottlenose dolphin strandings that were investigated during the 1990 Gulf of Mexico stranding event. This was due, in large part, to the state of decomposition of the stranded animals; only 32 (about 9%) of the stranded animals were in a condition suitable for a clinical necropsy. Another contributing factor to the paucity of clinical necropsies was the limited availability of personnel skilled in post-mortem examination of dolphins. Of the 13 clinical necropsy reports, four are brief pathological summaries and nine are lengthy reports. The reports are appended in Appendix VIII. General necropsies were performed on all of the 13 bottlenose dolphins. Histopathological analyses were done for five animals (samples were collected for two other animals, but were not examined), microbiological analyses for six and hematological analysis for one. The following direct causes of death were indicated: possible mastitis (1), a fisheries interaction mortality (1), bacterial pneumonia (gram-negative) (4) and septicemia (3) (three with lung congestion and edema which were thought to be agonal), and severe pancreatic fibrosis and nodular pneumonia with peripheral skin abscesses (1). In three animals the cause of death was not indicated, but was thought in one animal to be related to the fact that the most significant lesions were pulmonary animal; another was severely emaciated. There was moderate to severe post-mortem autolysis (PMA), preventing microscopic examination and determination of cause of death in three animals. Of these three, one was indicated as a possible calving associated mortality and another as pulmonary edema as cause of death or possibly drowning. Skin lesions (moderate to severe) were observed in nine animals; one was non- specific, five were listed as punctate, one as an ulceration, two were caused by septicemia, 88 one was related to severe local ischemic necrosis, one had a large nodular grey lesion on an auricula, and one was active but non-erupted. Multi-focal circulations with evidence of dermzil involvement were spread over the entire body of one animal, one other animal had numerous light grey irregular spots on its ventral abdomen and a third animal had white circular punctate scars distributed over its body as well as a few healed parallel scars (probably healed con-specific tooth-rake marks). An endometrial cyst and a Monorygma cyst were observed in one animal (the possible mastitis diagnosis), and a tan mass was observed on the ovary of another animal. Serosanguinous fluid was found in the abdominal cavity of one animal and in the thoracic and peritoneal cavities of another. A full-term fetus was found in two animals. One of these animals was the possible calving-associated mortality and the other was a non-indicated mortality. Adhesions were observed between the limg to the thoracic wall, and in the intestinal loops to each other, to the pseudopancreas and to the mesentery, in one animal, and between the intestinal loops and the liver and diaphragm (preventing diaphragmal reflection) in another. Mild to severe fibrosis was observed in the lungs, liver, lymph nodes, pancreas and pseudopancreas of six animals. Lymphoid atrophy was noted in one animal, and hyperplasia and lymphoid depletion were found in three animals. The following bacterial isolates were reported: enterics, Salmonella and gram-negative bacilli from skin samples; enterics, Clostridium, gram-negative bacilli, Pseudomonas and Aeromonas from lungs; enterics and Aeromonas from intestines; gram-negative bacilli and ciliated protozoans from the spleen; and an acute E.coli infection in a lymph node (suggesting immunocompromise). No viral isolates were found. Worn teeth (moderate to extreme), indicating an older animal, were observed in five animals. Minimal teeth wear was observed in two animals, one of which had immature testes, all indicating a younger animal. 89 SECTION X UMTTATIONS AND RECOMMENDATIONS Lany J. Hansen Southeast Fisheries Science Center Miami Laboratory 75 Virginia Beach Drive Miami, FL 33149 Limitations As with all investigations of unpredictable events of this nature, the scope of the investigation, and therefore it's ultimate usefulness, was limited to a degree due to available resources; including financial, human, and the existing baseline information. Some difiiculties in executing both the data collection and analytical phases of the investigation were encountered, which limited to some degree the scope of the investigation. The inability to recognize the anomalous mortality event, and the lack of a rapid response plan and a dedicated response team also hampered the investigation. Because of the timing and locations of stranded animals relative to available persormel, data collection was generally limited to areas where stranding networks were in place and had sufficient resources to deal with the increased workload. Because of this, after the mortality event was perceived as anomalous, the Texas network received considerable assistance from SEFC, but other areas did not. Lack of pre-existing standard and emergency tissue collection protocols was also a problem. After the event was recognized, SEUS network participants were asked to collect a specific set of tissues for the investigation. This lag in recognition of the event resulted in the protocol being established after about half of what has been defined as the anomalous mortality event occurred. Before the mortality event occurred, some data were lost from those animals investigated but for which no standard tissue sampling protocol was in place. The condition of the stranded animals also had a limiting effect on data collection. As is common in most investigations of stranded animals, few of the strandings were fi-eshly dead, and thus few provided tissues useful for histopathology and other pathological examinations (the available clinical necropsy and histopathology reports are presented in Appendix VIII). The inability to examine numerous live healthy and live "affected" dolphins limited the possibility of identifying disease agents or other potential causes of mortality. Few of the voluntary network participants have the skills required to conduct an adequate gross pathologic examination on marine mammals. The scope of the anomalous mortality event was not evident until about the end of February, 1990. At first, the event was somewhat obscured by the mass stranding in 90 Matagorda Bay during January, 1990. The delay in stranding recovery reporting (required within 30 days after recovery) resulted in a lag in recognizing the event. Although contacts with the SEUS network coordinator were in place before the event, SEFC contacts with the network were limited, and most were established during the mortality event. The lack of contacts had a impact on recognizing the event, and thus determining the scope of the event. The pre-existing database had a direct impact on the type and extent of analyses that could be done with the 1990 database. For example, the lack and/or inconsistency of life history tissue collection (teeth, reproductive tracts) limited the breadth of the age structure analysis and eliminated any examination of reproductive history. The inconsistency in stranding recovery efforts, and/or the lack of a measure of stranding reporting and recovery efforts, reduced the statistical strength of inter-aimual and other comparisons of stranding rates. Obviously, the lack of information on the effects and levels of contaminants and biotoxins in healthy bottlenose dolphins, and the lack of information on levels in the pre- 1990 sample constrained the extent of the analyses on contaminants and biotoxins. The SEFC implemented a bottlenose dolphin monitoring program in 1987 (Scott and Hansen 1989). This program is designed to detect a halving or doubling in abundance. The monitoring program did not detect such a decline in abimdance after the mortality event. However, smaller scale variations in abundance are much more difficult to detect. In addition, the scope of the aerial surveys conducted during 1990 was limited, and thus weakened any conclusions relative to 1990 and prior abundance levels. But it is not clear that "unlimited" surveys would have strengthened any conclusions. Although the investigation was not successful in conclusively identifying a single factor (or multiple factors) as a cause of the anomalous stranding event, several suspicious factors were identified. In addition, the investigation identified weaknesses, explored new methods and resulted in the implementation of several efforts which will improve future investigations. These include: a more thorough statistical treatment of stranding fi-equencies, correlations with envirormiental variables, and comparisons of two methods of assessing stranding frequency. Stranding protocols have been refined and further standardized, and the stranding networks have been augmented. The baseline data collected during this investigation will be invaluable in the evaluation of future episodes of mortality among bottlenose dolphins. The process of this investigation identified critical areas where the required information to understand patterns of bottlenose dolphin was lacking, and has resulted in the development of a plan of action which is described below. Recommendations and SEFC Program The 1990 bottlenose dolphin anomalous mortality event in the Gulf of Mexico illustrated that the SEUS network was not well prepared to mount an adequate response to increased strandings. Furthermore, gross inconsistencies in regular data collection and reporting among Network participants have resulted in a lack of consistent baseline information and the inability to monitor the stranding rate in a timely fashion. These 91 problems are primarily the result of relying on a partially organized, mostly ill-trained, and generally poorly equipped, volunteer Network. The SEFC has taken steps to improve the capabilities of the Network by assuming some responsibilities for reporting, by establishing collection protocols and providing collection materials, by providing training and arrangements for clinical necropsy of suitable specimens, by providing for analyses of tissue samples, and by informing Network participants on the results of their reponing and data collection efforts. The SEFC stranding response activities center on three areas: monitoring stranding rate, specimen necropsy, collection and analyses, and dissemination of results. The stranding rate is monitored by a system for rapid reporting of basic data on stranded animals. Consistent specimen collection is being accomplished by providing manuals, collection kits and training to Network participants. The SEFC is establishing pathways for ensuring clinical necropsy and tissue analyses of suitable specimens. Results will be disseminated to Network participants in a quarterly report produced in conjimction with the Network Coordinator. The most important component of all these activities is the development and maintenance of communication between the SEFC and the Network participants. The Network already had a system for reporting strandings, some data collection protocol, and for dissemination of results. However, the level of these activities was not sufficient to meet the SEFC information requirements. It should be clear that the SEFC is not attempting to takeover the Network, but that the SEFC is trying to supplement the Network by providing assistance for particular activities. Although the Network has been improved and efforts to further improve the Network continue, the system is not perfect. There will likely be difficulties, both anticipated and unanticipated, should another anomalous event occur. It is not possible to assure that adequate staff and funding will always be available to respond to anomalous events. The development of contingency plans and funding mechanisms at the national level for anomalous events was recommended by Wilkinson (1991). The establishment of a stranding emergency response team, much like the national oil spill response team, would eliminate many of the types of problems encountered during this investigation. Monitoring Stranding Rate The SEFC has estabhshed a system to receive stranding reports from the Network volunteers for near real-time monitoring of the stranding rate. Appropriate staff at each of the SEFC laboratories and the Regional Office have been identified as SEFC area representatives and have established contacts in their area with Network participants. The Network participants are required to report basic data (what, when, where and condition) to the SEFC area representative within 48 hours of a stranding event The area representative reports the basic data within 48 hours of receipt to the Miami Laboratory. A computer bulletin board system has been established for receiving basic data reports. The 92 Miami Laboratory area representative is responsible for reviewing the basic data reports and for weekly monitoring of the stranding rates throughout the southeast. This allows for rapid identification of anomalous stranding events and the transfer of this information to NMFS Headquarters and others in a timely maimer. Specimen Necropsy Collection and Analyses The Charleston Laboratory has developed necropsy protocols, specimen collection protocols and collection kit specifications. The protocols and kits have been distributed to the appropriate Network participants. The Miami Laboratory is presently identifying necropsy persoimel and necropsy facilities in the southeast. The SEFC area representatives will ensure that appropriate specimens are delivered to necropsy facilities. The SEFC area representatives receive, track, store and transfer collected samples. Arrangements have been made with the Armed Forces Institute of Pathology (AFIP) to conduct histopathological studies on appropriate specimuis. Other collected specimens are transferred for analyses when suitable investigators are identified (e.g., for genetic, food habits, aging, stock studies; some funding may be required and faculty appointments used to bring investigators onboard). These activities will ensure that adequate information is available to begin evaluating causes and potential effects of both normal and anomalous mortality events. Dissemination of Results A quarterly newsletter which provides stranding summaries, information on analyses underway or planned, and any noteworthy events or tips, is being prepared and will be distnljuted to each Network participant. The newsletter is being produced by the SEFC and the Network coordinator. Although this is a minor activity in terms of funding, it is critical for maintaining communication and cooperation between the SEFC and the Network participants. The primary purpose of this activity is to let the Network participants know that their efforts made to provide the SEFC with information and specimens are worthwhile. A biennial Stranding Network meeting should be held, sponsored by the SEFC and the Network coordinator. The meeting will provide a forum for reviewing the Network activities, providing training in necropsy and specimen collection, reporting related research findings, and for establishing and maintaining contacts between the Network participants and the SEFC. The proposed activities require varying amounts of staff time from each SEFC area representative. Initially, each area representative will spend a significant (probably 20 hrs or more per week for two to four weeks) amount of time identifying and contacting area 93 participants and clinical necropsy facilities and personnel. Subsequently, less time, probably one to five hours per week, will be required for reporting, delivering or transferring specimens, and maintaining contacts. Some area representatives may also participate in recovering stranded animals. The Miami Laboratory area representative was responsible for development and implementation of the computer bulletin board system. This required about one person-month. Approximately one-half of the Miami Laboratory area representative's time is spent on reviewing and analyzing reports, distributing specimens, reviewing results, maintaining and developing contacts, and preparing stranding program reports. Appendices DC and X provide more information on specific responsibilities and the planned implementation schedule for the improvement to the SEUS network. Literature Cited Geraci, J.R. 1989. Clinical investigation of the 1987-88 mass mortality of bottlenose dolphins along the U.S. central and south Atlantic coasL Final Report to NMFS, ONR, and MMC. Wilkinson, D.M. 1991. Report to: Assistant Administrator of Fisheries. Program review of the marine mammal stranding networks. National Marine Fisheries Service, Office of Protected Resources. 171 pp. 94 Appendix I. StraraM bottlanoM dolphirw docta^ttad during j««jiry-Jin>, 1990, aleno the U.S. Cutf of Mexico coMt. SEUS COLLECTION i C LEM 6LSS X C NO/DA ST LAT LOM TN BL MS KD LV M ST GO V SK EX CI BT CT FN HE 4762 CC083 4851 SE4851 «767 MNL-9001 4850 SE4850 4891 CA289 4768 »«L-9002F 47*9 11-0011 4793 90-11-0025-CH 47M SHCM-033 4763 CC084 4792 90-11-031 4892 aA290 4765 SHCM-a34 4766 SHCM-035 4981 PA184 4984 PA187 4982 PA1B5 4771 SHCM-036 4781 HS2-90 4947 P0113 4948 P01U 4949 P0115 4950 P0116 49S1 P0117 4952 P0118 4953 P0119 4934 PO100 4932 P0098 4941 PO107 4940 P0106 4945 P0111 4939 P0105 4936 P0102 4943 P0109 4935 P0101 4942 P0108 4931 PO097 4933 P0099 4946 P0112 4944 PO110 4938 P0104 4937 PO103 4849 SE4849 4782 MS3-90 4930 P0096 4929 PO095 492B P0094 4807 C90-11-0046CI( 4796 C90-11-0045 4893 CA291 4978 PA 181 4772 SHW-037 4769 90-04-0093FW> 4885 NAP-00a3 4954 PO120 4960 P0126 4753 MS190 4773 SHCM-038 4959 P0125 4958 P0124 4774 SHCK-039 5216 C90-01-0051CM 4783 MS4-90 4856 SP110 4775 SHCM-040 T 191 ■ U 5 T 201 ■ U 5 T 258 T 249 T 194 T 253 Tf 84 T 196 T 251 T 191 J T? 2S4 T 257 T 236 • T 270 T 2U T 164 • T 213 T 240 • T 229 T 191 T 282 • T 207 • T 216 ■ T 269 ■ T 261 T 254 T 262 • T 288 T 216 ■ T 225 ■ T 256 • T 7 ■ T 220 T 258 T 218 T 254 T 176 T 270 T 262 ■ T 256 T 275 T 245 T 188 T 247 T 240 T 235 T 196 T 137 T7 180 T 213 T 7 T 110 T 249 T 99 T 7 T 236 : T 235 T 164 T 245 4.9 T 163 0.9 T 154 ••- T 206 — T 234 --- T 240 15 T 99 ... 01/02 01/07 01/08 01/08 01/08 01/09 01/11 01/13 01/14 01/15 01/16 01/16 01/17 01/18 01/18 01/18 01/18 01/19 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/20 01/21 01/21 01/21 01/21 01/21 01/22 01/22 01/22 01/22 01/23 01/23 01/23 01/23 01/24 01/25 01/27 01/27 01/27 01/28 01/28 01/28 01/28 01/29 TX U FL U TO FL FL FL AL TO FL TO AL AL TO TO TO FL MS TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO TO LA NS TO TO TO FL FL TO TO AL FL NS TO TO MS AL TO TO AL FL NS TO AL 27^.0 29*47.0 27*24.5 29*47.0 2r47.8 27*38.0 29*43.0 30*24.1 30^7.5 27T2.5 30*24.1 29rS2.1 30*18.5 30*14.0 28*08. 27*42. 28*08. 30*17.5 30*12.1 28*38.6 28*38.9 28T9. 28^.3 28*39.6 2r41. 28*42.8 2r40.7 28*39.9 28*42.2 28*42.1 28*38.3 2r41.8 28*40.9 28*43.4 2r40.8 28*42.3 28*38.6 2r40.1 28*38.4 2ru.3 2r41.7 28*41.2 29*43.0 30*13.5 2r43.5 28*37.7 28*37. 30*25.7 30*20.9 28*53.9 27*50.1 30-13.5 27*31.2 • 28*21.3 28*31.2 30*21.0 30*25.5 28*32.2 28*32.7 30*14 J 29*49.7 30*12.1 29*37.4 30-14.0 97^8.0 93*10.0 8r39.5 93*10.0 95nK.7 82^.6 84*45.0 86*30.0 87T2.5 97*12.5 86*30.0 94*25. 87*31.0 88W.0 96*55. 87*28.0 88*28.1 95*53.8 95*53.4 95^2.6 95*52J 95*51.7' 95*49. 95*47.2 95*56^ 95<56J 95*54.7 95*55.2 95*55.4 95*55.9 95*56.7 95^1.5 95*56.8 95*54.6 95*57.1 95*56.2 95*54.1 95*49.4 95*56.1 95*56.6 9r47.0 88*54.3 95*41.8 95*53.9 95*55.6 86*30.0 87*24.7 95*21. 97*03.2 87*49.0 82*38.1 • 96^54. 96*09.5 88*30.5 87*54.5 96*07.2 96*06.3 88*14.0 8518.7 8r40.0 94*11.8 87*54.0 ♦ ♦ ♦ ♦ • ♦ ♦ ♦ ♦ ♦ T • -♦♦♦♦♦ ♦ -♦♦♦♦ - . . -. . « ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ • ♦ ♦ ♦ . ♦ ♦ ♦ • ♦ ♦ ♦ ♦ ♦ ♦ ♦ . ♦ ♦ ♦ . ♦ ♦ ♦ • ♦ ♦ ♦ - ♦ ♦ ♦ • ♦ ♦ ♦ • ♦ ♦ ♦ • ♦ ♦ ♦ • ♦ ♦ ♦ • ♦ ♦ ♦ . ♦ ♦ ♦ . ♦ ♦ ♦ • ♦ ♦ ♦ • ♦ ♦ ♦ • ♦ ♦ ♦ • ♦ ♦ ♦ • ♦ ♦ ♦ ♦ ♦ T Y T ♦ « ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ « ♦ ♦ ♦ ♦ ♦♦♦♦♦ ♦ ♦♦♦♦♦ ♦ ♦♦♦♦• T T T ♦ ♦♦♦♦♦♦♦ 95 A|]p«idix I. (canttnuad) SEUS aJLLECTIOW # G LEM CLCS X C W/OA ST UT 4957 P0123 1 r 229 2.5 M : I 01/29 TX 4784 W5-90 1 r 111 .... N . I 01/30 NS 4786 M7-90 1 r 246 .... f i I 01/31 NS 4785 »tS6-90 1 r 205 .... M i I 01/31 MS 4894 SA292 1 r 302 16 M i I 01/31 TX 4964 PO130 1 r 216 .... U i i 02/01 TX 4857 a>^^^ i r 254 12.5 M : ( 02/01 TX 4980 PA1B3 1 r 205 3 F ; I 02/02 TX 4895 CA293 182 0.9 M * • 02/03 TX 4896 CA294 1 r ? 13 u : S 02/03 TX 4809 9001 1 r 97 U i > 02/04 FL 4991 CC086 1 r 7 F i I 02/04 TX 4778 MK-1-90 1 r 108 M 2 S 02/05 FL 4996 PI035 1 r 241 F 3 t 02/05 TX 4805 SHCM-042 1 r 165 U ! > 02/07 AL 4791 90-2-1 1 r 7 F i > 02/07 U 4801 m900^ 1 r 220 F ] i 02/08 FL 4993 CC08a 1 r 216 4 M i . 02/08 TX 4961 P0127 1 r 179 2 M i > 02/08 TX 4813 90007 1 r 186 M : > 02/09 NS 4956 P0122 1 r 239 20 U 4 I 02/09 TX 4955 P0121 1 r 261 16 M : ( 02/09 TX 4977 P0143 1 r 241 9 M ] ( 02A)9 TX 4898 CA296 1 r 241 0 M : ( 02/10 TX 4897 GA295 1 r 268 N 2 ( 02/10 TX 5224 90-01-0070 1 r 137 N " f 02/11 FL 4983 PA186 ^ r 237 .... M i . 02/11 TX 4963 P0129 1 r 111 0 F 3 5 02/11 TX 4962 P012B 1 r 152 .... U ! > 02/11 TX 4994 CC089 1 r 183 M i . 02/12 TX 4900 CA298 1 r 239 15 F i . 02/12 TX 4899 CA297 1 r 200 5 U i . 02/12 TX 4803 SHCM-044 1 r 270 H : » 02/13 AL 4806 SHCM-046 1 r 7 U 2 S 02/13 AL 4802 SHCM-045 1 r ? U 2 S 02/13 AL 4808 9002 1 r 180 F 2 \ 02/13 FL 4873 MM-9003 1 r 205 F i . 02/13 FL 4872 MM9002 1 r 240 M I . 02/13 FL 4841 MS011-90 1 r ? U 2 i 02/13 NS 4842 MS012-90 1 r ? U 2 ( 02/13 NS 4901 CA299 1 r 184 1.5 U i . 02/13 TX 4902 CA300 1 r 7 U t . 02/13 TX 4903 GA301 1 r 239 17 F i . 02/13 TX 4815 90009 1 r 110 U 2 \ 02/14 NS 4858 SP112 1 r 260 M : » 02/14 TX 4995 CC090 1 r 234 F i , 02/14 TX 4874 HM-9004 1 r 212 U i . 02/15 FL 4905 CA303 1 r 140 U i . 02/15 TX 4904 CA304 1 219 30 T 2 1 02/15 TX 4859 SP113 1 241 M 2 1 02/15 TX 4906 CA302 1 r 262 17 M i . 02/15 TX 4875 MM-9005 1 r 190 F 2 ( 02/16 FL 4907 CA305 1 r 240 F : i 02/16 TX 5151 PI036 1 7 U 2 1 02/19 TX 5226 90-01-0082 1 183 F 1 f 02/20 FL 4839 MS015-90 1 2S2 M i ■ 02/22 NS 4860 SP1U 1 251 F 2 I 02/22 TX 4967 P0133 1 260 ir U 2 1 02/22 TX 4876 MH-9006 1 260 — M i > 02/24 FL 4974 PO140 1 97 — M 2 1 02/26 TX 5176 CC095 1 7 — U 1 02/26 TX 4910 CA308 1 121 0.8 H t . 02/27 TX LON TMILMSDLVWSTGOBKEXCirrCTFHME 2r38.3 ' 95*52.4' 30^2.5 ' 88*59.0' 30^4.5 8r53.2' 30M4.0 88*53.5' 2r58.3 9515.5' 2rJ7.2 «6*05.S' 29^.2 94*06.5 • 2r47. 97*06. • 29^4. 94*53. ' 29^9.3 94*U.6' 3019.5 8718.3' zrzT. 9718. • zera.2 8r48.3' 26*32.8 9716. ' 3013.8 87^.8' 29rS5.0 90*04.5' 2r42.1 82*28.4' 2719. 97*20. ' 28'39. 95*50.6' 3013.0 88*55.8' 2813.2 96*38.2' 2814. 96*36.7' 2r46.3 95*36.2' 29T3.7 94'43. ' 29*28. 94*37.1' 3010. ' 85*48. ' 27-50. 97*03. • 28T7.4 96*37.5' 28*27.8 96*24.6' 27*25. 9717. ' 2915.4 94*50.5' 29*00.8 9512.2' 30*15.0 87*39.5' 3016.2 87*34.8' 3017.0 87*32.0' 3018.2 87*23.5' 27T7,2 82*42.8' 2r49.7 82*31.0' 3012.0 88*26.0' 3012,0 88*29.0' 29*03.4 95*08.5' 29*04.1 95*08.2' 29*03.9 95*07.2' 3013.5 88*57.7' 29*39.0 94*07.5' 27*30. 9717. • 2r44.1 82*38.1* 29*32. 94*25.6' 29*27.8 94*36.2' 29*39.4 94*06.6' 29*28.4 94*35. ' 27*52.7 82*35.1' 29*22.2 94*49.6' 26*30.2 9715.1' 3015. 85*40. • 3312.7 88*57.7' 29*35. 9417.4' 28*49.1 95*30.9' 28*08. 96*45.7' 28^9.2 95*22.2' 28T)4.4 96*50.2' 27*54.7 82*31.8' 27*24. 96*30.2' zra.t 9712.1' 29*09.7 95*00.3' ♦ ♦♦♦♦♦♦♦ Y T T. - ♦ ♦♦♦♦♦♦ - T - T Y T T t - - - Y ♦ •♦♦♦♦♦ ♦ . • ♦ ♦ ♦ ♦ •♦♦♦♦ ♦ •♦♦♦♦ ♦ ♦ ♦ ♦ ♦ ♦♦♦«♦ T T Y Y C ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ Y T - Y - Y - Y Y Y • Y C 96 • THE REPORTED LENGTH FOR SPECIMEN GA304 NAY BE INCORRECT. THE REPORTED LENGTH IS ABOUT 20CN LESS THAN WHAT WOULD BE EXPECTED FOR A FEMALE OF THIS AGE (30 YRS). THE ORIGINAL DATA SHEET FOR THIS SPECIMEN WAS LOST AND THE LENGTH CANNOT BE VERIFIED. Appendix I. (continued) SEUS COLLECTION « G LEW CLCS X C MO/OA ST LAT LON 4877 MM -9007 T 265 --- - M 02/28 FL 2r45.3 ■ 82*45.7' 4909 CA307 T 97 -•- - M 02/28 TX 29°14.5 ' 94*52.3' 4852 MML-9004 T 215 --- - F 03/01 FL 27°38.0 • 82*33.6' 4888 MAP -0006 T 97 --■ - F 03/01 MS e 1 ■ 1 4881 MS020-90 T 190 --■ - U 03/01 MS 3ff23.5 ' 89°5«,2' 4887 MAP -0005 I 196 --- - F 03/01 MS 4838 MS013-90 I 198 --- ■ M 03/01 MS 30*14.6 ' 88*46.4' 4861 SP115 T 250 26 F 03/01 TX 29°37.1 94*12. ' 4880 MS019-90 r 91 --- • U 03/02 MS 30°16.7 89*22.4' 4970 P0136 r 160 1 F 03/02 TX 2831.1 96*09.6' 4969 P0135 r 211 7.9 F 03/02 TX 28T3.1 96*05.3' 4968 P0134 r 254 18 F 03/02 TX 28°33.1 96*05.2' 4878 MM-9008 r 137 --- ■ F 03/03 FL 2r42.2 82*40.2' 4840 HS17-90 r 7 --• ■ U 03/03 MS 30°18.0 88rS5.8' 4911 GA309 r 95 --- • M 03/03 TX 29^5.6 95*06.4' 48U SHCM-047 r 95 --- M 03/04 AL 30'14.7 88*12.0' 4845 SHCM-048 r 240 --- • M 03/04 AL 30*17.2 87*31.2' 5147 DRC-90-1 r 257 --- M 03/04 FL 24*30 . 81*49. ' 5010 MS027-90 r 112 --- U 03/04 MS 30*12.3 88*26.6' 4843 MS014-90 r 7 --- U 03/04 MS 30*12.0 88*28.0' 4913 GA311 1 r 233 27 F 03/04 TX 29*06. 95*07. • 5177 CC096 1 r 7 --- U 03/04 TX 26*54. 97*28.5' 4837 MS016-90 1 r 180 --- F 03/05 MS 30*25. 88*57. ' 4914 GA312 1 r 233 12 F 03/05 TX 29*00.7 95*12.5' 5203 SHCM-068 1 r 241 --- F 03/06 AL 30*14. 88*20. ' 5202 SHCM-067 1 r 279 --- M 03/06 AL 30*14. 88*20. ' 4853 MML-9005-B 1 r 66 --- U 03/06 FL 2ri7.8 82*34.0' 4883 MS022-90 1 r 109 --- U 03/06 MS 30*22.6 89T)2.6' 4882 MS021-90 1 r 102 --- u 03/06 MS 30*22.4 89*02.6' 4985 PA 188 1 r 95 -- F 03/07 TX 2r47.3 97TK. ' 4915 GA313 1 r 236 21 F 03/07 TX 29*25. 94*41.3' 4916 GA314 1 r 260 31 M 03/07 TX 29*13.9 94*52.9' 4972 PCI 38 1 r 274 20 F 03/08 TX 28*25.8 96*18. ' 4917 CA315 1 r 269 29 F 03/08 TX 29*25.7 94*40.7' 5152 PI037 1 r 235 ---■ U 03/08 TX 26*07. 97*09.9' 4918 GA316 1 104 U 03/08 TX 29*17.8 95*46.2' 4971 P0137 1 201 ---■ U 03/08 TX 28*37.7 95*54. ' 4986 PA189 1 198 ---■ F 03/08 TX 28*01. 98*10.8' 5221 90-01 -0109CM 1 114 ---■ F 03/09 FL 30*15. 85*57. ' 4973 P0139 1 7 28 U 03/09 TX 28*15.8 96*33.9' 4919 GA317 1 100 ••-• M 03/09 TX 29*29.6 94*32.2' 4846 SHCM-049 1 219 ---- F 03/10 AL 30*13.5 87*59.5' 4889 MAP -0007 1 122 •■•- F 03/10 MS • • 1 4921 GA319 1 109 0 M 03/10 TX 29*00.7 95*12.5' 4920 GA318 1 231 34 U 03/10 TX 29*28. 94*35.8' 5011 MS028-90 1 244 ---• U 03/11 MS 30*12.3 88*26.6' 5144 CC085 1 177 0.9 M 03/11 TX 27*29.3' 9ri7.3' 4862 SP116 1 7 U 03/11 TX 29*34.8' 94*18.2' 5130 11-90-0157 1 86 ---- M 03/12 FL 30*27. ' 86*35. ' 5216 90-01 •0122CM 1 137 •--■ U 03/12 FL 30*20. ' 86*15. • 4890 MAP -0008 1 241 •--- M 03/12 MS • 1 • 1 4863 SP117 1 253 - • U 03/12 TX 29*40. ' 94*04.4' 4864 SP118 1 237 23 u 03/12 TX 29*40.2' 94*04.2' 4922 GA320 1 249 9.3 u 03/12 TX 28*59.5' 9514.2' 4975 P0141 1 247 27 F 03/12 TX 28*34.1' 96*02.1' 4976 P0142 1 191 ---■ F 03/13 TX • 1 • 1 4927 GA325 1 220 U 03/13 TX 29*26.2' 94*54.5' 4923 GA321 1 162 --•- F 03/13 TX 29*29.2' 94*33.4' 4879 MS018-90 1 161 M 03/14 MS 30*22.9' 88*33.9' 4924 GA322 1 237 12 F 03/14 TX 29*16.1' 94*49.3' 4925 GA323 T 247 ---- F 03/14 TX 29*29.3' 94*32.4' 5370 SHCM-082 T 190 U 03/15 AL 30*16.5' 88*06.9' 4926 GA324 T 251 13 M 03/15 TX 29*27.9' 94*36. ' 5149 CC091 T 289 ■--• U 03/16 TX 27*31.1' 97*15.9' 5150 GA326 T 89 --■• U 03/16 TX 29*23. ' 94*43.3' TH BL MS KD LV BM ST GO BO SK EX CI 8T CT FH NE 4.. ..«.«. ..«.Y--- ♦ ---♦ ♦ ♦♦♦♦♦♦ Y- ♦ --♦♦♦♦♦ Y- ♦ -♦♦♦♦♦■♦■ Y- ♦ -♦♦♦♦♦♦♦- -Y-Y- ■♦-♦♦ YY--- - ♦♦. ♦ ♦♦♦■♦•♦♦♦• - -YYYYC ♦ ♦♦♦♦♦♦ Y- ♦ ♦♦♦♦♦♦♦♦♦•♦• *■*****■* Y-Y- ♦ •♦•♦♦♦♦♦ YYY- ♦ - ♦ ♦ ♦♦♦♦♦-♦----Y--- ♦ ♦ ♦--♦- ♦ ♦♦♦♦♦♦- - Y- ♦ ♦♦♦♦♦♦♦♦- - -YY- ♦ •---•♦ ♦ ♦•-♦ ****** YY-.. ♦ - ♦ ♦♦♦♦♦• -♦■••-Y-- ♦ ♦♦••♦ ♦ •♦♦♦ ♦ ♦♦♦♦♦♦-♦ ♦ - + -♦♦♦ ♦ ♦ + ♦♦♦♦♦ Y- ♦ ♦♦♦♦♦ ♦ --•-♦ ♦ ♦♦♦♦♦♦♦■ -YY-C - ♦♦♦♦♦♦♦- -♦YY- ♦ ♦♦♦♦♦♦♦♦ ♦ -♦♦♦♦♦-♦ ♦ ♦♦♦♦♦♦•♦ •♦• - *...... 97 Appendix I. (continued) SEUS COLLECTION « G LEN CLGS X C MO/OA ST LAT LON 4987 PA 190 T 213 ... - M 4 03/16 TX 27°50.5 ' 97*02. 7- A988 PA191 T 73 ... - M 4 03/16 TX 27^0.5 ' 97*02. 7' 5254 SE52W T 312 ... - U 4 03/17 U 29°45. ' 93T^. ' 5255 SE5255 T 167 ... - U 4 03/17 LA 29°46. ' 93*43. ' 5012 MS029-90 T 239 ... - M 3 03/17 HS 30*16. ' 88*41. • 4884 MM-9009 T 125 ... - H 4 03/18 FL 27^2.7 • 82*35.0' 5020 MS031-90 T 94 ... - U 4 03/18 LA 30'14.6 ' 89*46.4 • 5029 SHCM-054 f 163 ... - F 3 03/19 AL 30*14.9 88*04.9' 5025 SHCM-050 r 255 ... - F 3 03/19 AL 30*17. 8r45. ' 5027 SHCH-052 r 239 — - M 3 03/19 FL 30*22. 8ri4. ' 4997 MS023-90 r 7 ... - U 3 03/19 HS 30*19. 88*30. ' 5026 SHCM-051 r 86 — - M 4 03/20 AL 30*14.2 87^3.3' 5153 GA327 r 254 ... ■ U 4 03/20 TX 29*55.1 95*19.3' 5041 41650-1 r 183 ... - H 3 03/21 FL 29*40. 85*11. ' 5155 CC092 r 146 ... • U ? 03/21 TX 27*31. 9ri5.9' 5154 GA328 r 7 ... ■ U 4 03/21 TX 29*06.8 95*04.8' 5030 SHCH-055 r 160 ..." • F 3 03/22 AL 30*15. 87*49. ■ 5028 SHCM-053 r 266 ... ■ M 3 03/22 AL 30*18.6 88T)8.2' 5233 SE5233 r 232 ... ■ M 3 03/22 LA 29*46.5 93*35. ' 4998 MS024-90 r 109 — ■ U 2 03/22 MS 30*12. 88*25. ' 5164 PA193 r ? 13 U 5 03/22 TX 2n8.2 96*47.6' 5159 P0146 r 235 ... F 4 03/22 TX 28*16.8 96*37.2' 5160 P0147 1 r 7 ... U 5 03/22 TX 2n4.3 96*37.6' 5157 P0144 1 r 239 23 H 4 03/22 TX 2r46.9 95*35.1' 5156 SP119 1 r 114 0 H 3 03/22 TX 29*36.7 94*13.3' 5158 P0145 1 r 219 25 F 4 03/22 TX 28*48.6 95*31. 8' 5007 90-11-186 1 r 94 — M 3 03/23 FL 30*27. 86*30. ' SOU LA-TT-03 1 r 247 ... F 3 03/23 LA 29*03. 90*38. ' 5163 PA192 1 r 243 15 F 3 03/23 TX 27*57. 96*59.7' 5178 P0151 1 r ? — U 3 03/23 TX 28*25 96*22.7' 5162 GA329 1 r 260 ... U 4 03/23 TX 29*17.3 94*47.2' 5230 LA3 1 r 257 ... H 3 03/24 LA 29*45. 93*38.6' 5231 LA2 1 r 177 ... M 4 03/24 LA 29*46. 93*27. • 5232 LAI 1 173 ... F 3 03/24 LA 29*46.4 93*27.3' 5165 GA330 1 r 106 0 M 3 03/24 TX 29*26.2 94*36.6' 5132 90-11-0199-CM 1 168 ... U 7 03/25 FL 30*20. 87*20. ' 5042 LA-TT-01 1 251 ... F 3 03/25 LA 29*03. 90*23. ' 5043 LA-TT-02 1 265 ... N 4 03/25 LA 29*03. 90*28. • 5013 MS030-90 1 244 ... F 3 03/25 HS 30*13.8 88*40. ' 5008 MS025-90 1 229 ... F 3 03/25 HS 30*20.4 88*31.5' 5166 GA331 1 229 40 F 3 03/25 TX 29*20.1 94*43.7' 5179 P0152 1 ? ... U 3 03/25 TX 28*25 96*27.8' 5031 SHCM-056 1 240 ... F 3 03/26 AL 30*17.2 8r44.1' 5045 LA-TT-04 1 228 F 4 03/26 LA 29*03. 90*38. ' 5143 MS042-90 1 109 U 3 03/26 HS 30*18. 88*35. • 5009 MS026-90 1 272 H 3 03/27 HS 30*25. 88*51. ' 5022 MS033-90 1 234 U 3 03/28 MS 30*13.7 88*37.0' 5021 MS032-90 1 145 H 3 03/28 HS 30*14.6' 88*46.4' 5167 GA332 1 106 0 F 3 03/28 TX 29*13. 7- 94*53.7' 5168 PI038 1 247 10 F 3 03/28 TX 26*16.1' 9ri1.1' 5372 SE5372 1 7 7 U ? 03/29 LA 29*16.9' 91*19.1' 5371 SE5371 1 7 ? U 7 03/29 LA 29*13.2' 91*12.0' 5170 GA334 1 194 M 3 03/29 TX 29*11.5' 94*57.3' 5174 GA335 1 245 15 F 3 03/29 TX 29*10.2' 94*59.5' 5173 P0149 1 145 1 U 5 03/29 TX 28*38.2' 95*52.7' 5169 GA333 1 118 0 M 3 03/29 TX 29*15.8' 94*50. ' 5175 GA336 1 118 0 N 3 03/30 TX 29*26.4' 94*39.2' 5032 SHCM-057 1 130 H 4 04/01 AL 30*18.5' 87*30.8' 4989 CC094 1 158 N 2 04/01 TX 27*32. • 9ri3. ' 5023 MS034-90 1 91 U 4 04/02 HS 30*12.1' 88*28.0' 5024 MS035-90 1 254 M 4 04/02 HS 30*12.1' 88*28.0' 5148 PA194 T 110 F 3 04/03 TX 2r43.7' 97*07.8' 5249 SE5249 T 241 U 3 04/04 LA 29*34.4' 92*29.0' 5250 SE5250 T 232 U 4 04/04 LA 29*35.2' 9ri0.1' 5251 SE5251 7 326 u 3 04/04 LA 29*47.6' 93*22.5' TH BL HS KD LV BN ST GO BO SK EX CI BT CT FH NE ♦ -•--♦ ♦ ♦ ♦♦♦♦♦-♦•♦ ♦ ----■♦ ♦ ♦•♦♦-♦ ♦ --••♦ ♦ --•-•♦• ♦ ♦♦•«••♦ Y-- ♦ ----♦ ♦ ♦♦♦♦♦♦ Y-Y- ♦ ----♦ ♦ ♦ + + ♦♦ YY--- ♦ ♦♦♦♦•» YY--- ********* ♦ ♦♦♦♦♦ YY- ♦♦♦♦♦♦♦♦- ---YYYC ♦ ♦♦♦♦♦♦-♦- -YYYY- ♦ ♦♦♦♦♦♦♦♦♦♦•Y-Y- ♦ ♦♦♦♦♦♦- - - -YYYY- ♦ ♦♦♦♦♦ YYY-- ♦ ♦♦♦♦♦ YY-C 98 Appendix I. (continued) SEUS COLLECTION # G LEN GLCS X C MO/DA ST LAT 5247 SE5247 1 r 238 --- • u 04/04 U 29'41.0 92*51 .7' 5253 SE5253 1 r 250 -•- ■ M 04/04 LA 29°45.2 93*36.2' 5248 SE5248 1 r 226 --- • u 04/04 U 29T6.6 92*40.7' 5252 SE5252 1 r 246 --- u 04/04 LA 29°48.4 93*27.1' 5040 MML-9006F 1 r 173 ■•- F 04/07 FL 27°28.5 82*43.2' 5396 PA195 1 r 240 25 F 04/07 TX 27°43.7 97•V6.^' 5243 SE5243 1 r 151 ■•- F 04/08 U 29°47. 93*31. ' 5244 SE52U 1 r 245 -•- M 04/08 LA 29°46. 9r42. ' 5397 PA196 1 r 235 --- H 04/08 TX 27°46.6 97*05.9' 5398 GA338 1 r 105 0 H 04/08 TX 29°09.5 95*00.6' 5399 SP120 1 r 101 0 F 04/09 TX 29°39.8 94*04.9' 5401 SP122 1 r 213 --■ F 04/09 TX 29°35.7 94*15,5' 5400 SP121 1 r 117 0 U 04/09 TX 29°39.7 94*05.0' 5193 SHCM-058 1 r 269 --- H 04/11 AL 30°23.1 88*17.5' 5242 SE5242 1 r 249 --• H 04/12 U 29°46.2 93*24.2' 5194 SHCM-059 1 r 221 -•- F 04/13 AL 30^. 87*50. ' 5385 SE5385 1 r? 7 --• U 04/13 U 29°19.0 89*48.4' 5379 SE5379 1 r? 7 ■-- U 04/13 LA 29°14.3 89*59.3' 5381 SE5381 1 r? ? • - - U 04/13 LA 29°16.7 89*56.2' 5384 SE5384 1 r? ? --- U 04/13 LA 29°18.8 89°51.6' 5382 SE5382 1 r? ? --- U 04/13 LA 29-18.4 89*56.2' 5377 SE5377 1 r? ? -•- U 04/13 LA 29'08.5 90*07.5' 5376 SE5376 1 r? 7 •-- U 04/13 LA 29°05.2 90*13.5' 5374 SE5374 1 r? 7 --- U 04/13 LA 29°34.4 92*92.2' 5383 SE53a3 1 r? ? - - - U 04/13 LA 29*18.4 89*56.2' 5373 SE5373 1 r? ? - - • u 04/13 LA 29»16.6 91*18.8' 5378 SE5378 1 r? ? - - - u 04/13 LA 29°09.6 90*05.9' 5380 SE5380 1 r? ? - - - u 04/13 LA 29°14.6 89*58.7' 5375 SE5375 1 r? ? --- u 04/13 LA 29T)2.6 90*45.8' 5137 MS036-90 1 267 ■-- M 04/13 MS 30'15.2 88*42.9' 5402 PA 197 1 r ? --- U 04/14 TX 28*D4.4 97*02.1' 5196 SHCM-060 1 208 -•- F 04/15 AL 30*18.5 87^1. ' 5197 SHCM-062 1 93 --- F 04/15 AL 30*15.1 88*08.8' 5191 HS039-90 1 94 ... U 04/15 MS 30*13.2 88*52.4' 5403 GA339 1 244 32 F 04/15 TX 29*23. 94*43.4' 5196 SHCM-061 1 112 -■■ F 04/16 AL 30*16.2 87*33.8' 5186 FLGM41690-3 1 90 -•• H 04/16 FL 30*27. 86*30. ' 5240 SE5240 1 ? — M 04/18 LA 29*49.0 93*43.2' 5198 SHCM-063 1 171 -•- F 04/19 AL 30*18.2 8r44. ' 5404 SP123 1 208 4 H 04/19 TX 29*48.1 93*55.7' 5405 GA340 1 256 --- F 04/19 TX 29*21.4 94*43.3' 5199 SHCH-064 1 104 --■ F 04/21 FL 30*18.2 87*24. ' 5239 SE5239 1 r 113 •-- U 04/21 LA 29*47. 93*09. ' 5192 MS040-90 1 r 257 --- U 04/21 MS 30*11.6 88*59.3' 5406 GA341 1 r 97 0 F 04/21 TX 29*17.6 94*47. ' 5407 PA198 1 r 240 --- H 04/22 TX 28*04.7 97T)2.7' 5200 SHCH-065 1 7 --- H 04/23 AL 30*19.1 88*11. ' 5140 DSU90-01 1 250 --- F 04/23 FL 26*06.5 81*48.2' 5183 MS037-90 1 185 •-- F 04/24 AL 30*13.6 88*18.6' 5184 MS038-90 1 223 •-- F 04/24 MS 30*20.8 88*31.3' 5409 PA199 1 ? — F 04/25 TX 28*01.5 96*57.8' 5408 GA342 1 r 220 7 H 04/25 TX 29*30.8 95*10.8' 5201 SHCM-066 1 r 140 ■-- U 04/26 AL 30* sr 5138 HM9010 1 215 --- H 04/26 FL 27*53.2 82*28.2' 5234 SE5234 1 220 --- U 04/28 LA 29*45. 93*43. ' 5238 SE5238 1 r 240 --■ U 04/28 LA 29*U.2 93*41.5' 5209 SHCH-074 1 168 --- F 04/30 AL 30*14. 88*00.5' 5139 MM9011 1 r 7 •-• U 04/30 FL 2r4a.o 82*48.4' 5279 MS049-90 1 r 163 --- U 04/30 MS 30*13.3 88*30.4' 5210 SHCH-075 1 r 218 --- U 05/02 AL 30*14.5 87*54. ' 5211 SHCH-076 1 168 --- U 05/03 AL 30*14.4 87*53.7' 5204 SHCM-069 1 r 118 -•- H 05/03 AL 30*14.8 8r41.4' 5205 SE5205 1 r ? --- M 05/04 AL 30*18.7 88*08.2' 5206 SHCH-071 1 7 --- U 05/07 AL 30*13.9 87*54.2' 5142 MS041-90 1 r 150 •-- U 05/07 MS 30*13. 88*31. • LON TH BL MS KD LV BN ST GO BO SK EX CI BT CT FH NE ♦ ♦♦♦♦♦•♦•♦♦- -YYYC ♦ ♦♦♦♦♦•♦♦♦♦♦ ♦ ♦♦♦♦♦■•♦ ♦ •-••♦ ♦ •♦•♦♦♦♦♦-■♦■ ♦ •---♦ ♦ •♦•♦♦♦♦♦♦♦♦* ♦ ♦♦♦♦♦♦♦♦-♦-Y- ♦ --••♦♦ Y- ♦ ♦♦♦♦♦♦♦- •♦•Y- 4.4.4.. ...... ...... ♦ -- ♦ + ♦♦♦•♦ ♦ ♦♦♦♦♦ ♦ ♦♦♦♦♦--♦-. YYY-C 99 Af]pendix I. (continued) SEUS COLLECTION # G LEN GLGS X C MO/DA ST LAT LON 5207 SHCH-072 r 286 --■ - M 2 05/09 AL 30*13.6 87*49, 9- 5235 SE5235 r 179 --■ - F 4 05/10 LA 29°47. 93*30. ■ 5236 SE5236 r 261 -■■ ■ U 5 05/10 LA 29"47. 93*25. ' 5237 SE5237 r 202 --- • M 4 05/10 LA 29°47. 93*23. ' 5262 MS045-90 r 267 -•- • U 3 05/10 MS 30*19. 88*29. ' 5260 MS0«3-90 r ? ■•- • U 4 05/10 HS 30*19. 88*30. ' 5261 MS044-90 r 102 --- • U 4 05/10 MS 30*19. SBTSO. ' 5410 GA343 r 93 0 H 3 05/10 TX 29*19.4 94*U.5* 5263 MS046-90 r 168 --• U 4 05/13 MS 30*21. 88*24. • 5208 SHCH-073 r 215 --- • M 3 05/15 AL 30*32. 88*04, ' 5264 MS047-90 r 231 --- U 4 05/15 MS 30*20. 88*31. ' 5265 MS048-90 r 229 -•- U 4 05/15 MS 30*20. 88*31. ' 5282 MHL-9007F r 207 ■•- F 3 05/17 FL 26*54.7 82*21.2' 5271 MM9012 r 96 ■■- F 3 05/20 FL 27*43.8 82*44.6- 5571 PA r 7 --- U 5 05/21 TX zeroz.5 96'51.5' 5U6 DRC-90-2 1 r 244 --- U 1 05/22 FL 24*44.1 81*00.4' 5283 SHCM-077 1 r 261 --- H 2 05/23 AL 30*27.5 87*55. ' 5431 MCSM-90-r0009 1 r 251 --- F 4 05/24 MS 30*24. 88*54.5' 5272 MM9013 1 r 118 --- H 3 05/27 FL 27*53.2 82*28.2' 5267 CK-01-90 1 r 254 --- M 4 06/02 FL 27*59.0 8r48.5' 5617 90/11/466 1 r? 240 --- F 7 06/02 FL 30*23. 87*27. ' 5280 DSW-90-02 1 r 252 --- M 3 06/05 FL 26*38.3 82T)4.2' 5356 90-11-0491 1 r 229 --- H 7 06/07 FL 30*23. 86*30. ' 5281 FLGM60790-4 1 r 231 - - F 3 06/07 FL 30*23. 86*30. ' 5288 MS050-90 1 r 47 --- U 3 06/08 MS 30*13.5 88*54.3' 5411 GA3U 1 r 206 --- F 2 06/08 TX e • 1 5412 P0153 1 r 248 16 H 3 06/09 TX 28*08. 96*45.8' 5357 MML-9008F 1 r 203 --- H 3 06/10 FL 26*52.2 82*19.5' 5421 LA001-90 1 r 262 --- U 5 06/11 LA 30*07.7 89*25,8' 5289 MS051-90 1 r 206 ■-- H 3 06/11 MS 30*13.5 88*54.3' 5432 MCSM-90-F0010 1 r 126 ---• F 2 06/12 MS 30*20. 88*07. ' 5424 MS052-90 1 r 102 ---■ U 5 06/18 MS 30*22.1 88*50.2' 5133 MM9014 1 r 118 --•■ H 4 06/23 FL 27*55.7 82*32.0' 5366 SHCM-078 1 232 ---• M 4 06/25 AL 30*14.9 88*10,6' 5363 41650-26 1 r 167 ■--■ M 3 06/25 FL 29*40.5 8512. ' 5358 MML-9009F 1 r 89 -■-■ M 4 06/25 FL 26*58.5 82*23.1' 5433 MCSN-90-M0011 1 r 146 ---• M 3 06/26 MS 30*20. 89T)4. ■ 5413 CC097 1 280 ---■ H 4 06/26 TX 2ri2. 97*26. ' 5415 P0155 1 209 --- U 5 06/26 TX 28*07.7 96*46.2' 5367 SHCH-079 1 127 ---- H 3 06/28 AL 30*14.9 88*10.7- 5368 SHCM-080 1 250 ---- H 4 06/29 AL 30*14.2 88*16. ' 5369 SHCM-081 1 249 ---• F 3 06/30 AL 30*38. 88*01.7' TH BL MS KD LV BN ST GO BO SK EX CI BT CT FH NE - ♦-♦♦ ♦- -Y-C Y Y ♦ ♦♦•♦♦ ♦ Y Y Y Y Y Y Y Y C COOES: SEUS=archive #; COLLECTION it. Field Collection #; G, genus, T=Tursiops: LEM= length, cm; GLGS=age; X=8ex; C=condition; MO/DA=month/day; ST=state; LAT^latitude; LON=longitude; TH=teeth; BL«blubber; MS=»nuscle; KD»kidney LV«l iver; BN=booe; ST=stoniach; G0=gon8ds; BD=blood; SIC=skull; EX=other; CI, Y=tissues sent to MWFC; BT, Y=brevetOKin done; CT, Y«tissues sent to EPA; FH, Y=food habits done; NE, C=clinical necropsy 100 Aspendix II: Bottlertose dolphin strandings in the northern Gulf of Mexico. 1982-90. For Texas, the proportion listed is the proportion of Gulf strandings from Texas. YEAR STATE 82 83 84 85 86 87 88 89 90 82-9 TOTAL TX 29 .56 32 .51 84 .73 50 .59 116 .73 111 .57 100 .52 90 34 201 .42 612 .60 813 LA 0 0 6 0 6 17 14 0 49 43 92 MS 1 14 3 12 14 19 36 22 84 121 205 AL 1 0 4 1 0 7 9 11 58 33 91 FL 21 16 18 22 23 41 34 42 86 217 303 TOTAL 52 62 115 85 159 195 193 165 478 102 6 1504 101 Appendix HI: Overall sex ratios and sex ratios for animal <140cni by year for January-June Texas bottlenose dolphins strandings. YEAR I FNGTH TOTAL I FNGTH <140cin MAIF TOTAL MAf F <140cm FEMAIF TOTAL FEMAIf. <140cni M:F MF <140cin 1984 54 18 33 31 10 32 14 4 1.00: 0.45 1.00: 0.40 1985 31 4 .13 13 2 .15 12 1 .08 1.00: 0.92 1.00; 0.50 1986 82 29 35 37 15 .40 23 7 30 1.00: 0.62 1.00: 0.46 1987 114 35 31 52 15 .29 38 5 .13 1.00: 0.73 1.00: 033 1988 91 28 31 42 17 .40 22 3 .14 1.00: 0.52 1.00: 0.18 1989 59 15 .25 23 9 39 27 5 .18 0.85: 1.00 1.00: 0J5 1984-89 431 129 30 198 68 34 136 25 .18 1.00: 0.69 1.00: 037 1990 142 22 .15 58 13 .22 57 6 .10 1.00: 0.98 1.00: 0.46 102 Appendix FV. Report on aerial surveys of bottlenose dolphin abundance conducted in near- and offshore waters off the Texas coast during 1990. 103 Aerial Surveys Keith D. Mullin Southeast Fisheries Science Center Mississippi Laboratory Pascagoula Facility 3209 Frederic Street Pascagoula, MS 39567 Methods Survey Blocks Aerial surveys were conducted in response to two events: the 1990 bottlenose dolphin mortality event and the oil spill from the oil tanker MEGABORG. The surveys completed in response to the mortality event were conducted in block 154 during March, 1990. The surveys associated with the oil spill were conducted during Jime, 1990 in the vicinity of the MEGABORG and with one exception (block "B") duplicated survey blocks studied by Scott et al. (1989) (Figure 1): 152 - 1,296 km^, 153 - 1,588 km^, 053 - 16,292 km^ and 154 - 11,040 km^. Block "B", a 5,850 km^ area in the immediate vicinity of the MEGABORG was also surveyed (see Section II, Figure 1). Sampling methods for all surveys were similar. Sampling Aerial surveys using line transect methods (Bumham et al. 1980) were used to sample the survey blocks. The sampling strategy was similar to that presented by Scott et al. (1989). Transects were selected randomly and were placed perpendicular to water depth isobaths. Samples were designed to sample 7.5% of the surface area of blocks 152, 153, and 154, and 5% of 053. Survey flights were conducted during daylight hours from 22-24 March 1990 and 14-18 June 1990 when the weather was clear to partly cloudy and the Beaufort Sea State was 3 or less. The survey platform was a DeHavilland (DHC-6) Twin-Otter aircraft maintained and operated by NOAA's Aircraft Operations Center. The aircraft had a large plexiglas bubble window on each side which allowed for an unobstructed view of the transect line. Transects were surveyed from an altitude of 230 m (750 feet) at an airspeed of 204 km/hour (110 knots). The flight crew consisted of a NOAA pilot and copilot, and 3 experienced NMFS observers. While surveying transects, one observer was stationed at each bubble-window. The third observer entered data on a laptop computer. The computer was 104 interfaced with an aircraft LORAN system. A data acquisition program downloaded the time and date, and the latitude, longitude, speed and heading of the aircraft whenever sighting data was entered. The observers rotated positions about every 30 minutes. Observers and the flight crew commimicated through headsets via the aircraft intercom system. Observers searched for marine mammals, sea turtles and other marine life at the surface of the water from directly beneath the aircraft out to a perpendicular distance of 629 m. Whenever a sighting was made, the distance of the sighting from the transect hne was measured using calibrated marks delineating 7 perpendicular distance categories on each bubble window (40, 83, 132, 192, 273, 397, 629 m). When necessary, the aircraft was diverted from the transect line to make species identifications and to estimate marine mammal herd sizes. Density Estimation Bumham et al. (1980) recommended that sighting functions should be based on a minimum of 40 sightings, but stated 60-80 sightings were preferable. However, White et al. (1989) suggest that over 200 sightings may be required. Because only 94 cetacean herds were sighted (91 were bottlenose dolphin herds) within 629 m of the transect line during both surveys, the perpendicular distance sighting data were pooled with 1,523 bottlenose dolphin herd sightings coUected by the same survey team in the northern Gulf of Mexico from the same aircraft. These pooled data were used to construct a sighting histogram. To estimate !(0), the value of the probability density function evaluated at the transect line, a hazard-rate model (Buckland 1985) was fit to the histogram. The hazard-rate model was selected for two reasons: (1) the number of parameters in the model is fixed (there was no subjective decision making regarding the number of parameters), and (2) the model always has a shoulder near the transect line (distance zero). Bottlenose dolphin density for each survey block was estimated as the product of a herd density estimate and a estimate of mean herd size. Herd density was estimated separately for each survey block. In order to increase sample sizes and reduce variability of mean herd size estimates, all herds sighted in inshore blocks (152, 153, 154) were pooled as were all herds sighted in offshore blocks (054, B). [Bottlenose dolphin herds in the Gulf of Mexico may increase in size in deeper water.] Of the bottlenose dolphin herds sighted during both surveys, 89% were of 10 dolphins or less. However, three herds were sighted that were greater than 40. Because of relatively small sample sizes, these large herds had a tremendous influence on the means and substantially increased variability. Therefore data were trimmed from each end of the herd size distributions until the means stabilized. This generally occurred after a total of 15% of the data were excluded. The "trimmed" mean herd size for offshore and inshore blocks was estimated as the arithmetic mean. 105 The herd density, t)^, for each replicate transect, i, was estimated as where 1^ was the transect length and n was the number of herds sighted. To insure that no herds were coimted more than once (during each replicate), each transect was considered a replicate. The herd density, 6^, for each survey block was estimated from R replicate transects by and the variance of this estimate was approximated as Dolphin density, t)^, was calculated as ^a'^h the product of the mean herd size (fl) and estimated herd density. The variance of t)^ was estimated using Goodman's (1960) method of estimating the variance of a product v^r (Z?d) -^s% (4) 2+i5^s% (/f) ==-s% (i/) ^sfe (4) 2 where n was the number of herds used to estimate the mean. The standard error was estimated as The approximate 95% confidence interval, assuming lognormal error, was estimated as where 1.96 ./ln(i*(-^)*) C-e V ^ 106 Dolphin abundance was estimated as the product of sampling block surface area and the associated density estimate. Results Transects surveyed during the mortality-event-related surveys totaled 1,845 km. Over 2,200 transect kilometers were surveyed during the MEGABORG-related surveys. Forty-nine cetacean herds, all bottlenose dolphins, were sighted during the mortality event investigation surveys. A total of 42 herds of bottlenose dolphins were sighted during the MEGABORG surveys. Two herds of Atlantic spotted dolphins {SteneUa frontalis) and a dolphin that was probably a Risso's dolphin {Grampus griseus) were also sighted. Table L Estimates of bottlenose dolphio density and related parameien. Block R le(H) n t>. «e(l5h) R t>. •c(l5a) km' f^LSS fi «U« 22-24 March 1990 154 33 036 41 0.042 0.007 27 0.14 0.027 11,040 1,063 1,564 2048 14-18 June 1990 054 6S 1.67 8 0.033 0.010 4 0.21 0.083 1632 1,621 3,421 7,218 -B" 6.5 1.67 5 0.014 0.006 15 0.09 0.044 5350 213 527 1305 152 52 0.95 0 0 - 8 0 - 1,296 - 0 - 153 52 0.95 3 0.035 0.020 6 0.18 0.108 1,588 96 286 847 IS4 5.2 0.95 26 0.048 0.010 13 0.25 0.069 11.040 1.623 2.760 4.694 H - mean bottlenose dolphin herd size n - number of herds sighted C-b- bottlenose dolphin herd density (herds/km') R - number of replicate transects t>,- bottlenose dolphin density (dolphins/km^) km'. survey block surface area «L95. f^. I^U95 - bottlenose dolph lin abundance estimates. lower 95% interval bound, point estimate, and upper 95% interval bound, respectively An f(0) of 3.08 km"^ (se = 0.11 km"^) was estimated using the hazard-rate model. The estimated average bottlenose dolphin herd size used for density and abundance estimation ranged from 3.3 to 6.5 dolphins/herd (Table I). The largest herd sighted was of 52 bottlenose dolphins. Sighting conditions were less than optimal in block 152 and no bottlenose dolphin herds were sighted. In survey blocks where bottlenose dolphin herds were sighted, estimated dolphin densities ranged from 0.09 dolphins/km^ in the vicinity of the MEGABORG (block "B") to 0.25 dolphins/km^ in block 154 (Table I). 107 Literature Cited Buckland, S.T. 1985. Perpendicular distance models for line transect sampling. Biometrics 41:177-95. Bumham, ICP., D.R. Anderson and J.L. Laake. 1980. Estimation of density from line transect sampling of biological populations. Wildlife Monographs 72:1-202. Goodman, L~A- 1960. On the exact variance of products. Journal of the American Statistical Association 55:708-713. Scott, G.P., D.M. Bum, LJ. Hansen, and R.E. Owen. 1989. Estimates of bottlenose dolphin abundance in the Gulf of Mexico from regional surveys. National Marine Fisheries Service, Miami Laboratory, 75 Virginia Beach Drive, Miami, Fl 33149. White, G.C., R.M. Bartmami, L.H. Carpenter and RA. GarrotL 1989. Evjiluation of aerial line transects for estimating mule deer densities. Journal of Wildlife Management 53:625-635. 108 Appendix V. Gymnodinsum brtvt presence/absence' and quantitative cell counts^ SutioD BottoiD Sample Oymnodijuum breve Number Depth(m) Dcpth(m) Pre»cnce/Ab«cncc Quantitative + ± cellar' CaauDoils 10 11 13 24 30 34 36 32 32 30 28 24 26 0 3^ 8.9 12.7 0 + 10.4 + 16.5 + 22.8 0 + 14.5 + 20.5 + 273 + 0 + 16.6 + 23.1 + 32J + 0 + 15.8 + 24.6 + 33.5 + 0 + + 13.4 19J + 29.9 0 16.0 + 243 + 293 0 ++ 8.2 + + 13.7 ++ 28 0 11.2 17.2 27.1 0 11.1 + 183 23.4 + + 0 + 8.7 + 14.1 + 243 20 7 21 3 80 22 35 48 18 21 11 38 160/40 51 22 14 180^1 75 Sediment' Sediment Sediment Sediment 109 Appendix V. Continued. Station Bottom Sample Number Dcptli(m) DcptJi(m) Gynviodmjwn breve Prcscnce/AbscDce Quantitative + ± - cells r' Comments 12 13 14 15 16 17 18 19 20 21 24 22 17 17 12 14 17 10 11 0 + 8.4 + 15.8 + 23.4 + 0 + IZl 17.6 + 23.1 + 0 + 8.2 + 12.2 + 21.9 0 + S2 + 95 + 15.8 + 0 5.2 93 + 15.5 0 7.0 9.2 12.0 0 + 3.2 + 7.6 ++ n.i 0 5S + 82 16 0 Zl + 45 + 8.7 + 0 4.6 73 95 88 66 46 24 440/413 122 66 71 8 14 0 40/2 86 21 104 71 0/32 80/38 Sediment Sediment Sediment Sediment Sediment 110 Appendix V. Continued. SutioD Bottom Sample Number Deptli(m) Dcpili(m) Gymnodmhan breve Prcsence/Abceoce Quantitative + ± cells r' ConuDcnu 21A 22 24 25 12 14 20 16 13 0 0/19 3.4 + 240/45 7.9 + 11.2 + 0 38 53 32 10.2 ^ 1Z9 - 0 + 180/170 5.7 80 15.1 ± 193 ± 0 + «9 5.7 + 58 10.4 + 14.7 + 0 . 176 3.4 + 133 8.6 + 11.6 + 25A Surface water discolored NoaUuca appnxL 900,000 ceUs r> 25B 26 27 28 29 16 20 21 20 0 ± 8,000 r 3.2 + 2-4,000 r' 6J ± 9.2 + 0 on 4.1 5 9.9 - 14.8 + 0 45 3J 56 5.8 + 19.6 + 0 + 38 43 + 80/19 7.9 + 20J - 0 ± 10 6.7 + 51 143 ± 19.6 ± Noaihtca NoaHuca 111 Appendix V. Conltoued. SutiOD Bottom Sample OymFiodinaon brevt Number Depth(m) £>epth(m) Presence/Absence Oiuntiutive + ± cdl$r' Coauiiails 30 31 32 33 34 18 18 14 0 + S.0 + 10.6 + 17^ + 0 + 5^ lOJ + 17.0 0 + S2 + 92 \Z2 + 0 + \Ji + $2 8.4 0 A2 6.6 26 30 3 14 7010 5 0 7 Sediment Sediment Sediment Sediment 'initially, all samples were checked for presence/absence of Oymnodinium breve cells. This was a range finding pnxxdure to evaluate the techniques needed to do quantitative counts. These cunofy observations do not always agree with the quantitative observatioiu. Quantitative counts. See text for details on method for quantitative counts. ^Samples marked "sediment* had too much sediment to allow reliable ccamination. 112 Appendix VI. Contract report on brevetoxin analysis. Analysis and report prepared by the Chiral Corporation. 113 (Cover Sheet) Reference Order #: 40WCNF002505 Issuing Office: National Oceanic and Atmospheric Administration CASC Procurement, CC33 601 R 12th Street Kansas Oty MO 64106 Description: Analysis of bottlenose dolphin liver samples and fish samples for presence of brevetoxins as part of NMFS Emergency Investigation of Mass Mortality of Bottlenose Dolphins in the Gulf of Mexico. Issuing Date: April 19, 1990 Work Commencement Date: September 19, 1990 Work Completion Date: March 18, 1991 Report Date: March 18, 1991 U4 TABLE OF CONTENTS INTRODUCTION AND BACKGROUND 116 STATEMENT OF WORK 116 PRE-AWARD CONDITIONS 116 AWARD CONDITIONS H*^ PROTOCOL FOR LIVER ANALYSES 118 RATIONALE FOR METHODS 121 EXPERIENCE, EXPERTISE, AND PROHCENCY OF PERSONNEL 121 SUBMITTED SAMPLES 122 FEE SCHEDULE FOR BREVETOXIN ANALYSES 123 GENERAL OBSERVATIONS ON SAMPLES RECEIVED . 124 BIOASSAY DURING PURIHCATION 124 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 124 RADIOIMMUNOASSAY 125 POTENTL\L SOURCES OF ERROR 125 RECOMMENDATIONS 126 RADIOIMMUNOASSAY FIGURES AND CALCULATIONS OF BREVETOXIN EQUIVALENTS 127 115 ISTRODUCnON AND BACKGROUND The SEFC has reqxsnsibility for an Emergency Invesdgnion inio the eoent, cause and effea of the mass mortality of bottknose dolphins in the US Gulf of Mexico. Bottlenoce do^hins have been stranding at about 3 times the normal rate along the Gulf of Mexico from north central Florida to south Texas smce the fim of January (over 300 stranded dolphins have been observed). The majority of animals (circa 140) have stranded along the Texas coast. A biotoxin was identiSed as a possible cause of the 1987-88 east coast bottlenoce do^hin dieoS, and it is imperative that samples from the current dieoCT be analyzed for presence of biotcodns. Chiral Corporation was selected to cany out the assays for brevetoxins, based on expertise in the isolation and purification of these potent biotoxins. STATEMENT OF WORK The contractor shall complete analyses of bottlenose do^hin bver saiiq>les for brevetoxins for $2S0iX) per sample NTE S12400 in a term of 6 months. The contractor shaD analyze available marine mammal liver samples and fish samples selected by the NMFS OOTR, in consultation with the oontraaor and other researchers. The contractor agrees to conduct analyses of fifty (SO) san^)les for individual brevetoxins. A detailed schedule of activities wiD be provided by the contractor and agreed to by the OOTR before award. The contraaor also agrees to the following award conditions. Award of this contract is contingent upon acceptance by the CX)TR of the schedule and aU items identified by the OOTR below as award conditions before contract items 1 through S. Rnal paymem is oontingem vpoa acceptance by the OOTR of aD items identified below as Award Conditions Fioa] Report items 1 through 5. The contractor may submit biweekly invoices for saiiq>les analyzed, or may submit an invoice after all tissues are analyzed. The final invoice shaD consist of a draft final iqxm which nieets the specifications of aD items identified below as Award Conditions Final Rqiort items 1 throu^ 5. The draft final repon shaD be received by Dr. T. Siekicki of the Southeast Fisheries Science Center. Comments on the draft need to be incorporated into the final fqx>rt before final paymem can be made. NOTE: Subsequent to Award Notification, Dr. T. Siewicdd was replaced as OOTR by Dr. Sylvia Galloway. We operate under the assumption that Dr. Galloway shaO receive both the Draft and Revised Final Report PRE- AWARD CONDITIONS 1. Provide and agree to detaliled protocols for aD steps of sample preparation, extraction, purification, bioassay, and chromatography. Details should be provided to allow any competent researcher to repeat aD steps, (attached herein as ITEM #1] 2. Provide rationale for methods that sippon the effectiveness of the methods for detection and quantification of brevetoxins in marine mammal tissues, (attached herein as ITEM #2] 3. Identifywhat samples to analyze based on proximity of impacted area, confirmed bloom, freshness of carcass, case history (ind. pertinent Uological dau), proper handling and norage of the sample. AD samples wiD be coded and provided Mind to the contractor. A total of ten suitable control and qiiked-oontrol samples wiD be included in the total (attached as ITEM #3]. 4. Documentation of experience, expertise, and profiden^ of of brevetoxin analyses of tissue samples for aD persoiuiel must he provided. Similarly drtailrd documentation must be provided for any subcontractors to be used. Approval of subcontractois by the OOTR is required before award, (attached as ITEM #4] 5. Spediy amount of tissue needed for each analysis and any tpedai collection, handling or storage procedures required. U6 AWARD CONDITIONS 1 Provide quimiutive bioassay results for each cnidc extract and each preparation resuhing from major puriScation steps. 2. Include aS raw data for all procedures employed. 3. Provide quantitative results on each brevetaxin found in each sample analyzed (per unit sampkr weight). 4. Provide qualitative and quantitative resuhs from all procedures employed. 5. Identify any problems encountered and thoroughly describe any concerns or Umitations to the use. interpretations, or inferences made from the results. INTRODUCTION AND BACKGROUND. SOW, AND AWARD CONDITIONS were communicated by FAX, with follow-up hard-copy, on 7 June 1990. U7 Innovative Probes for Molecular Research PROTOCOL FOR LIVER ANALYSES BREVETOXINS IN DOLPHIN LIVER Spprif"" kems related to tamfde mhwhtioe are otmibered: (1) RECEIPT: Sampbi chaD be received Toesday-Tbursday (shipped Monday-Wedoetday) ai the Chira] CorporatioD Research and Developoeal Uboratory. The proper ^bippiag address is: Chira) Corporaiiao R&D Laboratory Uaivenity of Miami Rosenstid School of Marine and Atmoqiheric Scsence 4600 Rickeabadcer Causeway Miami norida 33149 ATTN: Daniel G. Bades The shipper should make every effort to retain portions of each sample shipped m case of Iocs of sh^nnent, unavoidable loss of sample, or for eonfirmatioe by additioB labocatories. (2) SIZE: Livers shaD be of "«"«i«"'"" size 45-60 f, and iwfividual samples shaD be padcaged in separate swirl packs or other DOs-water-solnUe p»'*^E™£ Other types of sample should be of similar size. (3) SHIPPING: Samples shaD be padced in dry ice, and shipping eootainers shaD be marked as appropriate for infectious agents, as idrntiftcd by the lATA. Appnyriate labels may include those pertaining to toxins, dry ice, and infectious agents. (4) CODING: All samples should be eoded in a random £ashiofi, with copies of the coding retained by the shipper. (5) SAMPLE QUALITY: Liver integrity is of principal importance in assays for brevetonns. No guarantee can be made for analyses resulting froin decomposed, improperly p«'*ig^'<. or otherwise non-pristine materials. (6) SAMPI ^. AGE All hough not qnantififid, cge may have Sipme bearing on the coocentratioas of toads remaining. We do not set a time limit oe sam|rie storage for data is incompleie, but draw anentioo to it as a possible complicating faaor in certain analyses. (7) CONTROLS: Appropriate eoded control amplrs should be submitted, mixed with authentic samples. Two coofirmed cootrcds should be marked as CONTROL, and shipped with the first set of samples, for batft'T confumation. Specific Protocol Steps are Numbered: (1) Upon receipt, fiver samples are nnparkfd, logged, weighed, and placed m a -20^ freezer. We shall analyze 10 samples per week, and based on shipments of this number, no sampk shaD be in our freezer for more than one week. AD samples are treated as iafectioos agenu as defined by HHS publication No. (NIH) 88-8395. (2) AD protocols ntifize pristine ^assware which has been nordtased medficaDvfor the proien md h».; not been used previousK. This is of predominant importance to prevent background lewds of tonn in re-used glassware. AU soh«Dts are of rea^at grade or better. (3) Individual livers an placed m SOO mL gUkt beakers, 300 mL acetone is added, and each is covered with a glass watchglass. Livers soak owemight at room temperature b acetone to dehydrate. The fiver suspension is homogenized using a double blade Vtrtis hoaogenizer operated at 2S00 RPM m an ice bath. The acetone/bver bomogeaate is powed off and is fihered throii^ a bochner funnel using Whatman #1 fiherpaper and eoltected 118 ■Bto SOO bL nde am fiasks aader vaaniiB atpiraiMCL Arrfopc tfttuiiotu arc truuicrrco u> mojviau^ loag ■ed SOO mL ranad boooB fiaikt and eadi ■ fiasb ewiponted oo a rotary evaporator operating at nduccd flrtsuxre. Reclaimed ioKeal ■ placed is five pBoe Bed drmas for appropriate rtnpnaal. (4) Hooogenate fiber caken, togetber with the fiber papert, are placed back iato their mdivuluil SOO mL beakerv muI ^OO mL chtorofarm it added. Hooogeaizaticxi and fibratioc it rcpeaiti (5) The cfalorofonn fihrates are added to the appropriate acetooe residues from ctep (3) and the chlorofons ■ remowed by rotary flasb-evaporation. [Note 1]. (6) Ib the SOO mL flasks «^n«ifm^£ the fiver residues, add 20 g dry silica gel (ICH chromatograpby grade or better) and follow with 100 mL acetone. Swirl each flask nitQ the silica gel scours the residue from the flask liie. and is uniformly tan to brown in color. Flash evaporate acetooe and apply the dried brown silica gel/liver enraa to the top of a 120 mL silica gel flash chromatography column (Baker Chemical). Add 2S mL acetone to the original round bottom flask to rinse any remaining material from the sides and pipette this toUuion oo top of the dried sibca gel in the column. R^ieat once. (7) Assemble the flash chromatography column resevoir and add Z7D oiL acetooe. Apply 4 psi nitrogen (99.999%) pressure to the column qiparatus and ooDect aD ehoed niateiial into dean SOO mL loqg neck round bonom flasks. Flash evaporate loivenl in each case. [Note 1]. (8) Repeat step (6). Substitute 20 mLehlorofonnimethanoLacetic add (100:10:1) flask rinse for the acetone rinse in step (6). (9) AssemUe the flash chromatogra|^ column resevoir and add 310 mL cfaloroformaaethanokacetic acid (100:10:1). Apply 4 pu nitrogen pressure to the column qiparatns and collect aD ehaed material imo a dean SOO mL loog neck round bottom flasL Flash evaporate. [Nou 1, Note 2]. (10) The residue is redissolved m L0-2J) mL solvent (either acetooe or cUoroform is fine) and is appfied to a preparative fluorescent silica gel presorbant thin-layer chrooiatqgraphy plate (20 x 20 cm., 1000 » **«i<*»»^». Whatman PK6F or equivalent) using an Applied ScicBcrs TLC breaking system, nates are chromatogn^ihed using 100 mL of mobile phase ***«i«*"£ ctf acetooedight prtrolrmn (30:70). [Note 1, Note 3). (11) Toxic fractions frt» the TLC plate are scrqied m a safety hood, groimd to a fine powder using a porcelis mortar and pestle [Note 4), and ehtted from the siEca gd in 30 mL sintered ^ass fibers using 2S mL afnot>f or methanol Eluted tooon is placed in individual SO mL round bottom flasks and flash evaporated. (12) Toadc frsefioBS tre redisstrfved m methanol, ud applied to semi-preparative fhioreseeat nlica gd thin- layer chromatography plates C^ x 20 bO., SOO a thirknr.w, Ar^hr^i Uoiplate or equivalenl) and chromatographed using 100 mL ethyl acetaieJight petrofeum (SChSO) as molnle phase. [Note 1, Note 3). (U) Repeal txep (11) for toobc fractions. (14) TooDC fractions are redissolved in methanol, and applied to semi-preparative fluorescent silica gel thin- layer chromatograiAy plates (20 x 20 cm., SOO a riiifknrti, Anahedi Uniplate at equivalettt) and chromatographed using 100 mL ethyl acetateJighi petroleinn (70-30) as mobile phue. [Note 1. Note 3J. (15) Repeat st^ (11) for tooac fraaioos. (16) Toobc fraetioes are £ssoh«d from SO mL round bottom flasks nsiag '"i"^*"*' (03-10 mL ^lic grade methanoL High performance liquid chromatography is performed in ana^ticd mode using a Ranun C-18 analytical reverse phase column (4j6 mm diameter x 2S ^ long) using isocratic 8S% methanol in water, 1.4 mL/minute flow rate. Effliimt is moohored at 215 am [Note 1). f^itmin are caEbrated with known cooceatrationt of analytical grade purified brevetoxins FbTx-L PbTx-2, and FbTx-3, the &ree predominant toxins m natural or cultured systems. Excepting note below and ""'**>^">£ steps, we «^ coofirm the identity of the |^)lc peaks by mixing equal proportioos of anthentv brevetooon and suspect brevttooun from samples. Mixed samples would then be sAjeaed to oo-migratioo in hplc analysis. Quantification of the amount of btevctoaan m the origina] unknown samples can be calculated from hplc data. 119 NOTE: Protocols beyood this aep are BOt withxa the eofltnaed S2S0X10 sunple. We will leek the achue of the Coetraaing Officer prior to imdaiakiqg apy mrrrrrirug a^(, and woold reqoetl approval prior to aaalyus Thuc procedum iDclude brevetosis radiobnaimoasuy, Fourier iraosforD infrared aaalyus, NMR, or bxmss tpeOTcmtuy. NOTES |1] Bioauay: Gombiuia it^intf, or moiqmto fish, are ittcd as bioasuy ^Kdmea for aD tte|&. Assays ■re cooduaed is 20 mL leawaier of 15% salinity uiiig 1 fish per 50 mL beaker. For lohitioas, cuspea toxxo ■ a>-absarbing bands. Remove lie plate from nv Ughi and cot two grooves from origin to solvent front, placed 03 cm qaait, qiprooomately 10 cm in from the sides of the plate. From origin to solvent fatrnt, cut 1 cm laments fatn the developed plate betwuji the two parallel score marks, and cnuh each 03 cm x 1 cm silica gel rmanglr into a powder to be added tobioassay beakers. f4] CAUTION: Brevetoxins are potent re^nratory irritants, especially when adsorbed oo siEca gel particles. Take aO precautions necessary for respiratary irritants &at are riwrififd as Class I poisanous ffuKtt"":* NIOSH certified panicle filtration masks may be worn in qiplicableiitnatiaas where expossre could occur. Al 1 scraping operations and grinding of silica gel powders tbould be canied out in an approved ventilation hood. 120 Rationale for Methods Hreveiniim Tc maieriils which becnae eoaceatraied in nmae titniet throngfa annwl feeAng trtjuit^t jf PP«f fwnittr<< rrfmniant Thc tonu, lynthrtTTT/l by thc marine faofUgellate ft>cfcadbaa b»rw «rc aorm*l)> fcKf^^n^wnuUte/i b fiher-feedtog orguisnu, but recent evidence hxfirnci that biomagnificatioc through the food T*i«iti by ahenaie roates oiay ako occur. The »<^»*«f« and parificatoB procedure and iafividna] protocols for brevetoiis from dolphio bvcrs is both jr^**^^ and laborious. Liver, bung the prindpa] drinrififatinn organ of «"»"""■>■■" ipcocs (and more ao in dolphins which lack a gaD bladder), is the organ we nprrt to find breveiams in their largest conccntratiom ■hould they east. Liver by its nature contains high coBceatratioBS of the eazynes necessary to detoxify organic ppH^ftwn isduding toodns. Also by its funetional nature is the ttor^ge depot for er»n«wim«tm to which the bviag doti^iin has been exposed. Iffitial aeps in the pnrificatioo seek to first denature any enzymatic machinery which may exist in th; frazen qiedsens using a modified acetOBe predpitatioo stq>. TUs ttep also tends to dehydrate the specimen and make e&raetioo of Iqiid soluble materiids easier (tlep 3 of protocols). Following dehydratioo, a noo-polar solvent (chloroform) is used to extract the brevetoxins from the tissue samfde. Brevetooons are freely sofa^ in the chloroform (step 4). Steps 5-16 are steps utthzed routinely and daily by Chira) Corporation in our protoctds for extradioo and purificatioo of brevetoiins from laboratory cultures of the **^***^^g'"**^. and as sudi are the results of 16 years of optimizatioa and progressioo from anal3^tical separation to large scale quantitative recovery. Flash chramatogra|diy (steps 6-9) is a routine and powerful way to separate toodc materials from fPfffftnmatmg oOs and pigments, the former of which is a massive probefan in marine mammal tissues. These initial steps are utlized in the order — and on the scale that thqr art 'to isolate miotigram quantities of toadn from gram quantities of interfering snlnianrrt Three sfqumtial thin-layer chromatograpfay oqs are utiEzed to isolate brevetoxins from one another, and are the exact trfhniqnrt that were used in our laboratory to first determine the individual nature of PbTi- 1, PbTx-2, and PbTz-3. When compared to the known migration of standardized brevetoaons, preliminary identity (tf suspect toxins can be made. The final step (tf high performance Equid riiTnmatogra|Ay is an analytical technique, «4iidi when ^ipiied in the parallel presence of standard brevetoasas, can ^ve a further jmjjiigj basis for ooofirming or denying brevetoxio identity. The Cambusia ajfinis fish bioassay is the most sensitive bioassay kimown for the brevetoxins, and individual brevetoiins exhibit lethal dose ranges in the nanooolar ooBcentratioB ranges (Le. aanomoles of toxio per liter of test water, fish are placed in 20 mL water «4iich yields a calculated sensitivity of 17.9 ^assay volume. We bebeve it is accessary to emphasize that '*' bioassay means the fish dies witUn the time period of observation. For most sensitive applications, observatioos are oiade over 48 hr, but positives fi«quently are exhibiied within hours of initial laqxKure. Those fraaions which test *•', Le. do death, are not pursued futher. Thus, a positive value does ajt necjsiarily iadi^tte br. noapn, bat a amative by these criteria does not contain brevet axis. Eadi step of the purification and analysis which tests positive lends increasiag support to the indications that brevetoxis are present in the sample. By back^4»««M2ii>f ased routinefy by the Corporation to purify brevetoaias; and (W) progressively affirm or deay the ideatity of toobc «i««tiTi«K as wevetoiins E)q>erience, Expertise, and Proficiency of Personnel The cnnicnhun vitae for Daniel G. Baden. President of Qiirs] Corporation, and Lloyd S. Schuhnaa, pan- 121 tne tr'*"^^-" for Chiral Carparatiae. art iadiided with tbete doamnrtt Dr. Bades has 17 ycMn qpciieace «i^ Uitiiuiim. aod Uoyd SchnlmaB has ewer four yean inrtmiral rrptiriwr wilb puriCcatioo aod aysuSijxMacm el hrwcuaaai. Submitted San^les (dupHratr^l oo Pratoco] iheeu) (1) RECEIPT: Saaplck ^aD be received Tuesday-Tbanday (chipped Moaday-WcdBctday) at the Chin] Con»oratjoo Reieardi and Dewelopncal Labocaioiy. The proper shippiqg addmt it CUral Corporatioo R&D Laboratoiy Usfvenity of Miami RoKsstiel Scbool of Marine and Atmospheric Sdeoce 4600 Rickeabuker Causeway Miami Florida 33149 ATTN: Daaiel C. Bades The chipper should make eveiy effort to retain portioes of each campk sh^iped in case of Iocs of shipnenl, Bnavoidable lou of campk, or for eonfirmatioo by additios laboratoriec (2) jgTTF- Liven chaD be of miaimnm cize 45^ f. and iwfividua] camples dull be pack«ged in separate twirl packs or other noo-water-cohible packaging Other types of cample cbonld be of cimilar cize. (3) SHIPPING: Samples duD be packed in dry iee, and chipring oootainen chaD be marked as appropriate for infectious agents, as identified by the lATA. Appropriate labels may indude those pmaining to toodns, dry ice, and infectious agents. (4) CODING: All camples choold be coded in a landom fashioB, with copies of the oodiqg retuned by the chipper. (5) SAMPLE QUALITY: Liver imegrity is of princ^ importance in assays for brevetoadns. No guarantee can be made for analyses resulting froa decomposed, improperly parkagrd, or otherwise noe-pristine materials. (6) SAMPLE AGE Ahhou^ not quantified, ^e may have come beariqg on the concrnrrations of tooDo remaining. We do not set a time bnit on cample ctorage for data is incomplete, bat draw anentioo to it as a posuble coofdicating factor m certain analyses. (7) CONTROLS: >^>propriate coded rootrol camples chould be cidmutted, miad with authentic camples. Two confirmed controls slxwld be marked as CONTROL, and chipped with the firtt cet of camples, for baseline (8) RIGHT OF REFUSAL: Chiral Corporatioo leaves biolopcal oceanqgr^diic questions of appropriateness of each cam|^ to the '■**'*«^^««g organizatioo(c). Our tuk is to affirm or deny the presence of brcnctooDns in the supplied camplft Chiral Corporation retains the right to refuse any roned or purifying camples, old or obviously decayed materials, bven of a less-than*pristine nature, or aaaterials whidi may pose a safety risk to our personnel Refused camples wfll be returned to the oootratxii^ agency. 122 FAXNombcr. (305)661-0140 Fee Schedule for BrevetoxiB Aiulyses Per Sample Costs (1) HomogenlMtlon, extractloii, and Cambusia fish bioassay of trade samples *l25.w (2) Solvent Partitioning, Silicic acid flash ^^ chromatography, and bioassay of crade extract $ 35.00 (3) -nun-layer Plate Silica Gel Chromatography of fractions from (2) * ^^ (4) High Performance Uqnid Chromatography of fra^ns , ,_ .^ from (3), compared with brevetoxin standards L2LBI Total Cost Per Assay $250.00 Additional Analytical Protocols Available hot not Included in above assay price (5) (6) (7) Radioimmnnoassay for Brevetoxins $ 20.00 Sodium Channel Receptor Assays for Brevetoxins $ 35.00 Fourier Transform Infc ared Analysis of purified materials $ 35.00 123 GENERAL OBSERVATIONS ON SAMPLES RECEIVED AQ samples ippeared to be of bver, with « fibrous constitution consistent with dolphin bver examined in the past. Samples ranged in size from a low mass of 12.411 g to a high mass of 110.614 g. Twenty-eight of the samples weighed below the ^jcdfied 45-60 g. Twenty-dght samples were judged to be of good condmon when received with no evidence of decomposition, eight were slightly freezer-burned, an additionaJ four were severely freezer-burned, and ten were received in homogenized condition, except as noted for sac. no sample was deemed unsuitable for testing. Samples were numbered in the laboratory as #]-#50 based on leoeipt and were not re-correlated with NMFS identification untS completion of assays. BJOASSAY DURING PURIFICATION Based on the size of a substantial number of the sanq>les, bioassay was not performed at neps (1) through (9) of the "Protocol For Liver Analyses: Brevetoxins in Dolphin Liver" [labeled ITEM #1J. Further, based on the consistent quality of the supplied samples, note [2] 'Aberrant Saiiq>les" did not tppYf. At step (10) in the protocols, the first preparative thin-layer silica gel diromaiogr^hic step, thirty-five (35) of the samples were non-taodc by Gambtaia affitiis fish bioassay and analyses were terminated in these cases. Negative sao^les at this stage were: GA342 SP112 MM9012 MM9008 SHCM0T7 PO095 MM9013 GA334 GA336 GA311 GA344 MS018-90 GA319 lAOOl GA332 PA1S3 GA321 SPlll GA314 P0123 GA335 PA192 P0134 SPllO F0125 GA313 GA315 GA339 GA302 P0135 MM-9007 2505-7 2505-8 The remaining sao^les tested positive by fish bioassay in at least one fraction of the panected thin- layer dut>matography plate. Nine of the samples were tone in multiple sections of the plate indicating a multiplicity of toxic materials. Of the samples which tested positive at the first TLC plate, twelve tested negative following thin-byer duomatography on the second plate (stq> 12). The samples «iuch tested negative at this step were: GA333 GA304 GAllO SP114 2505-1 2505-2 2505-3 2505-4 PA195 PIQ38 P0121 GA340 Following this diromatogn^hic step, five (5) san^les remained for further analysis. Of these, OO094, 2505-5, and 2505-6 were judged to be of limited quantity and were not subjected to the third thin-layer step (Rq> 14). Samples 2505-9 and 2505-10 possessed toxic fractions following cfaromatognqihy according to Step #14_ HIGH PERFORMANCE UQUID CHROMATOGRAPHY Based on migration of standard brevetoxins, san^les CX3)94, 2SQ5-S, and 2505-6 were negative by HPLC That is to say, they contain < 5 Mg toxinAotal original sample. Samples 2505-9 and 2505-10, when liPLCd, indicated the presence of PbTx-2 and this was confirmed by mixing with authentic PbTx-2 and re- injecting. Co-migration of the mixed samples further suggested authenticity. 124 RADIOIMhfUNQASSAY This procedure wis not within the oontncted SOW, and wis bsted in the Fee Schedule as ar Additional Ana)ytical Protocol. However, based on the hmhed number of samples reaching HPLC immunoassay of leveral samples wis undertaken to confirm identity, at our expense. At present, only brevetoxins and dguatoxin are known to cross-react in this radioimmunoassay. The %amp)es tested were: CC094 (podtivt through two TLC pises, negative by HPLQ 2505-5 (positive through two TLC plates— two fractions, negative by HPLQ 2505-6 (positive through two TLC ptates, negative ly HPLQ 2505-9 (positive through three TLC plates— four fractions, positive Yry HPLQ 2505-10 (positive through three TLC plates, positive by HPLQ T^ resuhs of RL\ are flhistrated in Rgures 1-5. An intemal standard displacement carve for unlabeled PbTx-3 as compcihor (against fixed trttiated PbTx-3) is used in each experiment. Based on these resuhs, and we feel these are unequivocal, each of the five samples indicated above was oontammated with brevetoxin to one degree or another. Based on RIA, the amooirt of brevttoxin contammatmg the ongmaJ samples was calculated to be as follows: cam 0390 Mg per 38i)36 g, or 102 Bg^ liver 2505-5 0.460 Mg per 41.255 g, or 12.1 ng^ bver 2505-6 0385 Atg per 43.482 g, or 933 ag^ liver 2505-9 im Mg per 46.639 g. or 240 Bg^ liver 2505-10 0.815 Mg per 45332 g, or 17jO ng/g bver POTENTIAL SOURCES OF ERROR We antic^>ated and were informed that random brevetoxin-spiked samples were induded amongst the 50 samples submitted for analysis, and that^amples fiom the previous Atlantic do^hin die-off of 1987-1988 might also be induded. In the former instance, that of ^iked samples, we expea to have little dif&uhy in identifying PbTx-2 and PbTx-3 (fairiy stable materials) and greater difBcuhy with Pbli-l or aiy of its oogeners (an unstable polyether backbone structure). We in fact expntsed our uncertainty with respeo to toxins with the PbTx-1 backbone prior to contract issuance. With reqject to samples from the previous Atlantic bottlenose die-off, we expressed a concern for the length of time in storage, whidi if it was not at -80*C might not be sufficiently cold to inhibh aO enzymatic activity. Our previous experience with a progressive reduction in dguatoxicity in shark bver is our basis for this concern. In these latter two cases, we might eipcfl to tee less or no brevetoxin in samples, whether q>iked or authentic Trace brevetoxin contamination within our R & D laboratories is a potential problem, there being brevetoxin purification progressing at any time. To alleviate the potential for oontamiiuaion, pristine glassware was purchased for these analyses, and was used only once. Thin-layer chromatography plates were newly unopened boxes and soWents were freshly opened prior to use. HPLC syringes were sequentially rinsed in an duotrophic series of soWents as were aniJytical HPLC cohimns prior to use. Fixed baseline at the absorbance sensitivity used for analyses were a pre-requisite. Ehition times which match authentic brevetoxin retention times are circumstantial and not ifieatiBactioa means in an authentic sense. Nor is oo-migration of authentic and unknown in the same sample. However, co-migration lends an iitcreased measure of certainty to the conclusions. Sensitivity of deteaion is prinqpaOy a function of ultraviolet absorbance, and this decreases from PbTx-2 to PbTx-3 to PbTx-9, and from PbTx-1 to PbTx-7 to PbTx-10. Thus, h wiD be more difficuh to detect PbTx-9 and PbTx-10, less difficuh to see PbTx-7 and PbTx-3, and relatively easy to see PbTx-1 and PbTx-2. 125 RadioimmuBoassay. again is not an analytical tool in the cxhct lense of the word, although posnive resihs in this test would tend to indicate a simDamy in stniauie more finely ascertained than TLC or HPLC migration. Being that brevetojms and dguatoxin are the only materials known to inhibit specific binding of brevetoxin to its ^ledBc antibody, one can condude with tome certainty that the five samples identified above contained brevetoodns or a very similar toodn filce dgiutonn. Concentrations in origina] samples are based on aliquot and cub-samples at cadi step, and are ultimately based on "PbTx-S^qtiivalents* in brevetoxin immunoassay. Efficacy of di^laoement of tritiated brevetoxin by unlabeled brevetoxin oogeners varies about 15-20 %. This obviously is a potential source of error. We can make no conclusions about the presence of toxin in any particular sample relative to ecologiuJ constderations, bloom conditions, proximity to contaminated fish sources or the like. All samples were received in a coded fashion, make-up and origin unknown to us. RECOMMENDATIONS (1) Continue collection of data and refine assaiys for detection. (2) Notify Chiral Corporation of identity of aD ^riked oontit^ and unknowns for our record- keeping and to aid us in refining our techniques. (3) Maintain a groi^ on alert to properly coDea and marie tissues (preferably fieeze-damped with dry ice to preserve). (4) Establish r^id nspoast protocols to aid in detection. (5) Establish protocols for handling of tissues (We treat aD samples as if they were infectious agents as defined by DHHS puUication No. (NIH) 88-8395.) 126 0.1 1.0 10.0 100.0 COMPETITOR CONCENTRATION (units) 1000.0 Figure 1. RIAof (•) 2505-9 fraction 4^ [plate 3], (^ 2505-9 fnctioii 73 \pi»te 3). (▲) 2505-9 HPLCpeak. (^) PbTx-3 intenia] nandard curve. 01 1.0 10.0 100.0 COMPETITOR CONCENTRATION (units) 1000.0 Figure 2. RIA of (•) 2S05-6 fraction 3 {plate 2), (^) FbTz-3 inteiiul standard curve. 127 I looi m u t2 80- u u Qu OQ D 60-- 40-- 20-- 0- Z u u £ -20 a. l.OE-2 0.1 1.0 10.0 100.0 COMPETITOR CONCENTRATION (units) 1000.0 Figure 3. RIA of (•) 2505-10 fraction 7 Iplate 31, (♦) PbTx-3 intenul fUndard anve. 0.1 1.0 10.0 100.0 COMPETITOR CONCENTRATION (iiniU) 1000.0 Figure 4. RIA of (•) OO094 fraakn 5 [plate 2), (#) PbTx-3 mtenu] stnadaid cnrve. 128 ^ 100 0.1 1.0 10.0 100.0 COMPETITOR CONCENTRATION (units) 1000.0 Figure 5. RIA of (•) 2505-5 fraaion 3 [plate 2], (T) 2505-5 fraction 5 [plate 2], PbTx-3 internal standard curve. 129 CALCULATIONS FOR RADIOIMMUNQASSAY PbTx-3 Unh concentrations ^ OJOl nMoies/bter to 100 nMoles/lher, or OJOl pMol/mL to 100 pMoI/mL 50% displacement (EDjg) of tmiated toxin occurs at IjO pMol/mL (0.895 pgrams) Unknowns Unit concentration « 125 fiL of iOOfd, original san^le (125%)« 100 Units 1 Unit » 0JQ12S% of sample Figure 1 ( #) 2505-9 fraction 4^ [plate 3] adjudged incondusive Iy) 2505-9 fraction 73 [plate 3] adjudged incondusive (A) 2505-9 HPLC peak 50% di^lacement at 11 Units or 0.1375 % at ehited peak (11 z 0i)125%) Therefore, 0.1375% * 895 pgram equivalents in HPLC peak fay oonqurison with standard curve for PbTx-3. 100% / 0.1375% - 72727. Yielding (895 x 727)- 650,655 pg. or 650 ng in total peak. The peak assayed was 50 mL/IOOO^L total, wfaidi « 650 ngx (1000/50)- 10 ug PbTx-3 equivalents in sample prior to HPLC Back calculations to original amount: three plates, using 42% or each plate for bioassay - 87i^ toodn remaining at HPLC stq>. (87.9% X 10 Mg equivalents calculated against standards) - apppiujumately 11 ^g brevetoxin in sample 2505-9. 'Based on 46.639 g of liver supplied, appioximately 024 fig toodn per g liver was presem in the sanq>le.* Figure 2 ( • ) 2505-6 fraction 3 [plate 2] 50% displacement at 20 units or 0253% of sample at this stage (20 x 0i)125%). Therefore, 0253% - 895 pgram equivalents in fraction 3 from the TLC plate by comparison with standard curves for PbTx-3. 100%/D253% - 39526, Yielding (895 x 39526) - 353,757 pg. or 353 ng in total sample. That 353 ng is equivalent to 91.8% of the total sanqile (100% z 0i^8 z 0.958 - 91 J%). Therefore, the sample was calculated to contain 385 ng brevetoxin (353 x 100%A)1.8%). 'Based on a liver we^t of 41255 g, appfoiimately 933 ng taadn per g fiver was present* 130 fisurej (•) 2505-10 fraction 7 [plate 3] 50% di^laoement at 10 unhs or 0.125% of sampie at this stage of puriScation (10 X 0.0125%). Therefore, 0.125% * 895 pg eqtiivaients in fraaion 7 from the TLC plate by comparison with standard curves for PbTx-3. 100%A3.125% - 800, Yielding (895 x 800) « 716,000 pg. or 716 ng in total sample. That 716 ng is equivalent to 87.9% of the total sample (100% x 0.958 x 0.958 x 0.958 = 87.9%) Therefore, the sample was calculated to contain 815 ng brevetoxin (716 x 100%/87.9%). •Based on 45^33 g of liver supplied, approximately 17 ng of brevetoxin per g Bver was present.* Figure 4 (•) CCD94 fraaion 5 [plate 2] 50% displacement at 20 unit or 025% of sample at this stage (20 x 0.0125%). Therefore, 025% « 895 pgram equivalents in fraction 5 from the tic plate l^ comparison with standard curves for PbTx-3. 100%/025% » 400, Yielding (895 x 400) ■ 358,000 pg, or 358 ng in total sample. That 358 ng is equivalent to 91 J% of the total sample (100% x 0.958 x 0.958 > 91.8%). Therefore, the sample was calculated to contain 390 ng brevetoxin (358 x 100%^1.8%). * Based on a weight of 38.036 g liver supplied, approximately 102 ng toxin per g liver.* Figure 5 ( •) 2505-5 fraction 3 [plate 2] 50% displacement at 100 units or 125% of sample at this stage (100 x 0.0125%). Therefore, 1 25% > 895 pg equivalents in fraction 3 from the TLC plate by comparison with PbTx-3 standard. 100%/125% > 80. Yielding (895 x 80) - 71600 pg. or 71 ng in sample. That 71 ng is equivalent to 91 J% of the total sample (100% x 0.958 x 0.958 - 91.8%). Therefore, the sample was catailatrd to contain 77 ng brevetoxin (71 x 100%^1.8%). 'Based on a liver mass of 412S5 g. approximately 19 ng brevetaxin ▼« present in the sample.* 131 (9) 2505-5 fraction 5 [pUte 2] 50% drqiUcrment u 20 or 025% of sample at this Rage (20 x Oi)I259b). Therefore, 025% ■ 895 pgram equivlaents in fraction 5 from the TLC plate by oompasion with standard curves. 100%yD25% - 400. Tielding (895 x 400) - 358.000 pg. or 358 ng in total sample. That 358 ng is equivalent to 91.8% of the total sample (100% x 0.958 x 0.958 * 91 J%). Therefore, the sample was calculated to contain 390 ng birvetoxin (358 x 100%^1.8%). * Based on a weight of 38i)36 g bver supplied, appimlmately 102 ng toxin per g bver.* 132 Appendix VII. 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'o. ^ "D E d d. d GL d cL 155 s CM cy] o p ^ ^ CM CO 9 o it E 3 0> c CO 8 ca o D. To 3 «. c c o ■E 8 § I 'a 1 E s o m I 1 CM a S M C z CO s Si n t^ o in iDioincMinar^MmcMCM o o ^oincviiDiomcMina 3 JS CO u Q. "a 3 5 m^>a»0«-«CMtn^l•no^>M▼n'OV ii>o»«o»a»*r>.pe>r:!P'"««'»^^« 8 2 2 R Si '^ 8 " Si « ^"8 MjrMacpcv|fD^CMamacwMMi«>^ £----- -CM o — CM r: « c c 55 .«>>««,.»«t^ S a> ^^ s.3i> Illlllfffililll III & & & S 2 fi O O w 1^ I JO- •8 s {: "S " <" - U 2 M K K ^ 1 i i 2 lA (;> in in in in in SSlnlnVin^-Ve S.^^'Oinvinin in V V •« T V* «> *" * *" InlnVin^-^c^v^^co^^mcop) inV(^in^VnT(^<^IX*^*^(^(^*^<^ Im V (m Im n hi CO p> (m* Ki hi Im hi hi hi hi kT cmcmcmcmcmcmcm'cm'cmcmcmcmcmcmcmcmcm 156 Appendix VIII. Available clinical necropsy and histopathology reports of bottlenose dolphins stranded in the U.S. Gulf of Mexico during Januaiy-June, 1990. 157 This page intentionally left blank. 158 TEXAS MARINE MAMMAL STRANDING NETWORK NECROPSY EXAMINATIONS SPRING 1990 Eight animals (all Tursiops truncatus) have recently been examined. Three (GA-321, PI-38, CC-94) were necropsied at the Texas Veterinary Medical Diagnostic Laboratory (TVMDL) in College Station, Texas and five (GA-287, PI-35, SP-114, PA-195, GA-342) were examined at the Galveston Marine Research Laboratory (GMRL) in Galveston, Texas. Veterinary affiliations of investigators: TAMO - College of Veterinary Medicine, Texas A&M University TVMDL - Texas Veterinary Medical Diagnostic Laboratory AFIP - Armed Forces Institute of Pathology NOSC - Naval Ocean Systems Center Sea World, Inc. In order of stranding date: GA-287 Date stranded: 12/11/89 Sex: Female (adult) Length: 242.7 cm Weight: 168.0 kg Pathology notes: Held in freezer prior to necropsy at GMRL. Necropsy by Dr. Linnehan (NOSC, San Diego) and Dr. Tarpley (TAMO) . Older animal - teeth fairly worn. Parallel rake marks over body -some fresh. Several punctate lesions on skin. Viscous yellowish exudate from central lacteal of right maimnary gland. SevereJ. nematodes in lung - not extensive. Several Nasitrema in left pterygoid sinus; one in right. Monorygma cyst at apex of urinary bladder. Corpora on both ovaries. Endometrial cyst in one uterine horn. Histopath tissues collected but not yet examined. Pathology summary: Possible mastitis. PI-35 Date stranded: 2/5/90 Sex: Female Length: 231.0 cm Weight: Pathology notes: Held in freezer prior to necropsy at GMRL. Necropsy by Dr. Magee (NOSC, Hawaii) and Dr. Tarpley (TAMD) . Moderate PMA and no remarkable gross lesions. Evidence of coyote scavenging around head. Lead pellet embedded in dorsal fin base but no sign of entry - probably ante-mortem. Pathology summary: No diagnostic conclusions. 159 SP-114 Date stranded: 2/22/90 Sex: Female Length: 234.5 cm Weight: 138.6 kg Pathology notes: Held in freezer prior to necropsy at GNRL. Necropsy by Dr. Tarpley (TAMD) . Many wraps of monofilament fishing line around left tailfluke blade. Blubber thickness 1.5 - 2.4 cm. Several punctate skin lesions over body. Minimal tooth wear. Essentially no parallel rake marks. Adhesion (17 X 15 cm) of cranial portion of right lung to thoracic wall. Extensive adhesions of loops of intestine with each other and with pseudopancreas and mesentery. No gross tissue reaction appeared to be associated with these adhesions. Pathology summary: Fisheries interaction mortality. 6A-321 Date stranded: 3/13/90 Sex: Female (juvenile) Length: 161.5 cm Weight: 40.5 kg Pathology notes: Held on ice and necropsied without freezing at TVMDL. This was a live stranding but received no treatment (died before TMMSN personnel arrived). Necropsy by Dr. Robinson (TVMDL) and Dr. Teirpley (TAMO). Microbiology by Dr. Whitford (TVMDL). Several rake marks (some fresh) and severe skin lesions randomly distributed. Skin lesions resulted from local ischemic necrosis caused by blockage of vessels by bacteria. Lymphoid hyperplasia in lymph node. Focal necrosis and inflaiOJiu'jory infiltration of lung tissue. Bacterial isolates: Enterics from skin and Aeromonas lung. No viruses isolated. Pathology summary: - Bacterial pneumonia and septicemia. PI-38 Date stranded: 3/28/90 Sex: Female (adult) Length: 246.5 cm Weight: Not weighed Pathology notes: Held on ice and necropsied without freezing at TVMDL. Considerable PMA. Necropsy by Dr. Fiske (TVMDL) and Dr. Tarpley (TVMDL). Histopath by Dr. Fiske. Microbiology by Dr. Whitford (TVMDL). Term fetus wrapped in amnion found free in abdominal cavity. A tear in the uterine wall may have been caused by knife cut when abdomen was punctured by unknotm person on beach. Bacterial isolates: Enterics and Aeromonas from intestine (no Salmonella) j Enterics and Clostri'dlum perf ringens) from lung. Pathology sunmiaryl Possible calving-associated mortality. l60 CC-94 Date stranded: 4/1/90 Sex: Male (juvenile) Length: 160.0 cm Weight: Not weighed . ^ .^w ^ * Pathology notes: Held on ice and necropsied without freezing at TVMDL. Necropsy by Dr. Jones (TVMDL) and Dr. Tarpley (TAMU) . Histopath by Dr. Robinson (TVMDL). Microbiology by Dr. Whitford (TVMDL). Several rake Barks and skin ulcerations with bacteria present in dermal vasculature. Pancreas very fibrotic with enlarged and granulated hepatopancreatic duct. Lymph node hyperplasia. Some portal fibrosis in liver. Lung contained fibrotic nodules and fibrosed terminal bronchioles. Salmonella isolated from skin lesion. Pathology summary: Severe pancreatic fibrosis. Nodular pneumonia with peripheral skin abscesses. PA-195 Date stranded: 4/7/90 Sex: Female (adult) Length: 240.0 cm Weight: 113.6 kg Pathology notes: This was a live stranding from Port Aransas region but died before TMMSN personnel arrived. Put in freezer initially but removed before freezing and transported to GMRL and held in cooler prior to necropsy. Necropsy by Dr. Tarpley (TAMD) and Dr. Schimeca (UTMB) . Histopath by Dr. Robinson (TVMDL). Microbiology by Dr. Whitford (TVMDL) . Extensive parallel rake marks (mostly fresh) and soiae dkin lesions with vasculitis and necrosis of epidermis. Older animal — considerable tooth wear. Generally emaciated. Congested lungs with regions of emphysema and -interstiticd fibrosis. Lymph node fibrotic and depleted of lymphoid cells. Spleen also has lymphoid depletion. Considerable portal fibrosis in liver. Pseudomonas and Enterobacter isolated from lung. Pathology summary: Possible bacterial septicemia associated with pneumonia. 161 GA-342 Date stranded: 4/25/90 Sex: Male (subadult) Length: 220.0 cm Weight: 94.5 kg Pathology notes: Live stranding from Galveston region. Therapy was limited to steroids (Solu-delta>corte£ 100 mg iv) on beach, fluids (water via stomach tube), vitamins (A&D combination, B12 and B complex - 5 cc of each IM) and Valium (100 mg IM) . Several blood samples were taken for clinical evaluation and culture. RBC and NBC counts low with no band neutrophils. Laboratory Submissions: Clinical Hematology — • St. Mary's Bospiteil, Galveston, TX Blood taken for CBC and chemistries (two samples) Clinical Toxicology ~ 3 cc serum to be sent to Dr. Ridgway (NOSC, San Diego) for heavy metal analysis. Microbiology Bacteriology and Mycology 1. St. Mary's HospitcJ. — blowhole swabs (2) , blood for culture (bacteria and fungi) , lung tissues (necrotic and hemorrhagic) , lung swabs, lymph node tissues. 2. The University of Texas Medical Branch in Galveston <~ blowhole swabs, heparinized blood sample for media inoculation. 3. TVMDL (Dr. Whitford) — lung and lymph node tissues for culture. Virology — Dr. Al Smith (Oregon State University), viral swabs taken from blowhole and anus and a seel- ion of active skin lesion and placed in media — saiu^les frozen at - 80 C at DTMfl to await shipment. Histopathology 1. APIP (Dr. Lipscomb) - set of tissues 2. TVMDL (Dr. Robinson and Dr. Jones) - set of tissues Necropsy performed immediately after death at GMRL by Dr. Lipscomb (AFIP) , Dr. Dover (Sea World, San Diego), Dr. Magee (NOSC, Hawaii) and Dr. Tarpley (TAMO). A young animal - minimal tooth wear and immature testes. Several punctate skin lesions. Several regular shark bites (not cookie- cutter) . One active lesion (not erupted to surface but swelling during the clinical course) with fibrin thrombi and neutrophilic vasculitis. Evidence of chronic bronchopneumonia (may be associated with lungworms or infectious organism) and acute septicemic pneumonia (short rod bacteria found in lung tissue and in pulmonary arteries) . Pulmonary lymph nodes and pseudopancreas contain 162 areas of necrosis (granulomatous inflammation) with multinucleated giant cells. Both lymph nodes and pseudopancreas appear depleted of lymphoid cells, other lymph nodes and spleen do not appear depleted. Congestion and portal fibrosis with mild fatty change in liver and lymphoid aggregates in portal areas and beneath liver capsule. Approximately 10 ulcers in mucosa of distal esophagus, a bleeding ulcer in fundic chamber near junction with forestomach and several longitudinal mucosal erosions in pyloric chamber. Lung and lymph node isolates from by both St. Mary's and TVMDL were E. coli (said by TVMDL to be hemolytic) . No bacterial isolates on blood culture (including no acid fast bacilli or anaerobes) — fungal culture still in progress. TVMDL commented that immunocompromise must be considered for a relatively non-pathogenic organism such as E. coli to have caused an acute effect. Pathology suomary: Hematogenous bacterial pneumonia. 163 Tursiops truncatus Fenale This dclDhin stranded in Karch 1990 and was held frozen in Galveston unt'il necropsy on 5 June 1990. GROSS NECROPSY EXTERNAL This appears to be an older dolphin with moderate tooth wear. However, there is ? remarkable absence of teeth, especially In three arcades (all but left upper). Crown wear in the teeth which are present does not appear to justify the complete loss of so many teeth. The alveolar sockets are healed, indicating that this loss was not extremely recent. The abdomen is rotund and suggests pregnancy. The genital groove is somewhat dilated in support of this. There are numerous light gray irregular spots in the otherwise unpigmented ventral abdomen. This is apparently normal coloration for this animal. There are several severe and recent rake marks fairly widely distributed but very evident around the genital area. Rake marks are present as well over the fluke blades, some on the flippers and dorsal fins and over the dorsum (especially in front of the dorsal fin caudal to the blowhole) and over the tail stock. The left eyelids are scarred and the dorsal eyelid has fibrotically adhered to tiie cornea. A few punctate skin lesions are randomly distributed. A few Xenobalanus are attached to the trailing edge of the left fluke blade near the notch. A tumor or papllloma-like growth extends from the left lateral of the tongue about midway along its length. The dorsal fin Is deeply notched (healed^ approximately midway along the caudal edge. A few Isolated lonoituctinal scars are present on the ventrum in front of the genital groove caudal to the umbilicus. There is a lengthy cut with some healing penetratina the epidermis at points along its course, running from the anterior insertion of the left flipper up and onto the lateral thoracic wall. A shorter, longitudinal scar runs parallel to this one at a distance of about 6 cm. Venipuncture site on ventral keel between fluke blades 3 cm deep and lies between two tendons.. ABDOMINAL An apparently full term fetus was found free In the abdominal cavity wrapped in amnion. The rostrum was oriented toward, and produced an indention upon, the caudal surface of the liver. The tailstock curved at the caudal limits of the abdominal cavity to make "U" so that the tailflukes pointed anteriorly. The body o* the uterus was ruptured along an anterior line which measured approximately 22 cm. The right uterine horn was slightly larger than the left and the supporting CL was on the right ovary. Both ovaries containec" many corpora. The right mammary gland measured approximately 1.3 an in height and 9.5 cm in width; however, there was nc mtcroscopic evidence of lactation. The abdominal cavity contained approximately 2-3 L of a dark reddish fluid. There were no stomach contents but the stomach was 11 gated and frozen for later formalin injection and use In rreparatlon of plastination 164 section. Fish remains were present In the esophagus. THORACIC The base of the left lung was adhered to the thoracic cavity near the pericardial sac. HEAD HISTOPATHOLOGY Not conducted due to frozen specimen. MICROBIOLOGY Not conducted due to frozen specimen. TOXICOLOGY Specimens of blubber (), muscle (), liver (). kidney () and bone () were collected and frozen for contaminant analysis. Liver (2 sections ^rom right lobe) were collected for brevetoxin assay. Stomach contents were frozen in situ. AGING Teeth were collected from the following arcades In formalin for aging: LL — 13th from rear LR — 10th, 12th. 13th, ISth from rear FEEDING HABITS Stomach was empty. SPECIMENS COLLECTED TMMSN archives Li qui pa k Drum ' Reproductive tract (entire Including external genitalia and ovaries) Brain with pituitary Intact Left mammary gland section Left tailfluke blade Left fluke blade swelling 165 Right kidney (entire) JdM imprint on left dorsum Longitudinal scarring from anterior flipper Insertion Jars Right mammary gland section Renlcular lesion from left kidney Adrenals (right and left) Venipuncture vessels In midkeel Left eye Teeth for aging Frozen Tissue sections for toxicology Blubber sections for G. Worthy Dorsal fin Stomach (entire, empty) MEASUREMENTS LENGTHS Total body length 234.0 cm Intestinal length 2990.0 cm Uterine body rupture 22.0 cm Fetal length 92.5 en TMMSN, Navy and G. Worthy blubber depth measurements taken WEIGHTS Total body weight 16B.3 kg Brain (with pituitary) 1228.4 g Kidney, right 639.4 g Kidney, left 516.9 g Adrenal, right 11.6 g Adrenal, left 11.8 g Stomach (with duodenal ampulla and contents) 3422.8 g Spleen 50.1 g Liver • 3635.0 g Heart (flushed) 997.7 g Lung, right 2787.5 g Lung, left 3116.9 g Lymph node (pulmonary at base of lung near diaphragm) .. 2.1 g Fetal weight 9446.3 g RATIOS Body length to body weight {) Brain weight to body weight (kg/kg) 0.007 Brain weight to body length (g/cm) 5.25 Intestinal length to body length (cn/cm) 12.78 166 NECROPSY NOTES SP nH General comments The most remarkable external finding Is the entanglement of many wraps of monofilament line around the left fluke blade. Severe lacerations are on the leading and trailing edges of the left fluke blade (see photos prints and slides). Possible single net marks are at the leading base of dorsal fin. There is minimal tooth wear. The two posterior crowns on the upper and lower arcades are moderately damaged. All teeth are present. Small lingual papillae which are fairly regressed are found on the lateral edges of thp tongue. A few white circular punctate scars are scattered over the entire body approximately Mmm In diameter (the appearance is similar to protozoal lesions). There Is a punctate scar about 8 mm in diameter that is approximately 8 cm dorsal to the cranial insertion of the left flipper. Another punctate scar Is present on the left tailstook and several punctate scars are present on the right side (see diagram for exact locations). A few well- healed parallel scars approximately 3 to 15 cm in length and 2 to li mm apart are noted in the right scapular region and on the dorsum above the scapular region are, but no reoent lacerations are detected. The thoracic cavity contains some serosangulnous fluid - the majority is frozen (ice formation). There is an area of adhesion of the cranial portion of the right lung to the thoracic wall about 170mffl by 150mm. There are 12 ribs on the left rib cage and the last (12th) is free. The first 11 ribs are attached to the vertebral column. The first four are double headed ribs. Abundant serosangulnoun fluid is present in the peritoneal cavity. Left ovary has a large round tan colored mass associated with the cranial pole. Both ovaries seem otherwise fairly inactive. Urinary bladder is empty. An oval lobulated spleen and a small accessory spleen is present. The loops of intestine are tightly adhered to each other and to the pseudopancreas. The mesentery is also tightly and almost entirely adhered to the intestines. Weights and Measures Heights (g) — L Kidney (with capsule) s 465.8 L Adrenal s ^t^,^ R Adrenal b 12.1 Spleen s 193.2 Liver = 6252.9 Measures (cm) — Intestine length s 123 x l8om Area of lung adhesion « 170mm x 150mm Punctate lesion acar s Smm # Anatomy Specimsns Formalin Gross — Brain 167 Tongue, larynx, hyoid bones, lungs , thyroids , epiglottic spout (in situ) Entire reproductive tract Blubber (8) 1 . anal girth 2. lOcD cranial to umbilicus 3> lateral body wall 4. lateral inbetween girth axilla - dorsal fin 5* 15cD caudal to blow hole 6. ventral Inbetween girth axilla - dorsal fin 7. dorsal inbetween girth axilla - dorsal fin 8. anal girth along lateral prooess F Eye L Adrenal R Adrenal L Ovary , H Ovary Histo — Kidney Frozen — Stomach Toxicology tissues Liver (1 left lobe; 2 right lobe) Blubber (4) Kidney (1) Bone (4) Muscle (4) Blubber samples for Graham Worthy Skeleton Fluid in peritoneal cavity Dorsal fin Pleural fluid L Eye 168 -sp\^^ HSCJtoPSY rrror.T sp 112 Multiple parallel linear aoars are pr<»sent ranging In aire from 3 en to 15 ea In leiigth and about 2 to ^ mm apnrt over the entire body (cicatrization). There ia a remarkable absence of fr*?. : parallel lacerations. The animal appears to be old and nutritionally compromised. Ee is emaciated, determined by his general body condition. Generalized over the entire body are multifocal circular ulcerations with evidence of dermal involvement rangin«r in size from 0.5 cm to 3.5 cm (see diagram for exact locations). No external parasites are present. The teeth show extensive wear with fractures present on numerous crowns over the entire mouth. No lingual papillae are' present. Tooth counts are as follows; Dpper right arcade: 24 slots with tooth #15* #16 and #23 missing. Upper left arcadet' 23 slots with tooth #24 missing. Lower right arcac'c: 23 slots with tooth #13 missing. Lower left arcadet 22 alots with no teeth missing. Intestinal adhesions predonlnate In the anterior portion of the intestines. Mutifocal neorotio areas about 3 om In diameter are present throughout the entire Intestinal tract. There are extensive adhesions between the liver and diaphragm so that the diaphragm cannot be reflected. The lung is firm, solid and heavy. Both lungs are flbrotlc with a diffusely mottled pink and white appearance. On out surface of the lungs white fibrous tissue is presei^t throughout the parenchyma. There are numeroua thin white nematodes present bilaterally. There are black as well as white nematodes present in the left lung, suggesting antemortem hemorrhage ingested by the worms, but only white nematodes are found in the right lung. There is a large nodular gray vegetative l^'ion In the left auricle. Weights and Measures Weig»-ts (c) — L Kidney (vlth capaulf) « 487.3 L Adrm-.l s 15.7 L Testicle with epidldymus s 534.4 L Testicle without epidldymus s 444.9 * R Testicle with epidldymus « 488.2 R Testicle without epidldymus s 435.2 R Kidney r *o9.2 R Adrenal « 11." R Lung = 3702.0 L Lung r 3004.2 Heart « 999.1 • Spleen s 94.0 Liver « 45?1.3 £pl»en « ?4,0 Eraln = 1260.9 Measures (cm) — L Testicle 25op x 8cm R T^st?.ele 23o»". r. Se- Intestines « 2r''4ei" 169 Anatomy Specimens Formalin Gross — L Adrenal R Adrenal Parathyroid and Thyroid Spleen (Whole) Histo — L Testicle Frozen — R Eye Toxicology Tissues Liver (»« left lobe; 2 right lobe) Blubber (1) Kidney (4) Bone (1) Muscle (4) Graham Worthy Blubber Samples Stomach with contents Blood from Heart Dorsal fin Skeleton 170 «E»»1.VT0 ATTEMTIONOF ARMED FORCES INSTITLrrE OF P/CTHOLOGY WASHINGTON. DC 20306-6000 September 2B, 1990 Larry Hansen Southeast Fisheries Center 75 Virginia Beach Drive Miami, FL 33155 Dear Larry, Reports on three Texas Gulf Coast dolphins are enclosed. Two other reports will be sent to you next week. I will then write up a summary of the findings in the five animals and forward it to you. . Best wishes. THOMAS P. LIPSCOMB, DVM MAJ, VC, USA Dept. of Veterinary Pathology 171 AHMtO FORCES INSTTTUTk 0»- CATHOLOCY WASHINGTON. DC 2030»-*0O0 KS'f^] J> ?iffK .T to Aiiutnon w PATIENT IDENTIFICATION ATIP ACCESSION NUMSEK 3362007-1 n.£ASC USE An^ ACCCSSKM CNccxoiaiT 01 SEQUENCE SSAN ASIUAL. CETACEl DOLPHIS Dr. 00130138 S T SUWICAL/AUTOWT PATH ACCtSSIOM rS Tarplcy PtEASE iwfoWM US or *nr PAtion iOE>fnr»cATX3N ehwows Dr. EAymond T*rpl«y TtxA« VAttrinAry Itodic&l C«nt«r Colltg* of VtttrlnAry lUdlcln* T«x«s ALU Unlvcrflty l_£oll«<« StAtion. TX 77843-44B8 n J CPU-V JMP/TPL/Jlp DATE 11 Jun* 1000 CONSULTATION REPORT ON CONTRIBUTOR MATERIAL AFIP DIAGNOSIS: 00120126 1. Lun|: Pn«UBonlA» aoutc, b«BorrbA|io, aniltifooAl. aodtrat*, with oolonlct of fraB-ntgativ* bAOiUl. AtUntlo bottlcnof* dolpbln (Iy£Elfi21 lllfflfiftlyi) • eatAOtan. 2. Lung: Bponcbopnauaonla, aout* to aubacuta, multlfooal, Bodarata, witb |pam-na|atlva baallli. 3. Lung: BronobopnauBonla, obronio-aotiva, aoa Inopbilic. Btultlfooal. Bodarata, witb aataatrongylld nanatodaa. 4. Lung: Pblabltia, acuta, Bultlfoaal, nlld, witb gram-nagativa bacilli. 5. Livar. bapatocytac: Vacuolar cbanga, ulerovaaicular, diffuaa, Bodarata. a. Livar: Hapatitia, lympbocytic. portal, pariportal and eapaular, Bultifocal, Bild. 7. Livar: Congaation. cantrilobular , diffuaa. Bild. Livar: Haaoaldaroaia, sultifocal, Bild. Livar: Extraaadullary baBatopoiaaia, sultifooal, Bild. Livar: Artaritia, acuta, focal, Bild. 6. 0. 10 11 Lyapbadanitia, Bild. Lyapboid daplation, diffuaa. »aantaric: lacroaia, acuta, Bultifocal, Lymph nodaa , pulBonary: nacroauppurativa, Bultifocal, 13. Lyapb nodaa, Baaantaric: Bodarata. 13. Lymph noda, Bild. 14. Lyapb noda, nacroauppurativa giant oallf. 15. Lymph noda, maaantaric: diffuaa, Bild. 16. Lymph noda, sadiaatinal; Bodarata, with hamoaidaroaia, 17. Lymph noda, aadiaatinal: diffuaa, Bild. 18. Lymph noda, axillary: HaBorrhaga and arytbropbagocytoaia, multifooAl, Bild. 10. Kidnay; Congaation, Bultifocal, Bodarata. 20. Stomach: aaatritia. aoalnophillo, diffuaa. Bild. with adama. Baaantaric: , Bultifocal Lympbadanitia, Bodarata, with aultinuclaatad Lympbadanitia, aoainopbilic, HaBorrbaga, diffusa, Lympbadanitia, aoainopbilic. AFIPF/L61 15 May 87 172 2282907-1 ANIMAL, CJTTMrEA DCaifHIN Dr. Tarpley 90120128 S T cro-v JMP/TPVjlP 11 June 1990 21. Stonach: Dloer, focal, noderate, with htguuiilizujii (jiiiinn diagnosis. 22. StoDacii, pylorus: Gastritis, lynrhnrytic, focal, mild. 23. Stonach, pylorus: Erosion, aaxte, fnmlly extensive, moderate. 24. Duodenum: Btteritis, lyntiyrfhl 1 1 ml nr, mil ti focal, moderate. 25. Spleen, c^sule: Ccngesticn and beonrxhage, multifocal, mild. 26. Trachea: Tradieitis, iyHY*vifttnir»ii^T- and eosinophilic, nultifocal, mild. 27. £sc|iiagus: Ulcer, acute, focal, moderate. 28. Skin: Dermatitis, acute to subacute, focally extensive, moderate, v±^ acute necxotizing vasculitis and fiisrin thrcnbi. Ihe cause of deatii is bacterial pneumonia. Ihe bacterial mocphology and staining characteristics are consistent with Escherichia ooli which was cultured frcm the Ixng. Ihe pulmonary lesions eure ocoplex. Ihe distribution of the hesmrxhagic pneumonia is consistent with hematogenous dissemination. Other findings that support septinpmjn are the presenoe of gram-negative bacilli within pulmonary veins, zuaite h^iatic arteritis and necxotizing vasculitis in the skin. Ihe bacterial bronchopneumonia %ras of longer duration than the very acute hemorrhagic pneumonia. The Ixmgworm a«pyici ntPti pneumonia was chronic and relatively mild. Ihe conbined effects of the lung lesions %«ere fated. Ihe microvesicular vacuolar change in hepatocytes is consistent vdth lipidosis. lesions of this type have been associated with a variety of toodns in other species. Specigd stains failed to reveal the cause of the necrotizing lesions in pulmonary and mesenteric lynph nodes. The gastric iiloer probably caused significant pain. The splenic congestion and raysular hanorxhage probably reflect the sqitioemia. Ihe causes of the other lesions were not ^parent. JOHN M. FLETCHER, EMI, MFH Colcnel, VC, USA Chairman, Department of Veterineury Pathology IHOAS P. LIF5GCHB, ZfM MAT, VC, USA Department of Veterinary Pathology 173 2297924-9 ANIMAL, CETACEA DOLPHIN C90101256 SBT Dr. Raymond J. Tarpley Dept. of Veterinary Anatomy Texas A&M University College Station, TX 77843 CPU-V jMP/TPL/mab 25 September 1990 C90101256 1. Spleen: Dermatitis, necroulcerative, acute, focal ly extensive, moderate, with numerous mixed gram-negative bacilli and ciliated protozoa, Atlantic bottlenose dolphin (Tursiops truncatus), cetacean. n Liver: Hepatitis, portal to periportal, lymphoplasmacytic and eosinophilic, diffuse, mild to moderate. 3. Liver: Congestion, centri lobular, diffuse, moderate. 4. Liver: Extramedullary hematopoiesis, multifocal, mild. 5. Lung, pleura: Fibrosis, focal ly extensive, moderate, with fibrous adhesions. 6. Lung: Congestion and alveolar edema, diffuse, moderate, with alveolar histiocytosis. 7. Lymph node: Lymphoid depletion, diffuse, moderate. 8. Lymph node: Lymphadenitis, eosinophilic, diffuse, mild. 9. Lymph node: Congestion, diffuse, moderate, with mild hemorrhage. Comment: The skin lesion is suggestive of cutaneous infarction. The presence of bacteria deep within the lesion raises the possibility that the lesion was caused by septic thromboembolism. However, bacterial invasion through the skin is also possible. Ciliated protozoa are found rather commonly in skin lesions of this type in dolphins. They are generally considered to be secondary invaders. The hepatic and pulmonary congestion are probably agonal. The pleural fibrosis and adhesions are probably the result of a resolved pneumonia and pleuritis. The causes of the other lesions are not apparent. JOHN M. PLETCHER, DVM, MPH Colonel, VC, USA Chairman, Department of Veterinary Pathology THOMAS P. LIPSCOMB, DVM MAJ, VC, USA Department of Veterinary Pathology 174 2297921-5 ANIMAL, CETACEA DOLPHIN 90092184 SBT Dr. Raymond J. Tarpley Dept. of Veterinary Anatomy Texas A&M University College Station, TX 77843 CPU-V JMP/TPL/mab 25 September 1990 90092184 1. Lung: Edema, alveolar, diffuse, moderate, with alveolar histiocytosis, Atlantic bottlenose dolphin (Tursiops truncatus), cetacean. T. Lung: Pyogranulomas, multifocal, moderate. 3. Lung: Fibrosis, multifocal, moderate. 4. Pancreas: Fibrosis and acinar cell atrophy, diffuse, severe, with mild multifocal chronic and chronic -active pancreatitis. 5. Liver: Vacuolar change, microvesicular, diffuse, moderate. 6. Liver: Sinusoidal dilatation, multifocal, moderate. 7. Lymph nodes: Lymphoid atrophy, diffuse, mild. 8. Thymus: Atrophy, diffuse, mild. Comment: Postmortem autolysis hindered microscopic evaluation. The pulmonary edema may have been agonal or caused by drowning. The pulmonary pyogranulomas were probably caused by metazoan parasites. The fibrosis in the lungs probably represents areas of resolved pneumonia. The caM«;e of the severe pancreatic fibrosis and atrophy was not apparent. Lesions of this type have been recognized previously, but generally in older dolphins. It is likely that both exocrine and endocrine pancreatic function were impaired. The cause of this lesion is unknown. Hepatic vacuolar change can be caused by a number of different factors. Microvesicular fatty change, which this vacuolar change resembles, has been associated with toxic hepatic injury in some species. The thymic atrophy may represent a normal physiologic process. The causes of the other lesions were not apparent. JOHN M. PLETCHER, DVM, MPH Colonel, VC, USA Chairman, Department of Veterinary Pathology THOMAS P. LIPSCOMB, DVM MAJ, VC, USA Department of Veterinary Pathology 175 ARMED POHCES INSTITUTE OF PATHOLOGY WASHrNGTON. DC 2030&-6000 REPIY TO V. October 11, 1990 Department of Veterinary Pathology Larry Hansen Southeast Fisheries Science Center National Marine Fisheries Service 75 Virginia Beach Drive Miami, TL 3J149 Dear Larry, Enclosed are the last two necropsy reports on Gulf of Mexico dolphins. I will get a summiury of the pathological findings in the five animals written up in the next week or so. Let me know if you need the report in a particular format. Sincerely, THOMAS P. LIPSCOMB, DVM MAJ, VC, USA Department of Veterinary Pathology 176 2298103-9 ANIMAL, CETACEA DOLPHIN C90073283 B Dr. Raymond 0. Tarpley Department of Veterinary Anatomy TVMC, Texas A&M University College Station, TX 77843-4458 CPU-V JMP/TPL/mab 11 October 1990 C90073283 1. Skin, dermis: Necrosis, coagulative, focally extensive, severe, with acute dermatitis and gram-negative bacilli, Atlantic bottlenose dolphin (Tursiops truncatus), cetacean. 2. Skin: Necrosis, coagulative, focally extensive, severe, with acute dermatitis, ulceration and ciliated protozoa. 3. Skin: Dermatitis, acute, focally extensive, moderate, with focally extensive dermal and epidermal necrosis, and fibrin thrombi . 4. Skin: Dermatitis, acute, focal, mild, with gram-negative bacilli. 5. Skin: Dermatitis, chronic, focally extensive, moderate. 6. Lung: Necrogranuloma, focal, moderate, with mineralization. 7. Lung: Congestion and alveolar edema, diffuse, mild to moderate, with alveolar histiocytosis. 8. Liver: Hepatitis, portal and periportal, lymphoplasmacytic, diffuse, mild. 9. Lymph node: Lymphoid hyperplasia, diffuse, moderate. 10. Lymph node: Lymphoid depletion, diffuse, mild. 11. Lymph node: Edema, diffuse, moderate, with mild hemorrhage. Comment: Gram-negative septicemia is considered the cause of death. Rarely, gram-negative bacilli were found in nonulcerated areas of acute dermatitis, strongly suggesting hematogenous dissemination of the bacteria. Several of the skin lesions were consistent with cutaneous infarcts. We believe that the majority of cutaneous lesions were caused by septicemia. Ciliated protozoa were found in some of the cutaneous lesions. This is a relatively common finding in dolphins; these protozoa are generally considered to be secondary invaders. The cause of the pulmonary necrogranuloma was not apparent; this lesion was not likely to have been clinically significant. The pulmonary congestion and edema were 177 2298103 Animal, Cetacea Dolphin Page two probably agonal. Histologically similar hepatitis has been seen previously in dolphins; the cause is unknown. The causes of the other lesions are also unknown. JOHN M. PLETCHER. DVM, MPH Colonel, VC, USA Chairman, Department of Veterinary Pathology THOMAS P. LIPSCOMB, DVM MAJ, VC, USA Department of Veterinary Pathology 178 2295794-8 ANIMAL. CETACEA DOLPHIN SA344 T Dr. Raymond J. Tarpley Department of Veterinary Anatomy TVMC, Texas A&« University College Station, TX 77843-4458 CPU-V JMP/TPL/maD n October 1990 6A344, C901 62225 1. Lung: Fibrosis, pleural and interstitial, multifocal, moderate, with mild multifocal chronic pneumonia, Atlantic bottlenose dolphin (Tursiops truncatus), cetacean. 2. Lung: Necrogranulomas, chronic, multifocal, moderate, with mineralization. 3. Lung: Congestion and edema, multifocal, moderate, with alveolar histiocytosis. 4. Liver: Congestion, centri lobular, diffuse, mild. 5. Liver: Fibrosis, portal and capsular, diffuse, mild. 6. Lymph nodes: Lymphadenitis, eosinophilic, necrotizing, multifocal, mild, with multinucleated giant cells. 7. Lymph node: Lymphoid hyperplasia, multifocal, moderate. 8. Lymph node: Lymphoid depletion, multifocal, moderate. Comment: The cause of death is not clear. The most significant lesions were pulmonary. The pulmonary fibrosis probably represents the resolution phase of a pneumonia; the cause was not evident. The pulmonary necrogranulomas were probably caused by parasites. The pulmonary congestion and edema could have agonal or caused by drowning. The hepatic congestion was probably caused by terminal cardiovascular collapse. Similar eosinophilic necrotizing lymphadenitis with multinucleated giant cells has been seen in other dolphins; the cause is unknown. Both hyperplastic and depleted lymph nodes were present. JOHN M. FLETCHER, DVM, MPH Colonel, VC, USA Chairman, Department of Veterinary Pathology THOMAS P. LIPSCOMB, DVM MAj, VC, USA Department of Veterinary Pathology 179 ARMED FORCES INSTTTUTE OF PATHOLOGY WASHINGTON. DC 20306-6000 »«EPl.r TO Sr. AmtfnoN Of ■*•'••». p. "•' November 2, 1990 Department* -G^ Veterinary Pathology Larry Hansen Southeast Fisheries Science Center National Marine Fisheries Service 75 Virginia Beach Drive Miami , FT. T?i4Q Sxunmary of Pathologic Findings in Atlantic Bottlenose Dolphins from the Giilf of Mexico. The Department of Veterinary Pathology of the Armed Forces Institute of Pathology received material from five Atlantic bottlenose dolphins that died from March through June 1990. All were from Texas waters. Two of the five had gram-negative septicemia. One of these two also had gram-negative bacterial pneumonia and £. coli was cultured from lung. Another of the five dolphins had cutaneous lesions suggestive of gram-negative septicemia. Gram-negative septicemia has not been found to be a common cause of death in dolphins, although Pseudomonas pseudomallei has caused septicemias in an aquarium in Hong Kong. Generally, grzun-negative septicemia tends to occur in the terminal stages of chronic diseases and in individuals with some basis for depressed immunity. The remaining two dolphins had various lesions but no clear-cut cause of death. A number of interesting lesions were found in these five dolphins. Diffuse hepatic microvesicular vacuolar change, consistent with microvesicular fatty change, vas found in two dolphi.ns. Although hepatic fatty change can be caused by a variety of physiologic and pathologic processes, diffuse microvesicular fatty change has been associated with a variety of toxins in other species. Lymphocytic portal hepatitis was present in two dolphins. This lesion has been previously described in dolphins from the Gulf of Mexico; its cause is unknown. Lymphadenitis, lymphoid hyperplasia and lymphoid depletion were all seen, occasionally in the same animal. The significance of these lesions is unknown. Significant amounts of pulmonary fibrosis were present in three dolphins; these probably represent areas of resolved pneumonia. One dolphin had severe pancreatic fibrosis and atrophy. Pancreatic lesions of this type have been recognized previously, but generally in dolphins that were older. (This dolphin was sexually immature.) Thomas P. Lipscomb, DVM MAJ, VC, USA Department of Veterinary Pathology 180 Sl-Hyocinthe, lll^lt^ 1 Mr. Lorry Hensen NMFS Miami Loborolory 75 Virginia Beech Drive Miami, Florida 33149 U.S.A. Dear Mr. Hensen, As requested by Mr. Charles A. Oravetz from the Protected Species Management Branch in his letter to Mr. Bruce McKay, Greenpeace Montreal, included with this letter, you will find a copy of the necropsy report of a male bottlenose dolphin I examined in Mobil, Alabama, in May 1990 (5HCM 072). If you need any information or have any question, don't hesitate to contact me, I will be glad to help you if i con. Sincerely yours. jlvain De Guise, D.M.V cc: Mr. Bruce McKay, Greenpeace Montreal C.P. 5000 Swit-Hyacmttw (QuMiac) J2S7C6 (514) 779-6521 — Mt«: 05-060505 Ttltcopwur (514) 773-2161 181 90-3530 Ey^grpaT examination: This is the fresh carcass of 8 1 15 kg Atlantic bottlenose dolphin on which there are a few external parasites, mostly on the tail. prp$s findings: -brain: No significant lesion, there are no parasites In the auditive canal. -mouth: All the teeth are worn, their surface being all even. -lungs: There is a severe extensive bilateral verminous pneumonia caracterised by whitish and greenish discoloration of the parenchyma on the cut section of the whole surface of both lungs associated with a heavy burden of parasites in the bronchi, with a mild to nrtoderete suppurative resction. The parasites are 2 to 3 cm long, whitish, with a thin longitudinal black stripe. There Is a mild brown to reddish exudate In the trachea and large bronchi. -heart: No significant lesion. -thymus: There is a small thymus remnant. -liver: There are a few slightly depressed lines of whitish discoloration on the surface of the liver with nothing significant on cut section. -kidneys: No significant lesion. -spleen: The spleen is about 5x4x4 cm, and a few daric red blackish foci of discoloration on the surface. The cut surface Is very regular. There are two small ectopic spleen of 1 and 0.5 cm In diameter. -pancreas: No significant lesion. -thyroid: No significant lesion. -adrenals: The adrenal cortex Is rather thin, with a rather thick medulla. -1st gastric compartment: No significant lesion. -2nd gastric compartment: No significant lesion. -3rd gastric comportment: There ore 3 foci of small pedonculated nodules 182 Appendix I. (conunueo; ottoched to the gostric mucosa. Those yellowish nodules ore 3 mm in diometer ond correspond to porosHes. -4th gastric compartment (duodenal ampula): No significant lesion. -intestine: No significant lesion. -testis- No significant lesion. -epididymis: No significant lesion. -seminal vesicles: -urinary bladder: No significant lesion. Macroscopic diagnosis: Verminous broncho-pneumonia Laboratory tests: Bacteriology: yes Virology: yes To><1cology: yes Parasitology: yes Histopothology: -general: All the tissues ore nfilldly to nfioderotely autolysed, and there are many clumps of bactehas In the lumen of blood vessels that sometimes occlude completely some small vessels. -brain: There Is a small to moderate amount of llpofuscin in the cytoplasm of some neurons of the brain, cerebellum and brainstem. -tongue: There is a moderate multifocal lymphocytic InftUratlon around the tongue mucus glands end their ducts under the Malplghlan epithelium. -lungs: We observe a moderate amount of nematodes In small bronchi, with a mild to moderate neutrophilic Infiltration around the parasite, often with a moderate Increase In the number of alveolar macrophages, and a moderate lympho-plasmacytlc Infiltration In the chorion. The same kind of inflammation extends in the surrounding alveolar lumen and wall, and In the wall of bronchi, bronchloll. We also note a mild multifocal 183 Appendix 1. (^conunuca; eosinophilic inflltrotlon In the well of some eWeoll end bronchi end sometimes In their lumen, with the neutrophils. Further from the moln inflammatory foci that contain the parasites, we often note alveolar edema and a mild infiltration of neutrophils and eosinophils. The muscles around the bronchioli are often hypertrophied, and we find a moderate amount of corpora amilacea (small calcified plagues) in the lumen of bronchi, bronchioli and alveoli, and sometimes incorporated into their wall, both of these lesions are not always seen in relation with the inflammatory foci. We also note ocasionnal giant cells in airways, with very few larvae, and a multifocal mild to moderate accumulation of Gram-negative bactehae sometimes associated with the lesions. -mediastinal and cervical lymph nodes: There Is a mild to moderate eosinophilic Infiltration In the mildly edematous cortex and In the medullary sinuses, where It Is accompanied by a few macrophages and some red blood cells. The cortex contains very few follicles. -heart: We note very few small foci of fibrosis In the myocardium, and a small to moderate amount of llpofuscln In the cardlomyocytes, around the nuclei. -aorta: No significant lesion. -diaphragm: No significant lesion. -thymus: Only some very thin thymus remnants are still there, and they ore poorly cellular. -liver In addition to a mild atrophy of the centrolobular hepatic cords, there Is a small amount of brown pigmet In the cytoplasm of the hepatocytes. -spleen: The white pulp It made of a large number of follicles that have a large germinal center with a very thin outer ring of mature lymphocytes. -pancreas: No significant lesion. -thyroid: Most of the follicles contain little colloid, but the autolysis is moderate. Some Intact follicles contain a normal amount of colloid. -adrenals: The cortex/medulla ratio Is low, and the junction Is Irregular. 184 -1st gostnc comportment: No significant lesion. -2nd gostnc comportment: There ie o emoll focol euperficiol erosion thot exudates mucus, while the rest of the mucosa is normal. -3rd gastric compartment: We observe some trematodes {Brounino cordjformis) attached to the mucosa by a thin layer of submucosa they incorporate in themselves, between their Inner and outer body. -4th gastric compartment (duodenal ampula): No significant lesion. -intestine: No significant lesion. -testis: No significant lesion, the spermatogenesis Is normal and active. -epididymis: No significant lesion, the tubules carry a good amount of spermatozoids. -urinary bladder No significant lesion, the epithelium Is preety high (not compressed). -muscle: A few muscle fibers are swollen. Final Diagnosis: -Severe verminous broncho-pneumonia. Comment: The moderate amount of bacteria sometimes found to be associated to the lung lesions suggests that a secondary bacterial infect/bn complicated the primary verminous pneumonia, a fact that could have precipitated death. SQlvain Oe Guise, D.n.V 185 I jtheast Fisheries Science Center representative system for the Man Mammal Stranding Network. Appendix EX. Proposal outline for Southeast Fisheries Science Center representative system for the Marir| 186 1 II .• •'■•• •i.» C Marine Mammal Stranding Network Representative System Organizational Woricsbop Miami Laboratoiy 75 VirgiiQa Beach E^ive Miami, FL 33149 May 7^ 1991 FISHERIES 187 Ma-tional Marine Fisheries Servicse Southeast Fisheries Science Center Marine Manual Stranding Metwork Representative System Organizational Moricshop Miami lAboratory 75 Virginia Beach Drive Miami, Florida May 7-8, 1991 188 Marine NasBal Stranding Network NMFS Area Representative Systea Southeast Fisheries Science Center Organizational Norkshop Miaai, Florida, Nay 7-8, 1991 ASSSDh Tuesday, Nay 7. 0900 Introductory remarks and introduction of participants Brad Bro%m. 0920 Overview of SEDS stranding netirark - Dan Odell. 0940 NMFS area representative authority at stranding site • Jeff Bro%m. 1000 Stranding of protected species. National perspective • Dean Wilkinson 1030 Coffee break. 1045 Discussion of NMFS stranding network area representative responsibilities - Larry Hansen. 1115 Discussion of basic aarine BaBmal stranding data reporting - Ben Blaylock. 1130 Denonstration of reporting software - Lee Weinberger. 1200 Break for lunch. 1330 Specinen and data collection protocols - Sylvia Galloway. 1430 Discussion of specinen storage and transfer - Larry Hansen. 1500 Coffee break. 1530 Ad hoc discussion of aspects of iapleaenting the reporting systea relative to individual NMFS laboratories . 1700 Adjourn. 189 Wednesday, May 8. 0900 Reconvene ... continue previous afternoon's discussion 1030 Coffee break. 1100 Ad hoc discussion period. 1200 Adjourn meeting. 190 WORKSHOP PARTTCTPAITPS Mr. Ben Blaylock Southeast Fisheries Science Center Miami Laboratory 75 Virginia Beach Drive Miami, FL 33149 (305) 361-4299/FTS 350-1299 Mr. Bill Bowen Southeast Fisheries Science Center Beaufort Laboratory Beaufort, NC 28516 (919) 728-3595/FTS 670-9740 Dr. Brad Brown, Center Director Southeast Fisheries Science Center 75 Virginia Beach Drive Mieuni, FL 33149 (305) 361-4286/FTS 350-1286 Mr. Jeff Bro%m Southeast Regional Office Management Division 9450 Koger Blvd. St. Petersburg, FL 33702 (813) 893-3366/FTS 826-3366 Dr. Charles Caillouet Southeast Fisheries Science Center Galveston Laboratory 4700 Avenue U. Galveston, TX 77550 (409) 527-6500/FTS 527-6500 Mr. Bill Fable, Jr. Southeast Fisheries Science Center Panama City Laboratory 3500 Delwood Beach Road Panama City, FL 32407 (904) 234-6541 Mr. Larry Hansen Southeast Fisheries Science Center Miami Laboratory 75 Virginia Beach Drive Miami, FL 33149 (305) 361-4264/FTS 350-1264 191 Mr. Wayne Hoggard Southeast Fisheries Science Center Mississippi LsUboratories P.O. Drawer 1207 Pascagoula, MS 39567 (601) 762-4591 Dr. Sylvia Galloway Southeast Fisheries Science Center Charleston Laboratory P.O. Box 12607 Charleston, SC 29412 (803) 762-1200 Mrs. Ann Jennings Southeast Fisheries Science Center Charleston Laboratory P.O. Box 12607 Charleston, SC 29412 (803) 762-1200 Dr. Dan Odell, SEUS Coordinator Sea World of Florida 7007 Sea World Drive Orlando, FL 32821 (407) 363-2158 Dr. Joe Powers, Laboratory Director Southeast Fisheries Science Center Miami Laboratory 75 Virginia Beach Drive Miami, FL 33149 (305) 361-4284/FTS 350-1284 Ms. Kathy Prunier Southeast Fisheries Science Center Miami Leiboratory 75 Virgina Beach Drive Miami, FL 33149 (305) 361-4596/FTS 350-1596 Dr. Gerry Sco-tt, Chief Oceanics and Pelagics Division Southeast Fisheries Science Center Miami Labora-tory 75 Virginia Beach Drive Miami, FL 33149 (305) 361-4530/FTS 350-1530 192 Mr. Lee Weinberger southeast Fisheries Science Center Data Management Division 75 Virginia Beach Drive Miami, FL 33149 (305) 361-4287/FTS 350-1287 Mr. Dean Wilkinson National Marine Fisheries Service Division of Protected Species 1335 East West Highway, Room 8259 Silver spring, MD 20910 (301) 427-2322/FTS 427-2322 193 Appendix 1. PreliMinaxy Proposal for Enhancing les required by the NMFS and f ortrard to the appropriate NMFS laboratory specified by Miami Lab. D. NMFS Area Representative will coordinate with participating veterinary clinical pathology laboratories for detailed necropsies of condition 1 & 2 carcasses and will forward 199 results to Miami Lab. E. NMFS will provide materials for specimen collection to SEUS Stranding Network and NMFS Area Representatives will accept collect telephone calls for reporting stranding. F. NMFS Miami Laboratory will manage NMFS Area Representatives, arrange for transfer of biological samples to analytical laboratories, coordinate final disposition of specimens, and with SEUS Network Director, produce newletter and assist in maintenance of regional organization. Miami Laboratory will maintain real-time cetacean stranding database for the NMFS southeast region. G. NMFS will not usxirp SEDS stranding network coordination and r espons ibi 1 it i es . gffng jgl^randlno Network A. Nettrork participants will report BASIC DATA (above) to SEDS Regional Coordinator by telephone within 24 hours of discovery of stranding as a requirement of the LOA. Voice confirmation of receipt of information is required. If SEUS Regional Coordinator cannot be reached on the first try, then the network participant will immediately telephone the NMFS Area Representative. B. SEUS Regional Coordinator will be required in LOA to report stranding to NMFS Area Representative within 24 hours of receipt from network participants. C. Network participants will perform general necropsies on carcasses to the extent that condition warrants. General necropsies will include morphological measurements, photographs, examination for fishery interaction and external pathology, and collection of specimens for life history, pathology, and genetics studies as detailed in necropsy manual and outlined herein. If the carcass is condition 1 or 2, and size permits, the «fhole carcass will be transported to a participating veterinary clinic for detailed necropsy. D. SEDS Stranding Network participant will deliver or coordinate delivery of NMFS-required specimens to the NMFS Area Representative and ensure proper storage of specimens until delivered to NMFS. E. SEDS Stranding Network participants will provide stranding data to SEUS Stranding Network Director as directed in the LOA. 200 TENTATIVE IMPLEMENTATION SCHEDULE A. BASIC: 1. NMFS area netirork representative selection...! March 1991. 2. Reporting requirements to SEUS nettrork participants via LOA 1 April 1991. 3. Collection and transfer of life history specinens iaaediately . 4. BASIC data reporting fully i]q>leBented 1 April 1991. B. PATHOLOGY: 1. Draft pathology xsanual 1 ^nril 1991. 2. Schedule training workshops ? C. GENETICS: 1. Collection of genetics speciaens iaaediately. D. FACILiry APPOINTMENTS 1. Pathology AFIP. 2 . Genetics ? 3. Life history a. Stonach content analysis Tursiops Sea Norld. Others ? b. Teeth for ageing ? c. Reproductive tracts and gonads ? d. Other ? 4. Other ? 201 SOUTHEAST REGIONAL STRANDING NETWORK DATA FLOW - SEUS STRANDING NETWORK PARTICIPANTS - TX LA,MS,AL NW FL. C-SW FL. KEYS-GA GA-SC BASIC DATA 24 Hoxxr Reporting Li ait NC GAL PAS SEUS REGIONAL COORDINATORS BASIC DATA 24 Hour Reporting Limit i NNFS AREA REPRESENTATIVES PAN ST. PETE. MIA CBASTON BEAD COMPLETE STRANDING RECORD 30 Day Reporting Limit. i SEUS Nettrork Coordinator T JOINT QUARTERLY REPORT TO SEUS AND NNFS NETWORK PARTICIPANTS BASIC DATA 48 Hour Reporting Limit i NNFS Miami Laboratory EMERGENCY REPORTS TO NMFS HQRTRS. AND MMC. 202 Appendix X. Report on the Southeast Fisheries Science Center Marine Mammal Stranding Network Representative System Organizational Workshop. 203 This page intentionally left blank. 204 ^ OF, ^ •n *«i«T 0» ' Report on the Southeast Fisheries Science Center Marine Mammal Stranding Network Representative System Organizational Workshop Southeast FisbariBS Scimacm Canter Miami, Florida May 7-8, 1991 I. Prepared by Staff Southeast Flsberiec Science Contribution KIA-91/92-24 December 1991 205 Report on the Soirtheast Fisheries Science Center Marine MaBsal Stranding Network Representative Systea Organizational Workshop, May 7-8, 1991 I. INTRODDCnOir The Marine Mammal Stranding Network NMFS Area Representative System Organizational Workshop was held at the Southeast Fisheries Science Center in Miami, Florida, May 7-9, 1991. The following participants attended the workshop: Mr. Ben Blaylock - Mizuni Laboratory Mr. Bill Bowen - Beaufort LeUooratory Dr. Brad Brown, Center Director - SETC Mr. Jeff Brown - Southeast Regional Office Dr. Charles Caillouet - Galveston Laboratory Mr. Bill Feible - Panama City Laboratory Dr. Sylvia Galloway - Chcurleston Laboratory Mr. Larry Hansen - Miami Laboratory Mr. Wayne Hoggeurd - Mississippi Laboratory Mrs. Ann Jennings - Charleston Laboratory Dr. Dan Odell, SEUS Coordinator - Sea World Dr. Joe Powers, Laboratory Director - Miami Laboratory Ms. Kathy Prunier - Miami Laboratory Dr. Gerry Scott - Miami Laboratory Mr. Lee Weinberger - Miami Laboratory Mr. Dean Wilkinson - Office of Protected Resources The workshop was convened to discuss methods to enhance the Southeast D.S. Marine Mammal Stremding Net%rork and to implement the Southeast Science Center Marine Meunmal Strtmding Network Area Representative System. It was recognized that the volunteer Stranding Network is non-uniformly organized and trained, and it is under-funded, resulting in two major problems. These are: (1) the Stranding Network is unprepared to mount an adequate response to increased stranding, and (2) gross inconsistencies in data collection and reporting have resulted in a lack of baseline information and in an inability to monitor the stranding rate in a timely fashion. The purpose of the SEPC Area Representative System is to supplement the Stranding Network by providing assistance to enhance the efficiency of the net«rork; however, it will not usurp SEUS Stranding Net«rork coordination and responsibilities. The system is designed to: Facilitate near real-time reporting of cetacean stranding to NMFS. Improve data collection quality, quantity, and consistency. 206 Foster information exchange and cooperation among Network Participants and NMFS Representatives. The SEFC Stranding Network Representative System will focus on three areas: (1) monitoring the stranding rate; (2) sp>ecimen necropsy, collection and analyses; and (3) dissemination of results . 7ntTT>ductorv l^f^mr^tR- Brad Brown The following are efforts designed to strengthen the Southeast Fisheries Science Center (SEFC) involvement in the activities of the SEUS Stranding Network: Improve funding for the stremding coordinator. Enlist and pay for veterinarians to perform necropsies. Assign responsible people at each laboratory to act as a major link to enhance collection of stranding information . This meeting occurred as a commitment to respond quickly to major stranding events. Although the SEFC has previously responded to stranding events analytically, it will become more active in the SEUS Stranding Nettrork. SEDS stranding Hetwork! Dan Odell There has been a lack of coordination in the SEUS Stremding Network and there is a perceived need for greater organization. The network began wii-h data cards and a mailing list involving Sea World and Marine Land (David and Melba Caldwell). The first Marine Mammal Stranding workshop %ras in Georgia in 1977, emd was sponsored by the Marine Meunmal Conoaission (MMC). In the first year of the network, less than 100 stranded mammals were reported, primarily Tursiops. Data increased greatly from 1978 to 1990. An individual field number is assigned to each stranded marine meusmal. It should be the primary number for tracking the specimen and oxir goal should be to identify a specimen with as few numbers as possible. There must be a standard developed to achieve iiniformity throughout the entire area. One suggestion is a series of numbers %rtiere the first two are symbolic of the state code, and the rest related to the area. However, it is importemt to not interfere with any previous long-term systems. The number of strandings depends on the amount of coastline in a given eurea and the number of people involved in the Net%rork. 207 There are approximately 250 people in the SEUS Stranding Network and although many state and local official agencies participate, there have often been problems in getting sufficient information from strandings. For instance, better coordination with the Marine Patrol is needed in parts of Florida. A training video is being created that will focus on the. data sheet used by the Stranding Network, with examples using real animals. Data collection sheets should be carried to each stranding, and everyone should keep a log book. It would be a good idea to package a log book and data collection materials for %rorkshop participants. Area Coordinators mxist collect and organize data. Reduction of time lags and duplicate information require more screening and organization of Network Peurticipants at the Area Coordinator level. There are two key forms used in the network, but only the short form is required by the Letter of Authorization (LOA). The information on the short form is entered into the SEUS Stranding Network computer information bank. II. NONirORING THE STRAMDIIIG RATE The SEFC has newly established a system for near real-time monitoring of the stremding rate. Appropriate staff at each of the SEFC laboratories and the Regional Office were identified as SEFC Area Representatives to esteiblish contact in their area with SEUS Network Participants. Each NMFS Area Representative must have back-up personnel assigned to receive stranding reports and specimens in their eibsence. A telephone emswering/message recording machine should be used during off-duty hours. The SEUS Network Participaa^ will report basic data to the SEUS Area Coordinator by telephone within 24 hours of discovery of the stremding as a requirement of the LOA. Voice confirmation of receipt of information is required. If the SEUS Area Coordinator cemnot be reached on the first try, then the Network Participant will immediately telephone the NMFS Area Representative. Completed SEUS data will be sent to the SEUS Network Coordinator within 30 days of collection as specified in the NMFS Letter of Agreement (LOA). The SEUS Area Coordinator will be required in the LOA to report strandings to NMFS Area Representatives within 24 hours of receipt from the Network Participants . A computerized system was established for transferring basic data reports to the Miami mainframe computer. NMFS Area Representatives will send stranding data to the SEFC NMFS Miami Laboratory within 48 hours of initial receipt from SEUS Area Coordinators (total elapsed time since initial discovery should not exceed 96 hours) using the computerized system. The basic 208 stranding data transmitted to the Miami Laboratory are: Field number. Species. Sex. Length. Year, month, day of first report of stranding. State. County . Condition (1-5). Fishery interaction (yes or no) . Time report first received by NMFS staff. SEUS Network Participant reporting to NMFS. Incidental remarks. The Miami Laboratory Area Representative is responsible for reviewing the basic data reports and for monitoring the stranding rates throughout the southeast. This system will allow for rapid identification of anomalous stranding events, the transfer of this information to NMFS Headquarters and others in a timely manner, and maintencmce of a real -time cetacean stranding database for the NMFS southeast region. The "chain of custody" within the 96 hour reporting system is from Seus Net%rork Participants to the SEUS Area Coordinator to the NMFS Representative to Miami. In Florida, there are three Area Representatives; however, the SEDS State Coordinator provides the real-time stranding reports directly to the Miami Laboratory. The NMFS Area Representatives are: • Bill Bowen • Ann Jennings • Ben Blaylock • Jeff Brotm • Bill Fable • Wayne Hoggard • Charles Caillouet Beaufort Charleston Miami St. Petersburg Panama City Pascagoula Galveston North Carolina So. Carolina, Georgia South Florida Central Florida Florida Panhandle Miss, Ala, La Texas III. SPECIMEN COLLECTION, NECROPSY, AMD ANALYSES Newly proposed regulations will require the collection of certain tissues from stranded marine mammals. This could lead to a decrease in coverage by volunteer participants of the stranding network. In order to maintain a cooperative atmosphere between the NMFS and SEUS Stranding Net%rork Participants, NMFS Area 209 Representatives should assist with code 1 and 2 strandings whenever possible to ensure that appropriate samples are collected. The Charleston Laboratory is currently developing necropsy and specimen collection protocols, and tissue collection kits which will be distributed to the appropriate personnel. The Mieuni Laboratory is presently identifying necropsy personnel and necropsy facilities in the southeast. The NMFS Area Representatives should enstire that appropriate specimens are delivered to the necropsy facilities. The SEFC Area Representatives will receive, track, store, and transfer collected samples as necesseury. Arremgements have been made with the AFIP to conduct histopathological studies on appropriate specimens. Specimens collected for genetic, food habit, aging, emd stock identification studies will be transferred for analyses when the appropriate investigators are identified. Some funding will be made available for faculty appointments to perform some of these studies. These activities should ensure that adequate information is available to begin evaluating causes and potential effects of both normal and anomalous mortality events. Official Responsibilities and Authority; Jeff Brown The three topics discussed were: who is on the network; non-government people and LOA's; and jurisdiction. SEDS Stranding Network There are approximately 250 participants in the network; all are listed in the Stranding Directory which is currently being updated. A suggestion was made to put the Area Representatives higher on the list, emd to alphabetize the list. Letters of Authorization (LQA) LOA holder qualifications vary. Potential peurticipants sxibmit a letter to the Regional Office stating %^y they are interested in participating in the Network, along with a resume indicating education and experience. Qualifications for LOA issuance are subjective and depend on the locality. Issuance of LOA's and compliance with rules and responsibilities of LOA holders is the responsibility of the Regional Office. It is important to keep the rules and the application process simple as the majority of people in the Stranding Network are volunteers. They must understand %fhy they are in the Network, that we do not impose anything on them that we trould not do ourselves, and that 210 proper procedures must be followed. The large turnover in peurticipants necessitates frequent review of the Network membership and LOAs are generally renewed annually. The renewal process consists of a letter from the Regional Office asking if there is a desire to renew, how many strandings they were called for, and how many they responded to. This information is reviewed and the renewal of the LOA is decided. A number of questions arose during this disciission. At a stranding can other people assist the LOA holder? The response was that legally, if a person touches an emimal they need an LOA. Obviously, LOAs cannot be issued for each person %rho helps in a stranding; thus, appropriate subjective judgement must be used for each situation. Section 109H (Appendix I) of the Marine Hanmal Protection Act, about the taking of meurine mammals is importcmt to read and understand. Section 112C (Appendix IZ) covers LOA holders and the federal government. Section 109H, concerning stranded or dead imimals, specifically states that federal, state and city employees need not have an LOA. Jurisdiction Theoretically, the first LOA holder or city or state official «rtio arrives at the stranding site has control; however, the stranding is ultimately under Federal jurisdiction and the NMFS may take control of the situation if necessary. He must deal tactfully with network members at strandings, but we also have a responsibility to ensure that the emimals receive humane treatment, including eutheuiasia, if necesseury. We must employ diplomacy emd coordinate with people on the scene. In the Florida Keys, it may be necessary to have a 2-3 person management team to make contacts; however, decision making must be restricted to 1 or 2 persons. Responsibility for disposal of carcasses varies regionally. For example, burial is not allowed in the Florida Keys. City agencies usually take care of carcass disposal for public health reasons «rtien a stranding occiirs near a municipality. A carcass may sometimes be left to rot in a remote eurea. NMFS Area Representatives should contact local authorities in advance and find out about carcass disposal policies and facilities. NMFS Area Representatives should become acquainted with key people in local and state agencies and coordinate these efforts. It is the job of the Area Representatives to ecttablish local contacts and work with them to facilitate retrieval of data and specimens and assist with carcass disposal if necessary. Local 211 veterinarians who work with large animals may provide assistance in carcass disposal. .qtrandina fi£ A PTPtggted SPCCJeS =. L National Persneetive; Dean Nilkinson A couple of regulatory chjmges have occxirred: The newly enacted requirement of proper registration and tracking of tissue taken from stranded animals. A revision of permit regulations concerning the public display of rehabilitated animals (not applicable to the NMFS Stranding Network Representative System) . Regional differences in LOA issuance and requirements currently exist; however, a national standard will be established later this year. An exciting development in the proposed fiscal year 1992 budget is $0.5 million to be split between the Stranding Networks and the National Marine Mammal Tissue Bank. This will provide approximately $20-25,000 to each region for basic Stranding Network support (ie. the prepeuration of guidebooks, training meetings, for equipment, and for responses to unusual stranding) if approved by Congress. There may be money funded for the SE region to hire a person to enhemce Stranding Network activities in the northern Gulf area. $100,000 will be withheld by OPR for unusual marine meunmal stranding. Si-ndi*»s id«»ntlf le*^ for gathering basic information on Tursiops and the use of stranded animals are: (1) an intra- tissue contaminant distribution study; (2) a conteuainant degradation study in 1992, to determine how many hours after death tissues can be used for the National Tissue Bank; and (3) a quality assurance program to obtain standard prep tissues for calibration. A training video by Dan Odell and a general stranding response manual by Dr. J. Geraci should be available by the end of 1991 for Stremding Network Members. ResponsibllltJgs fl£ AZfiA Reprcscntati veg i Larry Hansen Reporting: The primary goal of the enhancement project is to move the stranding information as quickly as possible. Presently, tiie Area Representative sends information to the state coordinator (if they're not the same person). The information is then passed 212 on to the Miami Lab. In Miami, Ben Blaylock or Kathy Prunier are the contacts to call about stranding reports. Lee Neinberger is in charge of the software organization in Miami. The report to Mieusi should occur within 48 hours of receipt by the NMFS Area Representative. The flow of information is from the SEUS state coordinator to the NMFS Area Representative to the Miami Laboratory. If the event seems unusual, the information will be passed to the SE Regional Office and the Office of Protected Resources to determine an appropriate response (e.g., should an emergency investigation be initiated?). Communication: It is importemt to commxinicate with area Nettrork Peurticipants the need for a better response to stranding and to give moral support. Jeff Bro%m will distribute a memo concerning legalities emd goals to the entire network. The memo will include the responsibilities of the Area Representatives, and the purpose of the program. It would be a good idea to include a copy of the 1987 Stranding Workshop Report with these letters. This will provide both information and feedback to participants. Approximately one week after the memo is sent, all SEDS Net%rork Participants should be personally contacted by the State Coordinators about the new protocol. Necropsies : Tissue collection from anything beyond Code 2 strandings should be collected as the situation warrants. It is a responsibility of the NMFS Area Representative to assist the SEUS Network Participants in tissue and data collection from Code 1 & 2 animals. Area Representatives are responsible for contacting qualified pai'hologists and v^terinnrian^ to see that quality necropsies take place. There are two methods to handle this. One is through a formal . contract with local veterinarians. There may also be local veterinarians who already SEUS Network Participants. The NMFS Area Rep. should make contact with these resources. Another is to set up a coordinated progreua through nearby veterinary schools. Small animal veterinarians may be helpful, but there is a big difference between small animals and large marine mammals. Hopefully, some formal veterinary necropsy arrangements will be made later this yeeur. There will be paid faculty appointments for basic life history studies with cost varying according to the event. Faculty appointees may be taxonomists, specimen analysts, emd/or veterinary pathologists. This will be easier if the appointee is affiliated with a university. In some instances faculty are allowed to consult but may not be allotted to use university facilities; however, there may be other facilities available. 213 NMFS Area Representatives should identify local available prospects for faculty appointments in advance. Expertise available in each area will also be investigated by the Miami LaOjoratory in consultation with the SEDS Network Coordinator. Our primary interest in each area should be to find people to do clinical necropsies and histopathological collection, and secondarily to do the other analyses. Area Representatives must ensure that Network Participants remain aware of the importance of data collection. Care must be taken in the collection of all data, as some participants are prone to collect only ceirtain data. The protocol being developed by Sylvia Galloway will clarify data collection requirements and procedxires . Discussion fi£ Specimen Storage aiul TmilBferL Larry Hansen Specimen Storage: It is important to know what facilities eure available. Ultra-cold freezers are necessary for storing tissues for toxin, biotoxin, and metal analyses. One cubic foot per animal is sufficient. If a regular chest freezer is used, place the specimens as far towards the back as possible before they are shipped to a facility which has an ultra-cold freezer. Sei^ile Care: A standard set of tissue will be collected. Life history seunples include teeth, stomachs, and reproductive organs. Contaminant analysis samples are organs such as the liver, kidney, and blubber. Detailed collection procedures will be in the protocol. Tissues will be sent to Miami or Charleston for distribution to the appropriate laboratory. Each facility must have temporary storage capability for both frozen and formalin- stored samples. Basic M*ii-i nm M»i—»»i Stranding I^ta Reporting; Ben Blaylock A primary purpose of this meeting was to establish the real- time marine mtunmal stranding reporting system. Prom data obtained within 96 hours of the discovery of a marine mammal strzmding, the Miami Laboratory will produce a summary of regional stremding activities, listing data such as species, year, state, sex, county, condition, and fishery interactions. To implement the system. Area Representatives received a set of computer disks %ihich allow automated reporting of level A data directly to the Mieuai Laboratory's mainframe computer, and a memual describing the reporting system. The data collected in 214 the progreuB were designed to facilitate the immediate reporting of data. The Miami Lab will produce a 7 and a 30 day report. Specimen and Cfi^ Collection Protocols: Sylvia Gallmfay Why do dolphins die? We would like to collect samples to explain how they die, and for metal and contaminimt analyses. A group convened by the NMFS reviewed the investigation of the 1990 Gulf of Mexico die-off and reached the consensus that a multi- disciplinary approach was necessary to gain adequate data from strandings. As a result, a data inventory list was prepared in advance of another large die-off. In addition, a kit was prepared in order to deal with unusual stranding events. These kits will go to a few trained individuals to begin building a database. The kits were designed by working with both experienced and inexperienced people in a triple necropsy. Peirticipants discussed each specimen and the requirements of each protocol, identified conflicts, and resolved most of the conflicts in a round-table discussion. The integrated protocol included a sample collection checklist. It was determined that a team is needed to perform a good necropsy on a code 2 animal, and a very fresh animal is necessary for microbiological analysis. Large pieces of tissue are needed for hi stopatho logical work. A veterinarian responding to a Code 2 stranding can obtain most of these data. It was decided to collect complete stomachs for pathology examination along with stomach contents. Locations and methods for obtaining blubber measurements needed to be specified. A compromise was to identify three points on the luiimal: one varieQ}le and dependent upon the species; one a standard point; and for the third, more information is needed before a determination can be made. Disposable supplies from the kits will be replenished by the user. These are not intended as field necropsy kits. Large tissues should be taken back to the lab for clean sub-sampling. There will be instructions for the appropriate use of the kits. Each Area Representative will maintain a fully-stocked kit. The kits are to be used to respond to an unxisual stranding event (as defined by a designated peuiel of experts), or Code 2 strandings. They should not be used for other strandings. Federal and army surplus stores cure good soxirces for general field necropsy supplies. i)ata Collection The NMFS will provide materials to the SEDS Stranding 215 Network for collecting samples specified by the NMFS (ie., life history siunples, tissues for contaminant analyses, histopathology samples, etc.) and NMFS Area Representatives should accept collect telephone calls for rep>orting strandings. SEUS Network Participants will F>erform general necropsies on carcasses to the extent that conditions weurxant. General gross necropsies include morphological measurements, photographs, examination for fishery interaction, external pathology. Specimens must be collected for life history, pathology, and genetics studies as detailed in the necropsy manual and outlined in this report. If the carcass is Code 2, emd size permits, the entire carcass should be tremsported to a participating veterinary clinic for detailed necropsy. The following specimens (condition code in parentheses) should be collected by SEUS Nettrork Participants emd delivered to, or picked up by, the NMFS Area network Representative. For Pathology: Necropsy reports from participating veterinary clinics (2). Histology specimens for AFIP (2). • Contaminants ( 2-4 ) . • Biotoxins (2-4). The Charleston Laboratory is currently establishing protocols for pathology specimen collection. For Life history specimens: Entire head if at all possible (2-5), otherwise collect lower jaw or 5 teeth from mid-lower jaw of each stranded specimen. • Gonads and reproductive tract (2-4). Stomachs ( 2-4 ) . Two mid-thoracic vertebrae ( 2-4 ) . For Genetics specimens: Blood (2-4). Liver (2). Heart (2). The SEUS Stranding Nettrork Participant will deliver or arrange delivery of NMFS-reguired specimens to the NMFS Area Representative and ensure proper storage of specimens until delivered to NMFS. The NMFS Area Representative will receive or retrieve biological samples required by the NMFS and forward them to the appropriate laboratory as specified by the Miami ledsoratory. The NMFS Area Representative will coordinate with participating veterinary clinical pathology laboratories for 216 detailed necropsies of Code 2 carcasses, and will forward tJie results to the Miami laboratory. The NMFS Miami Laboratory will manage NHFS Area Representatives, arrange for transfer of biological samples to analytical laboratories, and coordinate final disposition of specimens. The protocols in the manuals are very detailed and the check-off sheets are important to the procedures. Successful necropsies take time, practice, patience, and care. Each sample must be labeled. The SE Fisheries Science Center is willing to train, emd workshops are being planned. Acquiring contacts who will respond in the time of need is essential and NMFS Area Representatives are responsible for decreasing the response time to stranding. The MMC was pleased and has been complimentary about recent stranding data collection activities in the Southeast Region. Data Flow State Coordinators are a point source who feed information to the Area Representatives; howevar, the link between public authorities and the NMFS and the Marine Mammal Stranding Network needs to be strengthened, especially in particular areas. In some areas, using the marine patrol is advantageous and in others cooperation may be difficult to obtain. Posters advertising the Network are helpful in increasing public awareness. Often, whole fresh dolphin carcasses may be kept in local fish houses until arrangements cem be made for necropsy. The NMFS Area Representative should contact local and state authorities to increase their awareness of the system and enlist their help. General Discussion: Workshop Participants The following topics were brought up in a general round tzible discussion: As there are not many Code 2 necropsies, we should be able to get our necropsies performed using our protocol from local veterinary schools at their cost. Present contractual agreements allot $850.00 for each carcass. Me must determine how to appropriate veterinary expertise for fresh stremdings. The Miami Laboratory will contact veterinary schools and report to Area Representatives. Are there rehabilitation facilities in each state? There are no primarily rehabitative facilities in the southeast, but zoological park -type and oceanarium facilities have participated in the Net%rark. 217 How suitable are animals for the protocols when they have been reheibilitated for a F>6riod of time? The microbiological and viral information is lost but other information is still gained. Is there a protocol for the first 24 hours that a live animal is being treated? It would be important to get general clinical information. Pre-treatment protocol would include routine blood samples. An experienced veterineurian would likely do this; however, we need to specify our needs. Is it possible to determine whether a marine mammal has drown or suffocated? There eure both wet and dry dro%ming. Strontium in blood, liver, and kidney, and diatom levels in the lungs can be used to determine the type of death. These varieibles are used in human autopsies. Appropriate seunples to be collected from a stranded animal are outlined on page 24 of the necropsy protocol, in general it is as follows: (1) "clean" tissue collection protocol for code 1 and 2 animals; (2) stomachs and life history samples collected for all animals; and (3) for code 3-5 animals, protocols are being developed. If a rare animal has stranded. Network Participants should call their Area Representatives immediately. There will be specific protocols. The Area Representative should be prepared for this with appropriate information and specific questions to ask the network people. In this regard, the only normal stranding is single coastal bottlenose dolphins. IV. DISSEKXHATIOir OF RESULTS A quaurterly report will be produced jointly by SEUS/SEFC. It will include information of general interest to Stranding Network Participants, a quarterly summary of stranding activity in SEUS, information on analyses underway or planned, and any noteworthy events or tips. It will not duplicate the Smithsonian quarterly report; it is intended to be more of an informal newsletter. Although this is a minor activity in terms of funding, it is critical for maintaining communication and cooperation between the SEFC and SEUS Nettrark Participants. The primary purpose of this activity is to let Nettrark Participants know that their effozrts made to provide the NMFS with information and specimens are worthwhile. The NMFS Miami laboratory, in coordination with the SEUS Network Director, will both produce the newsletter and assist in the maintenance of regional organization. It was suggested that a biennial Stranding Network meeting should be held, sponsored by the SEFC and the SEUS Net«fork Coordinator. The purpose of the meeting would be to monitor the 218 Network activities, provide a forvun for reviewing Network activities, provide training in necropsy and specimen collection, report related research findings, and establish and maintain contacts between the SEUS Network Participemts and the NMFS. It should not be limited to stranding related research. V. AD HOC ITEMS Area Representatives should check into freezer storage and contact the Miami lab with this information. The Miami lab can then make the required arrangements. What communication is there between state agencies, NMFS, and the Network? Some states apparently have no regulations dealing with dead mzurine mammals, and in Louisiana and Florida, for example, permits are required to transport live animals. Hill there be a defined split in area boundaries in Louisiana? This question is under consideration. Network Participant responsibilities must be clear and well defined in the LOA. The LOA will be modified to include the 48 hour reporting requirement. Area Representative responsibilities will be detailed. Requirements unique to each area will also be included. There was a proposal that the SE region meet bi-amnually during the marine meunmal meetings, and that other stranding related persons and cetaceem reseeurchers from the SE region are invited. Regional gatherings every other year were also suggested. 219