Concurrent Exposure of Bottlenose Dolphins (Tursiops

truncatus) to Multiple Algal Toxins in Sarasota Bay,

Florida, USA

Michael J. Twiner1,2*

¤

, Spencer Fire1, Lori Schwacke3, Leigh Davidson1, Zhihong Wang1, Steve Morton1,

Stephen Roth4

, Brian Balmer5, Teresa K. Rowles6, Randall S. Wells5

1 Marine Biotoxins Program, National Oceanic and Atmospheric Administration/National Ocean Service, Charleston, South Carolina, United States of America,

2 Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, Michigan, United States of America, 3 Center for Human Health Risks at Hollings Marine

Laboratory, National Oceanic and Atmospheric Administration/National Ocean Service, Charleston, South Carolina, United States of America, 4 Coastal Ecology Program,

National Oceanic and Atmospheric Administration/National Ocean Service, Charleston, South Carolina, United States of America, 5 Chicago Zoological Society, Mote

Marine Laboratory, Sarasota, Florida, United States of America, 6Office of Protected Resources, National Oceanic and Atmospheric Administration Fisheries, Silver Spring,

Maryland, United States of America

Abstract

Sentinel species such as bottlenose dolphins (Tursiops truncatus) can be impacted by large-scale mortality events due to

exposure to marine algal toxins. In the Sarasota Bay region (Gulf of Mexico, Florida, USA), the bottlenose dolphin population

is frequently exposed to harmful algal blooms (HABs) of Karenia brevis and the neurotoxic brevetoxins (PbTx; BTX) produced

by this dinoflagellate. Live dolphins sampled during capture-release health assessments performed in this region tested

positive for two HAB toxins; brevetoxin and domoic acid (DA). Over a ten-year study period (2000–2009) we have

determined that bottlenose dolphins are exposed to brevetoxin and/or DA on a nearly annual basis (i.e., DA: 2004, 2005,

2006, 2008, 2009; brevetoxin: 2000, 2004, 2005, 2008, 2009) with 36% of all animals testing positive for brevetoxin (n = 118)

and 53% positive for DA (n = 83) with several individuals (14%) testing positive for both neurotoxins in at least one tissue/

fluid. To date there have been no previously published reports of DA in southwestern Florida marine mammals, however

the May 2008 health assessment coincided with a Pseudo-nitzschia pseudodelicatissima bloom that was the likely source of

DA observed in seawater and live dolphin samples. Concurrently, both DA and brevetoxin were observed in common prey

fish. Although no Pseudo-nitzschia bloom was identified the following year, DA was identified in seawater, fish, sediment,

snails, and dolphins. DA concentrations in feces were positively correlated with hematologic parameters including an

increase in total white blood cell (p = 0.001) and eosinophil (p,0.001) counts. Our findings demonstrate that dolphins

within Sarasota Bay are commonly exposed to two algal toxins, and provide the impetus to further explore the potential

long-term impacts on bottlenose dolphin health.

Citation: Twiner MJ, Fire S, Schwacke L, Davidson L, Wang Z, et al. (2011) Concurrent Exposure of Bottlenose Dolphins (Tursiops truncatus) to Multiple Algal

Toxins in Sarasota Bay, Florida, USA. PLoS ONE 6(3): e17394. doi:10.1371/journal.pone.0017394

Editor: Richard Unsworth, University of Glamorgan, United Kingdom

Received September 26, 2010; Accepted February 2, 2011; Published March 10, 2011

This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public

domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

Funding: The authors would like to acknowledge their funding sources, including the NOAA Marine Biotoxins Program (www.chbr.noaa.gov/habar/default.aspx),

NOAA Fisheries (www.nmfs.noaa.gov), the Batchelor Foundation, Dolphin Quest (www.dolphinquest.com), the Georgia Aquarium (www.georgiaaquarium.org),

Disney’s Animal Programs (disney.go.com/index), and the International Whaling Commission (iwcoffice.org). The funders had no role in study design, data

collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: Funding was received from Disney’s Animal Programs to help conduct this study; however, there are no restrictions on sharing of data

and materials.

* E-mail: mtwiner@umd.umich.edu

¤ Current address: Department of Natural Sciences, The University of Michigan-Dearborn, Dearborn, Michigan

Introduction

Harmful algal blooms (HABs) are commonly known for their

detrimental impacts on aquatic organisms (including marine

mammals), human health, and also local economies. Karenia brevis

blooms, often referred to as ‘‘Florida red tide’’, occur in the Gulf of

Mexico (Florida, USA) on a nearly annual basis [1]. This

dinoflagellate produces a suite of neurotoxins known as brevetoxins (PbTxs or BTXs), which are heat-stable, lipid-soluble,

polyether compounds [2,3,4]. The proximate pharmacological

target of PbTx is site 5 on the voltage-gated sodium channel [2],

where they bind with high affinity (Kd 1–50 nM; [5]. Once bound,

these toxins alter the voltage sensitivity of the channel by

interfering with the voltage sensor and inactivation gate, ultimately

resulting in nerve inhibition [6,7]

Human health effects of brevetoxins generally result from

neurotoxic shellfish poisoning (NSP) [8] and/or respiratory

illness caused by inhalation of aerosolized toxin [9]. The former

can occur following consumption of contaminated shellfish that

have accumulated sufficient levels of toxin while filter feeding the

algal assemblage including K. brevis. This illness typically affects

the nervous and gastrointestinal systems; however, all symptoms

are reversible and to date there have been no human deaths

associated with NSP [8,10]. Humans can also be exposed to

brevetoxins in aerosolized form when the fragile, unarmored K.

brevis cells burst due to wave action, releasing toxins into the air

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[11] and causing irritation and burning of the throat and upper

respiratory tract [12]. In addition to brevetoxins acting as potent

ichthyotoxins [13], Bossart et al. [14] observed brevetoxin

immunoreactivity in the lung, liver, and lymphoid tissues of

manatees collected during a 1996 mortality event, suggesting

that brevetoxins, at least in part, can be absorbed by mammals

via the inhalation of aerosolized toxins [14,15]. Although a

comprehensive understanding of brevetoxin trophic transfer is

lacking, it is clear that finfish [16,17,18] and certain types of

seagrasses (i.e., Thalassia testudinum) can accumulate or be

associated with brevetoxins and play a primary role in

brevetoxin-induced marine mammal Unusual Mortality Events

(UMEs) [18,19].

Members of the diatom genus Pseudo-nitzschia are associated with

production of domoic acid (DA), a neurotoxin that can cause

amnesic shellfish poisoning (ASP) in humans [20] and large-scale

mortality of sea birds [21], pinnipeds [22,23], and cetaceans [24].

DA is an analog of the neurotransmitter glutamate, and a partial

agonist that binds with high affinity to kainate receptors and

intermediate affinity to a-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) glutamate receptor subunits [25]. Trophic

transfer of DA via zooplankton into higher organisms has been

well documented in krill [26], shellfish [27], sand crabs [28], and

fish [23]. In the Texas region of the Gulf of Mexico, the presence

of DA was first reported in phytoplankton in 1989 [29] with

Nitzschia pungens f. multiseries (syn. P. multiseries) identified as the

putative producer of DA (2.1 pg DA/cell) [29,30]. A consortium

of Pseudo-nitzschia species was subsequently identified (P. pseudodelicatissima, P. delicatissima, P. multiseries, P. pungens, P. subfraudulenta) in

the nearby Louisiana coastal waters [31]. The most dominant

species, P. pseudodelicatissima, was shown to produce DA in situ (up

to 4.82 pg/cell) [31] and in culture (up to 36 fg DA/cell) [32];

levels that are not unlike Pseudo-nitzschia isolates collected from

other coastal regions susceptible to DA-related UMEs and/or ASP

events. A ‘hot spot’ of Pseudo-nitzschia spp. abundance has been

identified in the northern Gulf of Mexico, near coastal Louisiana,

near Mobile Bay, Alabama, and near Perdido Bay, Florida, that

appears to be highly influenced by high nutrient, submarine waters

[33].

As top predators, marine mammals such as bottlenose

dolphins can serve as important sentinels for coastal environmental health [34,35]. In the Gulf of Mexico, studies of the

effects of K. brevis and brevetoxins on dolphins have historically

focused on brevetoxin exposure and accumulation in carcasses

recovered during UMEs [15,18]. While data from carcasses are

useful for determining relative tissue distributions and estimating

amounts of toxin necessary to cause mortality, samples from live

animals provide more accurate animal data (i.e., toxin levels,

blood parameters, age class, sex, etc.) in conjunction with

environmental (i.e., HAB species, fish collections) and spatiotemporal data. As such, animal data from live dolphins can

provide insight into documenting non-lethal acute and/or

chronic exposure and the effects of these exposures on a variety

of other ‘health’ determinants not available from deceased

animals.

Dolphin health assessments in Sarasota Bay, Florida have been

ongoing since the 1980s. Because they have involved sampling

members of a long-term, year-round resident community of

dolphins observed since 1970 and spanning at least five

generations, these health assessments have provided a unique

opportunity to monitor long-term trends of a dolphin population

[36]. Samples obtained between 2003 and 2005 have shown

consistent exposure to brevetoxin when K. brevis was present in the

surrounding water [37]. The major route of exposure of these

animals to brevetoxin appeared to be via consumption of finfish

such as pinfish (Lagodon rhomboides), pigfish (Orthopristis chrysoptera),

striped mullet (Mugil cephalus), and spot (Leiostomus xanthurus) [17],

which are among the primary prey items for bottlenose dolphins in

the Sarasota Bay region [38,39]. Although K. brevis blooms have

been shown to affect fish population diversity and community

structure [40], detectable levels of brevetoxin have been identified

in shellfish and finfish for up to one year following a K. brevis bloom

[16,17,41]. In contrast to studies involving brevetoxin, there have

been no studies examining the presence or effects of DA or DAproducing Pseudo-nitzschia species on the dolphin population in

Sarasota Bay.

The primary objective of this study was to assess the exposure of

live dolphins from the Sarasota Bay area to both brevetoxin and

DA. Dolphin tissue/fluid and environmental samples were

obtained from an intensive sampling effort in May 2008 and

archived dolphin samples were retrospectively assessed back to

June 2000. These data have been used to determine the extent of

exposure of a marine mammal species to multiple algal toxins and

assess putative health effects that may have negative impacts on

this population.

Methods

Ethical treatment of animals

This study was carried out in strict accordance with the U.S.

Marine Mammal Protection Act. Protocols for the dolphin

capture-release and tagging were conducted under National

Marine Fisheries Service Scientific Research Permits No. 522-

1569 and No. 522-1785 issued to RSW. Research conducted

under these permits was approved through the Mote Marine

Laboratory annual Institutional Animal Care and Use Committee

(IACUC) reviews.

Phytoplankton cell abundance

Multiple-year Karenia brevis cell counts were determined on a

nearly daily basis at two designated sampling sites in the waters

adjacent to Mote Marine Laboratory (Bay Dock: lat./long.

27.33253/-82.57783 and New Pass: lat./long. 27.33382/

-82.57911) (Figure 1) and average cell counts served as proxy

indicators of HAB activity within the bay. Additional water

samples were collected for cell counts adjacent to various dolphin

capture-release sites in 2004 (February), 2008, and 2009. For all

samples, the direct microscopic cell enumeration method used had

a detection limit of 1000 cells/L. Prior to the May 2008 dolphin

health assessment, much of the data for Pseudo-nitzschia spp. cell

abundance were not quantified but limited to presence or absence

only. During the May 2008 and 2009 dolphin health assessments

phytoplankton were collected from surface waters either directly

into sample collection tubes or using a plankton net (model 9100-

10, Sea-Gear Corporation, Melbourne, FL, USA) with a 10 mm

mesh size. All plankton samples were preserved with ,1%

acidified Lugol’s iodine solution. Concentrated net tow samples

and whole water samples concentrated using Utermo ̈hl’s sedimentation chambers [42] were microscopically observed using an

Olympus BX-51 (Center Valley, PA, USA) microscope with

differential interference contrast (DIC). Samples with potential

HAB species were positively confirmed using scanning electron

microscopy (SEM) where samples were prepared using Simonsen’s

method for cleaning diatom frustules [43]. SEM samples were

prepared following the method outlined in Morton [44] and

examined with a JEOL 5600LV SEM (Tokyo, Japan) operated at

15 kV.

Algal Toxins in Florida Bottlenose Dolphins

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Benthic sediment and snail collections

To assess possible benthic routes of toxin trophic transfer, snails

and sediment samples were collected from several health

assessment sites using a 25 mm Fisher Scientific standard test

sieve. Approximately 500 g of the upper 5 cm of the sediment

layer was collected and drained of seawater, and whole snails (0.7–

1.6 mm shell length) contained in this sediment layer were

collected and stored frozen (220uC) in 50 mL polypropylene

centrifuge tubes. Fifty grams of the remaining sediment was

collected and stored in similar manner. Sediment samples were

subsequently centrifuged at 25006 g and again drained of

seawater prior to toxin extraction.

Fish collections

In 2008, various fish species were collected by cast net in the

study area on 5/1/2008 (set site 1, lat./long. 27.50841/

-82.69359) and 5/7/2008 (Chicago Zoological Society; lat./long.

27.33160/-82.57790) (see Figure 1B). In 2009, multiple fish

species were collected by cast net in the study area on 5/5/2009

(ca. set site 4, lat./long. 27.395157/-82.629472) and 5/7/2009

(ca. set sites 1 and 8, lat./long. 27.47256/-82.661086) (see

Figure 1B). Whole fish were identified, sorted according to

species, weighed, and frozen at 220uC. Whole viscera (all organs

in intracoelomic cavity) were dissected, weighed, and extracted

for biotoxins.

Dolphin samples

Dolphin samples (urine, feces, whole blood, serum, and gastric

fluid) for this study were collected from individuals sampled during

health assessments in June 2000, February 2004, June 2004,

February 2005, June 2005, June 2006, May 2008, and May 2009

(Figure 1A) according the methods of Wells et al. [34]. Briefly,

dolphins were captured by encircling them with a 500 m64 m

seine net deployed by a local commercial fisherman from a net

boat in shallow (,2 m) waters. May 2008 and 2009 capture and

release sites (‘‘set sites’’) are illustrated in Figure 1B. A team of

more than 50 biologists, veterinarians, and trained dolphin

handlers operating from up to eight other small boats was

involved to insure the safe handling of the animals. Once secured,

the dolphins were transferred one by one onto the padded, shaded

deck of a 9 m veterinary examination boat where a series of length

and girth measurements were taken, as well as weight. Whole

blood (,10 mL aliquot) was collected via venipuncture of the fluke

vasculature and was (1) separated by centrifugation into plasma

and serum for subsequent hematologic and biochemical analysis at

a diagnostic laboratory as previously described [34,45,46], and (2)

spotted onto blood collection cards for toxin analysis as described

by Maucher et al. [47]. Urine samples were collected via the

urethra using a sterile catheter. Gastric samples were collected

using a small tube inserted via the esophagus into the stomach,

from which stomach contents were drained and stored. Feces

Figure 1. Locations of live captured bottlenose dolphins (Tursiops truncatus) in Sarasota Bay, Florida, USA. Shown are (A) the captured

locations for all sampled animals between 2000 and 2009 and (B) the specific 2008 and 2009 set sites where additional water sampling was also

performed.

doi:10.1371/journal.pone.0017394.g001

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samples were taken during processing using a sterile catheter. All

samples were collected into sterile cryotubes or centrifuge tubes

and frozen until biotoxin analysis was conducted. Due to the

retrospective nature of this study, many pre-2008 samples were

initially stored at 220uC prior to permanent storage at 280uC.

Specific site and animal information for 2008 and 2009 are

outlined in Table 1.

Brevetoxin extraction and analysis

Urine samples were centrifuged at 130006 g for 10 min at

25uC, supernatant removed, and 0.45 mm filtered prior to analysis.

Fish viscera, dolphin feces and gastric samples were homogenized

and extracted in acetone (3 volumes) four times, filtered via a 1 or

0.2 mm Pall syringe filter, dried under nitrogen gas, resuspended in

80% aqueous methanol (30 mL), twice solvent partitioned with

hexane (30 mL), and the methanolic layer collected, dried under

nitrogen gas and resuspended in 100% methanol (0.5 or 5 mL,

depending on the weight of the original sample). Brevetoxins were

extracted from blood cards using a phosphate buffered saline

(94%)/methanol (6%) solvent (0.8 mL), followed by protein

precipitation in acetonitrile (2.4 mL) [47]. Briefly, samples were

centrifuged at 40006g for 15 min at 4uC and the supernatant was

collected. Plankton filter samples were collected by filtering

,250 mL of seawater through Whatman GF/F filters and storing

frozen at 220uC until analysis. Filters were twice extracted

overnight with 2.5 mL methanol (100%) and vortexing with five

short pulses [48] prior to centrifugation for 30 sec at 120006 g

through NanoSep MF (0.2 mm) spin columns. All extracts were

stored at 220uC until analysis.

The brevetoxin ELISA determined the presence of brevetoxin

and brevetoxin-like compounds based on cross-reactivity with an

antibody in a 96-well direct ELISA format [47]. In order to

eliminate matrix effects, minimum assay dilutions for each sample

type were: blood (1:10), serum (1:100), gastric fluid (1:50), feces

(1:50), and fish viscera (1:50). Sample calibration was performed

using a brevetoxin-3 standard curve and non-linear regression

analysis. The limits of detection (LOD) were: blood (0.2 ng/mL),

serum (9 ng/mL), gastric fluid (2.7 ng/mL), feces (0.8 ng/g), and

fish viscera (between 0.8 and 4.1 ng/g).

Table 1. Animal information and biotoxin tissue/fluid concentrations for the bottlenose dolphins (Tursiops truncatus) sampled

during the May 2008 and 2009 health assessments in Sarasota Bay, Florida.

Animal ID

(FB#)

Capture

Date Set #Sex Age

Length

(cm)

Weight

(kg) Brevetoxin Concentration (ng/g or ng/mL) Domoic Acid Concentration (ng/g or ng/mL)

Blood Serum Urine Fecal

Gastric

Fluid Milk Blood Serum Urine Fecal

Gastric

Fluid Milk

133 5/1/2008 1 F 9 225 148 ,dl ,dl ,dl 5.2 ,dl ,dl ,dl ,dl

224 5/1/2008 1 M 6 213 120 ,dl ,dl ,dl 13.6 ,dl ,dl ,dl 17.6 41.5 ,dl

203 5/2/2008 2 F 8 217 135 ,dl ,dl ,dl 5.6 ,dl ,dl 1.8 ,dl

155 5/5/2008 3 F 18 240 171 ,dl ,dl ,dl 30.0 6.5 ,dl ,dl 1.6 9.4 ,dl

205 5/5/2008 3 F 2 183 72 ,dl ,dl ,dl 4.1 ,dl ,dl ,dl ,dl

197 5/5/2008 3 F 5 210 118 ,dl ,dl ,dl 3.7 ,dl ,dl ,dl ,dl

175 5/6/2008 5 F 17 na na ,dl ,dl ,dl ,dl

207 5/6/2008 5 F 3 190 98 ,dl ,dl ,dl 5.4 ,dl ,dl 4.9 13.8

209 5/8/2008 8 F 4 198 92 ,dl ,dl ,dl 4.5 ,dl ,dl 6.1 ,dl

11 5/9/2008 9 F 24 257 204 ,dl ,dl ,dl 13.6 6.7 ,dl ,dl 1.0 10.3 ,dl

250 5/9/2008 9 M 3 190 89 ,dl ,dl ,dl 3.5 10.5 ,dl ,dl 4.4 8.5 ,dl

178 5/9/2008 9 M 13 257 201 ,dl ,dl ,dl 12.3 4.6 ,dl ,dl ,dl ,dl ,dl

188 5/9/2008 9 M 12 236 173 ,dl ,dl ,dl 2.3 4.3 ,dl ,dl ,dl ,dl ,dl

151 5/3/2009 1 F 9 228 130 ,dl ,dl ,dl ,dl ,dl ,dl ,dl 1.6 37 ,dl

55 5/4/2009 4 F 23 250 176 ,dl ,dl ,dl 3 ,dl ,dl ,dl ,dl trace 4 ,dl ,dl

213 5/4/2009 4 F 2 187 71 ,dl ,dl ,dl ,dl ,dl ,dl ,dl ,dl

138 5/4/2009 4 M 17 265 240 ,dl ,dl ,dl 3 ,dl ,dl ,dl 3.6 11.5 ,dl

92 5/5/2009 6 M 21 249 193 ,dl ,dl ,dl 9 ,dl ,dl ,dl ,dl 4 ,dl

252 5/6/2009 7 M 3 216 107 ,dl ,dl ,dl ,dl ,dl ,dl ,dl trace trace ,dl

151 5/6/2009 8 F 9 na na ,dl ,dl ,dl ,dl ,dl ,dl ,dl 1.6 37 ,dl

141 5/6/2009 8 F 19 232 149 ,dl ,dl ,dl ,dl ,dl ,dl ,dl ,dl ,dl 6.5 ,dl ,dl

217 5/6/2009 8 F 2 186 76 ,dl ,dl ,dl ,dl

215 5/6/2009 8 F 9 225 na ,dl ,dl ,dl ,dl

219 5/7/2009 9 F 14 255 203 ,dl ,dl ,dl 5 ,dl ,dl ,dl 4.6 36 2.2

254 5/7/2009 9 M .17 266 247 ,dl ,dl 9 ,dl ,dl 2.4 trace ,dl

125 5/7/2009 11 F 11 257 194 ,dl ,dl 32 ,dl ,dl ,dl 1.4 15 ,dl ,dl

256 5/7/2009 11 M 2 210 106 ,dl ,dl ,dl trace

198 5/7/2009 11 M 13 252 226 ,dl ,dl ,dl ,dl ,dl ,dl

doi:10.1371/journal.pone.0017394.t001

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The brevetoxin radioimmunoassay (RIA) determined the

presence of brevetoxin and brevetoxin-like compounds based on

a sheep antiserum prepared against a brevetoxin-2 conjugate

[49,50,51]. The RIA measured the competition between radiolabeled brevetoxin-3 (3

H-brevetoxin-3) and unknown samples for

the anti-brevetoxin-2 antiserum. Antibodies were filtered onto

25 mm glass fiber filters and the radioactivity of each filter was

measured to determine the amount of brevetoxin-3 equivalents in

each sample. The LODs were: blood (2.3 ng/mL), gastric fluid

(between 0.5 and 3.5 ng/mL), feces (between 0.5 and 3.5 ng/g),

and fish viscera (between 0.7 and 3.5 ng/g).

The brevetoxin receptor binding assay (RBA) measured

competition between radiolabeled brevetoxin-3 (3

H-brevetoxin-3)

and unknown samples for the voltage-gated sodium channel in a

rat brain crude membrane preparation, the pharmacological

target of brevetoxins, to determine the total brevetoxin-3

equivalent activity of the sample. Details of this assay are

described by Van Dolah et al. [52]. The LOD for feces samples

in this assay was ,100 ng brevetoxin-3-equiv./g.

The brevetoxin LC-MS/MS method measures the unambiguous structures of brevetoxin based on size, column retention and

fragmentation patterns. Brevetoxin liquid chromatography (LC)

separations were performed on a Luna C8(2) 15062 mm column

using an Agilent Technologies Model 1100 LC system. Mobile

phase consisted of water (A) and acetonitrile (B), with 0.1% acetic

acid additive. LC gradient: 2 min 35% B, linear gradient to 80%

at 30 min, 95% B at 35 min, held at 95% B for 7 min, returned to

35% B at 43 min, and held for 7 min before the next injection.

The mobile phase flow rate was 0.2 mL/min. The elutant from

LC was analyzed by an Applied Biosystems/MDS Sciex 4000

QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer equipped with a Turbo VTM source (Applied Biosystems,

Foster City, CA, USA). The detection of brevetoxin congeners and

metabolites by mass spectrometry was achieved by multiple

reaction monitoring (MRM) and selected ion monitoring (SIM)

[53,54]. The following brevetoxin congeners and their derivatives

were analyzed by comparison to commercial and/or in house

derivatized standards: brevetoxin-1, -2, -3, -7, -9, oxidized

brevetoxin-2, open A-ring brevetoxin-2, -3, -7, oxidized brevetoxin-2, cysteine-brevetoxin-A(B) and its sulfoxide, open-A ring

cysteine-brevetoxin-B, and glutathione-brevetoxin-A(B). S/N ratio

was about 26 for brevetoxin-3 standard at 1 ng/mL, 8 for

brevetoxin-7 at 5 ng/mL, 54 for open-A ring brevetoxin-3 at

5 ng/mL, 76 for cysteine-brevetoxin-B at 10 ng/mL, and 17 for

cysteine-brevetoxin-B sulfoxide at 10 ng/mL. The LODs for

brevetoxin in seawater were 0.01 mg/L and 1 ng/mL urine.

Domoic acid extraction and analysis

Dolphin urine and serum samples were centrifuged at 120006g

through a 0.22 mm filter column prior to analysis. Blood card

samples (100 mL whole blood) were extracted for DA using a water

(60%)/methanol (40%) solution (2.0 ml) according to the methods

of Maucher and Ramsdell [55]. Briefly, samples were sonicated

then extracted for 12 h at 4uC. Extracts were dried under nitrogen

gas and resuspended in 10 mM PBS/0.05 Tween (100 uL).

Gastric and feces samples were diluted 1:4 with aqueous methanol

(50%) prior to centrifugation for 10 min at 30006 g followed by

filtration through a Nanosep (0.45 mm) syringe filter. Fish viscera

were dissected from whole fish, homogenized and diluted 1:4 with

aqueous methanol (50%) prior to centrifugation for 10 min at

30006 g followed by filtration through a glass fiber filter (1 mm)

followed by a Nanosep (0.45 mm) syringe filter. Fish were not

allowed to thaw before the whole viscera was removed, in order to

avoid leakage of DA from the digestive tract into the adjacent

tissues [56]. Plankton filter samples were processed by filtering

,250 mL of surface seawater through Watman GF/F filters and

storing frozen at 220uC until extraction. Filters were extracted

with 5 mL aqueous methanol (10%) prior to centrifugation for

1 min at 60006g followed by filtration through a 0.22 mm syringe

filter. All extracts were stored at 220uC until analysis.

A direct competitive DA enzyme-linked immunosorbent assay

(ELISA) from Biosense Laboratories (Bergen, Norway) was used to

screen the dolphin serum, urine, and blood card extracts [55].

This assay measures DA in a sample through its competition with

DA coated onto microplate wells for anti-DA antibodies in

solution. Blood card extracts were diluted 1:10, serum samples

were diluted 1:100, and urine diluted 1:200 prior to analysis. The

limit of detection (LOD) of this assay was 0.12 ng/mL blood,

1.2 ng/mL serum, and 1.0 ng/mL urine.

Selected samples were analyzed for the presence of DA using

tandem mass spectrometry coupled with liquid chromatographic

separation (LC-MS/MS) [57]. This method utilized reverse phase

chromatography, using an Agilent 1100 HPLC coupled to an

Applied Biosystems/MDS Sciex API-4000 triple quadrupole mass

spectrometer equipped with a Turbo VTM source (Applied

Biosystems, Foster City, CA, USA). Chromatographic separation

was performed on a Luna C18(2), 5 mm, 15062 mm column

(Phenomenex, Torrance, CA, USA). Mobile phase consisted of

water and acetonitrile (ACN) in a binary system, with 0.1% formic

acid as an additive. The elution gradient was: 3 min of 5% ACN,

with a linear gradient to 40% ACN at 16 min, 95% ACN at

18 min, held for 5 min, then returned to initial conditions at

24 min and held for 5 min before the next injection. To reduce

mass spectrometer contamination, a diverter valve was used to

switch the LC eluant to the waste container except for the

6 minute window of LC eluant bracketing the retention time for

DA that was sent to the MS. Retention time of DA in samples was

determined based on the retention time observed with a certified

reference standard (NRC Canada, Halifax, Canada). The

detection of domoic acid by MS was achieved by multiple reaction

monitoring (MRM) method with Turbo ion spray interface in

positive ion mode. Four MRM transitions from protonated

domoic acid were monitored: m/z 312R266, m/z 312R248, m/

z 312R193, and m/z 312R161. The limits of quantification

(LOQ) for this method were ,0.5 ng/mL urine, 2 ng/mL gastric

fluid, 0.01 mg/L seawater, and 2 ng/g dolphin feces or fish

viscera, with a signal to noise ratio above ten.

Statistical Analysis

Statistical analysis was conducted to examine the relationship

between toxin (brevetoxin and DA) concentration and 16

hematologic and biochemical parameters. Hematology and blood

chemistry data were categorized into 3 panels of interest: Red

blood cell (RBC) indices, white blood cell (WBC) differential, and

liver and/or kidney associated enzymes. A generalized linear

model (GLM) was applied to examine the effect of toxin

concentration for each of the three panels. Each panel was first

analyzed using a multivariate GLM, and when the multivariate Fstatistic (Wilks lambda) was significant (p,0.05), univariate GLMs

(F-test) were conducted independently for each health parameter

in the panel. Age was included as a covariate for the WBC

differential and liver/kidney enzymes; sex was included as a

covariate for RBC indices. The inclusion of covariates was based

on previous examinations of factors influencing hematology and

blood chemistry parameters [45]. P-plots were examined to assess

normality of residuals, and variables were log-transformed when

necessary (specifically for neutrophil, monocyte, lymphocyte and

eosinophil counts) to meet model assumptions. Toxin concentraAlgal Toxins in Florida Bottlenose Dolphins

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tions from both urine and feces were included but in separate

analyses.

Results

In the Sarasota Bay region between 2000 and 2009, K. brevis cell

densities varied between below the detection limit (1000 cells/L) to

a maximum of 86107 cells/L (Figure 2). In each of these years,

with the exception of 2008 and 2009, K. brevis cell densities

exceeded 105 cells/L. On three occasions, dolphin health

assessments corresponded to periods in the bay when K. brevis

cell counts were greater than 105 cells/L (Feb 2004, Feb 2005,

June 2005), whereas the remaining four health assessments

occurred during periods when K. brevis was below the detection

limit (June 2000, June 2004, June 2006, May 2008, May 2009).

Unfortunately, K. brevis cell counts were not obtained at each set

site during the health assessments. In May 2008 and 2009,

seawater samples were analyzed for brevetoxin but all samples

were below the limit of detection (,dl) of ,0.01 mg/L (data not

shown).

Prior to 2008, there were essentially no data available in the

Sarasota Bay area on diatom presence, specifically Pseudo-nitzschia

spp. However, in mid-April just prior to the 2008 dolphin health

assessment, a Pseudo-nitzschia bloom occurred in the southern

region of Sarasota Bay (Bay Dock, New Pass, Longboat Key, and

Lido Key) (Figure 2) close to Mote Marine Laboratory with at least

one sample containing over 36106 cells/L (Valeriy Palubok, pers.

comm.). Not constrained to just the southern region of the bay,

water samples from each set site all contained Pseudo-nitzschia

densities in excess of 104 cells/L (Figure 3A). These same water

samples were used to positively identify the Pseudo-nitzschia cells as

P. pseudodelicatissima by SEM (Figures 3C and 3D). Water samples

containing P. pseudodelicatissima were confirmed in set sites 2, 5, and

8. Other harmful algal bloom species identified were Chaetoceros

spp. (Figure 3C) and Prorocentrum compressum (Figure 3E). All

seawater samples (n = 6) collected from each set site (Figure 1A)

during the May 2008 health assessment were confirmed positive

for DA with a mean particulate concentration of 101.7640.9 ng/

L (mean 6 SE; range: 16–289 ng/L) (Figure 3A). These

corresponded to cellular DA quotas of 5.1563.17 pg/cell (mean

6 SE; range: 0.54–17.57 pg/cell). Samples from set site 8, located

at the mouth of Palma Sola Bay just north of Sarasota Bay, had

the highest DA particulate and cell quota concentrations. Samples

from set site 9 were compromised during the cell count procedure

and not included.

Throughout much of 2009, low to moderate densities of Pseudonitzschia cells were observed at New Pass and Bay Dock but not

quantified (Figure 2). During the May 2009 health assessment,

Pseudo-nitzschia cells were observed at set sites 1, 6, 8, and 9 ranging

in densities between 6.66104 to 2.26106 cells/L with set sites 1, 6,

and 8 in the northern region of the bay and set site 9 in the

southern end (Figure 3B). Mean particulate domoic acid

concentrations were 80665.7 ng/L (mean 6 SE; range: ,dl405 ng/L) with three out of the six sites testing positive for DA (set

sites 1, 8, and 9) (Figure 3B). Samples from set sites 1 and 8 were

taken three days apart in Palma Sola Bay (northern region of the

greater Sarasota Bay; see Figure 1B), whereas set site sample 9 was

from the southern region of Sarasota Bay; suggesting patchy

domoic acid distributions. These toxin concentrations result in DA

cellular quotas of 0.4260.23 pg/cell (mean 6 range). At set sites 6

and 8, cells densities of Pseudo-nitzschia were in excess of 6.66104

cell/L but DA concentrations were ,LOD.

Viscera from seven different fish species (striped mullet, pigfish,

pinfish, striped mojarra, scaled sardines, mangrove snapper,

sheepshead) collected in the Sarasota region in 2008 and/or

2009 were analyzed for both brevetoxin and DA concentrations

(Table 2). Based on mean weight and calculated or measured

standard length, all fish were determined to be adults (www.

fishbase.org). In 2008, with the exception of the striped mojarra

that were collected by the Chicago Zoological Society, all other

fish were collected at set site 1 (Figure 1B). Mean brevetoxin

concentrations for these species (striped mullet, pigfish, pinfish,

striped mojarra, scaled sardines) ranged between 22 and 46 ng/g

viscera and mean DA concentrations ranged between 39 and

440 ng/g viscera. Fish specimens that were of sufficient size (i.e.,

striped mullet, pinfish, striped mojarra; n = 15 total) for analysis of

both toxins from the same homogenized tissues all tested positive

for both brevetoxin (8–60 mg/g) and DA (9–171 mg/g). In 2009,

striped mullet, pinfish, mangrove snapper, and sheepshead were

caught over a two-day period in Sarasota Bay. Brevetoxin

concentrations ranged between 3 and .20.3 ng/g viscera and

DA concentrations ranged between ,dl and 150.8 ng/g viscera.

Although all individual fish tested positive for brevetoxin by

ELISA and/or LC-MS/MS, only the striped mullet concurrently

tested positive for DA as well.

During the 2009 health assessment event, benthic samples were

taken for biotoxin analysis. All sediment samples (n = 3 total)

collected from set sites 1, 7, and 9 were negative for DA, but

positive for brevetoxin (by ELISA) ranging in concentration from

1–3 ng/g (Figure 4). At two sites, brevetoxin was identified at low

concentrations in benthic snails (3–6 ng/g whole tissue) (ELISA

with LC-MS/MS confirmation) with the concurrent presence of

domoic acid (range: 3–13 ng/g tissue).

During the May 2008 health assessment, several dolphin

samples (blood, serum, urine, feces, gastric fluid) from 13 animals

were obtained for biotoxin analysis. All feces (100%; 6/6) and

most gastric fluid (92%; 11/12) samples tested positive for

brevetoxin (2.3–30 ng/g or ng/mL) and a majority of urine

(58%; 7/12) and feces (67%; 4/6) samples tested positive for DA

(up to 17.6 ng/mL and 41.5 ng/g; respectively) (Table 1, Figure 5,

and Table S1). Feces samples contained the highest concentrations

of both biotoxins (brevetoxin: 2.3–30 ng/g; DA: ,dl-41.5 ng/g)

relative to other samples. Only one gastric fluid sample tested

positive for DA (8%; 1/12) and all blood and serum samples were

below the detection limit (,dl) for both toxins. With the exception

of FB175 where only blood and serum samples were available, 7 of

the remaining 12 dolphins (58%) concurrently contained both

brevetoxin and DA in at least one sample type (urine, feces, and/

or gastric fluid).

During the May 2009 health assessment, several dolphin tissue/

fluid samples (blood, serum, urine, feces, gastric fluid, milk) from

14 animals were obtained for biotoxin analysis. Many feces (67%;

6/9) samples tested positive for brevetoxin (,dl-32 ng/g) and a

majority of urine (67%; 8/12) and feces (100%; 9/9) samples

tested positive for DA (up to 4.6 ng/mL and 36 ng/g; respectively)

(Table 1, Figure 5, and Table S1). Only one gastric fluid sample

tested positive for DA (9%; 1/11) and all blood, serum and milk

samples were below the detection limit (,dl) for both toxins. Six

out of the 14 dolphins (43%) concurrently contained both

brevetoxin and DA in at least one sample type (most often feces

samples).

Archived samples from dolphin health assessments in the

Sarasota Bay area dating back to June 2000 (6 additional health

assessments) were obtained and analyzed for both brevetoxin and

DA (Figure 5, Table S1, and Table S2). All blood and serum

samples were negative for brevetoxin and DA except for several

brevetoxin-positive blood samples from February 2004. Nine out of

17 blood samples from February 2004 were positive for brevetoxin

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(53%; up to 1.3 ng/mL). Brevetoxin was detected in urine of

animals sampled in February 2004 (1/1), February 2005 (4/5) and

June 2006 (12/13) at concentrations up to 0.53, 7.1, 89.7 ng/mL;

respectively, but was not detected in urine from animals in June

2000 (0/1) and June 2004 (0/9). Similar to May 2008, feces samples

from June 2006 contained some of the highest brevetoxin

concentrations observed (up to 101 ng brevetoxin-3 equiv./g). DA

was detected in many of the urine samples collected in June 2000

(76%; 13/17), June 2004 (75%; 9/12), and June 2005 (83%; 5/6) up

to concentrations of 12.3, 21.8, 16.7 ng/mL; respectively, but was

not detected in urine samples from February 2005 (0/4) and June

2006 (0/15). Out of the 118 animals tested for brevetoxin over the

ten-year period (including individual animals that were repeat

samplings in different years), 43 were positive for brevetoxin (36%)

whereas 44 out of 83 (53%) animals tested for DA were positive.

Three animals in particular were exposed to brevetoxin on two

separate occasions (FB118, FB133 and FB188) while two individuals

were exposed to DA on two separate occasions (FB11 and FB92)

with a third animal exposed to DA three times (FB155) (Table S2).

Of the eight health assessments analyzed within this study, five of

these health assessments had several animals that tested positive for

brevetoxin and five of these health assessments had several animals

that tested positive for DA (Figure 5, Table S1, and Table S2). Over

the course of this study, 13 animals (all from 2008 and 2009) were

exposed to both brevetoxin and DA concurrently. Multivariate

GLMs for both urine and fecal DA concentration (Table 3) showed

a significant effect (p,0.05) on WBC differential and specifically on

eosinophil count (urine p = 0.031, fecal p,0.001) (Figure 6). In

addition, the effect of DA concentration in feces was significant for

total WBC (p = 0.001). The effect of DA concentration in urine was

significant for two RBC indices: red blood cell count (p = 0.002) and

mean corpuscular volume (p,0.001). However, DA concentration

in feces did not have a significant effect on RBC indices (p = 0.838).

Brevetoxin concentration did not have a significant effect on any of

the health panel indices.

Discussion

In this study we documented the repeated exposure of

bottlenose dolphins in southwest Florida to two marine algal

Figure 2. Representative K. brevis and Pseudo-nitzschia sp. cell count data for 2000 to 2009 in Sarasota Bay, Florida USA. Shaded

regions represent the times during which dolphin health assessments occurred and samples were obtained for analyses.

doi:10.1371/journal.pone.0017394.g002

Figure 3. Pseudo-nitzschia spp. and domoic acid in Sarasota Bay, Florida, USA. Cell densities of Pseudo-nitzschia spp. and domoic acid water

concentrations for various (A) 2008 and (B) 2009 set site locations. Scanning electron micrographs of three samples illustrating the presence of

potential HAB species: C) A mixed assemblage of phytoplankton cells including Chatocerous spp. and Pseudo-nitzschia spp. from 2008 set site 8, D) P.

pseudodelicatissima cells from 2008 sets sites 2, 5, and 8, and E) Prorocentrum compressum from 2008 set site 2. Note: Presence of P.

pseudodelicatissima was also confirmed in 2008 set sites 8 and 2. ‘na’ represents ‘not available’ and ‘,dl’ represents ‘below detection limit’.

doi:10.1371/journal.pone.0017394.g003

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toxins, brevetoxin and DA, over a ten year period (2000–2009)

and measured toxin concentrations in whole water samples as well

as in benthic samples and potential prey fish species that likely

serve as important vectors for toxin trophic transfer in this region.

We are the first to positively identify a known DA-producing

species of Pseudo-nitzschia (P. pseudodelicatissima) in Sarasota Bay, FL,

and to our knowledge, this is the first published report to describe

concurrent exposure of marine mammals to multiple algal toxins

in this region.

Karenia brevis blooms and brevetoxin

K. brevis blooms are a nearly annual event in the southwestern

region of Florida. Since K. brevis blooms are often patchy in their

spatial distribution, cell count data from two frequently sampled

sites (New Pass and Bay Dock) located at the southern end of

Sarasota Bay were used as a proxy for bloom activity within the

area. Although these data correlate with the cell count data in the

other parts of the bay, they cannot be used to accurately assess

toxin exposure to the dolphins or their prey items. During the

course of this study, every year except for 2008 and 2009 had K.

brevis cell densities greater than 105 cells/L. Most notable were

2005 and August through November 2006 when cell densities

exceeded 105 cells/L for months at a time. During this particular

period there was an ongoing multi-species UME (dolphins,

manatees and sea turtles) that began in September 2005 and

continued to July 2006 (www.nmfs.noaa.gov/pr/health/mmume/

). During the eight dolphin health assessments in southwestern

Florida that were used in this study, three of them occurred when

K. brevis cell densities were considered ‘bloom’ conditions ($105

cells/L), whereas during the remaining five assessments K. brevis

cell densities were at levels below detection (,103 cells/L).

The exposure of marine mammals from the Sarasota Bay region

to brevetoxin is not unusual or unexpected considering the intense

K. brevis blooms that have occurred in the area on a nearly annual

basis. In total, over a third of the animals (43 out of 118; 36%)

collected between 2000 and 2009 were brevetoxin-positive, at

concentrations similar to those published by Fire et al. [37]. The

occurrence of brevetoxin in dolphin samples often correlated

directly with the presence (February 2004, February 2005) or

absence (June 2000, June 2004) of K. brevis. However, there were

three occasions where low to moderate levels of brevetoxin were

observed in the animals (June 2006, May 2008, May 2009) even

though there was an absence of K. brevis. In fact, for each of these

occasions it had been over 4 months since blooms of K. brevis had

been detected in the area. This was in contrast to a previous study of

Sarasota dolphins that consistently found an absence of brevetoxin

in live dolphins during non-bloom conditions [58]. Nonetheless,

some of the highest brevetoxin concentrations observed in this study

were from animal samples collected in June 2006 (urine and feces),

and the highest proportion of brevetoxin-positive animals were from

May 2008 (92% animals positive) followed by May 2009 (43%).

However, it should be noted that more sample types were collected

and tested during the May 2008 assessment relative to the other

assessments. Nonetheless, there appeared to be additional factors

contributing to brevetoxin trophic transfer and bottlenose dolphin

exposure beyond the presence or absence of a concurrent K. brevis

bloom. Although some dolphin populations are migratory in nature,

the population within Sarasota Bay is quite endemic. The current

year-round resident population of dolphins in Sarasota Bay spans

five generations and includes two individuals initially identified in

1970–71 and still routinely observed in the Sarasota Bay region

[59]. The distribution of resightings of Sarasota dolphins suggests

long-term residency and a definitive localized home range to

Sarasota Bay [60].

Although the ichthyotoxic effects of brevetoxin are still being

described [13,16,61,62], it has been shown that blooms of K. brevis

Table 2. Concentrations of brevetoxin and domoic acid found in viscera of various fish species in Sarasota Bay, Florida, USA during

the May 2008 and May 2009 dolphin health assessments.

Capture

Date Common Name Genus species

Total No.

Fish

Total Fish

Weight (g)

Visceral

Weight (g)

Brevetoxin

(ng/g viscera)

Domoic acid

(ng/g viscera)

5/1/2008 Striped mullet Mugil cephalus 5 386649 72615 2264 (n = 5*) 90629 (n = 5*)

5/1/2008 Pinfish Lagodon rhomboides 5 3764 4.960.5 2465 (n = 5*) 39616 (n = 5*)

5/1/2008 Pigfish Orthopristis chrysoptera 7 4.360.2 0.2860.03 43620 (n = 2) 76621 (n = 5)

5/7/2008 Striped mojarra Eugerres plumieri 5 151642 1864 4566 (n = 5*) 65615 (n = 5*)

5/1/2008 Scaled sardines Harengula jaguana 10 1460.7 3.560.6 4664 (n = 5) 440692 (n = 5)

5/5/2009 Striped mullet Mugil cephalus 3 619665.4 72.765.0 all .20.3 (n = 3*) 5464 (n = 3*)

5/5/2009 Pinfish Lagodon rhomboides 3 8661.9 9.361.2 all .7 (n = 3*) ,dl (n = 3*)

5/7/2009 Mangrove snapper Lutjanus griseus 2 14765.5 760.2 361 (n = 2*) ,dl (n = 2*)

5/5/2009 Sheepshead Archosargus probatocephalus 1 562 45.8 10 (n = 1*) ,dl (n = 1*)

Note: Data shown are mean 6 SE (or 6 range where n = 2). An asterisk (*) represents the same fish specimens used for both toxin analyses.

doi:10.1371/journal.pone.0017394.t002

Figure 4. Domoic acid and brevetoxin in Sarasota Bay, FL

sediment and benthic snails. Various samples for biotoxin analysis

were collected in May 2009 that correspond to dolphin sampling set

sites (‘‘Sets’’). Note: ‘na’ represents ‘not available’ and ‘,dl’ represents

‘below detection limit’.

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reduce fish density and species richness in Sarasota Bay [40].

Finfish compose the majority of the diet of bottlenose dolphins

from the eastern Gulf of Mexico [38,39] and have been postulated

to play a role in brevetoxin trophic transfer to these mammals.

Undigested fish collected from the stomachs of dolphins that died

during the 2004 Florida Panhandle UME supported the link

between many species of planktivorous fish (presumably feeding on

K. brevis) and dolphin mortalities [18]. A subsequent study in the

same area has shown that over 40 species of live planktivorous,

omnivorous, and piscivorous fish contained brevetoxin in their

tissues; some for up to one year following the termination of a K.

brevis bloom [16]. Adult striped mullet (benthic omnivores that can

feed on decaying plant and algal material) [63] and scaled sardines

(pelagic omnivores feeding at least partially on copepods) [64,65]

contained some of the highest brevetoxin values, particularly in the

liver and gastrointestinal contents [16]. Similarly, viscera from

many of the fish in Sarasota Bay that are known to be prey for

resident dolphins [38] contained toxin concentrations ranging

between 68 and 190 ng brevetoxin-3 equiv./g in the absence of K.

brevis [17]. When K. brevis was present, brevetoxin concentrations

were much higher (pinfish.pigfish.mullet), averaging between 81

and 1313 ng brevetoxin-3 equiv./g viscera. During controlled

laboratory exposures of striped mullet to K. brevis, blood brevetoxin

concentrations varied according to the K. brevis/brevetoxin

exposure concentrations where maximal blood brevetoxin concentrations were ,19 ng/mL at 8 to 12 hr post-exposure with a

protracted retention time (T1/2) of over 11 days [66]. Studies

subsequently performed with Atlantic menhaden (Brevoortia

tyrannus) also demonstrated slow elimination rates with a half-life

of 24 days [67]. Although in our study the sample size was

relatively small (n = 1, 2 or 5 per fish species), these slow rates of

elimination probably cannot account for the observation that

brevetoxin was found in every fish species and each individual

collected in May 2008 and May 2009, many months since the

previous K. brevis bloom. As such, secondary routes of brevetoxin

trophic transfer might have been involved in these protracted

residency times of at least 5 months.

Omnivorous and piscivorous fish may also accumulate brevetoxins via indirect routes of trophic transfer [19]. As observed in

St. Joseph Bay, Florida, non-planktivorous fish accumulate

Figure 5. Biotoxin concentrations in urine, blood, and/or feces of Sarasota Bay, Florida dolphins between 2000–2009. (A) Brevetoxin

was analyzed by RIA, ELISA, RBA, and/or LC/MS, and (B) domoic acid was analyzed by ELISA and/or LC/MS. Brevetoxin values are reported in ng PbTx3 equiv./g or/mL. Data are median, quartile, and minimum/maximum values indicated by the midline, box, and whisker lines; respectively. Raw data

are available in Table S1 and Table S2. Note: ‘na’ denotes samples not available.

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moderate to high concentrations of brevetoxin in their tissues,

particularly the liver tissues of piscivorous fish such as red snapper

(Lutjanus campechanus; up to 16483 ng brevetoxin-3 equiv./g) [16].

One trophic route was demonstrated in laboratory studies where

pinfish and croakers (M. undulates) accumulated brevetoxin via

feeding on contaminated shellfish [16]. Brevetoxin trophic transfer

may also begin with herbivorous fish feeding on contaminated

seagrass. In 2002, 34 manatees died due to brevetoxicosis during a

UME in southwest Florida following consumption of brevetoxincontaminated seagrass such as Thalassia testudinum [18]. Organisms

feeding on (i.e., manatees, sea turtles) (D. Fauquier, pers. comm.)

living within (i.e., macrobenthic epifauna) [68], or associated with

(i.e., epiphytes, macroalgae) [69] seagrass may also be susceptible

to brevetoxin exposure. In addition, a benthic brevetoxin sink [70]

may play a role in toxin (re)cycling into benthic organisms or into

the water column during resuspension events as moderate

amounts of toxin can be found in coastal sediments at

concentrations up to 9.7 ng brevetoxin/g dry sediment [71]. This

finding is supported by the present study, which observed similar

levels in sediment (1–3 ng/g) and benthic snails (3–6 ng/g)

providing evidence that benthic brevetoxin is biologically

available.

Based on the comparable concentrations of brevetoxin for each

of the five fish species collected in May 2008 (22–46 ng brevetoxin

equiv./g viscera), it appears that in the absence of any recent K.

brevis bloom activity, these particular fish species pose an

approximately equal degree of risk for bottlenose dolphins. This

is unusual since the feeding behaviors of these fish species, all

adults based on size, are quite distinct. Scaled sardines are the

most likely to feed directly on K. brevis. Striped mullet feed on

decaying plant detritus and sediment which may include epiphytic

and benthic microalgae (such as dead or dying K. brevis), [63].

Pigfish, pinfish, and striped mojarra are more likely to be exposed

to brevetoxin via indirect routes such as feeding on crustaceans,

gastropods and/or polychaetes [64,72]. Pinfish in particular are

well known to associate with seagrass beds, and much of their diet

can be composed of vegetation [73]. Interestingly, scaled sardines

as well as other members of the clupeidae family (i.e., herrings,

shads, sardines, anchovies, menhadens) were shown to be

positively associated with K. brevis density while all other guilds

significantly decreased during K. brevis bloom events in Sarasota

Bay making clupeids potentially an even more vital dietary prey

item for bottlenose dolphins as well as an effective vector for

brevetoxin exposure [40]. The potential importance of striped

mullet as a source for biotoxin trophic transfer the Sarasota Bay

region was again identified in May 2009. Although all fish species

tested were positive for brevetoxin (striped mullet, pinfish,

mangrove snapper, sheepshead) (range: 3-.20.3 ng/g), the striped

mullet also concurrently contained DA (mean 6 SE: 5464 ng/g;

n = 3). In the Florida Panhandle, scaled sardines accumulate very

high concentrations of brevetoxin (.2400 ng/g liver and GI

contents) [16] and are not as susceptible to the ichthyotoxic effects

of K. brevis.

Pseudo-nitzschia blooms and domoic acid

In mid-April 2008, a Pseudo-nitzschia bloom was observed in the

southern region of Sarasota Bay with densities exceeding 1.5

million cells/L. P. pseudodelicatissima and DA were concurrently

identified in samples collected from three set sites within Sarasota

and Palma Sola Bays in early May 2008. Cell quotas for these

samples (5.15 pg/cell) were similar to field estimates of P.

pseudodelicatissima from Louisiana (4.82 pg/cell) [31] and P. multiseries from Texas (2.1 pg/cell) [29], but an order of magnitude

lower than some P. australis cells from California (7–75 pg/cell)

obtained during the 1998 sea lion UME [22]. In 2009, high

densities of Pseudo-nitzschia spp. were observed in Palma Sola Bay

(up to 2.26106 cells/L) with most samples in Sarasota Bay also

containing significant densities of Pseudo-nitzschia (.104 cells/L).

Table 3. Relationships between toxin concentrations and

various health parameters examined by p-values generated

from generalized linear model (GLM).

Health Parameters Domoic Acid Brevetoxin

Urine Fecal Urine Fecal

RBC Indices; Covariate: Sex 0.008 0.838 0.114 0.229

Red blood cells (RBCs) 0.002 na na na

Hematocrit (Hct) 0.245 na na na

Hemoglobin (Hgb) 0.355 na na na

Mean corpuscular volume (MCV) ,0.001 na na na

WBC Differential; Covariate: Age ,0.001 0.011 0.906 0.773

Total white blood cells (WBCs) 0.113 0.001 na na

Neutrophils 0.768 0.386 na na

Lymphocytes 0.612 0.127 na na

Monocytes 0.651 0.542 na na

Eosinophils 0.031 ,0.001 na na

Liver & Kidney Enzymes;

Covariate: Age

0.733 0.191 0.055 0.773

Blood urea nitrogen (BUN) na na na na

Creatinine na na na na

Alanine aminotransferase (ALT) na na na na

Aspartate aminotransferase (AST) na na na na

Gamma-glutamyl transferase (GGT) na na na na

Lactate dehydrogenase (LDH) na na na na

Creatine kinase (CK) na na na na

Note: Bolded data indicate results from multivariate F-test (Wilks lambda). When

multivariate p-value was less than 0.05, univariate F-tests were conducted for

each dependent (health) variable. ‘na’ denotes not available.

doi:10.1371/journal.pone.0017394.t003

Figure 6. Domoic acid concentration in feces versus eosinophil

count. Dashed green lines represent published reference thresholds

[45]. Regression analysis indicated a statistically significant increase in

eosinophil count (p,0.001), although none of the measured values

were outside of established reference thresholds.

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However, DA concentrations in the surface water varied greatly

(,dl to 405 ng/L) with low corresponding cell quotas (0.42 pg/

cell). Although no ASP events have been documented in the Gulf

of Mexico, clearly species of Pseudo-nitzschia have been established

in the northern region for many years, and we have now

demonstrated that toxigenic species of Pseudo-nitzschia are also

present in southwestern Florida.

In light of the fact that prior to May 2008 there were no

documented Pseudo-nitzschia blooms in southwestern Florida, this

study has demonstrated that since at least June 2000, dolphins in

this area have been exposed to DA. DA was detected in at least

one animal during five out of the eight health assessments. Over

our ten-year study period, more than half of the animals (44 out of

83; 53%) tested were positive for DA. Considering the intensity

and frequency of K. brevis blooms, it seems unusual that there were

a greater proportion of DA exposed animals than there were

brevetoxin exposed animals (36%). The pharmacokinetic properties of DA in mammals (i.e., rats) suggest that relative to

brevetoxin, DA is short-lived and quickly eliminated [74,75].

For DA to be observed so often in southwestern Florida dolphins,

DA-producing Pseudo-nitzschia spp. are likely present with greater

frequency and abundance than previously believed or cryptic

sources of DA (e.g., benthic) may re-emerge during non-bloom

periods. In three animal samples collected in May 2009, milk from

lactating females were all DA negative even in light of finding DA

in corresponding feces of those same mothers. The absence of DA

suggests that at that particular point in time, the potential for

maternal transfer of DA was minimal.

The trophic transfer of DA has been well documented with

growing evidence that both pelagic and benthic routes are

important. In the Sarasota Bay region, all fish collected in 2008

(n = 5 individuals of 5 species) were positive for DA. In 2009, only

the striped mullet were found to contain DA, whereas the other

fish species (pinfish, mangrove snapper, sheepshead) were all

negative. The benthic-feeding striped mullet (omnivore; M.

cephalus) and scaled sardines (pelagic filter feeder; Harengula jaguana)

contained the highest concentrations of DA, suggesting that toxin

content via direct exposure was at least partially explained by the

feeding behavior of these fish species. Although the DA

concentrations observed in the scaled sardines were still 2 orders

of magnitude less than what would be considered unfit for

consumption by FDA guidelines (20 mg/g or 20 ppm) and 1–3

orders of magnitude less than levels observed in anchovies and

sardines off the coast of California associated with UMEs (0.27–

223 mg/g viscera or gut) [22,23], it has been suggested that Gulf of

Mexico clupeid species become even more important dolphin

dietary items during and following K. brevis events [40]. This is

postulated to occur as the abundance of the dolphin’s normal

primary prey items (i.e., pigfish, pinfish, mullet) become

significantly reduced. As such, clupeids may play a significant

role in the trophic transfer of both DA and brevetoxin to

southwestern Florida dolphins.

Vertical fluxes of DA from blooms of DA-producing Pseudonitzschia blooms into sediments may in turn contaminate benthic

organisms [70,76] such as demersal fish [77], shellfish [27], and

crustaceans [28]. Benthic recycling or reemergence of DA in the

Sarasota Bay region may be a significant initiation point for new

trophic transfer pathways exposing dolphins to this toxin. In this

study, benthic snails collected from two sites contained 3–13 ng

DA/g. On the west coast of the US, DA has been observed in

other benthic organisms [77,78] presumably following rapid

downward transport of Pseudo-nitzschia cells and toxin from

ongoing pelagic blooms [70] and provide new dynamic routes of

exposure to marine mammals. Although sediments from Sarasota

Bay were below the limit of detection, DA has been shown to

adsorb to sediments. However, since the desorption rates of DA

have been shown to be quite rapid (i.e., minutes), in turn

maintaining equilibrium with the surrounding water [79], unless

DA was continually supplied, DA’s half-life in the sediment will be

relatively short.

Bottlenose dolphin health

Relative to brevetoxin concentrations reported for dolphins

from UMEs occurring in other regions of Florida [18], the

concentrations of brevetoxins observed in live Sarasota animals

were generally 1–2 orders of magnitude less. No Sarasota Bay

resident dolphins were known to have died as a result of exposure

to brevetoxin in 2005, even in the midst of a UME that

encompassed Sarasota Bay and surrounding waters. During these

UMEs, dolphin stomach contents typically contained the highest

concentrations of brevetoxin, ranging from ,dl to .54000 ng/g

(n = 53 animals) during the 2005/06 central west Florida UME

(unpublished data) and 136 to 6176 ng/g (mean = 2126; n = 30

animals) during the 2004 Panhandle UME [18]. Similarly, marine

mammals that have died during DA-related UMEs have had

much higher concentrations of DA within their tissues relative to

live Sarasota animals. In 1998, deceased California sea lions had

in excess of 1 mg DA/mL feces (up to 182 mg/mL) and between

0.12 and 3.7 mg DA/mL urine; 6- to 40-fold higher than any

concentration observed in this study. Nonetheless, the exposure of

these animals to either DA or brevetoxin at low concentrations

over long periods of time raises issues regarding potential chronic

health effects.

In this study we examined the relationship between measured

low-level biotoxin concentrations and panels of hematologic and

biochemical parameters. No relationship was found between

brevetoxin concentration and any of the health panels. However, a

weak but statistically significant effect of DA concentration was

determined for WBC differential; specifically there were increases

in total WBC and eosinophil counts. The effect of DA on

eosinophil counts was significant regardless of the matrix (urine or

feces) from which DA was measured. Yet even the highest

eosinophil count (7500 eosinophils/mL; DA concentration = 36.8 ng/g feces) was still lower than the established

reference threshold (8100 eosinophils/mL) [45] suggesting that

the low levels of DA are not perturbing hematological parameters

outside of the ‘‘normal’’ biological range. Nonetheless, these

findings were significant in that the results were consistent with

prior studies in mammals. A relatively large percentage of Florida

Panhandle dolphins (23%) that were exposed to DA (,dl -

201 ng/mL urine) had eosinophil counts that were well above the

established reference threshold [46]. The study further found that

the elevated eosinophils were associated with more sensitive

indicators of functional immune status, such as decreased Tlymphocyte proliferation and increased neutrophil phagocytosis.

Similarly, California sea lions with clinical symptoms of DA

toxicity had elevated eosinophil counts [80], which is further

supported by the observed immunomodulatory effects of DA on

mammalian WBCs [81,82]. As such, the results from this study

support the hypothesis that DA caused an immunomodulatory

response in these dolphins, although the implications for an

individual survival outcome and/or a population level health effect

require further elucidation.

Over the course of this study, 13 animals (14%) (all from 2008

and 2009) were concurrently exposed to both brevetoxin and DA.

Although neither of the toxin concentrations approached concentrations observed in UME animals, the presence of more than one

toxin in individual animals raises questions involving potential

Algal Toxins in Florida Bottlenose Dolphins

PLoS ONE | www.plosone.org 12 March 2011 | Volume 6 | Issue 3 | e17394
synergistic effects. Preliminary data investigating the effects of both

DA and brevetoxin suggested that there are no synergistic effects

of these toxins on mouse lethality following acute, single dose

exposures (J. Naar, pers. comm.) or neuroblastoma cytotoxicity (Y.

Bottein, pers. comm.). Nonetheless, stomach content, urine and/

or blood samples collected from 9 stranded animals during the

2004 Panhandle UME all contained low concentrations of DA

(,10 ng DA/g or mL) in addition to high concentrations of

brevetoxin (M. Twiner, unpublished data. One possible scenario is

that the presence of multiple toxins may first result in reduced

health such as immunosuppression, which in turn makes these

animals more sensitive to secondary stressors leading to death. In

addition to well-described neurological toxicities of DA and

brevetoxin, there is a growing amount of evidence that suggests

that both DA [82,83] and brevetoxin [84,85] also have

immunomodulatory effects that may aid in this sensitization. It

has recently been shown UME bottlenose dolphins off the coast of

Texas (USA) were concurrently exposed to both DA and okadaic

acid [86] and that the endangered North Atlantic right whales

(Eubalaena glacialis) are annually exposed to both DA and saxitoxin

via consumption of contaminated copepods (Calanus fimmarchicus)

[87] with postulated long-term effects on reproductive dysfunction.

It is generally acknowledged that there is an apparent increase

in the incidence and intensity of HABs [10,88] potentially

influenced by natural and anthropogenic factors, including

eutrophication [89] and global climate change [90]. If this

apparent increase represents an actual increase in bloom events

involving different HAB species producing unique toxic molecules,

there will likely be a greater chance of concurrent exposure of

aquatic organisms such as dolphins and humans to multiple HAB

toxins. It is therefore imperative that future studies attempt to gain

a better understanding of the potential effects of exposure to

multiple toxins.

Supporting Information

Table S1 Concentration of brevetoxin (ng/mL or ng/g)

in various animal samples.

(PDF)

Table S2 Concentration of domoic acid (ng/mL or ng/

g) in various animal samples.

(PDF)

Acknowledgments

Sample collection during bottlenose dolphin health assessments in Sarasota

Bay were made possible by the efforts of the staff, students, and volunteers

of the Chicago Zoological Society’s Sarasota Dolphin Research Program

along with collaborating scientists and veterinarians from a number of

institutions. Funding for health assessments and fish collections was

provided by the National Marine Fisheries Service, Dolphin Quest, and

Disney’s Animal Programs. The authors would like to thank Dr. Gary

Kirkpatrick of the Phytoplankton Ecology Program, Mote Marine

Laboratory, Sarasota, FL for generously supplying cell count data, Hannah

Giddens, Jen Maucher, Tod Leighfield, Seana Powers, Liz Symon, and

Valeriy Palubok for technical help, and Larry Fulford for dolphin capturerelease and fish collections. Disclaimer: This publication does not constitute

an endorsement of any commercial product or intend to be an opinion

beyond scientific or other results obtained by the National Oceanic and

Atmospheric Administration (NOAA). No reference shall be made to

NOAA, or this publication furnished by NOAA, to any advertising or sales

promotion which would indicate or imply that NOAA recommends or

endorses any proprietary product mentioned herein, or which has as its

purpose an interest to cause the advertised product to be used or purchased

because of this publication.

Author Contributions

Conceived and designed the experiments: MJT SF LS TKR RSW.

Performed the experiments: MJT SF LS LD ZW SM BB TKR RSW.

Analyzed the data: MJT SF LS ZW SR TKR RSW. Contributed

reagents/materials/analysis tools: MJT SF LS LD SR BB TKR RSW.

Wrote the paper: MJT SF LS RSW.

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PLoS ONE | www.plosone.org 15 March 2011 | Volume 6 | Issue 3 | e17394