# Interim Report

on the 

# Bottlenose Dolphin ( Tursiops truncatus ) Unusual Mortality Event Along the Panhandle of Florida March-April 2004Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### TABLE OF CONTENTS

I. Executive Summary

II. Introduction

A. Unusual Mortality Event (UME) Program and the W o rking Group on Marine Mam m a l Unusual Mortality Events B. Historic UMEs in the Gulf of Mexico and Florida Panhandle C. Historical Knowledge of Bo ttlenose Dolphin Populations and Strandings Along the Florida Panhandle

III. Methods and Results

A. Dolphin Sam p ling 1. Gross Necropsy Findings 2. Stom ach Content Analyses 3. Virology 4. Histopathology 5. Organochlorines 6. Biotoxins 7. Genetic Analyses B. Environm ental Sam p ling 1. Satellite Im agery 2. Harm ful Algal Bloom (HAB) Monitoring and Toxin Analyses 3. Mortality Reports in Addition to Dolphins 4. Shellfish Monitoring for Harm ful Algal Bloom Species

IV. Discussion and Future Plans

A. Com p arison with Other UMEs B. Insights into Dolphin Behavior and Feeding in the Florida Panhandle C. Continuing Studies D. Future Studies E. Evaluation of UME Response

V. Ack n owledgements

VI. References

VII. Tables and Figures

VIII. Appendices

A. Criteria f o r Determ ining an Unusual Mortality Event B. Fact Sheet on Brevetoxin and Red Tide C. Fact Sheet on Dom o ic Acid

2Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### I. EXECUTIVE SUMMARY

This interim report outlines the initial findings and the ongoing analyses in the investigation of the bottlenose dolphin ( Tursiops truncatus ) m o rtality event that began in March 2004. One hundred and seven (107) bottlenose dolphins stranded dead along the Florida Panhandle between March 10 and April 13, 2004. Hundreds of dead fish and

m a rine invertebrates were also discovered in the area. The National Marine Fisheries Service f o rm ally declared the dolphin deaths an “Unusual Mortality Event” (UME) af ter consulting with the W o rking Group on Marine Mam m a l Unusual Mortality Events. A

m u lti-agency investigation was initiated and is being conducted by federal and state

m a rine wildlife officials working in partners hip with private research organizations and universities.

Analyses conducted to date found brevetoxins (see Appendix B), naturally occurring neurotoxins produced by Karenia brevis , the Florida red tide, at high levels in the
stom ach contents of all dolphins exam ined to date and at variable levels in the tissues of these anim als. Metabolite concentrations in tissues of f

i sh or dolphins are not yet

com p leted. The concentrations of brevetoxins observed in the analyzed subsam ple of the
stom ach contents are greater than or equal to those observed in previous m a rine m a m m a l

m o rtality events associated with Florida red tides ( Karenia brevis bloom s) in the Gulf of Mexico. In m o st of the dolphins, the first cham ber of the stom achs was gorged with large

am ounts of fish, som e of which were partially whole and undigested indicating recent feeding. Fish (planktivorous, herbivorous, a nd om nivorous fish species) collected from St. Joseph Bay on March 18 tested positive f o r brevetoxins in stom ach contents and in

m u scle, liver, and gill tissues whereas f i sh collected on March 28-31 also tested positive f

o r brevetoxin but at m u ch lower levels (except in sea trout). Satellite im agery of the northern Gulf of Mexico indicated elevated chlorophyll levels in the UME area March 9- 11, 2004 but water sam p les collected March 11 and later in the area of the UME did not contain signif i cant quantities of Karenia brevis , although low levels of brevetoxins were detected. Thus, the source of the brevetoxins involved in this event rem a ins uncertain, but the presence of toxic fish and water suggests that there was an undetected bloom

som e where either in the bay itself or in waters in which the fish or dolphins were feeding. A sim ilar dolphin UME occurred in 1999-2000 in the sam e area of Florida and was correlated with a Karenia brevis bloom

.
In addition to brevetoxin, low levels of dom oic acid (see Appendix C) were detected in the stom achs, urine and feces of som e dolphins. Further, Pseudo-nitzschia delicatissima

, a diatom that can produce dom oic acid was present at low to m oderate levels in water

sam p les. However, the levels of dom oic acid detected were 1-2 orders of m a gnitude lower than those found in dom oic acid related m a rine m a m m a l m o rtalities on the California coast. Therefore, its role in the current event would appear to be secondary to brevetoxin if, in fact, it is involved at all.

The evidence based on gross and histological findings does not indicate an infectious process and viral testing has ruled out m o rbillivirus, a virus known to cause high

m o rbidity and m o rtality in dolphin populations. The lack of evidence of infectious disease, the wide age class spread of the m o rtalities, and the f act that m o st anim als had recently fed (dem onstrated by full stom achs) does im plicate a toxin of som e type as one

3Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### of the causes of the event. The Investigative Team is continuing to look at all potential causes of the m o rtality event and will conduct further analyses of the brevetoxin

m e tabolites, prey and stom ach contents, and genetic identif ication of the Florida Panhandle population. Additional work includes quantification of the estim ated dose of toxins received by the anim als, exam ination of any predisposing factors, and searching for evidence of any other toxicants that m i ght be in dolphin prey species. The fact that southwest Florida has significant bloom s of Karenia brevis alm o st annually without unusual num bers of bottlenose dolphin m o rtalities m a kes this investigation even m o re perplexing. In order to fully understand what is happening in these dolphin m o rtalities, the investigation will continue to work with an interdisciplinary team of scientists using an ecosystem approach to understand the factors that contributed to this m o rtality event.

II. INTRODUCTION

Between March 10 and April 13, 2004, 107 bottlenose dolphins ( Tursiops truncatus ) stranded dead along the Florida Panhandle coastline from Escam bia to Franklin counties. The m a jority of the anim als (70%) stranded in St. Joseph Bay and surrounding areas of Gulf County (Figure 1). On March 12, approxim a tely 26 dolphins had stranded within a 3-day period. The annual average of dolphin strandings along the Florida Panhandle is eight (8) anim als per year, so the high num ber of dolphin strandings in March 2004
prom pted the National Marine Fisheries Service (NMFS) to initiate consultation with the W o rking Group on Marine Mam m a l Unusual Mortality Event (W orking Group). The W o rking Group reviewed the available stranding data as well as historical records from the area for com p arison, and recom m e nded that NMFS declare the die-off an official “Unusual Mortality Event” (UME).

A. Unusual Mortality Event (UME) Program and the Working Group on Marine Mammal Unusual Mortality Events

Title IV of the Marine Mam m a l Protection Act (MMPA) established the “Marine Mam m a l Health and Stranding Response Program ” (16 U.S.C. 1421 et seq. ), and section 404 of the MMPA specifically directs the Secretary of Com m e rce (through NMFS) to adm i nister responses to UMEs. The MMPA characterizes UMEs as m a ss m o rtalities and/or strandings of m a rine m a m m a ls that are: (1) unexpected; (2) involve a significant die-off of any m a rine m a m m a l population; and (3) dem a nd an im m e diate response. Additional considerations include strandings of unusual species, locations, tim e of year,
etc.

Section 404 of the MMPA also established the W o rking Group whose role is to: (1) determ ine whether or not a UME is occurring; (2) determ ine if/when a UME has ended; and (3) help develop a contingency plan for UME response. The W o rking Group is

com posed of 12 scientific experts with experience in m a rine m a m m a l science, m a rine

m a m m a l veterinary science and husbandry, biom edical specialties, and/or m a rine conservation. There are seven criteria that the W o rking Group uses to determ ine if a

m a rine m a m m a l m o rtality event is “unusual” (see Appendix A). A single criterion or

com b ination of criteria m a y indicate the occurrence of an unusual m o rtality event.

4Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### B. Historic UMEs in the Gulf of Mexico and Florida Panhandle

Since 1992, eight m a rine m a m m a l unusual m o rtality events have been investigated in the Gulf of Mexico. Three of those involved m a natees and were associated with Karenia brevis bloom s and brevetoxins in tissues and gut contents. The rem a ining five UMEs involved bottlenose dolphins, and two of those occurred in the Florida Panhandle counties. In 1993-1994 an UME of bottlenose dolphins started in the Florida Panhandle and spread west with m o st of the m o rtalities occurring in Texas. That m o rtality event was caused by a m o rbillivirus epidem ic. Between August 1999 and February 2000, the Florida Panhandle experienced a second UME of at least 120 bottlenose dolphins coincident with Karenia brevis bloom s and fish kills. In both the 1999-2000 and 2004 UMEs other species of m a rine life such as fish, invertebrates and birds died. The current UME and the 1999-2000 UME both started in the sam e location (St. Joseph Bay) and extended along the seven counties of the Florida Panhandle (Escam bia, Santa Rosa, Okaloosa, W a lton, Bay, Gulf and Franklin Counties). The 1999-2000 UME persisted over a long period of tim e (9 m onths) and was spatially and tem porally concurrent with a K. brevis bloom that m oved from east to west. Brevetoxicosis was im plicated as the cause of the 1999-2000 UME, but carcass condition lim ited the investigation and no definitive diagnosis was m a de.

C. Historical Know ledge of Bottlenose Dolphin Populations and Strandings Along the Florida Panhandle

The historical average of bottlenose dolphin strandings for the Panhandle counties over the last 10 years is eight (8) anim als per year excluding years when a UME occurred. 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 our knowledge, no system atic research was perform e d prior to the 2004 UME to determ ine the daily or seasonal patterns of use of St. Joseph Bay by bottlenose dolphins, or their broader ranging patterns or genetic com position as these m i ght relate to stock
structure.

At other sites around the southeastern United States, a variety of techniques have been applied to determ ine stock structure, includi ng am ong others: (1) biopsy darting to obtain

sm all skin sam p les for genetic m a rking; (2) capture, sam p le, and release efforts to obtain genetic sam p les and apply tags or other m a rkings; (3) photographic identification and resighting using distinctive natural m a rkings on dolphin dorsal fins; and (4) radio-tagging and tracking. In the absence of specific em pirical data identifying stocks of dolphins in the coastal waters of the panhandle, NMFS has used the approach of designating each of the 33 bays, sounds or estuaries in the US Gulf of Mexico as separate putative stocks, until such tim e as better data becom e available clarifying stock identification. The NMFS U.S. Atlantic and Gulf of Mexico Marine Mammal Stock Assessments - 2000 (using 1993 survey data) reports that no dolphins were observed in St. Joseph Bay and surrounding waters during aerial surveys, so it was not designated as a putative stock.

5Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### The appearance of two die-offs of over 200 anim als in the last five years, as well as the recent biopsy darting and photographic identifi cation work with dolphins in St Joseph Bay and vicinity, indicate clearly that th e 1993 aerial survey findings of no dolphins present in the Bay are out of date and do not provide sufficient inform ation to determ ine im pacts of these m o rtality events on coastal populations or stocks.

III. METHODS AND RESULTS

A. Dolphin Sampling

The dolphin strandings were initially clustered tem porally (at least 30 dolphins within the first four days from March 10-13) and spatially along the Florida Panhandle, with the

m a jority of strandings (70%) in St. Joseph Bay (Figure 1). The first three dolphins were reported on March 10 and the last two dolphins were reported on April 13. All of the 107 dolphins involved in the UME were discovered dead and no live strandings were reported. A variety of sam p les were collected and analyzed by several collaborators (Table 1).

The dem ographics of the strandings indicate that both sexes and m o st age classes were involved. The sex distribution of the stranded anim als was approxim a tely equal: 36 fem a les and 46 m a les, with 25 carcasses of unknown sex. Age estim ates based on growth layer groups of teeth have not been com p leted, therefore for this report age estim ations are based on length to age curves f o r bottlenose dolphins in the Gulf of Mexico (Fernandez and Hohn, 1993; Read et al., 1993). Carcass m easurem ents of 100 anim als (not including two fetuses) show that length ranged from 76 cm (prem a ture parturition) to 280 cm (adult) (Figure 2). Com p aring the results of length classes with historic stranding records for this region (n=51, over 7 years), it is clear that a higher

num ber of anim als over 210 cm are represented in this die-off.

Gross Necropsy Findings:

Of the 107 dolphins involved in the UME, gross necropsies were conducted on 46 anim als, som e sam p les (e.g., teeth and skin) were collected from 44 anim als, and the

rem a ining anim als (n=17) were located and recorded but not sam p led due to logistical constraints and/or carcass condition. Blubber, liver and kidney were collected from four

m ildly decom posed anim als for the National Marine Mam m a l Tissue Bank.

Carcass decom position was classified into five categories: Code 2 - fresh (19 anim als); Code 3 - m ildly decom posed (39 anim als); Code 4 - m oderately decom posed (46 anim als); Code 5 - severely decom posed (2 anim als); and unknown (1 anim al). No live stranded anim als (Code 1) were found during the event. Most of the fresh anim als were detected in the first half of the event, and the last known Code 2 anim al was recovered on April 1.

Most anim als were found to be in good nutritional condition, however a few anim als were considered to be thin for this season as com p ared to winter season dolphin ranges in

6Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### Sarasota, Florida. Studies have shown that blubber thickness is affected by season in bottlenose dolphins in the Gulf of Mexico. Blubber thicknesses ranged from a low of 0.8

cm in a 268 cm m a le to a high of 2.3 cm in a 205 cm fem a le using the m i d-lateral

m easurem ent position from 19 anim als. Gross findings varied with each anim al, but pulm onary changes suggestive of pneum onia were noted in a num ber of anim als which was further characterized histologically. Som e anim als possessed m ild to m oderate parasite infestations which was character ized by individuals with pterygoid sinus
trem atodiasis, pulm onary nem a todiasis, gastric trem atodiasis and enteric cestodiasis. There was evidence in a few anim als of sedim e nt in the trachea and prim ary and secondary airways, a characteristic that is seen in som e anim als that strand live but die on the beach. Many anim als had evidence of generalized lym phoid stim ulation, which was characterized by m u ltiple enlarged lym ph nodes. A num ber of anim als had em pty urinary bladders or contained a scant am ount of opaque, pasty m a terial with few containing collectable urine for analysis (10 urine sam p les collected). The m a jority of anim als dem onstrated a distended stom ach with partially digested contents. Many anim als had intestines which contained scant ingesta (n=11).

Stomach Content Analyses:

Stom ach contents from 28 of 50 dolphins have been analyzed to date through the identification of fish rem a ins and hard parts. Gastric subsam ples were collected for biotoxin analyses from all 28 dolphins. Sem i -digested fish found in the dolphin stom achs were collected for toxin analyses: m e nhade n from 16 dolphins; a puffer fish from 1 dolphin; and spot, silver perch, kingfish, pi nfish and others from 11 dolphins. Two prelim inary findings stand out:

(1) Dolphins appear to have gorged on food prior to death, as m o st stom achs were packed to capacity. Mean stom ach content values were 773 g, with 10 sam p les weighing > 1 kg (m axim um : 2.258 kg). These figures are significantly higher than those obtained from the 2001 bottlenose dolphin UME that occurred in the Indian River Lagoon (IRL) system on the east coast of Florida. During the 2001 IRL event, dolphins were generally em aciated and had very little food m a tter in their stom achs (m ean of 36 g, n= 14) com p rised m o stly of fleshless fish bones. In addition, several of the IRL anim als had em pty stom achs. Com p ared with other studies in the southeastern U.S. (Figure 3), the 2004 UME dolphins from the Florida Panhandle stranded with considerably fuller stom achs than stranded dolphins in other areas. Data obtaine d from stranded dolphins in Texas (1987- 1988) also showed high stom ach m a ss values, but dolphins in that area are believed to gorge on fish discards from shrim p boats.

(2) Menhaden was the dom inant prey species in at least 14 of the 28 (50%) dolphins

exam ined, judging by the large num ber (dozens) of sem i -digested fish found in each stom ach. A sub-set (n= 19) of the undigested m e nhaden ranged from 9.5 to 16 cm , with m o st in the 14-15 cm size class. Sciaenid fish (e.g., silver perch, spot, kingfish) were dom inant in 11 (39%) of the other sam p les. A few dolphins had undigested shrim p rem a ins in their stom achs, and others had hundreds (possibly thousands) of fish ear bones (otoliths) of very sm all fish, m o stly

7Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### croakers of the fam ily Sciaenidae. These findings are sim ilar to those found for Texas dolphins, where feeding in association with shrim p ers is known to occur.

Virology:

Tissues obtained from 26 dolphins have been analyzed to date to determ ine pathogen presence using polym erase chain reaction (PCR) and reverse transcription PCR (RTPCR). To date, all sam p les have been negative f o r m o rbillivirus (50 sam p les f r om 24 anim als), influenza A virus (14 sam p les from 8 anim als), and influenza B viruses (14

sam p les from 10 anim als) as well as calicivirus (26 sam p les from 15 anim als), cetacean poxvirus (3 sam p les from 3 anim als), herpes virus (10 sam p les), and parapoxvirus (3

sam p les from 3 anim als).

Histopathology:

Histopathological exam inations were perform e d on partial to com p lete necropsy subm issions from 38 dolphins (14 fem a les and 24 m a les). No cause of death was evident histologically, and there was no histological ev idence of infectious disease. Autolysis and postm ortem bacterial overgrowth ranged from m oderate to advanced, occasionally lim iting the histological evaluations.

The vast m a jority of the dolphins appeared to be in good nutritional condition with m ild
to m oderate fat atrophy only present in 6 of the 38 anim als (16%) exam ined. Com m on histological lesions included prom inent eosinophilia within m u ltiple tissues (34/38), parasitic pneum onia (28/38), angiom atosis (20/ 38), m ild m yocardial f i brosis (11/38) and

m ild hepatic fibrosis (9/38). Less com m on lesions included m ild perivascular brain edem a (4/38), m ild chronic interstitial nephritis (4/38), lym phocytic laryngitis (2/38), lym phoid hyperplasia (2/38), m ild cerebral gliosis (1/38), m ild lym phocytic m e ningitis (1/38), m u ltif ocal chronic m yocarditis (1/38), and a pleural adhesion (1/38).

The prom inent eosinophilic inflam m a tion within m u ltiple tissues is m o st likely associated with parasitism

, a com m on f i nding in wild m a rine m a m m a ls. The changes within the liver are suggestive of a previous parasitic infection. Angiom atosis, a com m on proliferative lesion that occurs in the lungs and lym ph nodes of dolphins, is thought to be clinically insignificant. Mild m yocardial fibrosis is a fairly com m on finding in adult dolphins, and is usually clinically insignificant. The m ild perivascular edem a seen in a few dolphins is a com m on, nonspecific finding in the brains of stranded dolphins. Lym phoid hyperplasia is a nonspecific finding in response to antigenic stim ulation. The signif i cance of the m yocarditis is uncertain since the inf l am m a tion was not associated with degenerative changes within the m yocytes. The causes of the rem a ining lesions were not evident. They were all m ild, and not likely associated with the death of the dolphins. Gross and histological lesions of br evetoxicosis are not well established or are nonspecific except in the case in inhalation brevetoxicosis. Gross lesions from the

m a natees with inhalation brevetoxicosis included nasopharyngeal and pulm onary edem a and hem o rrhage. Histological lesions in m a natees with inhalation exposure included the above plus catarrhal rhinitis. No such gross or histological lesions were seen in the current investigation.

8Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### Organochlorines:

Concentrations of persistent organic pollutants (“POPs”, i.e. , polychlorinated biphenyl (PCB) congeners, DDT and its derivatives, and other organochlorine insecticides) in bottlenose dolphin blubber sam p les collected from these anim als were determ ined and

com p ared to those observed in free-ranging Sarasota Bay dolphins captured during health

assessm ent work. Only juvenile anim als were used in this com p arison because it has been shown that POP concentrations in juveniles are not statistically different between genders and juvenile anim als can be identified by their length. The use of juveniles was also an attem p t to control for age since that can influence POP levels in cetaceans.

Eighty PCB congeners, DDT and m e tabolites (DDE and DDD), oxychlordane, dieldrin and m i rex were determ ined in full depth blubber biopsies from 23 juvenile dolphins collected from Sarasota Bay (collection interval: 2000 to 2004) and seven full depth blubber biopsy sam p les collected from Code 2 dolphins that stranded during the UME event. Sam p les were analyzed by gas chrom a tography with m a ss spectrom e try at the NIST Charleston Laboratory. Total PCBs (sum of all m easurable congeners) and total DDT (sum of 2,4’- and 4,4’-DDD, -DDE, and DDT) were not statistically different between the UME and Sarasota anim als. The geom etric m ean of total PCBs was 18.5

m g /kg wet wt. (range 6.4 to 52.5 m g /kg wet wt.) and 16.5 m g /kg (range 12.3 to 24.7

m g /kg wet wt.) for the Sarasota and UME anim als, respectively. The geom etric m ean of total DDT was 9.7 m g /kg (range 3.7 to 25.9 m g /kg wet wt.) and 9.6 m g /kg (range 6.3to 21.8 m g /kg wet wt.) for the Sarasota and UME anim als, respectively. Based on the scientific literature, these levels are above those thought to have toxicological significance in delphinids (e.g., im m unosuppre ssion). Other organochlorine pesticides tended to be m u ch lower in the UME anim als than in the Sarasota anim als. For instance, concentration (geom e tric m ean) of dieldrin in blubber was 0.71 m g /kg (range 0.26 to 2.3

m g /kg wet wt.) in the Sarasota anim als versus 0.097 m g /kg the UME anim als (range 0.065 to 0.13 m g /g wet wt). Lipid content in blubber was not significantly different between the Sarasota dolphins and the six juvenile m a les from the UME analyzed to date, suggesting that these 6 anim als were not thin at the tim e of their deaths.

Biotoxins:

Tissue and fluid sam p les from 38 dolphins al ong the Florida Panhandle (Figure 4) were analyzed for brevetoxins using m u ltiple m e thods (com petitive ELISA, Radioim m unoassay, Receptor Binding Assay, LC/MS, and reversed phase chrom a tography coupled to tandem m a ss spectrom e try). All dolphins tested had high levels of brevetoxins in their stom ach cont ents and/or feces (Figure 5). Significantly lower concentrations were present in tissues. The pattern of m e tabolites in tissues has not been com p leted but will provide insights into m e tabolism and potential toxicity. Brevetoxins were also m easurable in the urine of m o st of the few anim als tested (n=10). Brevetoxin concentrations varied widely between individual anim als and between tissues but had a narrower range of values in 2004 than observed in the 1999-2000 dolphin UME along the Florida Panhandle. In addition, the levels found in stom achs of the 2004 UME dolphins were greater than those m easured in the 1996 and 2003 m a natee m o rtalities on the west coast of Florida (Figure 6). Although the toxic effect level of brevetoxin and its

9Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### m e tabolites rem a ins poorly defined, its presence at high levels in all anim als tested supports brevetoxicosis as a differential diagnosis for this m o rtality event.

Trace levels of dom oic acid (using receptor binding assay, ELISA and LC tandem m a ss spectroscopy) were found in the stom ach contents and feces of som e , but not all, of the stranded dolphins tested. Pseudo-nitzschia is not uncom m on in the waters along the Florida Panhandle coast and, as there is no data on the natural levels of dom oic acid in the food web from this part of the Gulf of Mexico, it is unclear if the trace am ounts of dom oic acid in the stom ach contents and feces of these anim als are significant to this

m o rtality event.

Genetic Analyses:

Genetic analyses were perform e d on 64 sam p led dolphins to determ ine whether the UME anim als belonged to coastal or offshore stocks of bottlenose dolphins using DNA sequencing of a portion of the m itochondrial DNA genom e (m ethods described in Rosel et al. 1999). Results indicate that all 64 anim als were of the coastal m o rphotype.

B. Environmental Sampling

Satellite Imagery:

Satellite ocean color im agery has been used effectively for over four years as part of a

m onitoring program for Karenia brevis to support state and federal activities. The prim ary sensor is on the Sea-viewing W i de Fi eld-of View Sensor (SeaW iFS). Data and analyses are typically provided to the state as a m onitoring and forecasting tool for the bloom s. These analyses have aided with other bloom s, such as Pseudo-nitzschia , but are

m o st ef f

ective with Karenia bloom s. Detection is lim ited to about 50,000 cells L -1 

### of Karenia , which is m u ch higher than that required for shellfish closures, but about the

m i nim u m at which hum an respiratory im pacts occur. Im ages of total chlorophyll concentration and im ages showing the presence of new bloom s are norm a lly used as part of the analyses (Figures 7-12).

On February 28, a chlorophyll feature (2-4 μ g/L) occurred about 12 nautical m iles SouthSouthwest of Cedar Key (Figures 8-9). In January 2004, a bloom of Karenia appeared near Clearwater Florida. Analyses of im agery and winds indicates that the bloom originated south of Cedar Key, m a king it possible that a K. brevis bloom was present in the Cedar Key area. This region is problem atic, with m a ny frequent bloom s.
On March 2, an area was flagged for new bloom s from Cape San Blas to near Tyndall Air Force Base. Extrem e elevated chlorophyll and a new bloom was evident in St Joseph Bay on March 9-11 (Figure 10). This characteristic of a bloom only in the Bay was observed in 1999 when a bloom of K. brevis occurred in St. Joseph Bay in August. Cell counts from water sam p les collected on March 11 contained Pseudo-nitzschia but not Karenia brevis (see HAB Monitoring), so it was assum e d that the elevated chlorophyll was the result of a Pseudo-nitzschia bloom . The presence of brevetoxins in water at low levels (March 12, see Toxicity Testing) and fish from St. Joseph Bay suggest that

10Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### K. brevis m a y also have been present but was gone by the tim e sam p les were collected for phytoplankton counts. By March 18, slightly elevated chlorophyll levels were observed from the east end of St. George Is land, offshore St. Joseph Bay and from Cape San Blas to 30 N.

Between April 12-15, a front passed through the UME area. Strong southerly winds followed by strong North-Northwesterlies caused m i xing in the St. Joseph Bay area and resuspension of benthic m a terial. This resulted in a rapid increase in chlorophyll that persisted for a few days and then dim i nished (Figures 11-12). Between April 16-18, two bloom patches were observed of f

s hore and f i sh kills that were reported in St. Joseph Bay and 20 m iles to the south of Cape San Blas.

Harmful Algal Bloom (HAB) Monitoring and Toxin Analyses:

HAB m onitoring Two dedicated cruises, one of which was funded by the CSCOR HAB Event Response Program and one cruise of opportunity, were conducted and water sam p les were collected at over 115 stations (Figure 13) from Marc h 11 to May 5, 2004. Microscopic (both light and electron m i croscopy) identification a nd enum eration of phytoplankton and toxin analyses were conducted on the sam p les. During the event, as often as was feasible,
water sam p les were collected in areas flagged by NOAA satellite im agery as potential HAB events.

No significant populations of Karenia brevis , were observed in 233 counts of sam p les from St. Joseph Bay and the surrounding offshore region in surface waters or at depth. Pseudo-nitzschia delicatissima was observed at low concentrations (0 to 1.7 x 10 4 

### cells L 1 

### ) outside St. Joseph Bay, and at m oderate concentrations (0 to 4.6 x 10 5 

### cells L -1 

### ) inside the Bay. All stations m easured had dissolved oxygen concentrations >7.2 m g L -1 

### , ruling out the involvem e nt of low oxygen concentrations in the event.

Toxin Analyses

Brevetoxins: 84 water sam p les and 60 fish (both live-caught and from fish kills, total of 128 sam p les) were analyzed for brevetoxin by com p etitive ELISA. All water sam p les collected outside of St. Joseph Bay were negative, while those collected inside the bay contained low levels of brevetoxin (0.72 – 1.1 μ g/L on March 12) in the absence of any apparent Karenia brevis cells.

Several species of f

i sh were collected live and f r om f

i sh kills in St. Joseph Bay. Most contained elevated levels of brevetoxin. Fish tissues exam ined included gill (64-798 ng/g), liver (865-4,655 ng/g), stom ach cont ents (51-9,000 ng/g) and m u scle (nd-473 ng/g). Fish collected live in St. Josephs Bay on March 18 contained variable levels of brevetoxin in their stom ach contents. Burrfish (carnivores), spot (om n ivores), and pinfish (herbivores) had high levels of toxin in their stom ach contents (>1000 ng/g), whereas Gulf flounder and sardine collected at the sam e tim e were negative. W h ether this reflects feeding habits of these species or individual variation is uncertain based on the sm all

num ber of sam p les analyzed to date. Live fish collected on March 28 and 31 (n=47)

11Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### included Spanish m ackerel, pinfish, flounder, s outhern codling, spot, sea trout, bluefish, and thread herring. Brevetoxins were m easurable but below 1000 ng/g in the stom ach contents of all fish except for the sea trout, which contained 4,000 ng/g. In addition brevetoxins were found in the flesh of som e fish.

Domoic Acid: Eighteen water sam p les were analyzed for dom oic acid by ELISA. Although Pseudo-nitzschia was present and identif ied as P. delicatissima , all water

sam p les were below the level of detection of the ELISA kit for dom oic acid.

Mortality Reports in Addition to Dolphins

Fish kills and other anim al m o rtalities were reported in St. Joseph Bay as f o llows:

03/11/04 Lim ited num ber of dead fish and horseshoe crabs were reported 03/14/04 Report of m a ny dozens of lightning whelk at the Bay entrance (m ay have been em pty shells) 03/18/04 Hundreds of dead horseshoe crabs; hundreds of dead jellyfish, one dead

corm orant; dead red drum ; num erous baitfish were swim m i ng in circles at the surface; one sick laughing gull and corm orant exhibited neurologic signs 03/23/04 Fish kill involving spot, bay anchovy, cowfish, and striped burrfish. Additionally horseshoe crabs 04/02/04 Red drum , hundreds of dead adult horseshoe crabs, two dead pelicans, one egret, and one loon 04/12/04 Red drum 04/19/04 Red drum 05/06/04 From Pig Island, SW corner of the Bay - red drum , Gulf killifish, spotted
sea trout

20 m iles south of Cape San Blas:

04/14/04 Red drum and thread herring

These reports were not necessarily verified in every case, nor are they necessarily separate events, nor were necropsies conducted on all anim als. Biotoxin analyses have not been com p leted for all the sam p les. General field observations and behavioral
sym p tom s of m o ribund or sick anim als are typical of exposure to Karenia brevis bloom s.

Shellfish Monitoring for Harmful Algal Bloom Species

The only shellfish harvesting area in the im m e diate vicinity of the UME is the St. Joseph Bay Shellfish Harvesting Area. The area was tem porarily closed at sunset on Novem b er 19, 2003 due to the detection of K. brevis . Therefore, no com m e rcial or recreational harvesting of shellfish was allowed following Novem b er 19, 2003 including the tim e that the UME occurred. W a ters surrounding the St. Joseph Bay Shellfish Harvesting Area are unclassified and com m e rcial and recreational shellfish harvesting is never allowed.

12Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### Results from sam p les of shellfish m eats for brevetoxins continued to be above the established regulatory level until April 29, 2004 (even though Karenia brevis cells were not present). The area was reopened to harvest at sunrise, on April 30, 2004 due to nontoxic bioassays for brevetoxin. Results from shellfish sam p les collected from the area showed that dom oic acid concentrations were well below the established regulatory lim it for public health protection. Therefore, recognized shellfish public health concerns were and continue to be adequately and appropriately addressed.

IV. DISCUSSION AND FUTURE PLANS

A. Comparison w i th Other UMEs

In the last decade there have been several UMEs of m a rine m a m m a ls that have been attributed to or correlated with harm ful algal bloom s. The m o st com m on harm ful algal bloom in the Gulf of Mexico is caused by the red tide organism Karenia brevis , known to cause Neurotoxic Shellfish Poisoning and respir atory irritation in hum ans. The biotoxin brevetoxin and its m e tabolites produced by Karenia brevis were im plicated in m a natee

m o rtality events in Florida in 1963, 1982, 1996, 2002, and 2003. Also, brevetoxin and its

m e tabolites were suspected as a possible cause f o r bottlenose dolphin die-of f events in Mississippi during 1996 and along the Fl orida Panhandle during 1999-2000. Although Pseudo-nitzschia spp. bloom s are known in the Gulf of Mexico, no m a rine m a m m a l

m o rtality events have been attributed to these bloom s or dom oic acid (the related biotoxin) in this region. W e m u st note that this is the first tim e that dom oic acid has been analyzed in tissues from m a rine m a m m a ls that have stranded in the Gulf of Mexico, therefore no baselines exist for this biotoxin in m a rine m a m m a ls in the Gulf of Mexico. However, Pseudo-nitzschia bloom s and subsequent dom oic acid intoxication have been associated with sea lion and dolphin m o rtalities in California (1998 to 2003) and hum pback whale m o rtalities on George’s Bank (2003). The concentrations of dom oic acid found in the bottlenose dolphin sam p les that were collected during this event were

m u ch lower than those found associated with previously docum ented m a rine anim al

m o rtalities. Theref ore dom oic acid intoxication is not considered as a prim ary cause of the 2004 dolphin UME, but has not been ruled out as a contributing factor.

B. Insights into Dolphin Behavior and Feeding in the Florida Panhandle

During Chicago Zoological Society/Florida St ate University biopsy sam p ling in AprilMay 2004, dolphins were frequently observed feeding on fish discarded from shrim p trawlers, while anchored or drifting and culling fish in St. Joseph Bay. Active trawlers were not observed in the bay -- it appeared that they trawled elsewhere and culled their catch in the sheltered waters of the bay prior to offloading their product at Port St. Joe. Croakers (Sciaenidae) are the predom inant finfish by-catch group in the Gulf of Mexico shrim p fishery, which targets organism s (e.g., shrim p, bottom fish) living on or near the bottom

. It has been suggested that the fact that som e dolphins in St. Joseph Bay obtain at least part of their daily diet from the fish caught outside of the bay by shrim p trawlers and provided to the dolphins in the bay m a y have potential im plications for evaluating the source of toxins in the stom ach contents of dolphins dying during the UME.

13Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### Menhaden, on the other hand, are caught in larg e num bers in purse-seine nets. The biopsy darting team also observed purse-seine vessels returning to Port St. Joe, but the boats were neither fishing nor culling in the bay at the tim e they were observed.

C. Continuing Studies:

Acoustic/Blast Trauma -- Local citizens reported that the Departm e nt of Defense was conducting m ilitary exercises off the coast of the Florida Panhandle in March 2004. Based on the gross exam inations, there was no physical evidence of blast or acoustic
traum a in the 22 freshest dolphin carcasses. However, heads and ears collected from a subset of these anim als will be exam ined specif i cally f o r blast and acoustic traum a using CT scanning m e thodology and histopathology. NMFS is working with the US Navy to determ ine what types of activities were conducted, and when and where they occurred, to

com p are with stranding data.

Life History – Dem ographics and life history param e ters are essential com ponents of UME investigations and provide trem endous insights into the real epidem iology of the event. Lif e history f actors that will be critical to this investigation include such f actors as: age determ ination, prey or feeding strategies (what do the anim als eat? ), and population or stock identification and assessm ent (who are the anim als and where do they go). Stable isotope analyses will be conducted to determ ine historic feeding habits. Age Determination -- Age estim ations of the dolphins involved in the UME will be obtained by analyzing growth layer groups of teeth and reproductive organs. Reproductive status will also be determ ined. Feeding (Stomach Content Analyses) -- Detailed dietary studies will be com p leted f o r all available UME sam p les (n= 50), including the contribution by biom ass of the m o st im portant prey species. In addition, sam p les obtained from the 1999-2000 UME (n= 70 to 80) will be analyzed f o r com p arative purposes. Genetics -- Subsequent to the die-of f

, genetic sam p les f r om 30 live bottlenose dolphins in St. Joseph Bay and vicinity were sam p led via biopsy darting. These sam p les will be

com p ared to the UME anim als using m itochondrial DNA sequencing and m i crosatellite analyses to help determ ine the origins of the dead dolphins. In addition, sam p les from the 1999-2000 event will also be analyzed and com p ared to the current m o rtalities.

Trophic transfer and metabolism -- There is increasing evidence that anim als can be exposed to brevetoxins by m u ltiple routes (inhalation and ingestion), though different vectors (prey, feeding habits, or directly consum ing the harm ful algae), and under different tim e scales. The m echanism s and tim ing of m ovem e nt of biotoxins through the ecosystem and food web are critical to understanding risks of exposure and risks to populations. In addition to exam ining prey types and biotoxin levels in prey, m e tabolism of brevetoxins through the various levels of the food web will also be exam ined.

Biotoxin Dosage Determination -- Additional work will be conducted to quantify the estim ated dose of toxins received by the anim als and search for evidence of any other toxicants that m i ght be in dolphin prey species and to com p are the results with previous

14Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### UMEs. The toxicity of m u ltiple biotoxin exposures in m a rine organism s using anim al

m odels will be useful to evaluate as part of long range studies.

D. Future Studies

This section outlines specific studies that need to be conducted to better understand this UME , the role of brevetoxins in UMEs, and the im pacts of repeat UMEs in this geographic area. Although needed, there is no identified funding specific for these investigations.

Stock Structure -- To date the stock structure of coastal and estuarine bottlenose dolphins in the northern Gulf of Mexico has not been rigorously investigated using genetic and photo identification techniques. This should be a priority. Since so m u ch is unknown about stock structure in this area, it will take som e tim e to understand the im pacts of these m o rtality events on a specific population(s) and risks from an increasing frequency of harm ful algal bloom s.

Health Assessments -- Health assessm ent indices should be developed and m onitored through the use of live captures, sam p ling and release exercises in specific areas where

anom alous m o rtality events have occurred (i.e., in St. Joseph Bay). Studies through live capture will be critical in determ ining risk factors in populations and which populations

m a y be at risk. These studies will be essential in understanding any contributing or predisposing factors in the population that puts this population m o re susceptible to biotoxins, inf ections or other m o rbidity or m o rtality f actors. This also includes

assessm ent of strandings that occur to obtain m o re baseline inf o rm ation on m o rtalities and life history in this area.

Feeding Ecology -- Understanding the trophic ecology of wild dolphins is crucial in the

m onitoring of UMEs in which feeding is suspected to play an im portant role, particularly in areas where algal bloom s are known to occur. A recom m e ndation is m a de that sam p les (dolphin stom achs) be collected from stranded anim als, so that baseline data on feeding and biotoxin concentrations can be obtained for the Florida Panhandle area.

15Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### E. Evaluation of UME Response

What went right?

• 

### Long-term stranding response data were available and identified the unusual level of

m o rtality alm o st im m e diately

• The response by the stranding network and colleagues was m o re organized and

com p lete than during the 1999-2000 event

• Multiple federal and state agencies and outside partners worked cooperatively on

a com p rehensive investigation

• Appropriate expertise for various analyses had been previously identified, resulting in efficient dissem i nation of sam p les for various analytical tests

What challenges were faced?

• Current efforts to m onitor bloom s are guided prim arily by the need to protect hum ans from Neurotoxic Shellfish Poisoning, not to protect m a rine m a m m a ls from Neurotoxin Fish Poisoning. Understanding the links between m a rine

m a m m a l m o rtality events in this area and the occurrence of HABs is not adequate to provide guidance for future m onitoring to predict m a rine m a m m a l events. Resources and equipm ent for the im m e diate response were lim ited

• Baseline inform ation on the health, distribution, dem ographics, and abundance of dolphins in the area of the UME was lim ited, m a king interpretation of results

m o re uncertain and tentative

• Baseline inform ation with regard to concentrations of biotoxins (brevetoxin, dom oic acid, and com b inations of the two) in plankton, food item s , digestive tract contents, body fluids, and tissues consistent with m o rbidity and m o rtality in bottlenose dolphins was, for the m o st part, lim ited or non-existent

• Baseline inform ation on the levels of m a n-m a de environm ental contam inants in this dolphin population and inform ation on how these m a y interact with natural toxins was not available

• Currently we continue to be lim ited in biotoxin detection and quantification (for

m e tabolites) by the state of the science and the lim itations on standards and techniques

• Training and m a intaining professional team s to respond to increasingly m o re

com p lex investigations anywhere in U.S. waters is difficult and requires substantial resources

16Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### V. ACKNOWLEDGEMENTS

NOAA/National Marine Fisheries Service Marine Mammal Health and Stranding Response Program: Angela Collins-Payne, Teri Rowles, Trevor Spradlin, Janet W h aley, Sarah W ilkin Southeast Fisheries Science Center, Miami: Laura Engleby, Ruth Ewing, Sarah Gom e z, Jenny Litz, Blair Mase-Guthrie Southeast Fisheries Science Center, Beaufort, NC: Aleta Hohn, Gretchen Lovewell, Karen Sayles Southeast Fisheries Science Center, Lafayette, LA: Sarah Kingston, Patricia Rosel Southeast Fisheries Science Center, Panama City, FL: Nancy Evou, Pete Sheridan

NOAA/National Ocean Service Center for Sponsored Coastal Ocean Research: Quay Dortch, Marc Suddleson, Center for Coastal Monitoring and Assessment: Richard Stum pf, Michelle Tom linson Center for Coastal Environmental Health and Biomolecular Research, Marine Biotoxins Program, Charleston, SC: Greg Doucette, Kristin King, Tod Leighfield, Jen Maucher, Brad Mitchell, Steve Morton, John Ram s dell, Fran Van Dolah, Zhihong W ong, Ricky W oofter Center for Coastal Environmental Health and Biomolecular Research, Marine Mammal Group, Charleston, SC: Leslie Gilroy-Burdett, W a yne McFee, Jessie Stenftenagel, Eric Zolm an

Florida Fish and Wildlife Conservation Commission/Florida Marine Research Institute Ken Arrison, Alex Costidis, Elsa Haubol d, Jessica Lightsey, Tom Pitchford, Tanya Pulf er, Butch Rom m e l, Leslie W a rd Jay Abbott, Susan Cook, Leanne Flewelling, Matt Garrett, Cindy Heil, Ryan Pigg, Bill Richardson, Paula Scott, Earnest Truby, Jennifer W o lny Ann Forstchen, Jan Landsberg, Em ilio Sosa, Christy Stephenson Stephanie Fahrny, Dayle Flint, Chad Hanson, Kelly Hooper, Ed Matheson, Stephanie McGrath, Bob McMichael, Missy Millender, S ky Rudloe, Sherri Thourot, Troy Tuckey, Jeff W r en Allison Bozarth

Armed Forces Institute of Pathology Dale Dunn, Michelle Fleetwood, Tom Lipscom b

Chicago Z oological Society, Sarasota Dolphin Project Jason Allen, Brian Balm er, Spencer Fire, Larry Fulford, Randall W e lls

Florida Department of Agriculture and Consumer Services Richard Davis, David Heil, Joe Shields

Florida Department of Environmental Protection T a mmy S u mme r s , K i m Wr e n

17Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### Florida State University Doug Nowacek

Gulf World Marine Park Ron Hardy, Cheryl Joyner

Harbor Branch Oceanographic Institute Greg Bossart

Hubbs-SeaWorld Research Institute Teresa Mazza, W e ndy Noke

Mote Marine Laboratory Nélio Barros, Mike Henry, Rich Pierce

National Institute of Standards and Technology Charleston Lab: Michael Ellisor, Jennifer Keller, John Kucklick, Rebecca Pugh, Bob Swarthout, Stacy Vander Pol

Sea World – San Diego Judy St. Leger

University of Florida Carlos Rom e ros
Working Group on Marine Mammal Unusual Mortality Events Val Beasley, University of Illinois Sylvain De Guise, University of Connecticut Dale Dunn, Arm e d Forces Institute of Pathology Frances Gulland, The Marine Mam m a l Center Ailsa Hall, NERC Sea Mam m a l Research Unit/University of St Andrews. Linda J. Lowenstine, University of California-Davis Pam e la A. Tuom i, Alaska SeaLife Center Frances Van Dolah, NOAA National Ocean Service Michael W a lsh, Sea W o rld-Orlando Randall W e lls, Chicago Zoological Society and Mote Marine Laboratory Sharon Young, Hum a ne Society of the United States Federal Agency Representatives: Teri Rowles, National Marine Fisheries Service Diane L. Bowen, U.S. Fish and W ildlife Service Tim Ragen, Marine Mam m a l Com m i ssion John Heisler, U.S. Environm ental Protection Agency International Participants: Canada: Ole Nielsen, Departm e nt of Fisheries and Oceans Canada Mexico: Lorenzo Rojas-Bracho, Program a Nacional de Mam í feros Marinos, Instituto Nacional de Ecología

18Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### VI. REFERENCES

Bossart, G.D., D.G. Baden, R.Y. Ewing, B. Roberts and S.D. W r ight. 1998. Brevitoxicosis in m a natees ( Trichechus manatus latirostris ) from the 1996 epizootic: gross, histologic and im m unohistochem i cal features. Toxicol

. Pathol

., 26: 276-282.

Ch’ng, M.M., T.A. Leighfield, M.A. Busm a n, F.M. Gulland, K. Matassa, M. Chechowitz, T. Rowles and F.M. Van Dolah. 2002. Analys is of dom oic acid involvem e nt in m a rine

m a m m a l m o rbidities and m o rtalities on the west coast of the U.S.: February-Aug 2000. Report to the W o rking Group on Marine Mam m a l Unusual Mortality Events. 18 pp.

Fernandez, S. and A.A. Hohn. 1998. Age structure, growth, and calving season of bottlenose dolphins stranded along the coast of Texas. Fish. Bull. 96(2):357-365.

Gulland, F.M. 2000. Dom o ic acid toxicity in sea lions ( Zalophus californianus ) stranded along the central California coast, May-October 1998. Report to the National Marine Fisheries Service W o rking Group on Marine Mam m a l Unusual Mortality Events. NOAA Tech. Mem o randum NMFS-OPR-17, 45 p.

Landsberg, J.H. and K.A. Steidinger. 1998. A historical review of Gymnodinium breve red tides im plicated in m a ss m o rtalities of the m a natee ( Trichechus manatus latirostris ) in Florida, USA. Pages 97-100 in B. Reguera, J. Blanco, M.L. Fernández and T. W y att, eds. Harm ful Algae. Xunta de Galicia, IOC, Paris, France.

O' Shea, T.J., G.B. Rathbun, R.K. Bonde, C.D. Buergelt and D.K. Odell. 1991. An epizootic of Florida m a natees associ ated with a dinoflagellate bloom

. Mar

. Mammal Sci

., 7: 165-179.

Read, A.J., R.S. W e lls, A.A. Hohn and M.D. Scott. 1993. Patterns of growth in wild bottlenose dolphins, Tursiops truncatus

. J. Zool., London, 231:107-123.

Rosel, P.E., S.C. France, J.Y. W a ng and T.D. Kocher. 1999. Genetic structure of harbour porpoise, Phocoena phocoena , populations in the Northwest Atlantic based on

m itochondrial and nuclear m a rkers. Mol. Ecol. , 8:S41-S54.

Scholin, C.A., F. Gulland, G.J. Doucette, S. Benson, M. Busm an, F.P. Chavez, J. Cordaro, R. Delong, A. De Vogelaere, J. Harv ey, M. Haulena, K. Lefebvre, T. Lipscom b, S. Loscutof f

, L.J. Lowenstine, R. Marin, III, P.E. Miller, W

. A. McLellan, P.D.R. Moeller, C.L. Powell, T. Rowles, P. Silvagni, M. Silver, T. Spraker, V. Trainer and F.M. Van Dolah. 2000. Mortality of sea lions along the central California coast linked to a toxic diatom bloom

. Nature , 403: 80-84.

Van Dolah, F.M., G.J. Doucette, F. Gulland, G. Bossart and T. Rowles. 2003. Im pacts of algal toxins on m a rine m a m m a ls. Pages 247-269 in J. Vos, G.D. Bossart, M. Fournier and T. O’Shea, eds. Toxicology of Marine Mam m a ls. Taylor and Francis, New York, NY.

19Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### Van Dolah, F.M., E.L. Finley, B.L. Haynes, G.J. Doucette, P.D. Moeller and J.S. Ram s dell. 1994. Developm ent of Rapid and Sensitive High Throughput Pharm acologic Assays for Marine Phycotoxins. Nat. Toxins, 2:189-196.

W oofter, R.T. et al. Environmental Health Perspectives , October 2003, volum e 111,

num ber 13, pages 1595-1600.

20Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### VII. TABLES AND FIGURES

Table 1. Sam p les collected for analysis during the 2004 Florida Panhandle Unusual Mortality Event

Anal yses Purpose Investi gator/ L aboratory No. of Animals Sampled Status

Gro ss Ex am in atio n and Necropsy Id en tificatio n o f gross causes of

m o rtality an d tissue sam p le collection Vari ous fi el d i nvest i g at ors Level 3* - 46 Level 2 – 44 Level 1 – 9 No Exam - 8 107 C o m p l e t e d

Aco u s tic trau m a To d e term in e

m o rphol ogy and pat hol ogy of

aco u s tic tissu es W oods Hole Oceanographic In stitu te 8 8 Pendi n g

Agi ng and Li fe History Estim ate age and reproduct i v e status NOAA Fisheries, Southeast Fisheries Science Center, Beaufort, NC 5 1 5 1 Pendi n g

Purdue Uni v ersi t y 2 2 Pendi ng B a c t e r i o l o g y B a c t e r i a l id en tificatio n to

exam ine in fectio u s processes and cause of death Hubbs Sea W o rl d Orl a ndo 2 2 Pendi ng

Florida Marine Research In stitu te Flo r id a Fish an d W ild life Co n s erv a tio n C o mmi s s i o n 1 1 1 1 C o m p l e t e d B r evet oxi n/ Dom o ic acid Tissue

ex am in atio n to id en tify an d quant i f y bi ot oxi ns M a ri ne B i ot oxi n Program NOAA/NOS C h arl e st on, SC 5 4 2 1 C o m p l e t e d 33 Pendi ng

Genetics DNA analy s is to det e rm i n e st ock id en tity NOAA/NOAA Fisheries Southeast Fisheries Science C e nt er B a t on R ouge, LA 6 5 6 5 C o m p l e t e d

Hi st opat hol ogy T i s s u e

ex am in atio n to identify disease
processes Arm e d Fo rces In stitu te o f Pat hol ogy 3 8 3 8 C o m p l e t e d

Im m unoperoxi d a s e E x a m i n at i on t o id en tify presence and di st ri but i on of brevet oxi n i n tissue sam p les Harbor Branch Oceanographic Institute TB D P e n d i n g

St om ach C ont ent s (Fish Species ID) To eval uat e di et M o t e M a ri ne Laborat ory 50 28 C o m p l e t e d In itial An alysis; 22 pendi ng (pl u s 1999-2000 anim als)

21Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004

Natio n a l In stitu te o f St andards and Technol ogy C h arl e st on, SC 3 3 2 6 Pendi n g 7 co m p leted fo r POPs Toxi col ogy Ident i f i cat i on and quant i f i cat i on of
t oxi ns i n cl udi ng p e sticid es, organochl ori d es and heavy

m e tals National Ocean Service Center for Coastal Envi ronm ent a l Heal t h and Biom olecular Research 5 1 C o m p l e t e d for water p e sticid es 4 Pendi ng

Virology V i r a l id en tificatio n / iso l atio n to

exam ine in fectio u s processes Carlos Rom e ro, Ph.D. Un iv ersity o f Flo r id a 3 1 2 1 C o m p l e t e d 10 Pendi ng



### * Level 3: full exam ination including necropsy and sam p le collection; Level 2: no necropsy, but som e sam p les collected (typically skin, blubber, m u scle and/or teeth); Level 1: basic data collected including length, sex and location.

22Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### Figure 1. Map of the Florida Panhandle indicating locations where the 107 reported bottlenose dolphins were recovered. Inse rt depicts Gulf County and Bay County including St. Joseph Bay where the m a jority of anim als stranded from March 10 - April 14, 2004.

23Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004

0 0. 02 0. 04 0. 06 0. 08 0. 1 0. 12 0. 14 0. 16

90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280
l e n g th (cm )proportion of total animals 97-03 (n=51)

2004 (n=80) New borns or

w i th in a fe w

m ont hs of bi rt h P r edi c t ed l engt h at 1 year of age J u veni l e (2-4 years ) S ubadul t A dul t (> 8-10 years fo r

fe m a le s; >10-12 years

fo r

m a le s)



### Figure 2. Standard length distribution (in cm ) of 80 stranded dolphins recovered during the Florida Panhandle UME March-April 2004, divided into age categories based on length, and com p ared to length data from stranded anim als reported in historical data (1997-2003).

24Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004

B o t t l e nos e D o l phi n S t om a c h C ont e n t B i om a s s

0 50 0 10 00 15 00 20 00 25 00

U M E 2004 P A N U M E 2001 I R L N O N

- U M E I R L ( 1 993- 2004) N O N

- U M E I R L ( 1 976-
1 992) U M E 1 990 T E X A S N O N

- U M E T E X

A S ( 1 987- 1 988) SA R A SO T A BA Y ( 1 984- 1 996)MeanStomachContent Wet Weight(g)

28

14 102 22

37

33 15



### Figure 3. Stom ach content biom ass com p arison.

25Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004

Dol p hin carcass

, brevetoxins confirmed in tissues ( n= 31 ) Dolphin carcass, tissues not tested (n= 76) 

### Gulf of M e xico

See Inset 

### Inset

GULF BAY ESCAMBIA

SANTA RO SA O KALO O SA WALTON HO LMES

W A S H INGTON

BAY

GULF JACKSO N

CALHOUN

LIBERTY

FRANKLIN WAKULLA GADSDEN

LEON

Gulf of Mexico

St. Joseph Bay



### Figure 4. Dolphin Carcass Recovery Locations (n =107) in the Panhandle Region of Florida (March 10 – April 13, 2004)

26Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### 0 20 0 40 0 60 0 80 0 100 0 120 0 140 0 160 0 180 0

s t o m

a c h c

o n t e n t s (2

7 ) fe c e s (7

)

u

r i n e (

1 0 )

b l o o d

( 1 2 )

l

i v e r ( 1 3 )

k i d

n e y ( 1 3 )

l u n g

( 1 3 )

c e r e b

e l l u m

( 1 3 )



### Figure 5. Brevetoxin concentrations found in stranded bottlenose dolphins. Concentrations in ng/g (tissues and feces) or ng/m l (urine and blood).

27Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004

0. 1

1 10 100 1000 1 000004 dolphin99 dolphin03 manatee96 manatee'04 dolphin99 dolphin03 manatee96 manatee04 dolphin99 dolphin03 manatee96 manatee04 dolphin99 dolphin03 manatee96 manateePbTx(ng/g)



### S t om ac h L i v er K i dney Lung



### Figure 6. Ranges of brevetoxin concentrations found in tissues of bottlenose Panhandle dolphins (’99 and ’04 m o rtality events) and m a natees (’96 and ’03 m o rtality events). Bars reflect high and low values for PbTx in ng/g.

28Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### Figure 7. Map of Florida Panhandle indicating locations referenced in report.

29Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### Figure 8. February 28, 2004, chlorophyll im age. Yellows and reds show high chlorophyll (1-5 μ g/L). High chlorophyll does not indicate the presence of harm ful algae.

Figure 9. February 28, 2004 im age showing new bloom s in red. HAB events are m o re

com m on as new bloom s.

30Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### Figure 10. March 9, 2004 im age showing the bloom in St. Joseph Bay.

Figure 11. April 14, 2004 showing the apparent bloom s all along the coast caused by the resuspension event. These apparent bloom s occur because algae at the bottom in shallow areas are resuspended with the sedim e nt.

31Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### Figure 12. April 14, 2004, chlorophyll im age.

32Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### St atio ns Sa m p led Dur i n g Dolphin M o r t alit y E v e n t ( M a r ch- A pril 2 004)

A r ea Shown

Gul f of Me x

i c o A t la n t ic Oce a n Pensac ol a

Gu l f of M e xi

co St. J o seph Bay Panam a C i ty

3 / 11- 3/19 (F D A C S , F D EP , F M RI) 3 / 20, 4/9, 4/28 ( v ol un teer )

4 / 7- 4/8 c r ui s e ( F M R I) Sam p l e date

3 / 22- 3/24 c r ui s e ( N OAA CSC O R H AB Ev ent R e spons e F und, F M R I ) 3 / 25, 3/31 ( F D EP, FM R I )

4 / 13, 4/16, 4/19 ( F D A CS, F D EP, F M R I ) Beas ley Par k

4 / 25- 4/29, 5/6 ( F M R I) 

### St atio ns Sa m p led Dur i n g Dolphin M o r t alit y E v e n t ( M a r ch- A pril 2 004)

A r ea Shown

Gul f of Me x

i c o A t la n t ic Oce a n Pensac ol a

Gu l f of M e xi

co St. J o seph Bay Panam a C i ty

3 / 11- 3/19 (F D A C S , F D EP , F M RI) 3 / 20, 4/9, 4/28 ( v ol un teer )

4 / 7- 4/8 c r ui s e ( F M R I) Sam p l e date

3 / 22- 3/24 c r ui s e ( N OAA CSC O R H AB Ev ent R e spons e F und, F M R I ) 3 / 25, 3/31 ( F D EP, FM R I )

4 / 13, 4/16, 4/19 ( F D A CS, F D EP, F M R I ) Beas ley Par k

4 / 25- 4/29, 5/6 ( F M R I)



### Figure 13. Stations sam p led by FMRI or associated personnel during the dolphin

m o rtality event, including the stations sam p led during the NOAA CSCOR HAB Event Response Program sponsored RV Sem i nole Cruise (red dots).

33Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### VIII. APPENDICES

A. Criteria for Determining an Unusual Mortality Event

1. A m a rked increase in the m a gnitude of strandings when com p ared with prior records. There is no set form ula for determ ining what m a gnitude would trigger a response. The NMFS Southeast Region has used a form ula of the historic m ean plus two tim es the standard deviation to determ ine a threshold level. The W o rking Group stated that m a gnitude m u st be weighed against other knowledge. As a pragm a tic m e thod, it was suggested that if a pulse in strandings is spread over an area or tim e fram e that strains the capacity of the Stranding Networks to respond, it should be cause for concern.

2. Anim als are stranding at a tim e of the year when strandings are unusual.

3. An increase in strandings is occurring in a very localized area (possibly suggesting a localized problem ), is occurring throughout the geographical range of the species/population, or spreads geographically with tim e.

4. The species, age, or sex com position of the stranded anim als is different than that of anim als that norm a lly strand in the area at that tim e of the year.

5. Stranded anim als exhibit sim ilar or unusual pathologic findings or the general physical condition (e.g., blubber thickness) of stranded anim als is different from what is norm a lly seen.

6. Mortality is accom p anied by behavior patterns observed am ong living individuals in the wild that are unusual, such as occurrence in habitats norm a lly avoided or abnorm a l patterns of swim m i ng and diving.

7. Critically endangered species are stranding. Stranding of three or four right whales, for exam ple, m a y be cause for great concern whereas stranding of a sim ilar num ber of fin whales m a y not.

34Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### B. Fact Sheet on Brevetoxin & Florida Red Tides

Source of Brevetoxin (BTX ): R e d t i d es i n Fl ori d a are caused by annual bl oom s of t h e di nofl a gel l a t e K a reni a brevi s whi c h have been recorded si nce at l east t h e m i d 1800s. K

a reni a brevi s is principally distribut ed throughout the Gulf of Mexico, with occasional red t i d es occurri ng al ong t h e m i d and sout h At l a nt i c coast

. The St at e of Fl ori d a cont i nuousl y m oni t o rs for K. brevis

, whi c h produces nerve t oxi ns (neurot oxi ns) known as brevetoxins that can cause serious pub lic health effects and significant anim al

m o rtalities.

T oxi n/ Mode of Acti on: B r evet oxi ns are pol y e t h er l a dder
neuro-t oxi ns t h at bi nd t o vol t a gesen s itiv e so d i u m ch an n e ls, an i m port a nt prot ei n st ruct ure of cel l

m e m b ranes. B i ndi ng resul t s i n persi s t e nt act i v at i on of neuronal cel l s , skeletal m u scle cells and cardiac cells. 

### K arenia brevis Source: FW C

Flo r id a Marin e Research In stitu te



### Brevetoxin Structure Source. http://www.cfsan.fda.gov/~mow/ brevotox.html Human Health Syndrome: Neurot oxi c Shel l f i s h Poi s oni ng (NSP) is caused by hum an consum pt i on of m o l l u scan (e.g. oy st ers, cl am s) shellfish contam inated by brevet oxins. NSP is characterized by parest hesi a (t i ngl i ng), reversal of hot -col d t e m p erat ure sensat i on,

m y al gi a (m uscl e pai n ), vert i go, at axi a (l oss of coordi nat i on), abdom i n al pain, nausea, diarrhea, headache, br adycardia (slow heart rate), and d ilated p u p ils.

Syndrome Distribution: Al t hough shel l f i s h poi soni ngs were known i n Fl ori d a si nce t h e 1880s, t h e cause was not i d ent i f i e d

as K. brevis until 1960. Shel l f i s h m oni t o ri ng t o prot ect t h e publ i c from NSP i s successful l y m a naged by t h e St at e of Fl ori d a

an d th ere h a v e b een n o h u m an fatalities attrib u t ed to NSP. New NSP cases m a y rarely occur when people consum e

u n r eg u l ated sh ellfish sp ecies, wh en sh ellfish are illeg a lly h a rv ested

, o r wh en b l o o m s ex p a n d to o t h e r u n m o n ito red g e o g r ap h i c areas. For exam pl e, i n 1987, an ent r ai ned K. brevis red tid e th at o r ig in ated o ff th e Flo r id a west co ast was tran sp o r ted to North Carolina waters, causing 48 NSP cases.

Other Human Health Effects: Peopl e can suffer from respi r at ory effect s when brevet oxi ns becom e aerosol i zed t h rough t h e di srupt i on of K. brevis cells b y b r eak in g wav e s, su rf o r o n s h o r e win d s . Th e first written rep o r t o f resp irato r y irritatio n cau sed by a Fl ori d a red t i d e was i n 1917.

Effects on Other Organisms : K a reni a brevi s red tides are responsible for the annual m a ss m o rtalities of thousands of fish. In som e years, notable m a ss m o rtalities of m a rine m a m m a ls, birds, and sea turtles have been reported. During 1946-1947,

o n e o f th e larg est K. brevis red t i d e event s on record occurred i n Fl ori d a wat e rs and was associ at ed wi t h cat ast r ophi c

m o rtalities of bottlenose dolphins, sea turtles, and num erous fi sh species from Tarpon Springs to Key W e st (150 m iles). Oth e r m a rin e m a m m a l an d b i rd m o rtality ev en ts with red tid e lin k s in clu d e :

• Brevetoxin involvem e nt suspected in the unpreceden ted die-off of m o re than 740 bottlenose dolphins, Tursiops
t r uncat us , from June 1987-February 1988 al ong t h e east e rn USA.

• During August 1999-February 2000, over 120 bottlenose dolphins stranded along th e Florida panhandle during a cooccurri ng red t i d e.

• Duri ng an Oct ober 1973-M a y 1974 red t i d e, l a rge num bers of bi rds were found m o ri bund or dead, part i c ul arl y doubl e-crest e d corm orant s , red-breast e d m e rgansers, and l e sser scaup.

• Subst a nt i a l num bers of si ck and dy i ng corm orant s co-occurred wi t h red t i d e out breaks al ong t h e west Fl ori d a coast duri ng 1995-1999.

• K. brevis was im p licated in m a ss m o rtalities o f th e en d a n g e red Flo r id a m a n a tee, Tri c hechus manat us l a t i r ost r i s, in 1963, 1982, 1996, 2002, and 2003 when hundreds of ani m al s di ed duri ng t h e wi nt er-spri ng i n sout hwest Fl ori d a.

35Interim Report on the Bottlenose Dolphin (Tursiops tr uncatus) Unusual Mortality Event, March-April 2004



### C. Fact Sheet on Domoic Acid

Source of Domoic Acid (DA): Pseudo-ni t s chi a , a di at om genus, can be found worldwide. Diatom s are m i croscopic m a rine p l an ts with silica sh ells. Sev e ral species of Pseudo-ni t s chi a produce the toxin DA. These toxic species have been found on t h e east and west coast s of t h e US, as wel l as t h e Gul f of M e xi co

.

T oxi n/ Mode of Acti on: DA is a
t r i carboxy l i c aci d t h at act s as a neurot oxi n. It bi nds t o gl ut am at e receptors, which are involved in

m e m o ry processing. W h en receptors are excessively activated, as occurs wi t h DA, dam a ge t o neurons l eads t o perm anent l o ss of neurol ogi cal funct i on.
Human Health Syndrome: DA causes
t h e sy ndrom e known as Am nesi c Shel l f i s h Poi s oni ng (ASP) . Mild cases arise wi t h i n 24 hours of consum pt i on of cont am i n at ed sh ellfish

. Sym p to m s in clu d e nausea, vom i t i ng, di arrhea, and abdom i n al cram ps. In m o re severe cases neurol ogi cal sy m p t o m s occur whi c h i n cl ude headaches, hal l u ci nat i ons, confusi on, short

- t e rm m e m o ry l o ss, respi r at ory di ffi cul t y , sei z ures, com a , and in extrem e cases, death. C h ai ns of Pseudo-ni t zschi a

mu ltiseries Sour ce: http://www.nwfsc.noaa.gov /hab/HABs_T oxins/HAB_Species/PseudoN/index. htm



### Dom o i c Acid St ruct ure Sou r c e : h t tp :/ / w ww

. c fsa n

. f da

. g ov/

~m ow / d omoi c

. ht ml

Syndrome Distribution: ASP was recorded for the first
t i m e off t h e At l a nt i c coast of C a nada i n 1987 when t h ree deat hs and over 100 confi r m e d cases of acut e i n t oxi cat i ons fol l o wed t h e consum pt i on of cul t u red

m u ssel s . M i l d er cases have subsequent l y been report e d.

Effect s on Other Organisms : S i nce t h e 1987 hu m an out break, DA has been i d ent i f ied as the causative agent in th e m a ss m o rtality o f p e lican s an d co rm o r an ts o ff o f C a l i f orni a, ext e nsi v e di e-offs of sea l i ons, dol phi ns, and
ot t e rs off of C a l i f orni a and whal e deat hs near Georges B a nk. The t oxi n i s t r ansferred t o hi gher t r ophi c l e vel carnivores via the food chain, usually from filter feeding fi sh or bi val v es.

Pseudo-ni t zschi a and DA i n the Gul f of Mexi co : Pseudo-ni t zschi a spp. are present, som e tim es at very hi gh abundance, i n coast a l wat e rs of t h e Gul f of M e xi co. There i s evi d ence t h at t h ei r abundance has i n creased as a

resu l t of eu t r oph i ca t i on from i n creased nu t r i ent supp l y t o

som e coast a l areas. Al t hough DA has been det ect ed i n nat u ral pl ankt on sam p l e s and i n cul t u res of Pseudonitzschia iso l ated fro m th e Gu lf, n o cases o f h u m an o r an im al illn ess o r d eath d u e to DA h a v e b een d o c u m en ted in th e Gu lf of Mexico. Source:http://www.whoi.edu/redtide/HABdi stribution/ICESm a p / U S A S P 9 3

- 0 2

. j p g

36