1502 VOLUME 114 | NUMBER 10 | October 2006 • Environmental Health Perspectives

Research

Puffer fish poisoning (PFP) is usually caused

by ingestion of tetrodotoxins (TTXs) found

naturally in certain species of puffer fish

(Halstead 1967; Mosher and Fuhrmann

1984). In Japan, 20–100 people die annually

from PFP, in spite of stringent controls by

authorities (Ogura 1971). TTXs can cause

fatal human poisoning, which is similar to

paralytic shellfish poisoning (PSP) caused by

saxitoxins (STXs). PSP is caused by the consumption of toxic shellfish (Shumway 1990)

and rarely by fish that have have become

toxic after feeding on STX-producing

microalgae (Maclean 1979). As well as

TTXs, STXs have also been found in at least

12 marine and freshwater puffer fish species

in Asia (Ahmed et al. 2001; Kodama et al.

1983; Kungsuwan et al. 1997; Nakamura

et al. 1984; Nakashima et al. 2004; Sato

et al. 1997, 2000; Zaman et al. 1997), but

their bioorigin has not been identified. 

TTXs are chemically distinct from STXs,

but both neurotoxins produce similar symptoms in mammals because they act on site 1

of the voltage-dependent sodium channel,

blocking the influx of sodium into excitable

cells and restricting signal transmission along

nerve and muscle membranes (Ahmed

1991). The symptoms of traditional PFP

from TTXs and of PSP from STXs include

tingling and numbness of the mouth, lips,

tongue, face, and fingers; paralysis of the

extremities; nausea; vomiting; ataxia; drowsiness; difficulty in speaking; progressively

decreasing ventilatory efficiency; and finally

in extreme cases, death by asphyxiation

caused by respiratory paralysis (Ahmed 1991;

Catterall 1985; Kao 1993). 

PFP cases in Europe (Kao 1993) and

Mexico (Nuñez-Vazquez et al. 2000) have

occasionally been reported. In the United

States, PFP has been associated with imports

of puffer fish [Centers for Disease Control

and Prevention (CDC) 1996]; rarely have

fatalities occurred after the consumption of

indigenous puffer fish. In Hawaii, white-spotted puffer fish, Arothron hispidus, were implicated in seven deaths (Ahmed 1991). Until

1974, seven PFP cases in Florida, outside of

the Indian River Lagoon (IRL), were caused

by the consumption of locally caught “blowfish” or puffer fish (Ahmed 1991; Benson

1956; Bigler 1999; Hemmert 1974; Mosher

and Fuhrmann 1984). These cases included

three fatalities, likely from TTX; for example,

one woman died 45 min after consuming

toxic liver from a checkered puffer fish

(Sphoeroides testudineus) (Benson 1956). The

toxins involved in the previous Florida PFP

cases were not characterized, but because PFP

is usually associated with TTX, investigators

likely assumed that TTX was the cause

(Benson 1956; Bigler 1999; Hemmert 1974).

Address correspondence to J.H. Landsberg, Fish and

Wildlife Research Institute, Florida Fish and

Wildlife Conservation Commission, 100 Eighth

Ave. SE, St. Petersburg, FL 33701 USA. Telephone:

(727) 896-8626. Fax: (727) 893-9840. E-mail:

jan.landsberg@myfwc.com 

We thank L. Sebastian, R. Paperno, D. Adams,

D. Tremain, S. Fisk, S. Stahl, S. Cook, J. D’Urso, and

A. Shurtleff, Florida Fish and Wildlife Conservation

Commission (FWC), for technical assistance and

D. Bodager and G. Jackow, Florida Department of

Health (FDOH), for specimen collection. 

Funding or support for this research was provided

by the FWC, FDOH, U.S. Food and Drug

Administration, Centers for Disease Control and

Prevention, and the National Oceanic and

Atmospheric Administration (NOAA). This article is

a result of research partially funded by the NOAA

Coastal Ocean ECOHAB Program under award

#NA03NOS4780196 to the FWC (ECOHAB contribution #152). 

The authors declare they have no competing

financial interests.

Received 11 January 2006; accepted 5 July 2006.

Saxitoxin Puffer Fish Poisoning in the United States, with the First Report 

of Pyrodinium bahamense as the Putative Toxin Source

Jan H. Landsberg,1 Sherwood Hall,2 Jan N. Johannessen,3 Kevin D. White,4 Stephen M. Conrad,2 Jay P. Abbott,1

Leanne J. Flewelling,1 R. William Richardson,1 Robert W. Dickey,5 Edward L.E. Jester,5 Stacey M. Etheridge,2

Jonathan R. Deeds,2 Frances M. Van Dolah,6 Tod A. Leighfield,6 Yinglin Zou,7 Clarke G. Beaudry,4

Ronald A. Benner,2 Patricia L. Rogers,2 Paula S. Scott,1 Kenji Kawabata,1 Jennifer L. Wolny,1,8 and 

Karen A. Steidinger1,8

1Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, St. Petersburg, Florida, USA; 2Food and Drug

Administration, Center for Food Safety and Applied Nutrition, Laurel, Maryland, USA; 3Food and Drug Administration, Office of the

Commissioner, Rockville, Maryland, USA; 4Food and Drug Administration, Center for Food Safety and Applied Nutrition, College Park,

Maryland, USA; 5Food and Drug Administration, Center for Food Safety and Applied Nutrition, Gulf Coast Seafood Laboratory, Dauphin

Island, Alabama, USA; 6National Oceanic and Atmospheric Administration, National Ocean Service, Center for Coastal Environmental

Health and Biomolecular Research, Charleston, South Carolina, USA; 7Key Laboratory of Science and Engineering for Marine Ecology

and Environment, First Institute of Oceanography, State Oceanic Administration, Qingdao, China; 8Florida Institute of Oceanography,

University of South Florida, St. Petersburg, Florida, USA

BACKGROUND: From January 2002 to May 2004, 28 puffer fish poisoning (PFP) cases in Florida,

New Jersey, Virginia, and New York were linked to the Indian River Lagoon (IRL) in Florida.

Saxitoxins (STXs) of unknown source were first identified in fillet remnants from a New Jersey PFP

case in 2002.

METHODS: We used the standard mouse bioassay (MBA), receptor binding assay (RBA), mouse

neuroblastoma cytotoxicity assay (MNCA), Ridascreen ELISA, MIST Alert assay, HPLC, and liquid chromatography-mass spectrometry (LC-MS) to determine the presence of STX, decarbamoyl

STX (dc-STX), and N-sulfocarbamoyl (B1) toxin in puffer fish tissues, clonal cultures, and natural

bloom samples of Pyrodinium bahamense from the IRL.

RESULTS: We found STXs in 516 IRL southern (Sphoeroides nephelus), checkered (Sphoeroides

testudineus), and bandtail (Sphoeroides spengleri) puffer fish. During 36 months of monitoring, we

detected STXs in skin, muscle, and viscera, with concentrations up to 22,104 μg STX equivalents

(eq)/100 g tissue (action level, 80 μg STX eq/100 g tissue) in ovaries. Puffer fish tissues, clonal cultures, and natural bloom samples of P. bahamense from the IRL tested toxic in the MBA, RBA,

MNCA, Ridascreen ELISA, and MIST Alert assay and positive for STX, dc-STX, and B1 toxin by

HPLC and LC-MS. Skin mucus of IRL southern puffer fish captive for 1-year was highly toxic

compared to Florida Gulf coast puffer fish. Therefore, we confirm puffer fish to be a hazardous

reservoir of STXs in Florida’s marine waters and implicate the dinoflagellate P. bahamense as the

putative toxin source.

CONCLUSIONS: Associated with fatal paralytic shellfish poisoning (PSP) in the Pacific but not

known to be toxic in the western Atlantic, P. bahamense is an emerging public health threat. We

propose characterizing this food poisoning syndrome as saxitoxin puffer fish poisoning (SPFP) to

distinguish it from PFP, which is traditionally associated with tetrodotoxin, and from PSP caused

by STXs in shellfish. 

KEY WORDS: dinoflagellate, Florida, harmful algae, puffer fish, Pyrodinium bahamense, saxitoxin

puffer fish poisoning, saxitoxins, Sphoeroides spp. Environ Health Perspect 114:1502–1507 (2006).

doi:10.1289/ehp.8998 available via http://dx.doi.org/ [Online 6 July 2006]
Tissues from Florida bandtail (Sphoeroides

spengleri), checkered, and southern puffer fish

(Sphoeroides nephelus) were found to be lethal

in the mouse bioassay (MBA) (Burklew and

Morton 1971; Lalone et al. 1963), but, again,

the toxins were not determined. 

Until January 2002 the harvest and consumption of puffer fish from the IRL was not

a risk to public health. Since then (until May

2004), however, 28 PFP cases occurring in

Florida (n = 21), New Jersey (n = 3), Virginia

(n = 2), and New York (n = 2) caused by

puffer fish originating from the IRL were

reported (Bodager 2002; CDC 2002a,

2002b). Analyses of toxins from unidentified

puffer fish fillet remnants from one of the

early 2002 PFP cases in New Jersey revealed

STXs (Quilliam et al. 2004), not TTXs, a distinction that alone could not be made on the

basis of consumer symptoms or traditional

screening methods (i.e., MBA). 

During 2002–2004, all PFP cases were

linked to puffer fish originating from the

northern IRL and the Banana River on

Florida’s east coast (Figure 1). Except for one

case, where puffer fish were commercially harvested and reached a New Jersey fish market,

puffer fish were caught recreationally [Bodager

2002; Florida Fish and Wildlife Conservation

Commission (FWC) 2004]. In April 2002,

state and federal officials issued health advisories, and the FWC banned puffer fish harvesting in the IRL, a ban that currently

remains in effect. In New York on 14 October

2002, two PFP cases were caused from fish

caught near Titusville, Florida, but frozen in

March 2002 before the harvesting ban

(Bodager D, personal communication). This

case demonstrated the stability of toxins in

puffer fish frozen for almost 9 months.

Because STXs had not previously been

identified in Florida’s marine waters and their

distribution, source, and origin were unknown

in April 2002 (Abbott et al. 2003; Landsberg

et al. 2002), we initiated an intensive survey of

biota in the IRL. In this article we present a

summary from 3 years of monitoring, as well

as the first report of the putative toxin source.

Materials and Methods

Field collections. From April 2002 through

April 2005, southern, checkered, and bandtail

puffer fish (n = 516) were harvested via a range

of fishing gear from the original source locations of the PFP incidents in the northern and

central IRL (Figure 1). The fish were shipped

biweekly or monthly on ice to the FWC’s Fish

and Wildlife Research Institute (FWRI) or to

the Food and Drug Administration’s (FDA)

Center for Food Safety and Applied Nutrition

Washington Seafood Laboratory and frozen in

individual sealable plastic bags until required

for toxicity testing.

Live phytoplankton samples were collected

routinely with a 62-μm mesh plankton net at

multiple locations along the IRL; also, a 1-L

water bottle was used to directly sample a

phytoplankton bloom. Water samples were

transported to FWRI at ambient temperature.

Live puffer fish. To determine if puffer fish

maintained toxicity once they were removed

from the putative toxin source, we kept puffer

fish in captivity. We obtained southern puffer

fish by rod and line or by seine net from the

IRL near Titusville (Atlantic coast) (n = 2) and

from Tampa Bay (Gulf coast), Florida (n = 2),

and transported them live in ambient seawater

to the wet laboratory at FWRI. Southern

puffer fish were individually held in covered,

80-L aquaria in 25 psu (practical salinity

units) artificial sea water (Instant Ocean;

Aquarium Systems, Inc., Mentor, OH) and

fed shrimp or squid that originated from nontoxic locations. We measured water quality

daily and routinely carried out 30% water

exchanges. After several weeks acclimation,

we tested fish skin mucus bimonthly by

lightly anesthetizing the fish [100 ppm tricaine methanesulfonate (MS-222; Sandoz

Pharmaceuticals Corp., Basel, Switzerland) in

4 L], placing the fish on a dissection board,

and collecting the mucus on a preweighed

47-mm–diameter, glass-fiber filter (Whatman,

Clifton, NJ) by gently rubbing the paper

along both sides of the body. 

Fish care. We conducted research in compliance with the Animal Welfare Act and

other federal statutes and regulations relating

to animals and experiments involving animals. All fish were treated humanely and with

regard for alleviation of suffering, according

to the Guide for Care and Use of Laboratory

Animals (Institute of Laboratory Animal

Resources 1996).

Preparation of tissues. Within several weeks

of collection, we thawed frozen puffer fish,

measured standard lengths and wet weights,

and removed skin, liver, stomach, intestinal

tract, muscle, and gonads. 

Pyrodinium bahamense cultures. We

established 11 clonal nonaxenic cultures of

P. bahamense from IRL samples, using the

micropipette technique to isolate single cells.

We maintained batch cultures in environmental chambers at near-ambient light and

temperature conditions (35 microEinsteins/

m2/sec, 25°C) and at salinities of 20–36 psu.

Growth media consisted of filtered, autoclaved natural offshore seawater enriched to

ES-DK (enriched natural seawater medium

modified by D. Kulis) (Kulis D, personal

communication; Kokinos and Anderson

1995) levels with the addition of 10-7 M selenium (as sodium selenite). 

Toxin detection. At various stages of

this survey, we tested puffer fish tissues for

STX bioactivity using the standard MBA,

Ridascreen ELISA (R-Biopharm GmBH,

Darmstadt, Germany), MIST Alert (Jellet

Biotek, Dartmouth, Canada) PSP kit, and

mouse neuroblastoma cytotoxicity assay

(MNCA, Neuro-2A) and receptor-binding

assay (RBA) [Association of Official Analytical

Chemists (AOAC) 1990; Cembella et al.

2003; Jellett et al. 2002; Luckas et al. 2003;

Powell and Doucette 1999; Ruberu et al.

2003; Usleber et al. 1991]. We also prepared

selected samples for toxin characterization and

confirmation by HPLC (Thermo Electron

Corporation, San Jose, CA), with postcolumn

oxidation and fluorescence detection and liquid chromatography-mass spectrometry

(LC-MS) (Waters Corporation, Milford, MA)

(Negri et al. 2003; Oshima 1995) using inhouse FDA reference standards. We split tissue

samples for interlaboratory calibrations and

then extracted them by one of two methods.

For bioactivity assays, tissue samples were

homogenized and weighed (wet weight) into

glass test tubes. Samples were extracted using

0.1 N HCl, adjusted to pH 2.5–4, boiled for

5 min in a boiling water bath, centrifuged at

3,000 × g for 10 min, and the supernatant

retained for toxin testing. For toxin characterization by HPLC and LC-MS, tissue splits

were extracted with 0.1 M aqueous acetic acid,

centrifuged, and the supernatants filtered

(0.22 μm). 

After the initial 2002 saxitoxin puffer fish

poisoning (SPFP) events, 11 southern puffer

fish were divided into the tissue compartments

(listed above), and tissue samples were extracted

by boiling in 0.1 N HCl (Washington Seafood

Laboratory) and analyzed for toxic activity

using three independent methods. MBAs

were performed at the Washington Seafood

Laboratory; MNCAs were performed at the

FDA Gulf Coast Seafood Laboratory; and

RBAs were performed at the National

Oceanic and Atmospheric Administration

National Ocean Service Center for Coastal

Environmental Health and Biomolecular

Research.

Saxitoxins in Pyrodinium bahamense in the United States 

Environmental Health Perspectives • VOLUME 114 | NUMBER 10 | October 2006 1503

SEMINOLE

ORANGE

OSCEOLA

Atlantic

Ocean

Atlantic

Ocean

Gulf of

Mexico

Area

shown

Pineda

Causeway

Cocoa

BREVARD

10 0 10 miles N

Titusville

Figure 1. Map showing locations (circles) in the

Indian River Lagoon, Florida, where toxic puffer

fish in the SPFP incidents originated (FWC 2004).

Sample collections of puffer fish and Pyrodinium

bahamense were conducted throughout this area

and further south to the St. Lucie River (not shown).
Clonal cultures and natural bloom samples of P. bahamense were filtered onto

25-mm glass-fiber filters (Whatman) or centrifuged at 3,000 × g for 5 min and then

extracted. Puffer fish mucus or Pyrodinium

samples on filters were homogenized in 0.1 N

HCl using a ground-glass tissue grinder and

treated as above. Pyrodinium extracts were

tested for toxicity by ELISA and MBA and

characterized for toxin profile using HPLC

and LC-MS. Puffer fish mucus was tested for

toxicity by ELISA. 

Electron microscopy. We prepared natural

field samples or clonal cultures of P. bahamense

for the scanning electron microscope (SEM)

using standard fixation methods (Truby 1997).

Pyrodinium samples were added to unacidified

Lugol’s at a dilution of 1:100 in suspension,

collected onto a 5-μm polycarbonate filter,

secondarily fixed with 4% paraformaldehyde

for 20 min, washed with water, dehydrated in

an ethanol series followed by a freon series,

critical-point-dried using carbon dioxide,

mounted onto aluminum stubs using carbon

adhesive tape, sputter-coated with gold/palladium, and photographed with a Cambridge

Stereoscan 240 SEM (Cambridge Instruments,

Cambridge, UK). 

Results

Puffer fish toxin analyses. During 36 months

of continuous monitoring after the initial

SPFP events, STXs were routinely detected in

the skin, muscle, viscera, and gonads of

516 puffer fish (southern, n = 402; checkered,

n = 105; bandtail, n = 9), which tested toxic in

MBA, RBA, Ridascreen ELISA, and MIST

Alert assays. By ELISA, maximum STX levels

in the muscle fillet were well above the action

level [80 μg STX equivalents (eq)/100 g tissue]

in southern puffer fish (maximum, 14,571 μg

STX eq/100 g tissue, mean = 938.4) and just

over the action limit in bandtail (maximum,

364.5 μg STX eq/100 g tissue; mean, 121.7)

and checkered puffer fish (maximum, 104.3 μg

STX eq/100 g tissue; mean, 6.9) (Table 1).

Maximum STX concentrations in the liver of

southern and checkered puffer fish were

1,443 and 51.1 μg STX eq/100 g tissue,

respectively. The highest tissue concentration,

22,104 μg STX eq/100 g tissue, was measured

in the ovaries of a southern puffer fish (data

not shown). 

All three assays (MBA, MNCA, and

RBA) confirmed elevated concentrations of

toxic activity in the muscle compared to the

liver (5- to 20-fold) of 11 southern puffer fish

(Table 2). By MBA, MNCA, and RBA,

ranges of STX concentrations in muscle were

197–5,264 (mean ± SD, 2,302.3 ± 1,539.3),

120–2,294 (957.7 ± 659.5), and 198–6,091

(2,439 ± 1,995.3) μg STX eq/100 g tissue,

Landsberg et al.

1504 VOLUME 114 | NUMBER 10 | October 2006 • Environmental Health Perspectives

Table 1. Comparison of saxitoxin concentrations (μg STX eq/100 g tissue) in muscle and liver of IRL puffer

fish species by ELISA.

Muscle Liver

Puffer fish species No. Mean ± SD Maximum No. Mean ± SD Maximum

Southern 402 938.4 ± 1,418 14,571 55 265.6 ± 393 1,443

Checkered 105 6.9 ± 11.4 104.3 3 20.3 ± 27.1 51.1

Bandtail 9 121.7 ± 117.9 364.5 0 — —

Table 2. Comparison of saxitoxin-like activity levels (μg STX dihydrochloride eq/100 g tissue) by LC-MS

in muscle and liver of southern puffer fish (S. nephelus) collected from the IRL after the first SPFP cases

in 2002. 

MBA RBA MNCA

Fish Muscle Liver Fold diff Muscle Liver Fold diff Muscle Liver Fold diff

1 5,264 1,034 5.1 4,136 711 5.8 2,294 420 5.5

2 4,697 376 12.5 6,091 304 20.0 1,230 280 4.4

3 2,986 242 12.3 2,433 280 8.7 1,947 160 12.2

4 2,804 203 13.8 1,423 147 9.7 1,100 120 9.2

5 2,564 149 17.2 5,253 297 17.7 844 110 7.7

6 2,153 135 15.9 2,911 173 16.8 790 240 3.3

7 1,970 263 7.5 2,257 142 15.9 750 150 5.0

8 1,216 254 4.8 805 154 5.2 350 140 2.5

9 1,098 221 5.0 1,089 180 6.1 480 110 4.4

10 376 83 4.5 198 16 12.4 630 70 9.0

11 197 149 1.3 231 50 4.6 120 60 2.0

Fold diff indicates fold difference of muscle compared with liver.

Figure 2. Toxin analysis of southern puffer fish muscle. HPLC chromatograms showing (A) dc-STX (7%) and STX (92%), and (B) B1 (1%). LC-MS ion chromatograms

(C, E) and mass spectra (D, F) of STX in reference standard (C, D) and Titusville puffer fish muscle (E, F).

5

4

3

2

1

0

0.20

0.15

0.10

0.05

0

0 5 10 15 20 25

0 5 10 15 20 25

Relative fluorescence (mV) Relative fluorescence (mV)

Time (min)

Time (min)

15.00 25.00

Time (min)

15.00 25.00

Time (min)

m/z

260 300 340 380

m/z

260 300 340 380

Intensity Intensity

 Relative intensity Relative intensity

314 100

152 100

A

B

C D

E F

STX

dc-STX

B1

STX

standard

20.79 300

301 282

300

282 301

20.77

STX in Titusville

puffer muscle
respectively. By MBA, MNCA, and RBA,

STX concentrations in liver were 83–1,034

(mean ± SD, 282.6 ± 261.5), 60–420 (169.1

± 106.2) and 16–711 (223.1 ± 186.5) μg

STX eq/100 g tissue, respectively (Table 2). 

Skin mucus of IRL southern puffer fish

held captive for 1 year was highly toxic

(2,407–9,039 μg STX eq/100 g) compared

with that of Florida Gulf Coast southern

puffer fish (6.25–140 μg STX eq/100 g).

Over a period of at least 6 months, STX levels

in the IRL southern puffer fish fluctuated but

remained at highly toxic concentrations.

Toxin profiles in unconsumed puffer fish

fillets (n = 4) from a 2004 PFP event were confirmed by HPLC (Figure 2A,B) and LC-MS

(Figure 2C–F) to be STX (92.4% ± 3.1),

decarbamoyl saxitoxin (dcSTX; 6.9% ± 2.4),

and N-sulfocarbamoyl B1 toxin (B1; 0.7% ±

0.7) as originally found in a 2002 PFP case

(Quilliam et al. 2004). 

We also detected TTX (quantified by MBA

and confirmed by LC-MS) in IRL checkered

puffer fish (n = 3) at concentrations of 1,553 ±

919 and 53,700 ± 19,212 μg TTX/100 g in the

muscle and liver, respectively (Figure 3A–D). 

Pyrodinium bahamense toxin analyses.

All clonal cultures (n = 11) and natural bloom

samples (n = 2) (> 3 million cells/L) of

P. bahamense (Figure 4) obtained from the IRL

tested positive for STX by HPLC, LC-MS,

Ridascreen ELISA, MIST Alert, and RBA

assays. Toxin concentrations for P. bahamense

isolates (n = 11) ranged from 1.68 to 25.57 pg

STX eq/cell (as determined by ELISA).

Further analysis of five of these isolates using

HPLC determined that the toxin profile was

composed of B1 (91.1% ± 2.2, mean ± SD)

and STX (8.9% ± 2.2), with integrated toxicity

values ranging from 2.02 to 12.74 pg STX

eq/cell. The HPLC toxin profile of a 2002

bloom sample at 3.28 pg STX eq/cell was

composed of STX (26%), B1 (73%), and

dcSTX (1%) (Figure 5A,B).

Discussion

PFP cases have been associated with STXs in

Asia (Ahmed et al. 2001), but the IRL incidents

are the first in which STX poisoning has been

confirmed in puffer fish originating in the

United States (Quilliam et al. 2004). The high

and low concentrations of STXs and TTX,

respectively, in the muscle of IRL puffer fish are

similar to those found in Philippine (Sato et al.

2000) and Japanese (Kodama et al. 1984)

puffer fish, although in the latter, visceral toxicity from TTX is high and fish-poisoning incidents usually occur after consumption of

fillet(s) contaminated due to improper preparation. Unlike the tissue distribution of TTXs

reported previously in various puffer fish species

(Kodama et al. 1984), STXs in IRL southern

puffer fish have been consistently much higher

in the muscle than in the liver and, in many

individual fish, were more than two orders of

magnitude above the action limit. Therefore,

even careful preparation of IRL puffer fish fillets

would not prevent intoxication in consumers.

Interestingly, the confirmation of extremely

high concentrations of TTX in the liver of

checkered puffer fish suggests that the earlierreported fatality from the consumption of this

species in south Florida (Benson 1956) was

likely caused by this toxin and not STX. 

The MBA, the traditional screening

method for PFP, does not distinguish between

STXs and TTXs. New reports in Asia (Ahmed

et al. 2001; Nakashima et al. 2004; Sato et al.

2000) have found both toxin groups co-occurring in puffer fish species previously thought to

contain only TTX. Both our results and these

reports suggest that STXs in puffer fish may be

more widespread than previously thought;

therefore, comprehensive analytical assessments

of PFP incidents are needed to distinguish

TTX from STX. We propose that the foodpoisoning syndrome caused by intoxication

from STX exposure from fish should be characterized as SPFP to distinguish it from PFP,

which is caused by—but not always verified to

be from—TTX, and to distinguish SPFP from

PSP associated with STXs in shellfish. 

In a 1960s toxicity study of IRL southern

puffer fish [erroneously identified by Lalone

et al. (1963) as northern puffer fish, Sphoeroides

maculatus, which are not found in the IRL

and occur only as far south as Jacksonville, FL

(Shipp and Yerger 1969a, 1969b; Tremain

and Adams, 1995)], muscle was demonstrated

to be toxic to mice by intraperitoneal injection. However, the toxins in these puffer fish

Saxitoxins in Pyrodinium bahamense in the United States 

Environmental Health Perspectives • VOLUME 114 | NUMBER 10 | October 2006 1505

Figure 3. LC-MS ion chromatograms (A, C) and mass spectra (B, D) of TTX in reference standard (A, B) and

checkered puffer fish liver (C, D).

10.00 20.00

Time (min)

Intensity 139

A TTX

standard

14.68

30.00

10.00 20.00 30.00

Time (min)

14.72

320

321 302

320

302 321

TTX in

checkered

puffer liver

Intensity

1,030

B

C D

m/z

240 290 330 380

m/z

240 290 330 380

100

100

Relative intensity Relative intensity

Figure 4. Scanning electron micrograph of two

P. bahamense cells isolated from the IRL (FWC

2004). Left cell, posterior-lateral view; right cell,

dorsal view. Bar = 20 μm.

Figure 5. Toxin analysis of P. bahamense by HPLC

chromatograms showing (A) dc-STX (1%) and STX

(26%), and (B) B1 (73%).

0

Relative fluorescence (mV) 1.5

1.0

0.5

0

A

STX

Time (min)

5 10 15 20 25

dc-STX

B

1.5

1.0

0.5

0

0 5 10 15 20 25

Time (min)

B1

Relative fluorescence (mV)
samples were not characterized. Of the tissues

investigated in that study, including skin,

liver, muscle, and testes or ovary, the muscle

was the most lethal to mice, similar to the pattern seen today. Although this anecdotal evidence suggests that southern puffer fish may

have been mildly toxic from STX in the IRL

for the past 45 years, there has been no indication that toxin levels were even close to the

order of magnitude observed since 2002 nor

was the FDOH informed of any poisoning

incidents from this area prior to this time. 

Globally, human food-poisoning incidents

from STX exposure are usually caused by toxic

marine shellfish (Kao 1993) that filter-feed on

STX-producing microalgae. PSP can be fatal

(Kao 1993), but the successful implementation of programs monitoring STX-producing

microalgae and STXs in shellfish has helped

minimize the risk of toxin exposure to

humans. In marine waters, PSP is caused by

toxic dinoflagellates, where STXs are produced

by more temperate Alexandrium species and

Gymnodinium catenatum and by tropical

Pyrodinium bahamense var. compressum (Kao

1993). PSP in the United States has been limited to New England and the Pacific West

Coast, including Alaska, and has only been

associated with STXs produced by temperate

Alexandrium spp. in these areas (Gessner

2000). 

The epidemiology of PSP incidents is

related to the global distribution of the various

STX-producing species and their toxigenic

strains. PSP outbreaks due to P. bahamense

have caused more fatalities than any other

microalgal species known (Usup and Azanza

1998). In 1987, PSP associated with P.

bahamense var. compressum in Champerico,

Guatemala, hospitalized at least 187 individuals and resulted in 26 fatalities (Rodrigue

et al. 1990). Before 1996, 1,768 cases of PSP

with 107 deaths had been reported in the

Philippines, mostly attributable to P.

bahamense var. compressum (Babaran et al.

1998). These fatalities were largely due to the

sudden appearance of P. bahamense in areas

previously unknown to contain toxic species,

because monitoring activities were not in

place or because hospital facilities had not

treated people in these previously unaffected

areas (Kao 1993).

In the present study we confirm unequivocally that puffer fish are a primary reservoir of

STXs in marine waters in Florida, and we

implicate for the first time the tropical western

Atlantic dinoflagellate P. bahamense as the

source of toxicity. We found the STX profile of

P. bahamense isolates from Florida to be similar

to, but proportionately different from, the toxin

profile of southern puffer fish fillet (Etheridge

et al. 2006; Quilliam et al. 2004), and we identified P. bahamense as the putative source of the

STXs. Confirmatory toxin-transfer studies from

Pyrodinium via shellfish to puffer fish are in

progress. Although many temperate marine

Alexandrium species, Gymnodinium catenatum,

and a few freshwater cyanobacteria species produce STXs (Kodama 2000), these organisms

have not been found in the IRL. 

In addition to the Caribbean and Gulf

coasts of Mexico, bioluminescent P. bahamense

blooms are found only along Florida’s Atlantic

and Gulf coasts (Badylak et al. 2004; Phlips

et al. 2004; Steidinger et al. 1980). However,

until the IRL SPFP incidents, the Atlantic/

Caribbean P. bahamense var. bahamense was not

known to be toxic (Steidinger et al. 1980),

unlike the Pacific P. bahamense var. compressum

found in Asia and the Pacific Coast of Central

America (Rodrigue et al. 1990; Usup and

Azanza 1998; Vargas-Montero and Freer

2004). The Atlantic P. bahamense var.

bahamense was separated from the Pacific

P. bahamense var. compressum based on morphologic criteria and evident lack of toxicity in

the former variety (Steidinger et al. 1980).

Based on our initial findings, we are testing

the hypothesis that this varietal distinction

may no longer be valid and that P. bahamense

is all one species. 

Florida has many toxigenic marine algal

species, but none were known to produce

STXs (Steidinger et al. 1999). It is conceivable

that STXs might have appeared in the IRL

because of one of several scenarios: a) toxigenic

populations of Pyrodinium have been introduced; b) ecologic conditions have changed

and have induced toxicity in a variety that was

previously nontoxic; c) toxic Pyrodinium was

present but produced toxins at undetectable

concentrations; or d) ecologic conditions have

changed and increased the food-web exposure

of susceptible biota to toxins. We believe that c

is the most likely scenario. In the IRL there is a

history of Pyrodinium (Badylak et al. 2004;

Phlips et al. 2004), and as mentioned previously, there is a historical precedent for lowlevel toxicity in puffer fish. 

In the past few years, the northern IRL has

experienced a number of unusual events: dolphin, manatee, fish, and horseshoe crab mortalities; increased tumor incidence in hard

clams; diseased shrimp; and reductions in the

natural recruitment of and increases in the

hatchery losses of hard clams (Bossart et al.

2003; Landsberg et al. 2002; Landsberg and

Kiryu 2005). To what extent, if at all, these

events are linked to the emerging issue of toxic

P. bahamense blooms remains undetermined.

The significant risk of SPFP and PSP from

saxitoxins in the IRL has been assessed and

management plans implemented accordingly.

Thus far, routine monitoring by Florida state

agencies has determined that STX levels in

shellfish, principally hard clams (Mercenaria

spp.), are not a significant risk to public health

(Landsberg et al. 2005). The extreme toxicity

of puffer fish fillet, well above the action level,

emphasizes the danger that puffer fish pose to

the public and supports the permanent ban on

their harvest in this area (FWC 2004). 

The widespread implications for public

health incidents from the tropical western

Atlantic P. bahamense remain unknown.

Public health officials and natural resource

managers should be aware of these new findings and remain vigilant to examine any potential association between the co-occurrence of

this species throughout its range and the

appearance of toxic food-poisoning incidents. 

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