Bottlenose Dolphins and Brevetoxins: 

A Coordinated Research and Response 

Plan 

Joseph K. Gaydos 

Epcot’s The Living Seas 

Walt Disney World Resorts 

Marine Mammal Health and Stranding 

Response Program 

Office of Protected Resources 

NOAA/National Marine Fisheries Service 

U.S. Department of Commerce 

National Oceanic and Atmospheric Administration 

National Marine Fisheries Service 

NOAA Technical Memorandum NMFS-OPR-32 

June 2006 

NOAA TECHNICAL MEMORANDUM NMFS-OPR-32 

Bottlenose dolphins and brevetoxins: a coordinated research and response plan 

Joseph K. Gaydos, VMD, PhD 

The SeaDoc Society, Wildlife Health Center, School of Veterinary Medicine, Davis, CA 

Orcas Island Office, 1016 Deer Harbor Road, Eastsound, WA 98245 

U.S. DEPARTMENT OF COMMERCE 

Carlos M. Gutierrez, Secretary 

NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION 

Vice Admiral Conrad C. Lautenbacher, Jr., USN (Ret), 

Under Secretary for Oceans and Atmosphere 

NATIONAL MARINE FISHERIES SERVICE 

William T. Hogarth, PhD, 

 Assistant Administrator for Fisheries 

June 2006 

Technical Memoranda are used for documentation and timely communication of preliminary results, interim reports, 

or special-purpose information. 
NOTICE 

The National Marine Fisheries Service (NMFS) does not approve, recommend or endorse any proprietary product or 

material mentioned in this publication. No reference shall be made to NMFS, or to this publication furnished by 

NMFS, in any advertising or sales promotion which would indicate or imply that NMFS approves, recommends or 

endorses any proprietary product or proprietary material herein or which has as its purpose any intent to cause 

directly or indirectly the advertised product to be used or purchased because of NMFS publication. 

Correct citation of this report is: 

Gaydos, J. K. 2006. Bottlenose Dolphins and Brevetoxins: A Coordinated Research and 

Response Plan. NOAA Technical Memorandum NMFS-OPR-32. 46 pp. 

Copies of this report can be obtained from: 

Office of Protected Resources 

NOAA, NMFS, F/PR2 

1315 Eaast West Highway 

Silver Spring, Maryland 20910 

Or on-line at: http://www.nmfs.noaa.gov/pr/health/ 
Table of Contents 

Introduction.......................................................................................................................... 1 

Insights Gained from the 2004 Panhandle Mortality Event........................ 2 

Coordinating Approaches ............................................................................................. 5 

Critical Research Needs ............................................................................................... 6 

Bloom Ecology and Dynamics ............................................................................................... 6 

Food Web Studies................................................................................................................... 8 

Toxin and Effects.................................................................................................................. 10 

Acute Impact on Bottlenose Dolphins .................................................................................. 12 

Acute Impact on Other Species............................................................................................. 16 

Impact of Chronic Brevetoxin Exposure on Bottlenose Dolphins........................................ 18 

Impact on Bottlenose Dolphin Populations .......................................................................... 19 

Improving Response to Future Brevetoxin-related UME’s........................ 21 

Moving Forward................................................................................................................ 26 

Acknowledgements:...................................................................................................... 27 

Literature Cited:............................................................................................................... 28 

Figures 

Figure 1: List of Reported Brevetoxin-related Marine Bird or Mammal Mortality Events ..... 31 

Figure 2: Red Tide Occurrence off of Florida’s West Coast.................................................... 32 

Figure 3: Shellfish Harvesting Areas in Florida ....................................................................... 33 

Appendices

Appendix I: Participant List for the Disney Workshop on Brevetoxins and Bottlenose 

Dolphins, December 13-15, 2004 ............................................................................................. 35 

Appendix II: HAB-related Tissue Disposition List for Marine Mammal Samples.................. 39 

Appendix III: Sample Tissue Checklist for Marine Mammal Necropsy .................................. 41 

Appendix IV: Samples to Take from Live Captured or Live Stranded Bottlenose Dolphins .. 43 
This Page Intentionally Left Blank 
1 

Introduction 

Bottlenose dolphins (Tursiops truncatus) are widely distributed medium-sized toothed whales. 

The Gulf of Mexico coastal stock complex of bottlenose dolphins inhabit coastal, nearshore, and 

estuarine habitats along the Gulf of Mexico. They are protected under the U.S. Marine Mammal 

Protection Act (MMPA), which also requires that marine mammal Unusual Mortality Events (UME) 

be investigated. 

 

One hundred and seven bottlenose dolphins stranded dead on the coast of the Florida 

Panhandle between March 10 and April 13, 2004 (Flewelling et al., 2005; NOAA, 2004). During this 

UME, hundreds of dead marine fish and invertebrates also were discovered in the area. Although 

no bloom was evident in the area concurrent with the mortalities, epidemiological evidence strongly 

suggests that naturally occurring brevetoxins produced by the dinoflagellate Karenia brevis (formerly 

Gymnodinium breve and Ptychodiscus brevis) were responsible. Brevetoxin involvement has been 

implicated in other marine mammal mortality events in the region (Figure 1), including a mortality 

event that killed 152 bottlenose dolphins in the Florida Panhandle from August 1999 to May 2000 

(NOAA, 2004; Mase et al., 2000) and a 1996 bottlenose dolphin mortality event in Mississippi 

(NOAA, 2004) that coincided with dense blooms of K. brevis. It is of note that blooms of K. brevis

are historically rare in the Panhandle region. In contrast, on the Southwest coast of Florida, K. brevis

blooms have been documented during 40 of the past 41 years. Yet, large scale red tide associated 

mortalities of bottlenose dolphins are comparatively rare in Southwest Florida with mortality events 

recorded only in 1946-47 and 1953-55 (Gunter et al., 1948; Rounsefell and Nelson, 1966 in 

Landsberg, 2002) and most recently during a prolonged bloom of K. brevis that occurred during the 

fall of 2005 and spring of 2006 (NOAA, Unpublished data). The bloom densities and cellular 

toxicities do not appear to differ between Southwest Florida and the Florida Panhandle. Overall the 

factors contributing to brevetoxin associated mortalities of bottlenose dolphins are poorly 

understood. Furthermore, the impact of repeated sublethal brevetoxin exposure to the health and 

fitness of bottlenose dolphins is unknown, and the consequences of repeated UMEs on bottlenose 

dolphin populations, particularly in the Florida Panhandle, has not been assessed. 

 This research and response plan summarizes the current knowledge of the circumstances 

resulting in red tide associated bottlenose dolphin mortality events and identifies data needed to 

better understand the acute and chronic impacts of brevetoxin exposure on individuals and 

populations of bottlenose dolphins. Because critical information needed to fill these knowledge 

gaps can primarily be obtained during these unpredictable red tide events, the response plan was 

developed to facilitate efficient data gathering and integration by the numerous organizations and 

agencies that participate in such investigations. This bottlenose dolphin – brevetoxin research and 

response plan is a product of a workshop held at Walt Disney World Resorts in Orlando, Florida 

between December 13 and 15, 2004 (see Appendix I for workshop participants) and outlines the 

steps needed to better understand: 

1. The role of brevetoxins in causing the 2004 bottlenose dolphin UME. 

2. The role of brevetoxins in causing historical and future bottlenose dolphin UMEs. 

3. The impact of repeat UMEs on bottlenose dolphin populations in the Florida Panhandle. 
2 

Insights Gained from the 2004 Panhandle Mortality Event 

Background 

 Four UMEs involving bottlenose dolphins have occurred in the northern Gulf of Mexico 

since 1993, almost as frequently as the occurrence of K. brevis blooms in this region of the Gulf. In 

1993-1994, an UME of bottlenose dolphins attributed to a morbillivirus epizootic started in the 

Florida Panhandle and spread west with most of the mortalities occurring in Texas (Lipscomb et al., 

1996). That mortality event was caused by morbillivirus as determined by histologic lesions, 

immunohistochemical demonstration of mobilliviral antigen, and detection of morbilliviral RNA by 

a reverse transcriptase polymerase chain reaction (Lipscomb et al., 1996). In 1996, dolphin 

mortalities coincided with an unusual bloom of K. brevis in Mississippi, but the role of brevetoxin in 

the fate of these animals was not confirmed (NOAA, 2004). Between August 1999 and May 2000, 

at least 152 bottlenose dolphins died in the Florida panhandle coincident with extensive and 

persistent Karenia brevis blooms (Mase et al., 2000; NOAA, Unpublished data). In 2004, 107 

bottlenose dolphins stranded dead on the coast of the Florida Panhandle between March 10 and 

April 13, 2004. In contrast with the 1999-2000 event, in 2004 a bloom of K. brevis was never 

detected, despite satellite remote sensing established in the region and extensive surveys funded 

through the National Oceanic and Atmospheric Administration (NOAA) emergency response 

funding. During both the 1999-2000 and 2004 bottlenose dolphin UMEs other species of marine 

life such as fish, invertebrates, sea turtles, and marine birds died. The 2004 UME and the 1999-2000 

UME both started in the same location (St. Joseph Bay) and extended along the seven counties of 

the Florida Panhandle (Escambia, Santa Rosa, Okaloosa, Walton, Bay, Gulf, and Franklin Counties). 

Unlike the 1999-2000 UME where a high proportion of dolphins stranded in Choctawhatchee Bay, 

only three animals stranded in this bay during the 2004 UME (NOAA, Unpublished data). 

Dolphin Ecology and Behavior in the Florida Panhandle 

 

The historical average of bottlenose dolphin strandings for the seven Panhandle counties 

(Bay, Escambia, Franklin, Gulf, Okaloosa, Santa Rosa, and Walton) over the last 10 years (1995-

2005) is 21.4 animals per year excluding 1999, 2000, 2004, and 2005 during which UMEs occurred 

(NOAA, Unpublished data). However, little is known of bottlenose dolphin behavior, ecology, and 

stock structure in the Florida Panhandle, including the region in and around St. Joseph Bay. To the 

best of knowledge, no systematic research was performed prior to the 2004 UME to determine the 

daily or seasonal patterns of use of St. Joseph Bay by bottlenose dolphins, or their broader ranging 

patterns or genetic composition as these might relate to stock structure. The occurrence of 2 dieoffs of over 200 animals in the last 5 years, as well as the recent biopsy darting and photographic 

identification work with dolphins in St. Joseph Bay and vicinity, indicate clearly that the 1993 aerial 

survey findings of no dolphins present in the Bay are out of date and do not provide sufficient 

information to determine impacts of these mortality events on coastal populations or stocks. 

Necropsy Findings 

For animals dying in the 2004 event, sex distribution was approximately equal: 36 females 

and 46 males, with 25 carcasses of unknown sex. Carcass measurements of 100 animals (not 

including 2 fetuses) show that length ranged from 76 cm (neonate) to 280 cm (adult). Comparing the 

results of length classes with historic stranding records for this region (n=51, over 7 years), a higher 
3 

number of animals over 210 cm are represented in this die-off. Most animals were found to be in 

good nutritional condition, however a few animals were considered to be thin for this season as 

compared to winter season dolphin nutritional condition in Sarasota, Florida. Many animals had 

evidence of generalized lymphoid stimulation, which was characterized by multiple enlarged lymph 

nodes with scarce cellularity. Lesions of the upper respiratory tract, indicative of aerosol exposure to 

brevetoxins as seen previously in manatees (Trichechus manatus latirostris), were not observed. 

Brevetoxins were found at high concentrations (average 1.8 μg/g, range 0.1 – 10 μg/g) in the 

stomach contents of all dolphins examined, which is a level greater than or equal to those observed 

in previous marine mammal mortality events associated with Florida red tides. In most of the 

dolphins, the first chamber of the stomach was filled with large amounts of fish, largely menhaden 

(Brevoortia spp.), a planktivorous herring-like fish. Some of the fish were partially whole and 

undigested indicating recent feeding. Mean stomach content values were 773 g, with 10 samples 

weighing > 1 kg (maximum: 2.258 kg). This suggests an acute intoxication through ingestion of 

menhaden that had ingested K. brevis cells. The fish themselves could have been compromised by 

brevetoxin intoxication (Steidinger et al., 1973; Lu and Tomchik, 2002) or could have been acting 

normally and just serving as vectors for the toxin after ingesting K. brevis cells. 

Although no bloom of K. brevis was identified, fish (planktivorous and omnivorous fish 

species) collected from St. Joseph Bay on March 18, 2004 tested positive for brevetoxins in stomach 

contents and in muscle, liver, and gill tissues. Fish collected on March 28-31, 2004 also tested 

positive for brevetoxin but at much lower levels. The identification of brevetoxin and brevetoxin 

metabolites in the tissues of fish for the first time identifies fish as a vector for transfer of 

brevetoxins up the food chain to top predators such as dolphins (Flewelling et al., 2005). 

Dr. Carlos Romero (University of Florida) tested samples collected during necropsy for viral 

nucleic acid using PCR and RT-PCR. All samples tested negative for: morbillivirus (50 samples from 

24 animals); influenza A virus (14 samples from 8 animals); influenza B viruses (14 samples from 10 

animals); calicivirus (26 samples from 15 animals); cetacean poxvirus (3 samples from 3 animals); 

herpes virus (10 samples); and parapoxvirus (3 samples from 3 animals). 

 

Concentrations of persistent organic pollutants (“POPs”, i.e., polychlorinated biphenyl 

congeners, DDT and its derivatives, and other organochlorine insecticides) in bottlenose dolphin 

blubber samples collected from the 2004 event were lower than those measured in free-ranging 

Sarasota Bay dolphins captured during health assessment work. Genetic analyses performed on 64 

sampled dolphins using DNA sequencing of a portion of the mitochondrial DNA genome indicated 

that all 64 animals were of the coastal morphotype. 

Conclusion 

 Overall, it appears that the 2004 bottlenose dolphin UME resulted from the transfer of 

brevetoxins from an undetected bloom through menhaden to dolphins via ingestion. The bloom 

possibly occurred offshore given that the usual signs of red tide were absent within St. Joseph Bay, 

including discolored water and aerosol effects. Bottlenose dolphins affected in this UME appeared 

to have gorged upon menhaden, a species not considered to be a major prey species for coastal 

bottlenose dolphins where their diet is known. The occurrence of brevetoxins in fish and seagrass 
4 

in St. Joseph Bay suggests that a bloom of K. brevis did occur in the region, possibly prior to the 

onset of the mortality event. Consistent with this hypothesis, large numbers of dead fish, jellyfish, 

and birds were documented during the same period in and around St. Joseph Bay. This UME 

identified for the first time the accumulation and persistence of brevetoxin in the muscle and viscera 

of fish, which can serve as a vector to higher trophic levels. The reason for the brevetoxin-related 

mortality of bottlenose dolphins in the Panhandle area of Florida, as compared to dolphins living in 

Southwest Florida, who are exposed annually to red tides, remains unresolved. 
5 

Coordinating Approaches 

 Harmful algal blooms (HABs) are complex in their effects as are the ecosystems in which 

they occur. Equally complicated is the array of scientists, managers, and private, state and federal 

agencies involved in studying and responding to HABs. In the U.S., the 1993 National Plan for Marine 

Biotoxins and Harmful Algae has served as a foundation to address HABs and marine biotoxins. Much 

has been learned about HABs and marine biotoxins since 1993 and agencies and organizations have 

changed. The national plan developed in 1993 was recently updated to reflect new findings and 

agency priorities and to redirect future resources to reflect current needs (HARRNESS, 2005). Many 

of the needs enumerated in the Harmful Algal Research & Response National Environmental Science Strategy 

2005-2015 document (HARRNESS, 2005) dovetail with needs required to better understand the 

impact of brevetoxins on bottlenose dolphins in the Gulf of Mexico. 

 

In the Gulf of Mexico, numerous private, state, and federal agencies respond to HABs and 

fish and wildlife related mortality events. No one agency or organization has the resources to address 

the multitude of research and monitoring needs required to understand the impacts of HABs on 

bottlenose dolphin populations and their significance to dolphin conservation and welfare. A 

coordinated effort is needed to answer the multitude of research and monitoring requirements. 

Historically, attempts have been made to bring together individuals and groups studying HABs and 

wildlife mortality events (e.g., the Gulf of Mexico Aquatic Mortality Network), however federal 

oversight and inclusion of groups and organizations working with cetacean strandings and pathology 

have been lacking. The December 2004 workshop held at Disney was the first time that many of 

these individuals and organizations have met, representing a critical first step in establishing the 

long-term professional cooperation needed to understand the impact of brevetoxins on bottlenose 

dolphins. Increased future collaboration and data integration are needed to develop this 

understanding of brevetoxicosis in dolphins. This document identifies the information, tools, and 

monitoring needs to do this, as well as the agencies and organizations who can accomplish them. 
6 

Critical Research Needs 

Bloom Ecology and Dynamics 

 In Florida, blooms of the dinoflagellate Karenia brevis most frequently originate offshore on 

the west Florida shelf where they can be transported inshore by currents and winds to the west coast 

between Tampa and Charlotte Harbor. Blooms tend to be seasonal, beginning in late summer and 

early fall and lasting into January or beyond (Steidinger, 1989). Over a century of data from the 

Florida Fish and Wildlife Conservation Commission suggest that blooms occur in the fall (Figure 2). 

Blooms in the Panhandle area are less frequent than along the southwest coast of Florida. As 

previously stated, we do not currently understand the factors that lead to brevetoxin-associated 

mortality in bottlenose dolphins during some blooms and not others. Improved understanding will 

require a more comprehensive monitoring of bloom ecology, particularly in the Panhandle region. 

 

In Florida, HAB monitoring for exposure assessment and physiological responses to exposure 

for the protection of public health from shellfish toxicity events is conducted jointly by the Florida 

Fish & Wildlife Conservation Commission’s (FWC) Fish and Wildlife Research Institute (FWRI), 

and the Florida Department of Agriculture and Consumer Services, Division of Aquaculture using 

fixed sampling sites associated with commercial shellfishing activities. This HAB monitoring 

program, which has successfully protected public health for 50 years, follows the guidelines of the 

Interstate Shellfish Sanitation Conference (a voluntary, cooperative association of states, U.S. Food 

and Drug Administration, National Marine Fisheries Service (NMFS), Environmental Protection 

Agency (EPA) and the shellfish industry). Shellfish beds are closed in the presence of 5,000 K. brevis

cells/L and reopened when shellfish contain <20MU/100g using the regulatory mouse bioassay. 

Test results and shellfish openings and closures are posted on the Internet 

(http://www.floridaaquaculture.com/RedTide/RedTideInfo.htm). Coastal areas not open to 

shellfish harvest are not closely monitored (Figure 3). Monitoring does not occur regularly in 

Florida Panhandle shellfish harvest areas unless there is a known bloom in the region. However, St. 

Joseph Bay is sampled monthly. 

 

An additional offshore red tide monitoring program for the Gulf coast of Florida has been 

instituted by FWC and utilizes volunteer charter boat captains to collect offshore water samples for 

red tide testing. Satellite imagery and red tide test results are routinely reported on the Internet 

(http://www.floridamarine.org/features/view_article.asp?id=9670#Rpt). On a larger scale, NOAA 

produces weekly HAB bulletins for the Gulf of Mexico posted on the Internet 

(http://coastwatch.noaa.gov/hab/bulletins_ns.htm). Satellite imagery, which detects grossly 

elevated chlorophyll levels (50,000 cells/L), has been successfully implemented as a forecasting and 

monitoring tool in Southwest Florida. However, the 2004 mortality event is an example in which 

extensive mortalities occurred in the absence of an observable bloom by either inshore sampling or 

satellite imagery. Better temporal and spatial documentation of K. brevis blooms are needed to 

understand their causal role in marine mammal mortality events in the Panhandle area. Therefore, it 

is recommended that additional bloom monitoring efforts be expanded in regions of the Panhandle 

that are prone to HAB-associated bottlenose dolphin mortality events. 

 The 2005 Harmful Algal Research and Response: A National Environmental Science 

Strategy (HARNESS) report calls for establishment of a coast-wide monitoring and data 
7 

management system through NOAA’s Integrated Ocean Observing System (IOOS). The Gulf of 

Mexico HAB community and developing regional IOOS regional associations have an existing 

infrastructure in the Harmful Algal Blooms Observing System (HABSOS) which integrates buoy 

data, remote sensing, cell counts, and volunteer monitoring. It is recommended that increased 

surveillance be implemented in the Panhandle region with particular emphasis on understanding the 

occurrence and food web effects of K. brevis blooms in this area. 

 Using receptor binding assay, ELISA, and LC tandem mass spectroscopy, trace levels of 

domoic acid were found in the stomach contents and feces of some, but not all, of the stranded 

dolphins tested from the 2004 event (FWRI, St. Petersburg, FL and NOAA/National Ocean Service 

[NOS], Charleston, SC). This was the first time that domoic acid had been documented in tissues 

from marine mammals that have stranded in the Gulf of Mexico, however the concentrations of 

domoic acid found were much lower than those found associated with marine animal mortalities 

documented in other regions, such as California (Gulland, 2000). Additionally, Pseudo-nitzschia

delicatissima was present in water samples, although domoic acid concentrations were below levels 

detectable by ELISA (FWRI, St. Petersburg, FL and NOAA/NOS, Charleston, SC). Shellfish 

samples collected from the area had domoic acid concentrations well below the established 

regulatory limit (20 μg/g) for public health protection. Domoic acid intoxication is not considered as 

a primary cause of the 2004 dolphin UME. In response to the mortality, a preliminary study aiming 

to identify possible synergistic effects on mammals of concurrent exposure with domoic acid and 

brevetoxins was carried out. This study revealed that in mouse models and at the toxin levels 

observed in dolphins, presence of domoic acid did not increase the potency of the brevetoxins and 

do not result in synergistic effects (Naar and Flewelling, unpublished data). Studies on Pseudo-nitzschia

bloom ecology should be combined with efforts to better understand K. brevis bloom ecology and 

dynamics. It is important to note that domoic acid has never been documented to be a problem in 

the Gulf of Mexico, however proactive efforts to better understand Pseudo-nitzschia ecology and the 

potential for domoic acid toxicity in the region will provide important baseline information this 

historical situation change. 

 Work is needed to better understand the potential for oceanographic and other factors to 

cause brevetoxin-related bottlenose dolphin strandings to be over-represented in Gulf County, 

Florida (or at least in St. Joseph Bay). St. Joseph Bay is a small embayment (29.80° N 85.36° 

W),which is partially isolated from the Gulf of Mexico by St. Joseph Peninsula/Spit, which extends 

from Cape San Blas in the south to St. Joseph Point in the north. It is the only body of water in the 

eastern Gulf of Mexico not influenced by the inflow of fresh water. In non-UME years, bottlenose 

dolphin strandings are extremely rare in St. Joseph Bay and on the Coast of Gulf County. However, 

the 1999-2000 and the 2004 brevetoxin-associated UMEs started in St. Joseph Bay. During the 2004 

event, 70 percent of strandings were localized in St. Joseph Bay (NOAA, 2004). 

 Work is also needed to better understand why brevetoxin-related dolphin strandings were 

over-represented in Choctawhatchee Bay during the 1999-2000 UME, as well as during the recently 

declared 2005-2006 Bottlenose Dolphin UME (NOAA, Unpublished data). Choctawhatchee Bay lies 

west of St. Joseph Bay within the counties of Okaloosa and Walton (30.45° N 86.45° W). 

Freshwater flow to the bay comes from the Choctawhatchee River, Pea River, Wrights Creek, Sandy 

Creek, Pine Log, Seven Run, Holmes Creek, and Bruce Creeks. The bay is separated from the Gulf 

of Mexico along most of its length, but connects through the Pensacola and East Passes entering 

through the south at Destin Pass. 
8 

 In addition to increasing baseline monitoring for HABs and background brevetoxin levels in 

these bays (discussed in detail under Food Web Studies), detailed bathymetric mapping of the region 

combined with passive oceanographic drift card studies are needed to characterize the bays’ 

oceanographic features as they might relate to brevetoxin bloom or toxin persistence. 

Food Web Studies 

 Evidence from the 2004 dolphin mortality event suggests that brevetoxin can be 

concentrated in shellfish and fish in the absence of detectable blooms, resulting in dolphin exposure 

that is delayed or remote from Karenia blooms (Flewelling et al., 2005). Therefore, improved efforts 

to monitor the duration and extent of biotoxins in marine waters also need to document the extent 

and duration of biotoxins in living marine organisms, including fish and shellfish. Active sampling of 

organisms within and proximal to defined bloom areas is probably the most effective way to do this. 

 St. Joseph Bay has been the apparent epicenter of two brevetoxin-induced dolphin mortality 

events and both St. Joseph Bay and Choctawhatchee Bay have been over-represented with 

strandings during recent brevetoxin-associated UMEs. Increased baseline monitoring for HABs in 

these bays is warranted. A suite of biological indicator organisms, including fish and shellfish, 

should be considered for monitoring background brevetoxin levels. Due to the presence of K. brevis, 

shellfish harvest in St. Joseph Bay was closed on November 19, 2003, four months prior to the 2004 

bottlenose dolphin mortality event. Oysters collected and tested on February 2, 2004 were still too 

toxic to re-open and were not tested again until after the dolphin UME was underway. At that time, 

levels were <20MU/100g but due to the on-going dolphin mortality in the region, shellfish harvest 

remained closed until April 29, 2004 for precautionary reasons. Real-time data on toxin levels in 

shellfish can provide important information on brevetoxin persistence in the Bay. Expanding 

within-bloom shellfish monitoring to include other organisms such as fish feeding at different 

trophic levels would provide valuable data on brevetoxin persistence within the ecosystem and help 

better document potential avenues for bottlenose dolphin or human exposure.

Priority Research and Monitoring Needs: Bloom Ecology and Dynamics 

• Increase temporal and spatial coverage of routine HAB 

monitoring in the Florida Panhandle region 

• Implement the use of buoys for remote monitoring in and 

offshore of the St. Joseph Bay and Choctawhatchee Bay areas 

• Incorporate the Florida panhandle into the existing HABSOS 

monitoring network 

• Compile or develop a detailed understanding and GIS analysis 

of St. Joseph and Choctawhatchee Bays’ oceanographic features 

(bathymetry, wind, currents, etc.) and how they influence bloom 

dynamics and toxin and carcass distribution 
9 

 Experimental tracing of brevetoxins from dinoflagellates through copepods grazers to 

juvenile fish has been conducted, but work needs to be done looking at this transfer in natural 

settings (Tester et al., 2000; and Landsberg, 2002). Presently it appears that we see parent toxins and 

metabolites in planktivorous and carnivorous fish (note: PbTx-2, which is rapidly metabolized, is not 

observed in carnivorous fish; Flewelling et al., 2005 and Flewelling, pers. comm.). Experimental 

exposure revealed that brevetoxin accumulates in the muscle and viscera of all fish types tested, 

however levels are higher in viscera than in muscle (Flewelling et al., 2005). It is not known how 

long fish can remain toxic and what concentrations of which metabolites cause acute or chronic 

disease in bottlenose dolphins. Field work on biotoxins in the food chain should be closely linked 

with laboratory work on the toxins and their effects. 

 The trophic transfer of brevetoxin in the marine ecosystem and the mechanism of 

bottlenose dolphin exposure could provide important information about when and where K. brevis

blooms cause brevetoxicosis in bottlenose dolphins. It has been hypothesized and is understood 

that manatees can be exposed to brevetoxins via ingestion or inhalation (O’Shea et al., 1991; Bossart 

et al., 1998; Landsberg and Steidinger, 1998; and Flewelling et al., 2005). Unlike the 1996 manatee 

mortality event where upper respiratory tract lesions suggested inhalation as a route of brevetoxin 

exposure for manatees (Bossart et al., 1998), data from the 1999-200 and 2004 dolphin mortality 

events suggest that the route of exposure in bottlenose dolphins was likely oral (Flewelling et al., 

2005; Mase et al., 2000). In a 1987-1988 bottlenose dolphin mortality event where morbillivirus was 

the primary etiology, brevetoxin was detected in dolphin liver samples, as well as in the viscera of 

migratory menhaden. It was hypothesized that menhaden were likely sources of brevetoxin for 

dolphins (Geraci, 1989; Steidinger, 1989). Spanish mackerel (Scromberomorus maculatus), a preferred 

food item for dolphins, prey on menhaden and were hypothesized to be another route through 

which dolphins ingested brevetoxin (Steidinger, 1989). During the 2004 event, preliminary works 

show that menhaden was the dominant prey species in at least 50 percent of the dolphin stomach’s 

examined. Shrimp, sciaenid fish, and other fish also were found in the stomachs of dead dolphins. 

Menhaden are not considered a significant dietary item for bottlenose dolphins off the coast of west 

central Florida (Barros and Wells, 1998), east central Florida (Barros, 1993), North Carolina 

(Gannon and Waples, 2004), nor at other locations in the Southeastern U.S. (Barros and Odell, 

1990). Despite this, menhaden seem to be over-represented in the stomach contents from dolphins 

in the 1999 and 2004 brevetoxin-associated mortality events. 

Clearly, it is important to better understand the preferred diet of bottlenose dolphins inside 

and outside of regions where brevetoxin causes mortality in dolphins. Understanding the trophic 

transfer of brevetoxin and its metabolites in the marine ecosystem is also needed. Evaluating the 

stomach contents of stranded dolphins is one mechanism by which to learn more about dolphin 

diets, especially when animals die acutely or are determined to die of brevetoxicosis. Stomach 

contents from all stranded dolphins, however, will likely not be characteristic of preferred prey in 

many cases, especially if animals show signs of chronic disease. Therefore, it is still critical to 

identify otoliths and boney remains in the stomachs of all stranded dolphins. Whole stomachs 

should be sent to a laboratory where contents will be weighed and accurately identified. Currently, 

this is being performed at Mote Marine Laboratory. When present, gastric juices and fleshy remains 

of prey will be submitted for brevetoxin analysis with samples being alternately sent to a 

NOAA/NOS laboratory and the FWC FWRI. Additionally, a project using biopsy and necropsy 

samples and fatty acid or stable isotope analysis should be developed to learn more about the 

trophic level at which different dolphin stocks are feeding. Finally, during HAB events with 

documented dolphin mortality, efforts should be made to recover and analyze brevetoxin levels in 
10 

fish in dolphin stomachs, fish caught near mortality sites, dead fish on the beach, and fish bought 

from local fish markets. 

Toxin and Effects 

 Brevetoxins are neurotoxins and hemolysins produced by Karenia brevis (a dinoflagellate) and 

Chloromorum toxicum (Thomas et al., 2005; Landsberg, 2002). Brevetoxin-like compounds have been 

identified in another Karenia sp. and in the raphidophytes C. antique, C. marina, Heterosigma akashiwo, 

and Fibrocapsa japonica (Landsberg, 2002). Historically, brevetoxin-producing Karenia blooms have 

occurred more frequently and impacted a greater range of species than raphidophyte blooms, which 

seem to be primarily ichthyotoxic. Brevetoxins are complex lipid soluble polycyclic ethers. The nine 

brevetoxins that have been isolated from K. brevis (PbTx-1, 2, 3, 5, 6, 7, 8, 9, and 10) have varying 

degrees of toxicity and are differentiated into two groups based on whether they have a 10-ring 

(PbTx-1 type) or 11 ring backbone (PbTx-2 type) (Baden, 1989; Schulman et al., 1990). Twenty-one 

toxin metabolites also have been identified (Wang et al., 2004). 

 The mechanism of toxin action is sodium channel activation: toxin binds to site 5 on the 

voltage gated sodium channel, resulting in channel opening at normal resting potential and 

prolongation of their open state, leading to uncontrolled Na+

 influx into the cell (Poli et al., 1986; 

Gawley et al., 1992). Toxin metabolism is not as well understood. In rats, PbTx-3 (Brevetoxin-3) 

injected intravenously was cleared (90 percent) within 1 minute, but a sub-lethal oral dose remained 

in serum up to 192 hours post consumption, with maximum concentration found at 48 hours (Poli 

et al, 1990). However, differences in the rate and mechanism of clearance of PbTx-2 versus PbTx-3 

from blood are seen in rats. PbTx-2 is rapidly transformed to a polar metabolite of reduced 

biological activity that appears in the blood and is retained for up to 4 hours, yet is cleared entirely to 

Priority Research and Monitoring Needs: Food Web Studies 

• Increase monitoring for HABs and background brevetoxin and 

domoic acid levels in the St. Joseph Bay region, including 

monitoring of toxin levels in seagrass, shellfish, and fish from the 

region 

• Understand the feeding ecology of bottlenose dolphins in the 

region of St. Joseph and Choctawhatchee Bays as compared to 

dolphins in other regions that do not seem to be as impacted by 

brevetoxin, especially sites that appear to have more K. brevis

blooms, but less brevetoxin-associated dolphin mortality 

• Compare stomach contents between dolphins diagnosed with 

brevetoxicosis and dolphins dying from other causes 

• Determine the tissue distribution of toxins and metabolites in fish 

that are positive for brevetoxin 
11 

the urine within 24 hours in the form of cysteine adducts (Radwan et al, 2005). At the same dose, 

only about one-fifth as much PbTx-3 was cleared to urine. The routes of toxin clearance in orally 

exposed mammals (rats) appear to be fecal, then renal; probably with most metabolism and highest 

levels occurring in the liver (Cattet and Geraci, 1993). In vitro studies with hepatocytes identified the 

involvement of cytochrome P450 and glutathione in generating metabolites of PbTx-2 in rat 

(Radwan and Ramsdell, In Press) Toxin metabolism, the pathogenesis of toxicosis, and the 

mechanism of toxin clearance have not been studied in bottlenose dolphins and more research is 

needed. Experimental dosing of bottlenose dolphins is not an option, so alternate non-invasive and 

opportunistic strategies such as immunohistochemistry tracking of metabolites in tissues of dead 

stranded dolphins will have to be employed. 

 During the 2004 mortality event, metabolites in stomach, liver and kidney were identified in 

approximately six animals. PbTx-3 and -9 dominated in liver and kidney, with the cys-conjugate also 

present in stomach contents, indicative of the presence of PbTx2 (Radwan et al., in prep). Efforts 

should be made to compare the PbTx (brevetoxin) types and concentrations in other mortality 

events to gain further insight into the levels and form that functionally compromise and adversely 

affect bottlenose dolphins. In addition, the identification and quantization of toxin metabolites 

present in different dolphin tissues will help to confirm if uptake, metabolism, and clearance 

patterns are similar to those in rodent models and may yield insight into the contributions of oral 

versus aerosol routes of exposure. Improved immunohistochemical detection of brevetoxin in 

tissues of intoxicated dolphins will further serve to refine the understanding of its mechanism of 

toxicity in stranded animals. Collectively, these data will help to create a case definition for 

brevetoxicosis in bottlenose dolphins and potentially allow the definition of sub-acute or chronic 

exposure. Additionally, a better understanding of PbTx types and metabolism should help the 

understanding of toxin movement and metabolism through marine trophic levels (see food web 

studies). 

In addition to research on brevetoxin metabolism and clearance, additional tools are needed 

to assist in monitoring of toxin associated with dolphin mortalities. In particular, the investigation 

of brevetoxin associated mortality events would be greatly facilitated by the development of a rapid 

portable test to confirm presence/absence of brevetoxin in urine or blood. Confirmation of 

brevetoxin exposure in a stranded animal by field personnel would aid in initiating collection of the 

proper evidence of blooms (which may not be evident from shore), could potentially help 

responders treat live-stranded dolphins, and would facilitate collection of critical tissue samples for 

the quantitative analyses described above in dead-stranded animals. 
12 

Acute Impact on Bottlenose Dolphins 

 Just as better documentation of the temporal and spatial distribution of K. brevis blooms is 

needed (see Bloom Ecology and Dynamics), so are better documentation and investigation of 

cetacean mortality events. The relationship between the blooms and dolphin mortality events will be 

better understood only with improvement in both of these areas. Marine mammal mortality events 

in Florida are monitored by NOAA’s Marine Mammal Health and Stranding Response Program, 

which includes a Stranding Network comprised of other government agencies, non-governmental 

agencies, aquaria, and universities. The historical average of bottlenose dolphin strandings for the 

seven Panhandle counties (Bay, Escambia, Franklin, Gulf, Okaloosa, Santa Rosa, and Walton) over 

the last 10 years (1995-2005) is 21.4 animals per year excluding 1999, 2000, 2004, and 2005 during 

which UMEs occurred (NOAA, Unpublished data). Excluding brevetoxin-associated UMEs, 

information is not available on the level of brevetoxin (background or other) in stomach contents, 

feces, or tissues from bottlenose dolphins in the Florida Panhandle. In fact, brevetoxin levels in 

dolphins stranded off the coast of South Carolina were used as references during the 2004 UME 

(NOAA, 2004). Despite frequent and numerous documented K. brevis blooms in the Gulf of Mexico, 

few brevetoxin-related bottlenose dolphin mass mortality events have been documented (Figure 1). 

More work is needed to determine why some K. brevis blooms in the Gulf of Mexico result in 

bottlenose dolphin mortality, and why the majority apparently do not. 

 The Florida coast along the northern Gulf of Mexico (including the Panhandle and 

Northwest Florida) currently has some areas where stranding response is well-covered, and others 

where it is either limited or have no coverage. It is imperative that efforts be made to: (1) improve 

coverage and response to marine mammal strandings in the region; (2) provide additional training 

for responders to collect Level B and C data, with an emphasis on collecting samples for prey 

identification and brevetoxin and domoic acid analysis; and (3) make every effort to determine the 

cause of death and the level of brevetoxin and domoic acid involvement in all regional marine 

Priority Research and Monitoring Needs: Toxin and Effects 

• Understand the relative toxicities of brevetoxins and their 

metabolites in bottlenose dolphins using non-invasive opportunistic 

investigations or in-vitro techniques 

• Understand metabolism, retention and clearance of brevetoxins in 

bottlenose dolphins using non-invasive techniques or a biomarker 

that acts as a proxy for brevetoxin clearance 

• Develop toxin-specific biomarkers of exposure and effects for 

assessing sub-lethal and chronic exposure 

• Develop a case definition for brevetoxicosis in bottlenose dolphins 

• Develop a field test for rapid assessment of brevetoxin exposure in 

bottlenose dolphins
13 

mammal strandings. Opportunities (realistic and idealistic) to improve marine mammal stranding 

response in under-served areas include: 

 Fund a stranding response person at the NOAA Panama City laboratory whose job it will be 

to coordinate more effective response to strandings. 

 Pay for weekly aerial surveillance to establish a stranding baseline and facilitate retrieval of 

carcasses while they are still reasonably fresh (estimated cost of $2,000-4,000/week). 

 Develop a standard fly-over and reporting schedule utilizing the U.S. Coast Guard or local 

Air Force Base (Eglin Air Force Base) to survey and report stranded marine mammals. 

 Develop and implement a sustained outreach campaign to educate the public about the 

importance of reporting live and dead strandings, and how to report stranded marine 

mammals. 

 Provide necropsy training for more Stranding Network volunteers. 

 Provision and fund a rapid response necropsy team to assist local stranding response groups 

with mass strandings. 

 Provide freezer storage space to store the volume of tissues that often result from a UME. 

 Build capacity for stranding response in Northwest Florida, in areas where there is no 

stranding response. 

In addition to enhancing routine marine mammal stranding response, efforts should be made to 

enhance surveillance for strandings during documented HABs, which could improve diagnosis of 

low level, HAB-related mortality. There is concern that Karenia spp. other than K. brevis are present 

in the Gulf of Mexico and that they can produce brevetoxin-like compounds. Because there is 

interest in the impact of Pseudo-nitzschia spp. and other toxin-producing algae on bottlenose dolphin 

populations, all identified HABs, and not just K. brevis blooms, need to be investigated for associated 

marine mammal mortality. 

 To adequately monitor HABs for associated cetacean mortality there needs to be a 

mechanism for alerting management agencies and targeted surveillance groups that a bloom has 

been identified in their region and that they need to heighten their surveillance efforts. An old multistate HAB alert network called GMNET (Gulf of Mexico Aquatic Mortality Network) sponsored by 

the EPA used to alert natural resource managers about mortality events and potential or diagnosed 

HABs. It was suggested that NOAA or the Department of Homeland Security should be asked to 

resurrect GMNET or possibly ask the EPA if they are interested in resurrecting GMNET. A fish-kill 

hotline (1-800-636-0511) is currently in operation and is run by the FWRI’s Fish and Wildlife Health 

(FWH) Group. The public can call this number to report fish kills, and often use it to report other 

wildlife mortality events. Callers are either referred to the appropriate agencies or the appropriate 

agencies are contacted after FWC receives the call. It could potentially be officially expanded to 

include reporting of wildlife mortality and integrated with the current system for reporting and 

responding to marine mammal strandings. As appropriate for the relevant species, unofficially FWRI 

FWH staff call the pre-established NOAA number for reporting marine mammal mortalities (1-888-

404-3922), but perhaps a more official memorandum of understanding should be developed. 

 An electronic discussion group that alerts people to the presence of HABs and provides a 

venue for discussion of fish kills also exists and is maintained by the FWC/FWRI. Electronic mail is 

sent out weekly alerting participants of HAB status and detailed HAB status reports and maps are 

frequently attached. Weekly maps of HAB distribution are posted on the FWC/FWRI website at 

http://floridamarine.org/. Marine mammal stranding networks should be linked into the FWC’s 
14 

discussion group as a means of better understanding when HABs are present in their region. In 

addition, stranding network responders should sign up to receive the HAB bulletin, which is 

electronically sent to subscribers twice weekly and reports developing blooms and changes in the 

location and extent of existing blooms (see website at http://www.csc.noaa.gov/crs/habf/). 

Electronic communications reporting HAB locations and requests for people to be alert for wildlife 

mortalities should also target members of state parks, wildlife reserves, wildlife rehabilitation 

organizations, county sheriff departments, beach watcher programs, the Florida Marine Patrol, and 

others that have routine beach exposure or are frequently alerted to marine mammal mortality. 

These people could use the pre-established NOAA number for reporting marine mammal 

mortalities (1-877-433-8299). Ultimately all cetacean stranding reports should go to NOAA 

Fisheries. All marine mammal mortality investigations should culminate in a report that is sent to a 

central clearinghouse, which needs to be established. A dedicated person or group of people should 

create and maintain a database of HABs with and without reported marine mammal mortality (and 

wildlife mortality; see next chapter), as well as a database of causes of marine mammal mortality, 

including information on what proportion are HAB-related. There is on-going work with on 

electronic databases with the FWC, the Florida Department of Health, and NOAA and new 

initiatives should build upon these efforts. 

 In addition to receiving complete post-mortem examinations that include histopathology 

and ancillary testing directed to determining cause of death, all cetaceans collected for necropsies 

(presumed HAB-associated and presumed non-HAB associated) should be examined according to 

similar standardized testing for acute and chronic exposure to HAB-related toxins. This should 

include toxicological testing performed on a suite of similar tissues, as well as immunohistochemistry 

on a suite of standardized paraffin-embedded tissues, which help define the presence, abundance, 

and distribution of brevetoxins in tissues. A standardized necropsy form should be used that 

includes sampling guidelines, a handling guide for tissues (packaging, shipping, and chain of 

custody), photographic requirements, and morphometric requirements. Appendix II contains a 

preliminary description of samples requested by researchers for certain tests and Appendix III 

contains a list of samples to be collected from every animal that is necropsied. 

 HAB testing proposed in Appendix II should be limited to all marine mammals in carcass 

condition 2 or 3. Captive bottlenose dolphins in Florida that die also should be tested for exposure 

to brevetoxin and domoic acid, especially if locally caught fish make up some portion of their diet. 

Necropsy and test results (final reports) for every case should be submitted to a central database as 

described above, which will permit differentiating HABs that have associated fish and wildlife 

mortality and those that do not. 

 In addition to expanding the existing HAB electronic discussion group in an effort to 

identify HAB-associated marine mammal mortality, work needs to done to enhance the recovery 

and necropsy of stranded marine mammals even when HABs are not identified in the region. 

Individual stranding networks could be empowered to do this as they see fit or a regional advertising 

campaign could be considered. All non-HAB related stranded marine mammals also should be 

subjected to the suite of testing described in Appendix II. 

 The pharmacokinetics, residence time, and pathogenesis of brevetoxin in bottlenose 

dolphins are unknown. A validated bottlenose dolphin specific immunohistochemical test for 

determining the presence of brevetoxin in paraffin-embedded tissues would greatly improve the 

understanding of the pathogenic mechanisms of lethal and sub-lethal chronic exposure in this 
15 

species. Immunohistochemistry that utilizes a primary goat anti-brevetoxin has been developed and 

has been utilized to identify brevetoxin in tissues of manatees (Bossart et al., 1998). This is a 

polyclonal antibody that reacts with all nine brevetoxins. It has also been modified to eliminate 

nonspecific background and used to test tissues of double-crested cormorants (Kreuder et al., 2002). 

Work is underway to further modify and utilize the technique on tissues of bottlenose dolphins 

(Bossart, unpub. data). Immunohistochemistry identified brevetoxin in macrophages and 

lymphocytes from multiple tissues in manatees (Bossart et al., 1998) and in lymphoid cells (spleen), 

macrophages (spleen and lung), tracheal mucosa, heart, and brain of double-crested cormorants 

(Kreuder et al., 2002). Once the technique is perfected for bottlenose dolphins, 

immunohistochemistry performed on brevetoxin and non-brevetoxin associated dolphin mortalities 

may increase the understanding of pharmacokinetics, residence time, and pathogenesis of brevetoxin 

in this species, especially if a method is developed to distinguish between toxic and non-toxic 

metabolites. 

 Although not discussed in detail at the Orlando meeting, live-stranded bottlenose dolphins 

provide an additional opportunity to learn more about the acute effects of brevetoxins. The Marine 

Mammal Stranding Network responds to live stranded animals and rehabilitation facilities including, 

but not limited to, Gulf World Marine Park, Mote Marine Laboratory, and Clearwater Marine 

Aquarium will take these animals into rehabilitation when appropriate. Developing and using a 

sampling protocol for live-stranded dolphins in rehabilitation centers offers a great opportunity to 

study naturally occurring brevetoxicosis-related morbidity in bottlenose dolphins. It also permits 

comparisons between dolphins in Sarasota and the Panhandle, which differ dramatically in the 

number of dolphins dying of brevetoxicosis. A protocol should be developed that permits testing of 

all live-stranded dolphins for acute brevetoxin and domoic acid exposure, including length of time, 

course of metal excretion, and clinical symptoms. Also, they may be tested for antibodies to 

brevetoxins if a test is available. Animals collected during live-capture, sample, and release health 

assessment projects should also be similarly evaluated when possible. 
16 

Acute Impact on Other Species 

 While monitoring for marine mammal mortality, it is also important to monitor documented 

HABs for associated mortality in birds, sea turtles, fish, and other species. The FWC has been 

monitoring HABs and networking with local resource agencies, academia, and volunteer groups for 

associated non-cetacean wildlife mortality events and these efforts should be enhanced. In order to 

adequately monitor HABs for associated fish and wildlife mortality there needs to be an enhanced 

mechanism for alerting management agencies and targeted surveillance groups (beyond the FWC) 

that a bloom has been identified in their region and that they need to heighten their surveillance 

efforts to document and diagnose associated fish and wildlife mortality (see Acute Impacts on 

Priority Research and Monitoring Needs: Acute Impact on Dolphins 

• Improve marine mammal stranding response in under-served 

areas and enhance routine marine mammal stranding response 

• Develop toxin-specific biomarkers of exposure and effects for 

assessing acute (lethal and sub-lethal) and chronic exposure 

including protein biomarkers in serum and urine as well as the 

expression of brevetoxin antibodies in serum 

• Increase testing of code 1 and 2 stranded bottlenose dolphins 

for brevetoxin and domoic acid in non-UME situations. Liver, 

stomach, urine and feces should be tested. 

• Perform standardized postmortem examinations on all code 2 

bottlenose dolphins including contaminates, infectious 

diseases, and presence or absence of brevetoxins and domoic 

acid. 

• Develop a case definition for brevetoxicosis in bottlenose 

dolphins 

• Develop improved immunohistochemistry methods for 

diagnosis of brevetoxicosis in paraffin-embedded marine 

mammal tissues 

• Develop a kinetic model for brevetoxicosis in bottlenose 

dolphins 

• Establish a lethal effect level for brevetoxin in bottlenose 

dolphins 

• Coordinate the reporting of marine mammal disease testing 

into a central database or sharing of data to permit 

retrospective analysis of region-wide marine mammal 

mortality 

• Develop and utilize a sampling protocol for testing livestranded dolphins for acute and chronic brevetoxin exposure 
17 

Bottlenose Dolphins). If they are not already doing so, regional fish, bird, and turtle biologists 

should be encouraged to join an electronic discussion list or join the HAB bulletin distribution list 

so that they might have a heightened awareness of mortality during HAB events. 

 Bird mortalities in the state of Florida are reported to the FWC (see the website at: 

http://myfwc.com/bird/default.asp). Staff from FWC is available to respond to potential marine 

bird events and to test for potential HAB involvement. Marine bird mortality calls reported through 

the fish kill hotline are transferred to Dr. Danielle Stanek (FWC,FWRI) who coordinates response 

efforts and diagnostics with collaborating organizations including FWC, University of Florida 

College of Veterinary Medicine (Drs. Spalding and Forrester), the Southeastern Cooperative Wildlife 

Disease Study (Dr. Kevin Keel, Athens, GA), FDACS Animal Diagnostics laboratory (Kissimmee, 

FL), Mote Marine Laboratory (Dr. Deb Fauquier, Sarasota, FL), or the National Wildlife Health 

Center (Dr. Grace McLaughlin, Madison, WI). Bird rehabilitation facilities in the Florida Panhandle 

should be educated about brevetoxicosis and other HABs in fish-eating marine birds. A list of 

regional bird rehabilitation facilities could be developed and guidelines could be written directing 

these facilities to submit appropriate carcasses of fish-eating marine birds to a pre-determined 

postmortem laboratory or pathologist for necropsy or at least to submit tissues for brevetoxin 

testing. 

 

 Sea turtle mortality should continue to be reported to NMFS or to FWC (Allen Foley, 904-

573-3930). Non-marine mammal wildlife collected for necropsies (presumed HAB-associated and 

presumed non-HAB associated) need to receive standardized testing for acute and chronic exposure 

to HAB-related toxins, just like marine mammal mortality investigations. Due to the sheer numbers 

of fish kills and bird mortality reported, HAB testing proposed in Appendix II should be limited to 

mass mortality of fish-eating marine birds (>5 birds) and all sea turtles. 

 Fish, bird, and sea turtle mortality and HAB reports are separately housed in the FWC, and 

plans are being made for integration. These data could also be linked to a federal database that 

contains marine mammal mortality information. This could be done by linking marine mammal, 

bird, sea turtle, and fish mortality databases with a HAB database, which would permit evaluation of 

HABs on causing fish and wildlife mortality. 

Priority Research and Monitoring Needs: Acute Impacts on Other Species 

• Appropriate agencies should continue to investigate the impact of 

brevetoxins as a mortality factor for birds, turtles and fish and 

improve coordination of findings with marine mammal managers 

• Fish, bird and turtle mortality reports should be integrated into a 

federal database with marine mammal mortality information. 
18 

Impact of Chronic Brevetoxin Exposure on Bottlenose Dolphins 

The lipophilic nature of brevetoxins suggests that they are likely metabolized and stored in 

the tissues and/or blubber of dolphins for some time after an acute exposure. However, the body 

burden of brevetoxin in bottlenose dolphins exposed repeatedly to brevetoxin on the Southwest 

Florida coast has not been established and may be critical to establishing the toxic effect level in 

acute exposure cases. The use of blood collection cards has shown promise for monitoring exposure 

in living animals exposed to red tides (Fairey et al., 2001). However, the timeframe in which this 

method is useful following exposure has not yet been determined. 

An alternative to testing toxin itself in living animals is to use protein biomarkers as a proxy 

for toxin exposure. The development of biomarkers specific for brevetoxin exposure is feasible 

with the use of toxicogenomic (microarray) analyses or plasma following exposures in or proteomic 

analyses in serum or urine of exposed animals. Protein biomarkers would be expected to provide a 

longer-lived signal of toxic exposure or effect than the toxin alone. 

The consequences of chronic and repeat exposures to oral brevetoxin exposure have not 

been investigated, even in rodent models. Brevetoxin-exposure should be evaluated as a risk factor 

for chronic diseases identified in stranded animals. Aerosol exposures in manatees (Bossart et al., 

1998) and rats (Benson et al., 2005) have previously been shown to cause immunosuppression. 

Many examples exist to suggest that immunosuppression brought on by acute toxicities may 

facilitate the establishment of secondary infections. Thus investigating the possible 

immunosuppressive effect of oral exposure to brevetoxin in a rodent model would provide insight 

into its possible adverse effects in dolphins. 

Conversely, given the episodic exposures experienced by dolphins in Southwest Florida, an 

obvious question arises as to whether repeat exposure confers some level of resistance to 

brevetoxicosis that the Panhandle dolphins lack. Although the brevetoxin molecule is somewhat 

small to be predicted to be antigenic, nonetheless exploration of the possibility of an adaptive 

immune response in Southwest Florida dolphins is warranted and could be addressed using a 

competitive ELISA to assess anti-PbTx antibodies in dolphin serum. 
19 

Impact on Bottlenose Dolphin Populations 

 Although the information is integral to understanding brevetoxin-associated mortality and 

population impacts of brevetoxin UMEs on bottlenose dolphins in the Florida Panhandle, little is 

known about bottlenose dolphin stock structure in the region. Currently, NMFS designates each of 

the 33 bays, sounds, and estuaries in the in the Gulf of Mexico as separate stocks, along with 3 

separate coastal stocks. This does not include St. Joseph Bay, where dolphins were not observed 

during the 1993 aerial surveys that were used to designate these putative stocks (NOAA, 2004). 

Little information exists about the natural history of bottlenose dolphins in the Florida Panhandle. 

A better understanding of home range and movement patterns for dolphin stocks in the region will 

allow a more accurate assessment of which stocks could be impacted by blooms. Brevetoxicosis, 

however, is more complex than dolphin temporal and spatial association with blooms and 

information is needed beyond just spatial and temporal movement of dolphins. Evidence from the 

2004 UME strongly suggests that dolphins orally ingested brevetoxin by feeding on fish that 

accumulated toxin through the food chain. If bottlenose dolphin feeding ecology varies significantly 

between stocks, knowledge about preferred diets in different regions or at different times of the year 

combined with better knowledge of toxin movement through the food chain could give a better 

understanding of why some dolphin populations are impacted by brevetoxin while others are not. 

 Work is underway to characterize the bottlenose dolphin population in the St. Joseph Bay 

region and this information should be included in a comprehensive re-evaluation and possible restructuring of bottlenose dolphin stocks along the Florida panhandle. All stocks that could 

potentially be affected by a HAB need to be described. This description should include a 

description of the stock's geographic range, a minimum population estimate, current population 

trends, current and maximum net productivity rates, and optimum sustainable population levels. In 

addition, baseline information about the behavior, ecology, and health status of these stocks should 

Priority Research and Monitoring Needs: 

Impacts of Chronic Exposure on Dolphins 

• Establish brevetoxin body burdens in dolphins repeatedly exposed 

to brevetoxin 

• Develop toxin-specific biomarkers of exposure and effects for 

assessing sub-lethal and chronic exposure including protein 

biomarkers in serum and urine as well as the expression of 

brevetoxin antibodies in serum 

• Utilize developed tests to investigate potential chronic impacts 

• Evaluate the immunological integrity and potential suppression of 

brevetoxin-exposed and non-exposed bottlenose dolphin 

populations 

• Determine if brevetoxin exposure has reproductive effects in 

bottlenose dolphins 
20 

be gathered. Information such as diet, seasonal home range, and baseline brevetoxin exposure 

could be instrumental in determining why brevetoxin-associated UMEs have occurred frequently 

along the coast of Florida’s Panhandle, and rarely in areas such as along the Florida west coast. 

 With the exception of gathering biopsies and conducting live captures of dolphins, detailed 

research plans for redefining the stock structure and learning more about the natural history of 

bottlenose dolphin populations in the Florida Panhandle were not discussed. If the sample size is 

sufficient and genetic markers are adequate, biopsies collected from free-ranging animals along the 

coast could help provide a genetic picture of the region’s stocks. Genetic data from stranded 

animals could then be compared to the genetic picture developed from free-ranging dolphin samples 

to confirm animal origin and hopefully get more reliable information on stock identity of stranded 

animals. 

The health assessment of bottlenose dolphins in the Panhandle region using live-capturerelease techniques is important to determine other potential health issues and additional biological 

and chemical toxin exposures that could make them more susceptible to mortality events. Data from 

these baseline health assessments can be compared to data being collected from other U.S. coastal 

sites to determine if the Panhandle dolphins differ from dolphins in other sites, such as Sarasota 

Bay, where the populations do not appear to be as vulnerable to mortality events. Collection of 

samples listed in Appendix III from live-captured animals will provide important baseline data on 

background biotoxin levels, pathogen exposure, immune function, hematology, and serum 

chemistry, as well as genetic material for stock assessment. This data, combined with other 

information, will help to better understand: (1) underlying health issues of Florida Panhandle 

dolphins; (2) potential hemolytic actions of brevetoxins; (3) lethal brevetoxin doses for bottlenose 

dolphins; (4) whether or not acute or chronic sub-lethal brevetoxin exposure can have reproductive 

impacts or increase dolphin susceptibility to infectious diseases; (5) if sub-lethal exposure confers 

immunity to subsequent brevetoxin exposure; and (6) how brevetoxin is metabolized, and even if 

differences in prey exist (or at least if differences trophic level of feeding exist). Additionally, live 

captures will enable biologists to affix radio-transmitters and time depth recorders to dolphins to 

learn more about dolphin movements, seasonal habitat use, feeding behavior, and other natural 

history characteristics. 

 To adequately determine the impacts of brevetoxin-associated mortality and UMEs on 

bottlenose dolphin populations on the coast of the Florida Panhandle, changes in the population 

size over time need to be identified. Spikes in mortality do not necessarily correspond to declines in 

the population, if the population is growing proportionally to the increases in mortality. Because a 

true census is not feasible, some sort of population index needs to be established and used to 

evaluate changes in the population over time. This index should be repeated annually if possible. As 

future UMEs occur, changes in annual population indices can be evaluated in light of mortality. This 

was not discussed at the Orlando workshop, but bears consideration. 
21 

Improving Response to Future Brevetoxin-related UME’s 

Immediate Needs 

 There are numerous action items that could improve response efforts during future 

brevetoxin-associated cetacean UMEs and resources need to be identified to make these happen. 

These action items include the assessment of existing resources, filling identified gaps, and 

establishing memorandums of understanding between NOAA and other potential partners such as 

the U.S. Coast Guard. Although these ideas were discussed at the Workshop, many were not 

fleshed out regarding responsibility, cost, or benefit, and currently funding needed to make these 

things happen has not been identified. Items discussed to be done as parts of a preparedness plan 

include: 

1. Designate Marine Mammal Leads who will be responsible for overseeing response efforts in 

different regions. These people could correspond to pre-established stranding networks and 

response structure. Alternates should be established in the case that the primary Lead is 

Priority Research and Monitoring Needs: Impacts on Dolphin Populations 

• Determine stock structure of bottlenose dolphins along the Florida 

panhandle 

• Better understand biological and chemical toxin and pathogen 

exposures of bottlenose dolphins along the Florida panhandle 

• Examine differences in health parameters (e.g. immunological 

measures) between Florida panhandle dolphins and other coastal 

dolphin populations that appear less vulnerable to mortality events 

• Better understand the spatial and temporal movements of 

bottlenose dolphin stocks that repeatedly experience brevetoxinassociated mortality 

• Develop toxin-specific biomarkers of exposure and effects for 

assessing sub-lethal and chronic exposure including protein 

biomarkers in serum and urine as well as the expression of 

brevetoxin antibodies in serum 

• Utilize brevetoxin-specific biomarkers to investigate differences in 

brevetoxin exposure and susceptibility in different bottlenose 

dolphin populations 

• Develop an indexing method to determine changes in dolphin 

populations that experience brevetoxin-associated mortality 

• Utilize new information to develop a long-term risk assessment for 

dolphin populations experiencing brevetoxin-related mortality
22 

unavailable. These designated Leads should help to conduct the additional pre-outbreak steps 

designated below as they need to be thoroughly knowledgeable about normal stranding patterns 

and regional stranding response capacity. 

2. Develop a list of names and contact numbers for people who are willing and potentially available 

to respond to a mass mortality event. This should be two-tiered, with the first level being the 

routine responders and the second level being additional volunteers and professionals who are 

willing to help. Although routine marine mammal stranding response capabilities need to be 

enhanced in certain regions as discussed previously, a mass mortality event would overwhelm 

these resources and require participation of increased numbers of people to assist in search and 

recovery efforts, conducting necropsies, sample recording and shipment, media relations, etc. 

Representatives from the National Institute of Standards and Technology (Charleston, SC) 

should be requested to attend all UMEs to collect samples for the National Marine Mammal 

Tissue Bank. 

3. Develop a similar set of names and contact numbers for people who could make up a HABfocused team. During documented HAB-related bottlenose dolphin mortality this group would 

be responsible for monitoring HAB movement using water samples, imaging, and dedicated 

ships. 

4. Conduct a complete survey of marine mammal stranding resources in the Florida Panhandle 

region. This should include a map of areas covered by specific stranding networks, names and 

contact numbers of people who participate in those networks, and lists of facilities, freezers, 

four-wheelers, trucks, refrigerated trucks, boats, planes, and necropsy supplies maintained or 

available to each network. Gaps in resources should be filled. For example, the Big Bend area 

has been identified as an area with poor road access where most strandings go unreported. 

Additionally, it was reported that most first responder partners (Apalachicola National Estuarine 

Research Reserve, St. Joseph Peninsula State Park, St. Joseph Buffer Preserve State Park, Gulf 

World, or NMFS Panama City Laboratory, etc.) have limited equipment for stranding response. 

A list of potential incident command centers should be established. Refrigerators, freezers, and 

necropsy facilities should be upgraded at each facility (or be ready to be upgraded) for events. 

Telecommunication options should be evaluated for each potential incident command center. 

For beach areas where cell phone coverage is poor, plans should be made for communication 

via radio or satellite phones. Carcass disposal options also should be designated for each facility. 

5. Provide search and collection and necropsy training for all stranding participants in the region, 

as well as for all people who are willing and potentially available to respond to a mass mortality 

event. 

6. Establish standardized aerial survey techniques for enhancing identification and recovery of 

marine mammals stranded in remote locations. Aerial surveys could be conducted by NOAA 

contracted pilots, volunteer pilots, the U.S. Coast Guard, or others. This does not have to be 

done by a single entity and could be done regionally for each stranding network. This should be 

linked to research efforts previously discussed to determine non-event baseline levels of 

stranding. 
23 

7. Develop a standardized necropsy form and tissue handling guide. The form that exists should 

be modified to include diagrams of marine mammals as well as all of the tissue and other 

samples listed in Appendix II of this plan. 

Note: It is probably a good idea to replicate steps 1-4 for Alabama, Louisiana and Mississippi 

where stranding response is weak. NOAA Fisheries may also want to consider making these 

pre-outbreak preparations in Alabama, Louisiana, Mississippi and Texas. 

 

Event Response Plan 

 A well-developed response plan could help improve response as well as information learned 

during future brevetoxin-related bottlenose dolphin mortality events. Such a plan was developed for 

dealing with manatee mortalities (Geraci and Lounsbury, 1997) and has proved very successful. 

Described HAB monitoring, marine mammal stranding response, and routine testing of stranded 

cetaceans for exposure to brevetoxins should adequately alert the NMFS’ Marine Mammal Health 

and Stranding Response Program of a brevetoxin-associated cetacean mortality event in the Florida 

Panhandle. Although a standard case definition for brevetoxicosis in bottlenose dolphins does not 

exist, evidence from the 2004 UME suggests that brevetoxicosis acutely kills animals of all age 

classes and both sexes leaving no pathopneumonic gross or histopathological lesions except levels of 

brevetoxin exceeding 200 ng/g in stomach contents and feces (NOAA, 2004). Further work must 

be done to help better understand background brevetoxin levels in bottlenose dolphins and 

brevetoxin pharmacokinetics, residence time, and pathogenesis. However, a working case definition 

for brevetoxicosis in bottlenose dolphins might be: any bottlenose dolphin that dies with 

postmortem evidence that the mortality was acute, no significant underlying pathology is present 

that could explain death in the animal, and stomach and or fecal brevetoxin levels exceed 200 ng/g. 

When the Marine Mammal Stranding Network determines there is an increase in strandings, the 

network participants and investigators should be alerted and sample analyses should be rushed to 

confirm or deny brevetoxin and domoic acid involvement. 

Additionally, the following items need to happen: 

1. All of the standard responders, as well as the list of potential additional responders, should be 

contacted by phone or electronic mail, warning them that reported regional strandings are 

increased or a dolphin has been diagnosed with brevetoxicosis and that they should be on alert 

for a potential UME. At this time, it also would be helpful to advise them of the location and 

status of a red tide, if one has been identified. 

2. The Marine Mammal Lead or backup person for the area (both to be determined) needs to 

discuss the location of the diagnosed mortality with lead HAB people (to be determined) who 

can provide up to date information on HAB monitoring, including the defined area any 

identified blooms in the region and levels of brevetoxin in any biological organisms (shellfish or 

other) being monitored. 

3. The Marine Mammal Lead should then alert all stranding response groups in the potentially 

affected and adjacent regions to mobilize their search and collection people and report findings 

back to the Marine Mammal Lead. Depending on the location of the bloom, wind direction, and 
24 

potential drift time, search and collection response might need to be repeated every day or every 

other day for multiple days. 

4. If mortality is identified, routine but temporally expedited response, necropsy, and testing should 

be performed. All findings at every stage should be reported to the Marine Mammal Lead. 

Depending on number of carcasses located and gross necropsy findings (multiple age classes, 

both sexes, no evidence of chronic disease), the Marine Mammal Lead might want to consider 

having the UME working group appraised of the situation and determine what other 

information they need to declare the mortality event a UME. 

5. The exact timing of the next step will vary for each unique situation, but at some point, the 

Marine Mammal Lead will need to determine if the situation is going to overwhelm standard 

stranding response capacity. If it is, then a command center should be established in reasonable 

proximity to the center of the mortality event and all response efforts should then be conducted 

from that site. People with needed skill sets should be called from the previously developed list 

of potential additional responders. 

6. If it is possible, all stranded carcasses should be permanently marked in the field (roto-tags or 

other identification) and transported to the command center for processing. Refrigerated trucks 

would be ideal for transportation as they also could double as temporary carcass storage facilities 

pending necropsy. The lead pathologist present should triage the carcasses, conducting 

necropsies on carcasses in better condition first and refrigerating marginal carcasses for later. All 

efforts should be made to collect and archive the uniform set of samples described in Appendix 

II. Samples taken do not necessarily have to be tested, but samples not taken during necropsy 

can not be taken later. 

7. A person responsible for all record keeping should be designated. This person should develop a 

system for recording all information for each animal, including: spatially explicit stranding 

location; permanent number assigned in the field and utilized in labs; carcass measurements; 

photographs; gross necropsy findings; which samples were taken; location of samples; and which 

samples were shipped. These datasheets should be created prior to a mass mortality event. 

8. Ideally, a person trained in identifying stomach contents should be at the command center with 

the equipment needed to rapidly identify stomach contents. If it is possible to transport 

analytical machines from the NOAA/NOS laboratory or the FWC FWRI, they too should be 

moved to the command center to analyze samples on site. It was recommended that a mobile 

HAB analytical lab be created (or designed for creation in a rented vehicle when needed) so 

equipment and personnel could do on-site testing. 

9. For monetary and personnel limitations, people often wonder when testing of the standard set 

of tissue samples, recommended in Appendix II, should be stopped. As discussed earlier, if it is 

possible all samples should be collected and archived so they can be tested at a later date if 

determined to be important. Important aspects of all epidemiological investigations are the 

temporal and spatial descriptions of the mortality event. For this reason, at a minimum routine 

samples should be tested from all carcasses that expand the known spatial and temporal aspect 

of the die-off. For example, if all carcasses are coming from St. Joseph Bay and one or two 

animals also show up in Pensacola Bay or Cedar Key, they definitely should receive complete 

testing as they might or might not be associated with the event in St. Joseph Bay. Additionally, if 

10 carcasses a week are found for a month, it is important to periodically run a complete set of 
25 

test on a subset of animals in good condition each week. This will establish continuity of the 

outbreak by fulfilling case definition criteria and also provide important material for research 

concerning dolphin diet over the outbreak, levels of toxin relative to time of exposure, and a 

whole suite of other potentially important information. 

10. During a documented HAB-related dolphin mortality event, the HAB-focused response team 

should be called to monitor HAB movement using water samples, imaging, and dedicated ships. 

This information should be reported daily to the Marine Mammal Lead, who then could use it to 

direct daily search and collection efforts. It is imperative that during an event the extent and 

duration of brevetoxin in the marine ecosystem, as well as the extent and duration of bloom, are 

determined. Sampling should systematically determine where brevetoxin is present in the water, 

as well as which species of fish and invertebrate contain toxin and where and for how long is the 

toxin present in these species. This information on toxin duration and trophic transfer will aid 

our understanding of why certain K. brevis blooms are associated with dolphin mortality while 

others are not. As an ancillary sampling technique, it was recommended that during a 

documented brevetoxin-bottlenose dolphin mortality event, fish should even be purchased at 

local fish markets and tested. 

11. During a brevetoxin-associated bottlenose dolphin mortality event, marine mammal stranding 

responders and pathologists are not likely to have the time to collect and necropsy co-localized 

dead birds, sea turtles, and fishes. Currently, these animals are collected and sampled by the 

State of Florida. Ideally, dedicated parallel response groups for different taxa should work 

cooperatively during mortality events to ensure that all carcasses collected receive a similar suite 

of tests and data from all species mortalities are used to define the events. This could be done 

formally or informally and bears further consideration. 

12. Media relations during the outbreak can help to generate volunteers to help, educate the public, 

and galvanize popular support for marine mammal stranding work and ocean health. A specified 

media relations person (or group) should be identified as soon as an increase in strandings in 

noted. The FWC/FWRI has history of dealing with HABs, aquatic animal mortality events and 

has designated point responders and media people. The Marine Mammal Lead could work 

closely with FWC/FWRI personnel and the media contact person. All new information should 

be filtered through these people so that the media knows who to contact and the message 

coming from the response is coordinated and uniform. 
26 

Moving Forward 

 As the database of mortality-free HAB events and HAB-associated marine mammal 

mortality grows, it should be periodically evaluated for spatial, temporal, and other trends that could 

suggest why some K. brevis blooms in the Gulf of Mexico result in bottlenose dolphin mortality, 

while others do not. The background brevetoxin tissue levels and stomach content levels, 

immunohistochemistry patterns, differences in disease and toxin exposure, differences in fish 

identified in gastric contents, as well as other necropsy and test results also should periodically be 

evaluated. These data (and data collected from live-caught and captive bottlenose dolphins) will 

provide insight into background brevetoxin levels, guidelines for diagnosing brevetoxicosis in 

bottlenose dolphins, and a better understanding as to why and when brevetoxicosis occurs in 

bottlenose dolphins. This will not happen without somebody to lead this effort. Ideally, a lead 

person should be contracted to periodically review all new research findings, compare marine 

mammal strandings and HAB data, and update research and response needs. An alternate strategy 

would be to assemble a small working group that could meet periodically to perform these tasks. 

Regardless of the option or combination of options chosen, funding will be necessary to either pay a 

lead person for their time or at a minimum, pay the travel and meeting expenses of a working group. 

 Better tools and approaches are needed for understanding the impacts of HABs. The 

recently published national research and response strategy (HARRNESS, 2005) updates the current 

state of the HAB problem and identifies the research needs and priorities necessary for better 

understanding and managing HABs. All of the needs required to better understand the impact of 

brevetoxins on bottlenose dolphins in the Gulf of Mexico fit with the nation’s critical needs for 

HAB research and response. In that light, elucidating the priority research and monitoring need 

enumerated in this document will not only benefit marine mammals, they also will gather 

knowledge and data required by the nation to understand HABS and the impact they have on 

human health, wildlife health, and ecosystem health. 
27 

Acknowledgements: 

This research and response plan would not have been possible with the help of many people. 

Support for this project came from a collaborative effort between the Marine Mammal Health and 

Stranding Response Program, Office of Protected Resources, NOAA/National Marine Fisheries 

Service and Disney’s Animal Programs who helped sponsor the workshop held at Walt Disney 

World Resorts in Orlando, Florida between December 13 and 15, 2004. T. Rowles and A. Stamper 

were the people who made this happen. Many people took the time to attend this workshop and 

contribute their knowledge and ideas (Appendix I) and many workshop participants provided 

comments that improved the quality of this plan including N. Barros, G. Bossart, L. Engleby, R. 

Ewing, S. Fire, L. Flewelling, J. Landsberg, J. Naar, P. Rosel, T. Rowles, L. Schwacke A. Stamper, F. 

Van Dolah, R. Varela, and R. Wells. Other people also provided important information and help 

including L. Garrison, F. Gulland, J. Litz, and B. Mase. T. Spradlin did an incredible job 

coordinating the project. None of this would be possible with the help of the volunteers and staff 

who make up the Florida Marine Mammal Stranding Network and the scientists that study 

bottlenose dolphins, harmful algal blooms and all the other disciplines that come together in this 

plan. 
28 

Literature Cited:

Baden, D. G. 1989. Brevetoxins; unique polyether dinoflagellate toxins. FASEB Journal 3; 1807-

1817. 

Barros, N.B. 1993. Feeding ecology and foraging strategies of bottlenose dolphins on the central 

east coast of Florida. Ph.D. Dissertation. University of Miami. Coral Gables, FL. 328 pp. 

Barros, N. B. and R. S. Wells. 1998. Prey and feeding patterns of bottlenose dolphins (Tursiops 

truncatus) resident in Sarasota Bay, Florida. Journal of Mammalogy 79: 1045-1059. 

Barros, N.B. and D.K. Odell. 1990. Food habits of bottlenose dolphins in the southeastern United 

States. Pp: 309-328 In Leatherwood, S. and R.R. Reeves (eds). The Bottlenose Dolphin. 

Academic Press: San Diego, CA. 

Benson, J. M., F. F. Hahn, T. H. March, J. D. McDonald, A. P. Gomez, M. J. Sopori, A. J. 

Bourdelais, J. Naar, J. Zaias, G. D. Bossart, and D. G. Baden. 2005. Inhalation toxicity of 

brevetoxin 3 in rats exposed for twenty-two days. Environmental Health Perspectives 113: 

626-631. 

Bossart, G. D., D. G. Baden, R. Y. Ewing, B. Roberts, S. D. Wright. 1998. Brevetoxicosis in 

manatees (Trichechus manatus latirostris) from the 196 epizootic: gross, histologic, and 

immunohistochemical features. Toxicology Pathology 26: 276-282. 

Cattet, M. and J. R. Geraci. 1993. Distribution and elimination of ingested brevetoxin (PbTx-3) in 

rats. Toxicon 31: 1483-1486. 

Fairey, E. R., N. G. Shuart, M. Busman, P. D. R. Moeller, J. S. Ramsdell. 2001. Biomonitoring 

brevetoxin exposure in mammals using blood collection cards. Environmental Health 

Perspectives 109: 717-720.

Flewelling, L. J., J. P. Naar, J. P. Abott, D. G. Baden, N. B. Barros, G. D. Bossart, M-Y. D. Bottein, 

D. G. Hammond, E. M. Haubold, C. A. Heil, M. S. Henry, H. M. Jacocks, T. A. Leighfield, 

R. H. Pierce, T. D. Pitchford, S. A. Rommel, P. S. Scott, K. A. Steidinger, E. W. Truby, F. 

M. Van Dolah, and J. H. Landsberg. 2005. Red tides and marine mammal mortalities. Nature 

435: 755-756. 

Forrester, D. J., J. M. Gaskin, F. H. White, N. P. Thompson, J. A. Quick, G. E. Henderson, J. C. 

Woodard, and W. D. Robertson. 1977. An epizootic of waterfowl associated with a red tide 

episode in Florida. Journal of Wildlife Diseases 13: 160-167. 

Gannon, D. P. and D. M. Waples. 2004. Diets of coastal bottlenose dolphins from the U.S. MidAtlantic coast differ by habitat. Marine Mammal Science 20: 527-545. 

Gawley, R. E., K. S. R, M. Kinoshita, and D. G. Baden. 1992. Binding of brevetoxins and ciguatoxin 

to the voltage-sensitive sodium channel and conformational analysis of brevetoxin B. 

Toxicon 30: 780-785. 

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 National Marine Fisheries Service 

and U.S. Navy, Office of Naval Research and Marine Mammal Commission, April 1989. 

Geraci, J. R. and V. J. Loundsbury. 1997. The Florida manatee: Contingency plan for health-related 

events. Prepared for Florida Department of Environmental Protection, Florida Marine 

Research Institute. St. Petersburg, FL. Cntract NO. MR199. 101 pp. 

Gulland, F., 2000, Domoic acid toxicity in California sea lions (Zalophus californianus) stranded along the 

central California coast, May-October 1998, NOAA Technical Memorandum, NMFS-OPR, 

17. 45 pp. 
29 

Gunter, G., R. H. Williams, C. C. Davis, and F. G. Walton Smith. 1948. Catastrophic mass 

mortalities of marine animals and coincidental phytoplankton bloom on the west coast of 

Florida, November 1946 to August 1947. Ecological Monographs 18: 309-324. 

HARRNESS. 2005. Harmful Algal Research and Response: A National Environmental Science 

Strategy 2005-2015. Rumsdell, J. S., D. M. Anderson and P. M. Glibert (Eds.), Ecological 

Society of America. Washington DC. Pp. 96. 

Kreuder, C., J. A. Mazet, G. D. Bossart, T. E. Carpenter, M. Holyoak, M. S. Elie, S. D. Wright. 2002. 

Clinicopathologic features of suspected brevetoxicosis in double-crested cormorants 

(Phalacrocorax auritis) along the Florida gulf coast. Journal of Zoo and Wildlife Medicine 33: 

8-15. 

Landsberg, J. H. 2002. The effects of harmful algal blooms on aquatic organisms. Reviews in 

Fisheries Science 10: 113-390. 

Landsberg, J. H. and Steidinger, K. A. 1998. A historical review of Gymnodinium breve red tides 

implicated in mass mortalities of the manatee (Trichechus latirostris manatus) in Florida USA. In: 

Harmful Algae, pp. 97-100 (B. Reguera, J. Blanco, M. L. Fernández, and T. Wyatt, eds.). 

Paris: Xunta de Galicia IOC of UNESCO. 

Layne, J. N. 1965. Observations on marine mammals in Florida waters. Bulletin of the Florida State 

Museum 9: 131-181. 

Lipscomb, T. P., S. Kennedy, D. Moffet, A. Krafft, B. A. Klaunberg, J. H. Lichy, G. T. Regan, G. A. 

Worthy, J. K. Taubenberger. 1996. Morbilliviral epizootic in bottlenose dolphins of the Gulf 

of Mexico. Journal of Veterinary Diagnostic Investigation 8: 283-290. 

Lu, Z. and S. M. Tomchik. 2002. Effects of a red-tide toxin on fish hearing. Journal of Comparative 

Physiology 188: 807-813. 

Mase, B., W. Jones, R. Ewing, G. Bossart, F. VanDolah, T. Leighfield, M. Busman, J. Litz, B. 

Roberts, T. Rowles. 2000. Epizootic in bottlenose dolphins in the Florida panhandle: 1999-

2000. In Proceedings of the American Association of Zoo Veterinarians and the 

International Association of Aquatic Animal Medicine (Baer ed.). New Orleans, LA 2000: 

522-525 

NOAA. 2004. Interim report on the bottlenose dolphin (Tursiops truncatus) unusual mortality event 

along the panhandle of Florida March-April 2004. 36 pp. 

O’Shea, T. J., G. B. Rathbun, R. K. Bonde, C. D. Buergelt, and D. K. Odell. 1991. An epizootic of 

Florida manatees associated with a dinoflagellate bloom. Marine Mammal Science 7: 165-

179. 

O’Shea, T. J. and G. B. Rathbun. 1982. Summary report on a die-off of the West Indian manatee 

(Trichechus manatus) in Lee County Florida, Spring 1982. 3 volumes. Sirenia Project Report, 

Gainesville, Florida. 

Poli, M. A., T. J. Mende, and D. G. Baden. 1986. Brevetoxin, unique activators of voltage-sensitive 

sodium channels, bind to specific sites in rat brain synaptosomes. Molecular Pharmacology 

30: 129-135. 

Poli, M. A., C. B. Templeton, W. L. Thompson, and J. F. Hewetson. 1990. Distribution and 

elimination of brevetoxin PbTx-3 in rats. Toxicon 28: 903-910. 

Radwan, F. F. and Ramsdell J.S. In Press. Characterization of In Vitro Oxidative and Conjugative 

Metabolic Pathways for Brevetoxin (PbTx-2). Toxicological Sciences. 

Radwan, F. F., Z. Wang, and J. S. Ramsdell. 2005. Identification of a rapid detoxification mechanism 

for brevetoxin in rats. Toxicological Sciences 85: 839-46. 

Schulman, L. S., L. E. Roszell, T. J. Mende, R. W. King, and D. G. Baden. 1990. A new polyether 

toxin from Florida’s red tide dinoflagellate. In Toxic Marine Phytoplankton, 407-412 (E. 

Graneli, B. Sundstrom, L. Edler, and D. M. Anderson, eds.) New York: Academic Press. 
30 

Steidinger, K. A., M. Burklew, and R. M. Ingle. 1973. The effects of Gymnodinium breve toxin on 

estuarine animals. In Marine Pharmacognosy: Action of Marine Toxins at the Cellular Level, 

pp. 179-202 (D.F. Martin and G. M. Padilla, eds.) New York: Academic Press. 

Steidinger, K. A. 1989. Implications of 1986-87 Ptychodiscus brevis red tide and 1987-88 mass 

bottlenose dolphin mortalities. In Toxic dinoflagellates and marine mammal mortalities, 56-

65 pp. ( D. M. Anderson and A. W. White, eds). Woods Hole Oceanographic Institution 

Technical Report CRC-89-6. 

Tester, P. A., J. T. Turner, and D. Shea. 2000. Vectorial transport of toxins from the dinoflagellate 

Gymnodinium breve through copepods to fish. Journal of Plankton Research 22 47-62. 

Thomas, C. R., A. Bourdelais, L. E. Tomas, and A. O. Tatters. 2005. Growth, nutrient utilization 

and evidence for toxin production by the new toxic flagellate Chloromorum toxicum. In 

Proceedings of the 3rd Symposium on Harmful Algal in the U.S. 

Wang, Z., S. M. Plakas, E. Said, and R. Kathleen. 2004. LC/MS analysis of brevetoxin metabolites in 

the Eastern oyster (Crassostrea virginica) Toxicon 43: 455-465. 
31 

Figure 1: List of Reported Brevetoxin Related Marine Bird or Mammal 

Mortality Events 

Started 

(month/year) 

Ended 

(month/year) Species affected Location Reference 

November 1946 August 1947 

Bottlenose dolphins, 

marine fish and 

invertebrates 

Florida (Sarasota 

to Keys region) Gunter et al., 1948 

Unknown 1953 Unknown 1955 Bottlenose dolphins, sea 

turtles, and marine fish 

Tarpon Springs to 

Key West 

Rounsefell and Nelson, 

1966 in Landsberg, 2002 

Unknown 1963 Unknown 1963 

Manatees (7), turtles, 

cormorants, fish, and 

possible raccoon 

Unknown Layne, 1965; Landsberg, 

2002 

October 1973 May 1974 

Lesser Scaup (approx 

2,000), double-crested 

cormorants, red-breasted 

mergansers 

Florida Forrester et al., 1977 

Unknown 1982 Unknown 1982 Manatees (39) Florida –Charlotte 

Harbor 

O’Shea and Rathbun, 

1982; O’Shea et al., 

1991 

June 1987 March 1988 Bottlenose dolphins 

(>740) Atlantic Coast 

Geraci, J. R., 1989; 

Steidinger, K. A. 1989 

(morbillivirus epizootic; 

brevetoxin detected in 

stranded animals) 

Unknown 1995 Unknown 1999 Double-crested 

cormorants (360) 

Florida - Gulf 

Coast Kreuder et al., 2002 

March 1996 April 1996 Manatees (149) Florida 

Bossart et al., 1998; 

Landsberg and 

Steidinger 1998 

Unknown 1996 Unknown 1996 Bottlenose dolphins Mississippi NOAA, 2004 

August 1999 May 2000 

Bottlenose dolphins (at 

least 152), other cetacean 

species, marine birds, 

fish, sea turtles and 

invertebrates 

Florida Panhandle 

Mase et al., 2000; 

NOAA, 2004; NOAA 

Southeast US Marine 

Mammal Stranding 

Database; FWC unpub. 

data 

Unknown 2002 Unknown 2002 Manatees (34) Florida 

FWC unpub data; 

NOAA, 2004; 

Flewelling et al., 2005 

Unknown 2003 Unknown 2003 Manatees (96) Florida 

FWC unpub data; 

NOAA, 2004; unpub. 

data 

March 2004 April 2004 

Bottlenose dolphins 

(107), marine birds, fish 

and invertebrates 

Florida Panhandle NOAA, 2004; 

Flewelling et al., 2005 

July 2005 Unknown 2006 

Manatees, bottlenose 

dolphins (132 as of April 

06), birds, fish, sea turtles 

Southwest Florida NOAA and FWC, 

unpub. data 

September 2005 Unknown 2006 

Bottlenose dolphins (93 

as of April 2006), marine 

birds, fish (sturgeon, gar, 

and anchovies) 

Florida Panhandle NOAA and FWC, 

unpub. data 
32 

Figure 2: Red Tide Occurrence off of Florida’s West Coast 

(Courtesy of Florida Fish and Wildlife Conservation Commission) 

119 Years of Red Tide off 

Florida’s West Coast 

(1878-1997) 

0

5

10

15

20

25

30

35

 

 53% of Florida West Coast Red Tide Blooms occur September-December. 

46% of the blooms are initiated in September, 21% in October

Jan Feb Mar Apr May June July Aug Sept Oct Nov Dec 
33 

Figure 3: Shellfish Harvesting Areas in Florida 

(numbered and colored to distinguish sites) 

(Courtesy of http://www.floridaaquaculture.com/SEAS/SEAS_SHAmap.htm) 
34 

This Page Intentionally Left Blank 
35 

Appendix I: Participant List for the Disney Workshop on Brevetoxins 

and Bottlenose Dolphins, December 13-15, 2004 

Nelio Barros, Ph.D. 

Mote Marine Laboratory 

1600 Ken Thompson Parkway 

Sarasota, FL 34236 

Tel. 941.388.4441 

nbarros@mote.org 

Gregory D. Bossart, V.M.D., Ph.D. 

Director and Head of Pathology 

Division of Marine Mammal Research and 

Conservation 

Harbor Branch Oceanographic Institution 

5600 US 1 North 

Ft. Pierce, FL 34946 

Tel. 772.465.2400 

gbossart@hboi.edu 

Quay Dortch, Ph.D. 

ECOHAB Program Coordinator 

NOAA/National Ocean Service 

1305 East-West Highway 

Silver Spring, MD 20910 

Tel. 301.713.3338 

Quay.Dortch@noaa.gov 

Laura Engleby 

Southeast Regional Office 

NOAA/National Marine Fisheries Service 

9721 Executive Center Drive N 

St. Petersburg, FL 33702 

Tel. 727.551.5791 

Laura.Engleby@noaa.gov 

Ruth Ewing, DVM 

Southeast Fisheries Science Center 

NOAA/National Marine Fisheries Service 

75 Virginia Beach Dr. 

Miami, FL 33149 

Tel. 305.361.4233 

Ruth.Ewing@noaa.gov 

Spencer Fire 

Mote Marine Laboratory 

1600 Ken Thompson Parkway 

Sarasota, FL 34236 

Tel. 941.388.4441 

sfire@mote.org 

Leanne Flewelling 

Florida Fish and Wildlife Conservation Commission 

Florida Fish and Wildlife Research Institute 

100 8th Avenue SE 

St. Petersburg, FL 33701 

Tel. 727.896.8626 ext.1572 

leanne.flewelling@MyFWC.com 

Joseph K. Gaydos, V.M.D., Ph.D. 

UC Davis School of Veterinary Medicine 

Wildlife Health Center – Orcas Island Office 

1016 Deer Harbor Road 

Eastsound, WA 98245 

Tel. 360.376.3910 

jkgaydos@ucdavis.edu 

Elsa M. Haubold, Ph.D. 

Program Administrator 

Florida Fish and Wildlife Conservation Commission 

Fish and Wildlife Research Institute 

100 8th Ave. SE 

St. Petersburg, FL 33701 

Tel. 727.896.8626 x 1902 

Elsa.Haubold@myfwc.com 

Aleta Hohn, Ph.D. 

Southeast Fisheries Science Center 

NOAA/National Marine Fisheries Service 

101 Pivers Island Road 

Beaufort, NC 28516 

Tel. 252.728.8797 

Aleta.Hohn@noaa.gov 

Jan Landsberg, Ph.D. 

Research Scientist 

Fish and Wildlife Health 

Florida Fish and Wildlife Conservation Commission 

Fish and Wildlife Research Institute 

100 8th Ave. SE 

St. Petersburg, FL 33701 

Tel. 727.896.8626 

Jan.Landsberg@myfwc.com 
36 

Tod Leighfield 

Center for Coastal Environmental Health and 

Biomolecular Research 

NOAA/National Ocean Service 

219 Fort Johnson Road 

Charleston, SC 29412 

Tel. 843.762.8631 

Tod.Leighfield@noaa.gov 

Ed Matheson 

Florida Fish and Wildlife Conservation Commission 

Fish and Wildlife Research Institute 

100 8th Avenue SE 

St. Petersburg, FL 33701 

Tel. 727.896-8626 

Eddie.Matheson@MyFWC.com 

William A. McLellan 

Biological Sciences 

University of North Carolina at Wilmington 

601 S. College Rd. 

Wilmington, NC 28403 

Tel. 910.962.7266 

mclellanw@uncw.edu 

Keith D. Mullin 

Southeast Fisheries Science Center 

NOAA/National Marine Fisheries Service 

3209 Frederic Street 

Pascagoula, MS 39567 

Tel. 228.762.4591 

Keith.D.Mullin@noaa.gov 

Jerome Naar, Ph.D. 

Research Faculty 

Center for Marine Science 

University of North Carolina at Wilmington 

5600 Marvin K Moss Lane, 

Wilmington NC 28428 

Tel. 910 962 2367 

naarj@uncw.edu 

Douglas Nowacek, Ph.D. 

Asst. Professor, Oceanography Dept. 

Florida State University 

509 OSB, West Call St. 

Tallahassee, FL 32306 

Tel. 850.645.1547 

nowacek@ocean.fsu.edu 

D. Ann Pabst, Ph.D. 

Biological Sciences 

University of North Carolina at Wilmington 

601 S. College Rd. 

Wilmington, NC 28403 

Tel. 910.962.7266 

pabsta@uncw.edu

Richard H. Pierce, Ph.D. 

Senior Scientist and Director 

Center for Ecotoxicology 

Mote Marine Laboratory 

1600 Ken Thompson Parkway 

Sarasota, FL 34236 

Tel. 941.388.4441 

rich@mote.org 

Patricia Rosel, Ph.D. 

Southeast Fisheries Science Center 

NOAA/National Marine Fisheries Service 

646 Cajundome Blvd, Suite 234 

Lafayette, LA 70506 USA 

Tel. 337.291.2123 

Patricia.Rosel@noaa.gov 

Dave S. Rotstein, DVM, MPVM, Dipl. ACVP 

Assistant Professor, Department of Pathobiology 

College of Veterinary Medicine 

University of Tennessee 

2407 River Drive, Room A201 

Knoxville, TN 37996-4542 

Tel. 865.974.8205 

drotstein@mail.ag.utk.edu 

Teri Rowles, D.V.M., Ph.D. 

Marine Mammal Health and Stranding Response 

Program 

Office of Protected Resources 

NOAA/National Marine Fisheries Service 

1315 East-West Highway 

Silver Spring, MD 20910 

Tel. 301.713.2322 

Teri.Rowles@noaa.gov 

Lori Schwacke, Ph.D. 

Cooperative Center for Marine Animal Health 

NOAA/National Ocean Service 

219 Ft. Johnson Rd. 

Charleston, SC 29412 

Tel. 843.762.8541 

Lori.Schwacke@noaa.gov 
37 

Trevor Spradlin 

Marine Mammal Health and Stranding Response Program 

Office of Protected Resources 

NOAA/National Marine Fisheries Service 

1315 East-West Highway 

Silver Spring, MD 20910 

Tel. 301.713.2322 

Trevor.Spradlin@noaa.gov 

M. Andrew Stamper D.V.M., Dipl. A.C.Z.M. 

Research Biologist/Clinical Veterinarian 

Epcot’s The Living Seas 

Walt Disney World Resorts 

2020 North Avenue of the Stars 

Lake Buena Vista, FL 32830-1000 

Tel. 407.560.5576 

Andy.M.Stamper@disney.com 

Fran Van Dolah, Ph.D. 

Center for Coastal Environmental Health and Biomolecular Research 

NOAA/National Ocean Service 

219 Ft. Johnson Rd. 

Charleston, SC 29412 

Tel. 843.762.8529 

Fran.VanDolah@noaa.gov 

René A. Varela, M.S., V.M.D. 

Medical Manager and Assistant Research Scientist 

Division of Marine Mammal Research and Conservation 

Harbor Branch Oceanographic Institution 

5600 US 1 North 

Ft. Pierce, FL 34946 

Tel. 772.465.2400 

RVarela@HBOI.edu 

Current contact information: 

Ocean Embassy 

6433 Pinecastle Blvd., Suite 2 

Orlando, Florida 32809 

Ph. 321.299.6936 

rvarela@oceanembassy.com 

Randy Wells, Ph.D. 

Chicago Zoological Society 

c/o Mote Marine Laboratory 

Mote Marine Laboratory 

1600 Ken Thompson Parkway 

Sarasota, FL 34236 

Tel. 941.388.4441 

rwells@mote.org 

Timothy Wynne 

Center for Coastal Monitoring and Assessment 

NOAA/National Ocean Service 

1305 East-West Highway 

Silver Spring, MD 20910 

Tel. 301.713.3028 x173 

Timothy.Wynne@noaa.gov 
38 

This Page Intentionally Left Blank 
39 

Appendix II: HAB-related Tissue Disposition List for 

Marine Mammal Samples 

This is a sample wish-list. The following are suggestions for sample to be collected from all 

marine mammals (code 2 and 3); all mass mortality (>5 birds) of fish-eating seabirds and all sea 

turtles. If there are fresh dead fish proximal to mass mortality of sea turtles, birds or mammals – 

put the entire fish in a Ziploc bag and freeze; if the fish is too large, freeze the stomach contents 

o Collect the following samples (ideally at least 50 gm each where applicable) in a whirl pack 

or Ziploc bag and store at -20C: submit alternately to NOAA/NOS laboratory (Charleston, 

SC) and FWC FWRI (St. Petersburg, FL) for PbTx analysis: 

• Feces, urine, vitreous humor, brain, lung, liver, spleen, kidney (freeze separately) 

• Note: the entire stomach (forestomach, glandular compartment, and pyloric stomach) 

should be submitted first for contents analysis; when present, gastric juices and fleshy 

remains of prey will be submitted o NOAA/National Ocean Service lab (Charleston, 

SC) and FWC FWRI (St. Petersburg, FL) for PbTx analysis 

• Collect postmortem heart blood for PbTx antibodies – spin off serum and freeze at -

20C; send to a 1⁄2 ml aliquot to Jerome Naar (Center for Marine Science, Wilmington, 

NC - address in Appendix I) 

o Collect and submit 50gm liver sample to Mote Marine Laboratory for fatty acid profiles or 

stable isotopes 

o Collect the following tissue samples, fix in 10% neutral buffered formalin, and embed in 

paraffin within 6 days. Have pathologist read slides and submit a subset of the paraffinembedded tissues for immunohistochemistry to Greg Bossart (Harbor Branch Oceanographic 

Institution). 

Important note: tissues fixed in 10% neutral buffered formalin should be processed 

(embedded into paraffin) within 10 days of complete formalin fixation. This is critical. 

• For Histopathology: Skin, umbilicus, mammary gland, blubber, tongue, thymus, 

thyroid, vitreous humor, brain (cerebrum, cerebellum, brainstem), spinal cord, 

trachea, lung, pulmonary lymph node, heart, liver, spleen, kidney, bladder, adrenal 

stomach (forestomach, glandular compartment, and pyloric stomach), small intestine, 

large intestine, mesenteric lymph node, inguinal lymph node, skeletal muscle, 

reproductive tissues, bone marrow, and multiple other lymph nodes. 

• For Immunohistochemistry, submit the following subset of tissues embedded in 

paraffin to Dr. Bossart: brain, lung, liver, kidney, spleen, multiple lymph nodes (at 

least 3 sites), forestomach, fundic stomach, small intestine and large intestine 

o Take photographs (high resolution if digital) of the carcass being sure to get the entire body 

from above and both sides of the dorsal fin. 

o Collect the following water samples at the mortality site; store as described: submit 

alternately to NOAA/NOS laboratory (Charleston, SC) and FWC FWRI (St. Petersburg, FL) 
40 

• Take 1 liter of water and freeze it for toxin analysis 

• Take three 25 ml water samples (at stranding site and 100 meters to either side of site) 

and fix for algae identification (using kit provided) 

o Not discussed, but should consider: It would be helpful to get a skin biopsy for genetics from 

all Code 2, 3, and 4 stranded bottlenose dolphins (probably should contact Renee Varela, 

Harbor Branch Oceanographic Institution). 

Note: Additionally, every effort should be made to get a spatially explicit exact reference point 

(GPS location) for every animal collected for necropsy. 
41 

Appendix III: Sample TissueChecklist for Marine Mammal Necropsy 

Tissue 

10% 

buffered 

formalin 

HAB 

samples 

(Whirl Pack -

frozen) 

Bacteriology, 

virology, etc. (Whirl 

Pack -frozen) 

Other 

Skin  

Genetics – 20% 

DMSO saturated 

sodium chloride 

Umbilicus  

Mammary gland  

Blubber   D L V

Milk  

Tongue  

Thymus 

Thyroid  

Vitreous Humor  (red-top 

tube - freeze) 

Brain - cerebrum     (freeze in foil for 

contaminants) 

Brain - cerebellum     (freeze in foil for 

contaminants) 

Brain - Brainstem     (freeze in foil for 

contaminants) 

Spinal cord (thoracic)   

Trachea  

Lung    

L.N. (pulmonary)   

Blood from heart 

 

(1/2 ml 

serum) 

 Red-top tubes – 

freeze serum 

 Blood card 

Heart  

Liver    

 (freeze in foil for 

trace metals) 

 (freeze in foil for 

fatty acids / stable 

isotopes) 

Spleen   

Kidney        (freeze in foil for 

trace metals) 

Bladder  

Urine    Red top tube - 

freeze 
42 

Tissue 

10% 

buffered 

formalin 

HAB 

samples 

(Whirl Pack -

frozen) 

Bacteriology, 

virology, etc. (Whirl 

Pack -frozen) 

Other 

Adrenals   

Stomach 

(forestomach, 

glandular 

compartment, and 

pyloric stomach) 

    Ziploc bag 

with contents 

Note: send entire 

stomach for prey 

identification; will 

aliquot HAB samples 

from there 

Small intestine  

Large intestine  

Fecal sample    Parasitology 

L.N. (mesenteric)  

L.N. (inguinal)  

Uterus  

Testes  

Muscle (skeletal)  

Bone marrow  

Note: where two boxes are present () take 2 samples; please submit desired samples to 

researchers listed above paying close attention to the following recommendations: 

 Label each whirl pack, Teflon bag, vial or aluminum foil packet with the following: 

 Necropsy Number 

 Species 

 Tissue 

 Date of collection 

 When taking samples for 10% Buffered Formalin remember to take thin (1cm) sections and 

be sure there are 10 parts formalin to 1 part tissue in the vial. Note: all tissues collected in 

10% neutral buffered formalin must be embedded in paraffin within 6 days! 

 Be sure to store all tissue samples as recommended. 

 Please call ahead prior to shipping any samples. 
43 

Appendix IV: Samples to Take from Live Captured or Live Stranded 

Bottlenose Dolphins 

Note: Contact Dr. Randy Wells for a complete list of samples as well as shipping and 

handling requirements. This is an abbreviated list 

Blood: 

o Serum from red top, thrombin or serum separator tubes; freeze in 1⁄2 ml aliquots at -20C: 

• Serum chemistry including iron 

• Test for antibodies to PbTx: send a 1⁄2 ml aliquot to Jerome Naar (Center for Marine 

Science, Wilmington, NC - address in Appendix I) 

• Test for antibodies to other select pathogens - including marine Brucella (National 

Veterinary Services Laboratory) Caliciviruses (A. Smith, Oregon State); Eastern 

Equine Encephalitis; Erysipelas rhusiopathiae; Herpesvirus; Influenza A and B; 

Leptospirosis serovars; Morbilliviruses (CDV, PDV, CMV; J. Saliki, Oklahoma State 

Diagnostic Laboratory); West Nile Virus; others 

• Hormones: cortisol, T3, T4, fT4, aldosterone, estradiol, progesterone 

• Mercury / Selenium 

• Immunology testing 

• PbTx ELISA – S. Fire’s work 

o EDTA-anticoagulated blood (purple top) 

• Complete blood count with differential 

• Trace elements 

• Mercury / Selenium 

o Heparin-anticoagulated blood (green top): 

• Blood gasses (I-stat machine) 

• Organochlorines 

• Mitochondrial DNA for microsatellite markers 

o Citrate-anticoagulated blood 

• Fibrinogen 

o Whole blood: 

• Trace elements 

o Blood collection card (provided by Tod Leighfield, NOAA/ National Ocean Service, 

Charleston, SC); collect whole blood, allow to dry then store at room temperature until 

submission. These are used to test for the presence of brevetoxins in blood (Fairey et al, 

2001) 
44 

o A PAX gene tube 

• Fran Van Dolah, NOAA/NOS laboratory, Charleston, SC 

• Ryan’s RNA work 

• Warr’s RNA work 

Biopsy Samples: 

o Histology 

o Stable Isotopes 

o Stress proteins 

o Trace elements 

o Mercury and selenium and other contaminant concentrations 

o Genetics 

Dorsal Fin Plug: 

o Genetics 

o Stress proteins 

Milk Sample: 

o Contaminants 

o Fatty acids 

o Composition 

o PbTx ELISA - Spencer Fire’s work 

Urine: 

o Urinalysis 

o Urine electrolytes 

o Toxicology 

o Reproductive status 

o PbTx ELISA - Spencer Fire’s work 

o Leptospirosis 

o Biotoxin analysis 

o Contaminants 

Tooth: 

o Age 

Blowhole swabs 

o Nasitrema sp. cytology 

o Microbiological culture 

Exhalation sample: 

o Microbiological culture 

o Red Tide work 

o Breath analysis 
45 

Fecal Sample: 

o Toxicology 

o PbTx ELISA - Spencer Fire’s work 

o Lungworm analysis 

o Microbiological culture 

o Diet and ageing study 

Gastric sample: 

o Cytology 

o Red Tide Analysis -Discussed, but not deliberated: This should be frozen and submitted 

for PbTx analysis. This would serve as a baseline for levels of brevetoxin present in 

stomach contents of bottlenose dolphins that appear healthy. Fluid collected should be 

frozen at -20C until shipped to one of the labs equipped to test for PbTx (FFWCC and 

Charleston NOAA lab) and tested by Spencer Fire at Mote Marine Laboratory. 

o If ingested material (otoliths or other bony structures are collected, they should be frozen 

and sent for identification) 

Whole body photograph, dorsal fin photographs (both sides) and routine measurements 

Discussed, but not deliberated: It would be beneficial to get a small liver biopsy from all livecaptured dolphins. A liver biopsy could be divided between Fatty Acid analysis / stable isotope 

analysis, one of the two labs equipped to test for PbTx (FWC and Charleston NOAA/NOS 

laboratory) and analyzed for PbTx via ELISA by Spencer Fire. It was recommended that a 

group of medical professionals experienced in cetacean liver biopsy discuss the risk versus merit 

of this procedure. 
46 

This Page Intentionally Left Blank