Ciguatera: A public health perspective

Robert W. Dickey*, Steven M. Plakas

U.S. FDA Center for Food Safety and Applied Nutrition, Office of Food Safety, Division of Seafood Science and Technology, Gulf Coast Seafood Laboratory,

1 Iberville Drive, Dauphin Island, AL 36528, USA

article info

Article history:

Received 10 January 2009

Received in revised form

15 September 2009

Accepted 18 September 2009

Available online 23 September 2009

Keywords:

Ciguatera

Ciguatoxins

abstract

Ciguatera fish poisoning is a seafood-borne illness caused by consumption of fish that have

accumulated lipid-soluble ciguatoxins. In the United States, ciguatera is responsible for the

highest reported incidence of food-borne illness outbreaks attributed to finfish, and it is

reported to hold this distinction globally. Ciguatoxins traverse the marine food web from

primary producers, Gambierdiscus spp., to commonly consumed fish in tropical and

subtropical regions of the world. Ciguatoxins comprise 12 known congeners among

Caribbean and tropical Atlantic fish and 29 reported congeners among Pacific fish.

Expanding trade in fisheries from ciguatera-endemic regions contributes to wider distribution and increasing frequency of disease among seafood consumers in non-endemic

regions. Ciguatoxins produce a complex array of gastrointestinal, neurological and cardiological symptoms. Treatment options are very limited and supportive in nature. Information derived from the study of ciguatera outbreaks has improved clinical recognition,

confirmation, and timely treatment. Such studies are equally important for the differentiation of ciguatoxin profiles in fish from one region to the next, the determination of

toxicity thresholds in humans, and the formulation of safety limits. Analytical information

from case and outbreak investigations was used to derive Pacific and Caribbean ciguatoxin

threshold contamination rates for adverse effects in seafood consumers. To these threshold

estimates 10-fold safety factors were applied to address individual human risk factors;

uncertainty in the amount of fish consumed; and analytical accuracy. The studies may

serve as the basis for industry and consumer advisory levels of 0.10 ppb C-CTX-1 equivalent toxicity in fish from the tropical Atlantic, Gulf of Mexico, Caribbean, and 0.01 ppb

P-CTX-1 equivalent toxicity in fish from Pacific regions.

Published by Elsevier Ltd.

1. Introduction

Ciguatera fish poisoning is a food-borne disease

endemic to tropical and subtropical coral reef regions of the

world. It is contracted by consumption of finfish that have

accumulated lipid-soluble toxins produced by microalgae

(dinoflagellates) of the genus Gambierdiscus. The vectors

and symptoms of this disease in the Caribbean and South

Pacific have been described in the literature since the 18th

century, with mentions of illness consistent with ciguatera

dating back to the 16th century (e.g. Halstead, 1967).

Current estimates of ciguatera prevalence in endemic

regions range from less than 0.1% of populations of continental land masses (e.g., Queensland, Australia; Florida,

USA) to greater than 50% of populations of small islands of

the South Pacific and Caribbean (see reviews by Lewis,

1986a; Lange, 1994; Fleming et al., 1998, 2001). Ciguatera

has over time become a hazard to consumers in nonendemic regions because of expanding international trade

in seafood from tropical fisheries. In 2007, the European

Union and the United States imported greater than 80% of

their fishery products to meet consumer demand. In the

U.S., approximately 2.4% of fishery imports originated from

the islands of Oceania (excluding Australia and New

* Corresponding author. Tel.: þ1 251 690 3368; fax: þ1 251 694 4477

E-mail address: robert.dickey@fda.hhs.gov (R.W. Dickey).

Contents lists available at ScienceDirect

Toxicon

journal homepage: www.elsevier.com/locate/toxicon

0041-0101/$ – see front matter Published by Elsevier Ltd.

doi:10.1016/j.toxicon.2009.09.008

Toxicon 56 (2010) 123–136
Zealand) and 2.0% of fishery imports originated from

tropical Atlantic and Caribbean sources (NMFS, 2008).

Oceania and the tropical Atlantic and Caribbean encompass

ciguatera-endemic regions and, consequently, the incidence of ciguatera disease in the continental U.S. has been

linked to these sources. The recognition of ciguatera in nonendemic regions led public health institutions worldwide

to rank ciguatera as the most common food-borne disease

related to the consumption of finfish (De Fouw et al., 1999;

Lehane, 2000).

On a global level, the collection of ciguatera epidemiological data has been inefficient. The public health impact

of the disease is significantly underestimated because of

a generalized reticence to report illness; this reflects the

lack of conviction that anything can be done to cure the

disease or ameliorate symptoms. In non-endemic regions,

underreporting appears to result from the lack of diagnostic recognition of ciguatera poisoning by consumers and

medical practitioners (McKee et al., 2001). It is estimated

that less than 20% of ciguatera illnesses are reported. Lewis

and Sellin (1992) estimated that over 25,000 people

worldwide are affected annually by ciguatera. Fleming et al.

(1998) ventured a broader estimate of 50,000–500,000

poisonings per year worldwide, while Tosteson (1995)

suggested that 20,000–40,000 illnesses per year occur in

Puerto Rico and the American Virgin Islands alone. The

incidence and worldwide distribution of ciguatera is suggested to be on the increase (DeHaro et al., 2003; Levine,

1995; Lehane and Lewis, 2000; Poon-King et al., 2004),

paralleling a worldwide increase in harmful algae bloom

(HAB) events (Anderson, 1989; Hallegraeff, 1992, 1993;

Lechuga-Deveze and Sierra-Beltran, 1995). Recent observations consistent with geographic expansion of ciguatera

include the first reports of consumer illness and detection

of ciguatoxic fish from the Canary Islands of the eastern

Atlantic (Perez-Arellano et al., 2005), the western Gulf of

Mexico (Villareal et al., 2007), and the eastern Mediterranean (Bentur and Spanier, 2007). Implicated meal

remnants of the fish caught within the respective regions

were confirmed to contain ciguatoxins and species of

Gambierdiscus were identified from the respective coastal

waters. Additional evidence consistent with expansion of

ciguatera into new regions of the globe include observations of Gambierdiscus species at Crete Island (Aligizaki and

Nikolaidis, 2008); Rio de Janeiro, Brazil (Nascimento et al.,

2008); Cau Island, Viet Nam (The et al., 2008); and Hong

Kong (Lu and Hodgkiss, 2004). It was suggested that HAB

intensification and expansion are linked to anthropogenic

(Ruff, 1989) and naturally occurring environmental

changes, including global warming and increased nutrient

loading (Smayda, 1989). Increases in sea surface temperature (SST) were associated with increased dinoflagellate

abundance and fish toxicity in Puerto Rico (Tosteson et al.,

1998; Tosteson, 2004). Chateau-Degat et al. (2005) reported a positive correlation between SST and Gambierdiscus

abundance in Tahiti and used those data and human case

incidence to develop a predictive model for disease. Several

explanations were suggested for this relationship between

elevated temperatures and increased ciguatera incidence,

including the enhancement of denuded coraline substrates

for Gambierdiscus through coral bleaching. Physical

disturbances of coral reefs (e.g., dredging, harbor

construction) were also associated with increased Gambierdiscus abundance (Lewis, 1986a; Brusle ́, 1997) and

outbreaks of ciguatera (Ruff, 1989; De Sylva, 1999). More

recently, petroleum production platforms and state-sponsored artificial reef programs were shown to provide

substrates that support coral and other components of the

tropical benthos, including Gambierdiscus (Villareal et al.,

2007). The introduced structures created new habitat in the

upper euphotic zone and served as fish aggregation points,

thereby accelerating toxin transfer from Gambierdiscus to

prized food fish. The authors suggested that the structures

could have unintended consequences for human health,

particularly if rising SST over the next century alter benthic

biota distributions and fish migration patterns. These

concerns also extend to proposals for off-shore aquaculture

operations or off-shore wind farms which would also add

new substrate for benthic flora.

Although recognition of disease incidence in nonendemic regions has raised general concern, the public

health and economic impacts of ciguatera have long existed

and are particularly high in remote island nations and

territories of the Pacific (Lewis, 1986b, 1992a, b) and

Caribbean. Many of these affected communities are at

marginal socioeconomic levels and fish represent an

important source of protein and income. High disease

incidence rates in some of the smaller islands of the South

Pacific (Lewis, 1986a) and discouragement of commercial

fisheries development due to the threat of ciguatera in

export markets (e.g. Sadovy, 1999; Wong et al., 2005)

significantly hinder human welfare in many small island

communities. The risk of fish poisoning is broadly recognized by resident populations in endemic regions. In most

cases, smaller reef fish are preferred because smaller fish

are considered less likely to be poisonous. The odds are

better for smaller fish to be less toxic or nontoxic, but

smaller species and smaller specimens of apex predators

can be as toxic as larger species of known repute. Island

residents waste very little of the fish they catch when

preparing meals (e.g., fish stew with head and viscera),

a practice that negates the margin of safety sought by

selection of the smaller species because of higher toxin

concentrations in organ tissues. Many residents of the

ciguatera-endemic Caribbean and Pacific consume subthreshold levels of toxin on a regular basis (e.g. Goodman

et al., 2003; Glaziou and Martin, 1993). The toxins accumulate in their systems until that point where toxicity

threshold is reached and symptoms appear. The afflicted

then stop eating fish for a time, are treated using traditional

remedies (e.g., Pink Pepper, Schinus terebenthifolius, in New

Caledonia and Vanuatu; Bourdy et al., 1992; Garrec et al.,

2005) and, when symptoms subside, they resume fish

consumption.

While the distribution of ciguatoxic fish is often

described as global between latitudes 35north and 35

south, the actual locations of toxic fish within this broad

geographic range are discrete and heterogeneous. There are

many areas that are relatively free of ciguatera, that are

often found in close proximity to areas of high risk for

ciguatera. Lewis (2006) describes, for example, the

southern reef of Tarawa and the western reef of Maraki in

124 R.W. Dickey, S.M. Plakas / Toxicon 56 (2010) 123–136
The Republic of Kiribati as high risk areas for ciguatera,

while the remaining reefs of these atolls are low-risk areas.

Indeed, the toxicity of tropical fish depends on where the

fish are caught, and because toxin is accumulated through

trophic transfer from the primary production level, the

length of time fish reside and feed in a toxic area is an

important variable. The heterogeneous distribution of toxic

reefs is consistent with the territorial nature of most

tropical fish species. The same surmise can be made for the

semi-pelagic species of fish known for ciguatera (e.g.,

Scomberomorus cavalla and Caranx latus). For the fisherman

or consumer, there are no methods to discern how long

a fish has lived in a particular area. Experience and historical knowledge of toxic reefs within any particular region

appear to be the only meaningful mitigating factors for the

avoidance of toxic fish.

2. The source of ciguatera toxins

Building on the pioneering works of Randall (1958),

Helfrich and Banner (1963), Helfrich et al. (1968) and

Banner et al. (1960), Banner and Helfrich (1964), Yasumoto

et al. (1971, 1976) selected Ctenochaetus striatus (Surgeonfish) with known reputation for causing ciguatera for

detailed study. They correlated disc-shaped dinoflagellates

in Surgeonfish stomach contents with toxicity of viscera

extracts. Extracts of the dinoflagellate collected from populations epiphytizing calcareous algae and encrusting dead

coral in the Gambier Islands yielded fractions toxicologically and chemically consistent with those extracted from

ciguatoxic fish. Examination of field-collected and laboratory cultured samples of this dinoflagellate confirmed the

benthic link to ciguatera toxins (Yasumoto et al., 1977, 1979,

1980; Bagnis et al., 1980). The dinoflagellate was designated

a new genus and species, Gambierdiscus toxicus (Adachi and

Fukuyo, 1979). Since the initial discovery of G. toxicus,

several new species have been added to the genus

including G. belizeanus (Faust, 1995), G. yasumotoi (Holmes,

1998), G. pacificus, G. australes, and G. polynesiensis (Chinain

et al., 1999a). However, recent work by Richlen et al. (2008)

and Litaker et al. (2008) using detailed morphological and

rDNA sequence data question the original assumption of

a single cosmopolitan G. toxicus species and the more

recent designation of new species based upon morphological characteristics alone. Genetic clustering suggests

that a wide-ranging complex of multiple cryptic species

exists.

Gambierdiscus spp. are single-cell algae (division Phyrrophyta) that occupy benthic and epiphytic niches in

ciguatera-endemic regions. These dinoflagellates differ

from better known open-water ‘‘red-tide’’ species in that

they do not form conspicuous surface blooms. Gambierdiscus spp. are frequently found in sparse populations

associated with macroalgae or encrusting denuded coraline

surfaces, and they can form dense submerged populations

in protected locations. Significant variation in toxin

production occurs within the genus. This variation is

reflected in regional differences in ciguatera toxin profiles

associated with fish and the symptoms reported in

outbreaks in the three major ciguatera-endemic regions,

(Caribbean Sea and the Pacific and Indian Oceans). The

distribution of ciguatoxic finfish, often characterized as

spatially irregular, is consistent with the distribution of

benthic dinoflagellate communities in low energy systems;

e.g., mangrove systems in the Caribbean and leeward sides

of coral reefs and atolls (personal observations; Tindall and

Morton, 1998; Stewart, 1988) where physical structures

provide some degree of protection from energetic water

movement. The protected environments in which Gambierdiscus spp. are found in abundance are, in some cases,

nursery grounds for fishes and invertebrates which ultimately populate higher energy coral reef areas. Dietary

intake of G. toxicus was shown to disturb the equilibrium of

fish; as a result predation escape abilities might be

diminished (Davin et al., 1986, 1988; Kohler et al., 1989;

Lewis, 1992a, b; Gonzalez et al., 1994). The toxins also

impair embryonic and larval stages of development in fish,

decrease larval survivability, and may expedite toxin

transfer from Gambierdiscus spp. to higher trophic levels

(Ungerer and Thomas, 1996; Edmunds et al., 1999; Colman

et al., 2004). The aforementioned observations of adverse

effects on intermediate trophic levels are consistent with

a rapid 3 month transit of ciguatera toxins from peak

densities of Gambierdiscus spp. to peak number of cases of

ciguatera reported in French Polynesia (Chateau-Degat

et al., 2005).

Laboratory studies show considerable variation in

toxicity within the genus Gambierdiscus (Sperr and Doucette, 1996). Although extracts from field collections of G.

toxicus from French Polynesia yielded toxins which were

chemically consistent with ciguatoxins isolated from fish

(Bagnis et al., 1980; Yasumoto et al., 1971), very few clonal

cultures from those collections reproduced the same

results. Holmes et al. (1991) reported that the production of

toxins was limited to certain strains of G. toxicus and that

wild cells produced 100-fold greater quantities of toxins

than cultured cells. Variations in toxin yield were also

observed in field populations in the Caribbean (McMillan

et al., 1986) and Pacific (Chinain et al., 1999b) where G.

toxicus biomass did not correlate with toxicity. This led to

the supposition that ciguatera outbreaks occur when

environmental conditions favor the growth of highly toxic

genetic strains within a population (Holmes et al., 1994;

Holmes and Lewis, 1994; Chinain et al., 1999b). Laboratory

studies suggest that individual G. toxicus strains were

adapted to particular environmental regimes. Bomber et al.

(1988) reported reduced growth rates at high irradiance

and increased toxicity at higher temperatures (>28 C) for

a number of strains. Morton et al. (1993) showed a 200-fold

difference in G. toxicus toxicity depending on the light,

salinity, and temperature conditions used during culture.

More recently, studies in genetic speciation suggest that G.

toxicus is not a single cosmopolitan species but a wideranging complex of multiple cryptic species. This could

partly explain the seemingly random patterns of toxicity

(Richlen et al., 2008). Few G. toxicus strains have been the

focus of toxicity studies and limited data has been gathered

for the newer species designates. The toxicity of G. belizeanus is not known, and the description of G. yasumotoi

included a brief mention of maitotoxin-like activity. Chinain et al. (1999b) reported ciguatoxin-like activity from G.

pacificus, G. australis, and G. polynesiensis, with low levels of

R.W. Dickey, S.M. Plakas / Toxicon 56 (2010) 123–136 125
activity in G. pacificus and G. australes, and ‘‘exceptionally

high’’ activity in two G. polynesiensis strains. All of the

known species except yasumotoi were reported to occur in

Belize (Faust, 1995; Chinain et al., 1999a). Because the

different Gambierdiscus spp. co-occur and appear to vary in

toxicity, changes in the proportion of Gambierdiscus spp. in

a population appear to contribute to variable toxicity in

natural populations.

The first ciguatoxin structure elucidated from G. toxicus

was designated CTX-4B (M þ Hþ, m/z 1061.6) and was

consistent with a deoxy precursor to CTX-1 from moray eel

(Murata et al., 1989, 1990). Subsequent studies identified,

from G. toxicus and fish, a C-52 epimer of CTX-4B designated CTX4A; 52-epi-54-deoxy congener of CTX-1; and

a series of congeners containing oxocine E-rings including

a deacylated-deoxy congener CTX-3C; 51-hydroxy-CTX-3C;

and 2,3- dihydroxy-CTX-3C (Satake et al., 1993, 1997, 1998).

All appear to be precursors to oxidized CTX-1 congeners

identified from toxic fish (Murata et al., 1990; Lewis and

Holmes, 1993). The less polar CTX-1 precursors identified

from G. toxicus exhibit median lethal potencies in the

mouse model approximately 10-fold less potent than CTX-1

from fish. In addition to the lipid-soluble ciguatoxins, G.

toxicus produces the water-soluble toxins, maitotoxin,

gambierol and gambieric acids (see review by Wright and

Cembella, 1998). These compounds are not thought to be

significant contributors to ciguatera fish poisoning due to

their water-soluble nature and low oral potency.

3. Assimilation and metabolism of toxins in fish

Ciguatera toxins in finfish comprise larger assemblages

of ciguatoxin congeners. The Pacific and Caribbean ciguatoxins differ slightly in structure and toxicity. The more

abundant and potent Pacific ciguatoxins are piscine

metabolites of Gambierdiscus toxins and are assimilated

and metabolized through multiple trophic levels of the

marine food web (Lewis and Holmes, 1993). Similar

evidence for the Caribbean ciguatoxins is lacking in the

absence of structure elucidation of Caribbean Gambierdiscus toxins but most surmise the same must be true. The

principal and most toxic Pacific ciguatoxin was first isolated

from moray eels in 1967 (Scheuer et al., 1967; Tachibana

et al., 1987). Structure elucidation of the principal Pacific

ciguatoxin and its precursor-toxin CTX-4B from G. toxicus

was completed in 1989 (Murata et al., 1989, 1990). The

ciguatoxins are lipid-soluble polyether compounds with

skeletal structures of 13–14 transfused ether rings. They are

odorless, colorless, devoid of heteroatoms other than

oxygen, and bear few conjugated bonds. These physical

features made them very difficult to detect by conventional

means (e.g. UV-absorption) at the time of elucidation. CTX1, has a mass of 1110.6 daltons and a molecular formula of

C60H86O19 (Murata et al., 1989, 1990). It is a potent sodium

channel agonist and exhibits extreme lethality in mouse

models with a median lethal dose of 0.25 mg/kg. Investigations of ciguatoxic fish from the central and western

Pacific, led by Yasumoto (Japan), Lewis (Australia) and

Legrand (France), determined that multiple CTX-1 congeners were present in toxic fish. The body of work also

suggested that a select few were truly abundant, and, in

most cases, CTX-1 dominated toxin profiles. The contribution of CTX-1 to composite toxicity in mice, and, by

extension, to humans, was estimated at 90% (Lewis et al.,

1991a, b). Although numerous precursors, isomers, and

congeners were detected, insufficient amounts of the less

abundant toxins were recovered for structural elucidation

by NMR. However, using fragmentation patterns of the

structures of CTX-1 and CTX-4B as templates many

congeners were identified by a combination of FAB/MS/MS

and synthetic conversions to known structures (Yasumoto

et al., 2000). Approximately 29 congeners were identified

from fish and comprise products of acylation, epimerization, hydroxylation, and oxidation of CTX-4B and CTX-3C.

Similar studies were performed in the Caribbean

(Crouch et al., 1995; Vernoux and Lewis, 1997) and, in 2002,

the isolation and characterization of Indian Ocean CTX was

reported by Hamilton et al. (2002a, b). Caribbean ciguatoxin (C-CTX-1) was first described by Lewis et al. (1998)

with a mass of 1140.6 Da and a molecular formula of

C62H92O19. Like Pacific CTX, it is a potent sodium channel

agonist and exhibits a median lethal dose in the mouse

model of 3.7 mg/kg; it is approximately 10-fold less potent

than P-CTX-1. The case for dominance of a single Caribbean

ciguatoxin appears less convincing than is the case in the

Pacific. A single, and apparently dominant, Caribbean

ciguatoxin (presumptive MHþ, m/z 1123.6) was isolated

and partially characterized from Sphyraena barracuda and

Caranx latus (Crouch et al., 1995). This dominant ciguatoxin

and at least two minor toxins were also observed in

Scomberomorus cavalla (greater amberjack) meal remnants

from a ciguatera outbreak among U.S. soldiers in Haiti in

1995 (Poli et al., 1997). In the latter two studies, the toxins

were not obtained in sufficient quantities for structural

elucidation by NMR, and there was some question as to the

mass assignment of the principal toxin (i.e., m/z 1123.6). In

1997, five ciguatoxins were isolated from C. latus (Vernoux

and Lewis, 1997) and the structures of two, (C-CTX-1: MHþ,

m/z 1141.6 and its C-56 epimer C-CTX-2) were elucidated by

NMR and MS (Lewis et al., 1998). It was estimated that CCTX-1 and C-CTX-2 contribute 40–60% to total fish toxicity

in the waters of St. Barts in the French West Indies (Pottier

et al., 2002). Seven additional C-CTX congeners and three

additional isomers of C-CTX-1 or C-CTX-2 were identified

(Pottier et al., 2002). The isomers and congeners identified

comprised products of epimerization, hydroxylation, and

oxidation of C-CTX-1 or C-CTX-2. A system for annotation of

ciguatoxins from the Caribbean and Pacific was proposed

by Vernoux and Lewis (1997) to distinguish CTX source and

structural variants in which the acronym CTX is used with

a letter prefix to indicate region of origin (i.e., P, for Pacific;

C, for Caribbean; and I, for Indian) and a postscript

numbering system to indicate the chronological order of

reporting.

4. Symptoms, diagnosis, and treatment of ciguatera

Variation in symptom patterns in the Pacific Ocean,

Indian Ocean, and Caribbean Sea regions have been

attributed to the different suites of ciguatoxins identified

from those regions (Lewis, 2000). In the Caribbean,

gastrointestinal symptoms and signs are characteristic in

126 R.W. Dickey, S.M. Plakas / Toxicon 56 (2010) 123–136
the acute phase (Lawrence et al., 1980), and are followed

closely by neurologic, especially peripheral, neurologic

symptoms. In the Pacific and Indian Ocean regions, the

neurological symptoms and signs are more pronounced in

the acute phase with occasional reports of more severe

neurologic effects, including coma (Bagnis et al., 1979).

Onset of symptoms and signs typically begins within 0.5–

12 h of eating a toxic fish, and the acute phase often abates

within 24 h (Hokama, 1988). Cardiovascular problems

(generally a combination of bradycardia with hypotension)

may be present during this acute period. In the Pacific and

Indian Oceans, there are reports of rapid progression to

respiratory distress, coma, and occasionally death (DeFusco

et al., 1993; Lange, 1987; Habermehl et al., 1994). More

extensive and subjective neurological signs may emerge in

a few hours to two weeks after exposure. Paresthesias,

numbness and tingling of perioral region and extremities and

temperature related dysesthesias (i.e., hot and cold temperature sensation reversals) are considered characteristic

symptoms of ciguatera fish poisoning (Pearn, 2001). Studies

suggested that the unusual sensations represent tingling or

‘‘electric shock’’ pain rather than a true reversal of hot and

cold perception (Cameron and Capra, 1993). Cameron and

Capra (1993) concluded that the ciguatera toxins induced

abnormal discharges in the peripheral A-delta and peripheral

C-modal nociocepter fibers involved in temperature sensation and may also be involved in the intense itching that

patients report. Nerve conduction studies in a limited

number of patients with acute illness, demonstrated

a generalized disturbance in sensory and motor conduction

with significant prolongation of the refractory periods and

the supernormal period of excitability (Cameron et al., 1991).

Other subjective neurological symptoms include

metallic taste, pruritis, arthralgia, and myalgias (muscle

aches, especially in the legs) and sensations of loose teeth

(Poon-King et al., 2004). Cerebellar signs and tremors may

present up to 10 days after initial exposure (Chungue et al.,

1977). Headache, which appears non-localized, intense,

and prolonged may be a presenting sign (Pearn, 2001).

General weakness, hyporeflexia, and dysphagia may also be

found. While cranial and peripheral nerves appear to

dominate the clinical picture, Karlis et al. (2000) reported

that 11% of the neurological symptoms observed were

indicative of central nervous system involvement (paralysis, ataxia, stupor, confusion) which is usually indicative of

the most severe cases (Cameron and Capra, 1993). A wide

range of neurobehavioral symptoms were reported from

the Pacific and Indian Ocean regions following ciguatera

poisoning, including fatigue, anxiety, depression, hysteria,

‘‘neurosis,’’ memory disturbance and mental inefficiencies

(Bagnis et al., 1979; Gillespie et al., 1986; Lipkin, 1989;

DeFusco et al., 1993; Quod and Turquet, 1996; Karlis et al.,

2000; Arena et al., 2004; Friedman et al., 2007; Williams

et al., 2008). Depression in many patients accompanies

slow regression of the paresthesias, weakness, fatigue, and

complaints of general malaise, suggesting a secondary

reaction to chronic illness. In contrast, neurocognitive

complaints (memory, mental inefficiencies) may be the

direct result of neurotoxic exposure, i.e., a general cognitive

inefficiency as a result of diminished cerebral functioning

or secondary to fatigue, depression, or general malaise.

Acute gastrointestinal problems typically resolve within

24–28 h and cardiovascular disorders reverse within 48–

72 h (Butera et al., 2000; Hokama, 1988). Recovery from

neurologic symptoms is longer and less predictable,

ranging from 1 week to 6 months (Butera et al., 2000; Lange

et al., 1992; Morris et al., 1982a, b; Poon-King et al., 2004).

Chronic illness may occur in a subset of patients, and is

characterized by a vague and poorly defined combination

of recurring neurologic and neuropsychological symptoms.

Pruritus, arthralgia and fatigue can persist for months or

years (Gillespie et al., 1986). The fatigue can be so debilitating that it resembles a Chronic Fatigue Syndrome (Pearn,

1996, 1997) and evidence of causal linkage to chronic phase

lipids was suggested (Hokama et al., 2003a, b, 2006, 2008).

Chronicity may reflect lengthy persistence of ciguatoxins in

the body (Chan and Kwok, 2001) or lowered neural

thresholds responding to dietary or behavioral stimuli

unrelated to ciguatoxins. The persistence of symptoms in

some patients for several years is not unusual. In one study

(Lange et al., 1992), 65% of the patients had symptoms for 6

months or longer with recurrence up to two years.

Based on observations that repeated exposures to

ciguatera toxins might be associated with a more severe

clinical illness, it was hypothesized that the fat-soluble

ciguatoxins accumulate in humans and lower threshold

tolerances (Bagnis et al., 1979; Glaziou and Martin, 1993;

Hokama, 1988). Increasing age and weight, presumably

associated with greater lifetime exposures and greater

capacity for toxin storage, have also been linked to the

duration and severity of symptoms (Katz et al., 1993). An

alternate explanation is that the ciguatoxins cause irreversible sub-clinical damage. Physical or dietary behaviors

(e.g., exercise, alcohol consumption, or excessive caffeine)

or repeat exposure to sub-threshold levels of ciguatera

toxins may induce symptom recurrence.

Diagnosis of ciguatera by medical professionals remains

a matter of exclusionary assessment. There are currently no

reliable biomarkers that can be used to confirm diagnosis of

ciguatera in clinical settings, although studies using

animals (Morey et al., 2008; Ryan et al., 2007; Dechraoui

et al., 2005a, b, 2007) suggest that detection of ciguatoxin

in clinical samples may be possible in the near future. Using

a murine model, progress was made in the use of blood

collection cards for sample collection and highly sensitive

in vitro assays for detection of ciguatera toxin activity were

optimized for blood matrix (Dechraoui et al., 2005a). At

present, however, diagnosis is based on presenting symptoms and time course, dietary history of reef fish

consumption, and the exclusion of other diagnoses that

could account for symptoms that are common to other

etiologies (e.g. Perkins and Morgan, 2004). Ciguatera has

some symptoms in common with illness caused by

neurotoxic shellfish poisonings, scombroid poisoning,

pathogenic bacteria and enteric viruses (Ting and Brown,

2001), organophosphate pesticide poisoning, eosinophilic

meningitis, multiple sclerosis and other neurologic conditions (Friedman et al., 2008). However, variation occurs in

the clinical syndrome and severity of disease. This appears

to reflect individual risk factors among patients in different

parts of the world, and the geographic differences in

ciguatoxin profiles within and among species of ciguatoxic

R.W. Dickey, S.M. Plakas / Toxicon 56 (2010) 123–136 127
fish. The current standard for diagnosis of ciguatera

includes confirmation of ciguatoxins in the consumed fish

by laboratory methods (e.g. see Detection in Fish, below). A

outbreak investigation that reports multiple individuals

consuming the same fish, and with symptoms and time

course consistent with ciguatera, is strongly supportive of

the diagnosis.

In the Pacific and Caribbean, a variety of traditional

herbal medicines and remedies have been used to treat

ciguatera. Some of the plants and preparations identified

(Bourdy et al., 1992) were found to ameliorate ciguatoxin

effects in animal models (Amade and Laurent, 1991).

Extracts of Argusia argentea and Schinus terebenthifolius had

beneficial effects on mice fed toxic moray eel liver. Davallia

solida acted like mannitol on isolated frog axonal preparations exposed to ciguatoxin (Benoit et al., 2000). However,

active agents in many plant preparations have not been

identified and there are no controlled studies on their

efficacy and safety for treating ciguatera. Various medical

treatments for specific symptoms have been tried, but with

variable success (reviewed by Friedman et al., 2008).

Fluoxetine for chronic fatigue (Berlin et al., 1992); amitriptyline for paresthesias, pruritis, and headaches (Lange et al.,

1992; Davis and Villar, 1986); acetaminophen and nifedipine for headaches (Calvert et al., 1987; Berlin et al., 1992);

and gabapentin was used to treat pain (Perez et al., 2001). In

all cases, randomized trials to assess fitness for purpose are

lacking. Obstacles to randomized clinical trials of prospective treatments for ciguatera include the spontaneous

nature of disease occurrence, a general lack of resources for

conducting clinical trials in those places where it occurs

with greater frequency, the lack of biomarkers and clinical

tests to confirm the diagnosis of ciguatera, typically small

patient cohorts, variability among patients in time to

treatment, and differences between Pacific Ocean, Indian

Ocean, and Caribbean toxin profiles in fish.

Palafox et al. (1988) first described treatment of ciguatera by intravenous (IV) mannitol with complete resolution

of symptoms in 17 of 24 patients within 48 h of infusion;

these included two cases of coma and one case of shock

that improved within minutes. Similar results (Pearn et al.,

1989; Blythe et al., 1992) suggested that treatment with IV

mannitol abbreviated morbidity and chronicity. IV

mannitol infusion is the most studied therapy for ciguatera,

and the only therapy assessed by randomized clinical trials

(Bagnis et al., 1992; Schnorf et al., 2002). IV mannitol is

administered at 0.–1.0 g/kg body weight over a 30–45 min

period within 48–72 h of ingestion of toxic fish (Palafox

et al., 1988; Blythe et al., 2001). Beneficial effects were

reported for up to several weeks after intoxication (Blythe

et al., 1992, 1994; Schwarz et al., 2008). The effect of

mannitol infusion was thought to be mediated by the

osmotic reduction of neuronal edema (Pearn, 2001) and

sodium channel dependent reduction in binding effects

(Birinyi-Strachan et al., 2005a, b). In the latter study and

that of Bagnis et al. (1992), mannitol effects were distinguished from control treatments including hyper- and isoosmolar D-sorbitol, sucrose, glucose, vitamin B, calcium

gluconate and free radical scavenging agents Troloxand

L-ascorbic acid. Contradictory findings were reported,

however, by Schnorf et al. (2002) in a double-blind and

randomized study in which saline was found to be as

effective as mannitol in ameliorating the symptoms of

ciguatera. Nicholson and Lewis (2006) noted that clinical

use of mannitol may induce excessive loss of fluids in

patients suffering from acute diarrhea and vomiting, and

that patients experiencing bradycardia and hypotension are

at higher risk of cardiac failure if infused with high doses of

mannitol. Lewis and King (1996) suggested that mannitol

should not be administered until the patient is adequately

rehydrated. Mannitol therapy has been recommended for

two primary goals: reduction of acute neurologic symptoms

and prevention of chronic neurologic symptoms.

5. Pharmacology of ciguatoxins

Symptoms of ciguatera were long recognized as indicative of central and peripheral nervous system injury. Early

studies identified sodium dependent, and tetrodotoxin

sensitive, excitable cell depolarization in a variety of isolated nerve and muscle tissue preparations (e.g., Rayner,

1970; Ohshika, 1971; Miyahara et al., 1979). Voltage-clamp

studies suggested that ciguatoxins cause spontaneous, and

enhance evoked, action potentials by lowering activation

thresholds and delayed repolarization of voltage-gated

sodium channels (Benoit et al., 1986; Bidard et al., 1984;

Molgo et al., 1990). A continuous and large body of work

further elucidated cellular effects and molecular mechanisms of ciguatoxin actions including selective binding and

competition with another polyether toxin, brevetoxin, for

‘‘site 5’’ on voltage-gated sodium channels (Bidard et al.,

1984; Poli et al., 1986, 1997; Sharkey et al., 1987; Lombet

et al., 1987; Lewis et al., 1991a, b; Gawley et al., 1992;

Trainer et al., 1994; Dechraoui et al., 1999); elevation of

intracellular calcium levels (Lewis and Endean, 1986;

Molgo et al., 1993); stimulation of spontaneous and evoked

neurotransmitter release from synaptosomes and motor

nerve terminals (Molgo et al., 1990; Hamblin et al., 1995;

Brock et al., 1995); axonal and schwannn cell edema (Allsop

et al., 1986; Benoit et al., 1996; Mattei et al., 1999); induction of tetrodotoxin-sensitive leakage current in dorsal root

ganglion neurons (Strachan et al., 1999); and blockade of

voltage-gated potassium channels (Hidalgo et al., 2002;

Birinyi-Strachan et al., 2005a, b). While both ciguatoxins

and brevetoxin elicit membrane depolarization, ciguatoxin

induces unique spontaneous single channel events in

sensory neurons (Hogg et al., 1998). Birinyi-Strachan et al.

(2005a, b) observed ciguatoxins to induce a blockade of

voltage-gated potassium channels contributing to neuronal

excitability in rat parasympathetic neurons. Other reports

suggested that ciguatoxins prolong action potential duration after hyperpolarization via blockade of voltage-gated

potassium channels (Birinyi-Strachan et al., 2005a, b;

Benoit and Legrand, 1992) and induce leakage current

(Strachan et al., 1999). Hogg et al. (2002) also observed that

ciguatoxins induce unique oscillations in membrane

potential and action potential firing in rat cultured dorsal

root ganglion neurons. Dechraoui et al. (2006) noted the

differential expression of voltage-gated sodium channel

isoforms of nervous, skeletal and cardiac tissues and

assessed the relative affinity of brevetoxins and ciguatoxin

in the rat and the fish Centropristis striata models by

128 R.W. Dickey, S.M. Plakas / Toxicon 56 (2010) 123–136
radioligand (tritiated brevetoxin-3) competitive binding.

Results indicated a species related resistance of heart

voltage-gated sodium channels in the rat and comparable

sensitivity between the species for brain and skeletal muscle.

Although the neurological symptoms observed in clinical

cases of ciguatera poisoning are believed to be consistent

with the direct interaction of ciguatoxins with voltagegated sodium channels (see review by Lehane and Lewis,

2000), Kumar-Roine et al. (2008) argued that the unique

effects of ciguatoxins on voltage-gated sodium channels

did not fully explain the spectrum of symptoms elicited by

ciguatoxins. They cited a number of studies where sodium

channel activator affects, and ciguatoxin activation of Ltype calcium channels, were mediated by nitric oxide (NO)

via the neuronal nitric oxide synthase (nNOS) pathway, and

that nNOS and induceable NOS (iNOS) were also implicated

in nocioceptive and inflammatory responses to other types

of envenomation. They observed that Pacific ciguatoxin-1B

induced prolonged dose- and time-dependent NO production in murine macrophages through modulation of iNOS

expression. Based on these observations and studies

showing elevated levels of NOS-induced inflammatory

cytokines, L-citrulline and neopterin in the urine and serum

of ciguatera patients, the authors hypothesized a cascade of

excessive N-methyl-D-aspartate (NMDA) receptor activity

due to prolonged voltage-gated sodium channel activity.

This might result in calcium influx, with activation of

constitutive NOS (cNOS) and production of NO; the latter

reacts with superoxide radicals to produce peroxynitrite

anion creating an oxidative stress that activates the transcription factor NF-KB, and stimulating iNOS gene transcription leading to further NO production. While direct

evidence is lacking for ciguatoxin activation of NMDA

receptors, it has been shown for brevetoxins (Dravid et al.,

2005; Berman and Murray, 1999; Massensini et al., 2003).

The hypothesis appears to be consistent with free radical

scavenging activity reported for mannitol and active agents

of traditional herbal remedies (e.g. mangiferin in D. solida).

Depressed body temperature in mice following intraperitoneal administration of toxic fish extracts was noted in

early studies (Doorenbos et al., 1976; Hoffman et al., 1983;

Sawyer et al., 1984; Lewis et al., 1993). Peng et al. (1995)

presented evidence for CNS effects of ciguatoxin and brevetoxin on thermoregulatory centers of the brain based

upon localization of Fos in the medial preoptic (MPO)

nucleus in mice and perhaps reflecting changes in core

body temperature. Ciguatoxin activity on sodium channels

of excitable membranes and the thermal dysthesia in

patients was reported to result from alteration of C-polymodal nociceptors (Cameron and Capra, 1993). More

recently Gatti et al. (2008) in a retrospective study of

ciguatera cases in French Polynesia reported body

temperature depression under 36.5 C in 48 of 80 documented patient files. The latter observation appears to be

a first report and is consistent with CNS activity reported in

prior studies (see review by Gordon and Ramdell, 2005).

6. Detection of ciguatera toxins

The ideal method for the detection and quantification of

ciguatoxins in fish, a method that is simple, rapid and

accurately measures toxicity relative to human susceptibility is yet to be developed. Over the written history of

ciguatera a wide variety of methods have been devised to

detect and reject toxic fish. Many traditional methods such

as discoloration of silver coins or copper wire or the

repulsion of flies and ants were readily discredited (Banner

and Helfrich, 1964). Other in vivo methods including the

mongoose assay (Banner et al., 1960) and cat assay (Lewis,

1987; Bagnis et al., 1985a, b) were simple but cumbersome,

non-quantitative and ethically objectionable. The brine

shrimp (Granade et al., 1976; Hungerford, 1993), mosquito

(Bagnis et al., 1985a, b, 1987) and diptera larva (Labrousse

and Matile, 1996) assays also were fairly simple but

ambiguous and unconvincing. Isolated tissue assays

including the guinea pig ileum assay (Dickey et al., 1982),

the guinea pig atrium assay (Lewis, 1988; Lewis and

Endean, 1986), the isolated frog nerve assay (Benoit et al.,

1986), and hemolytic assays using human and mouse blood

cells (Escolana De Mota et al., 1986) showed greater

sensitivity but in most cases technical difficulty or ethical

objections precluded them from wider application.

The mouse bioassay, based on the method described by

Banner et al. (1960), refined and described in detail by

Yasumoto et al. (1984), is presently the most widely used

assay for the detection of ciguatoxins in fish. The method

specifies intraperitoneal injection of serially diluted semipurified fish extracts into mice and 24 h observation for

signs of toxicity. Characteristic signs of toxicity qualify

ciguatoxins in extracts and the relationship between dose

and time to death is used to quantify toxicity. Total

lethality is expressed in mouse units (MU). Calculation of

fish toxicity (Lewis and Sellin, 1992; Lewis et al., 1991a, b)

is approximated by log MU 1⁄4 2.3 log (1 þ T  1), where MU

is the number of mouse units of ciguatoxin injected and T

time to death in hours. One MU is the LD50 dose for a 20 g

mouse which is equivalent to 5, 48 and 18 ng of Pacific

CTX-1, CTX-2 and CTX-3, respectively. Additional purification of herbivorous fish extracts can be performed to

separate the polar maitotoxins from lipid-soluble ciguatoxins (Yokoyama et al., 1988; Holmes et al., 1991; Holmes

and Lewis, 1994; Legrand et al., 1992a, b) since the maitotoxins exhibit low oral potency, and do not appear to

accumulate in piscivorous fish causing ciguatera in

humans. The mouse assay has been used extensively in

research settings and to a lesser extent in monitoring

market fish. It shares some of the disadvantages noted for

other in vivo assays, including maintenance of animal

colonies, cumbersome execution and ethically compromised in addition to questions of acceptable levels of

sensitivity and specificity to be preferred in modern

methods of public health importance.

Alternatives to in vivo testing were developed to detect

ciguatoxins using sodium channel specific cytotoxicity

(Manger et al., 1993, 1995) and sodium channel receptor

binding in rat brain synaptosomal preparations (Lombet

et al., 1987; Lewis et al., 1991a, b; Poli et al., 1997). The

assays can detect subpicogram levels of ciguatoxins in fish

extracts and provide qualitative and quantitative estimates

of toxicity. Both assays can be formatted for high sample

throughput (e.g. Van Dolah et al., 1994). The neuro-2a

mouse neuroblast cell assay (Manger et al., 1993, 1995) is

R.W. Dickey, S.M. Plakas / Toxicon 56 (2010) 123–136 129
a ouabain-veratridine-dependent in vitro screening

method for sodium channel specific algal toxins. Assay

specificity is achieved by pretreatment of neuroblasts with

veratridine to initiate sodium channel gating and ouabain

to block the sodium-potassium pump which would otherwise permit cells to compensate for sodium ion flux. Neuroblasts that are not treated with veratridine and ouabain

are not affected by sodium channel specific toxins. Both

treated and untreated neuroblasts, however, are susceptible to toxins acting through other vital metabolic mechanisms. Through appropriate experimental design the cell

assay can discriminate between toxins that activate (e.g.,

brevetoxins, ciguatoxins) or block (e.g., tetrodotoxins,

saxitoxins) voltage-gated sodium channels, and also

distinguish between sodium channel specific toxins and

those acting through other modes of action. The assay

provides a composite response to all sodium channel

specific toxins in an extract and it cannot discriminate

molecular species acting in the same manner or determine

what other modes of action may be where toxicity is not

sodium channel specific. The neuro-2a mouse neuroblast

cell assay is used routinely for research and outbreak

applications in some laboratories (e.g., Dickey et al., 1999;

Dickey, 2008; Dechraoui et al., 2005a).

The receptor binding assay is based on binding

competition between ciguatoxin standard or sample with

tritiated brevetoxin for the sodium channel receptor. The

bound and unbound toxins are separated by centrifugation

or filtration, and the amount of bound radiolabeled brevetoxin is measured by liquid scintillation counting.

Samples are quantitated by comparison with a standard

competition curve, generated by addition of increasing

concentrations of unlabeled ciguatoxin or brevetoxin to

a mixture of receptor and tritiated brevetoxin. Binding

competition can be carried out in traditional test-tube

format or adapted to a microplate format and quantified

using a microplate scintillation counter to permit high

sample throughput (Poli et al., 1997; Van Dolah et al., 1994;

Dechraoui et al., 2005a; Darius et al., 2007). Receptor assays

provide a quantitative estimate of composite toxicity much

like the cytotoxicity assay where all toxins in an extract

bind to the sodium channel receptor. Relative binding

affinities correlate with their relative toxic potencies.

Ciguatoxin detection limits are at the pmol range. A

disadvantage to the application of receptor assays for

ciguatoxins, and other algal toxins, is that they are technically complex, and require the use of radiolabeled materials

with attendant licensing requirements. Modifications of

receptor assays to colorimetric or fluorometric end points

have generally not been successful because colorimetric or

fluorometric tags tend to decrease binding affinity of the

toxin. However, a membrane potential-based assay for

brevetoxins was developed using synaptoneurosomes isolated from mice. The assay detected the depolarizing effect

of brevetoxins as enhancements of the veratridine-dependent increase in fluorescence of the voltage-sensitive

fluorescent probe rhodamine 6 G. The assay was relatively

rapid and detected brevetoxin activity in the nanomolar

range. The synaptoneurosomal technique was shown to

compare favorably with the cytotoxicity assay, the receptor

binding assay and HPLC/MS (David et al., 2003).

The development of immunoassays for ciguatoxins in

fish tissues appear to have the best potential for meeting

the utilitarian requirements of speed, simplicity and accuracy in the measurement of toxicity relative to human

susceptibility. Initial development if immunoassays for

ciguatera began with a radio immunoassay format using

polyclonal antibody to a partially purified ciguatoxin

preparation (Hokama et al., 1977). In succeeding iterations

of polyclonal and monoclonal antibody based assay

developments (Hokama et al., 1984, 1998; Hokama 1985,

1990; Park 1994), specificity, i.e. cross-reactivity with other

toxins including okadaic acid, maitotoxin and brevetoxin,

and unacceptable rates of false positives and false negatives

(Dickey et al., 1994; Wong et al., 2005) remained obstacles

to success (see review by Van Dolah and Ramsdell, 2001).

The later monoclonal antibody assays developed for

ciguatera were also reported to react with abnormal lipids

in sera of chronic fatigue syndrome, chronic ciguatera fish

poisoning, hepatitis B and cancer patients suggesting

a causal link (Hokama et al., 2003a, b, 2006, 2008). More

recently reported developments of mouse and chicken

antibodies specific to synthetic fragments of ciguatoxin

were reported to produce reliable and accurate results in

screening fish populations for ciguatoxins (Campora et al.,

2008a, b). The alternative strategy to produce anti-ciguatoxin antibodies using protein-conjugated synthetic fragments of ciguatoxins (Pauillac et al., 2000, 1998) appeared

to be successful and sufficiently sensitive for identifying

ciguatoxins in fish tissue but the antiserum was reportedly

lost before thorough evaluation could be performed. Tsumuraya et al. (2006) noted the extremely low content, and

difficulty in recovery, of ciguatoxins in contaminated fish

necessary for anti-ciguatoxin antibody development. They

used synthetic haptens in producing anti-ciguatoxin

monoclonal antibodies against both ends of ciguatoxin

CTX-3C (Oguri et al., 2003). The paired antibodies were

used to develop a sandwich enzyme-linked immunosorbent assay (ELISA) that detected CTX-3C at the ppb level

without cross-reactivity to other related marine toxins,

including brevetoxin A, brevetoxin B, okadaic acid (OA),

and maitotoxin. This report was followed closely by the

generation of monoclonal antibodies against the right wing

of ciguatoxin CTx-1B and 51-hydroxy-CTX-3C (Tsumuraya

et al., 2006), also without cross-reactivity to the other

marine toxins tested for the anti-CTX-3C preparation.

The application of mass spectrometry (MS) played

a critical role in the structure elucidation of most of the

ciguatoxin congeners recovered in trace quantities from

toxic fish and Gambierdiscus. Using as templates the fragmentation patterns of those ciguatoxins obtained in sufficient quantity for NMR structure elucidation, the structures

of the many congeners were deduced by a combination of

FAB/MS/MS and synthetic conversions to known structures

(Yasumoto et al., 2000). More recent advancements in MS

technology have dramatically improved the analytical

capabilities, reduced expense, size and technical

complexity of instrumentation, to afford wider access to

the analytical power of the technology. Prior to these

developments MS was utilized in relatively few laboratories and required extensive purification and concentration

of ciguatera toxins recovered from toxic fish (e.g. Lewis and

130 R.W. Dickey, S.M. Plakas / Toxicon 56 (2010) 123–136
Sellin, 1992, 1993). The application of MS to ciguatoxin

detection in crude extracts at sub-part per billion levels

required for public health relevance was first reported by

Lewis et al. (1999). They described a method for gradient

reversed-phase HPLC/tandem mass spectrometry (HPLCMS/MS) of P-CTX-1 and C-CTX-1 in spiked and naturally

incurred crude extracts of fish. Levels equivalent to 0.04

ppb P-CTX-1 and 0.10 ppb C-CTX-1 were detectable, and

using P-CTX-1 as an internal standard, the analysis of fish

extracts from the Caribbean Sea suggested an estimated

risk level of >0.25 ppb C-CTX-1. Following this development other laboratories began to incorporate LC-MS/MS in

research and in some cases regulatory analysis of fish for

ciguatoxins content (Dickey, 2008). However, the analytical

precision and specificity of the technology was also found

to be a weakness of the technique as applied to regulatory

surveillance and response requirements. The physical

detection and identification of specific ciguatoxin congeners, and their quantification where standards are available, is not readily linked to human thresholds of

susceptibility. This circumstance might also be relevant to

the application of immunoassays where inference of

toxicity through epitopic (i.e. structural) recognition of

toxin by antibody is made less definitive where affinities for

multiple toxins or congeners are known, or likely to be,

variable; where toxin or congener profiles in fish are

known to be variable; where relative toxic potencies are

poorly known; and where toxin or congener standards are

not widely available. Accordingly, it was found that in the

assessment of toxic fish for consumer protection a two-part

protocol comprising in vitro mouse neuroblastoma cell

assay to measure toxic potency and LC-MS/MS to confirm

the molecular presence of ciguatoxins provided the most

appropriate information for decisions of public health and

economic importance.

7. Ciguatoxin adverse affect levels

The availability and analysis of meal remnants directly

linked to ciguatera disease is infrequent. Most often,

implicated meal remnants are discarded and the only

samples available for analysis are from implicated ‘‘lots’’ of

fish. In commercial practice the term ‘‘lot’’ can mean fish

caught at the same time from a particular location; fish sold

as a single grouping and perhaps co-mingled with others

from different capture locations; or fish sold by a particular

vendor with little or no record of origins. Therefore,

traceback to a specific lot may not be helpful in identifying

the source of toxic fish. While the availability of fish from

implicated lots is potentially useful for circumstantial

inference, the documented variability of ciguatoxin burden

in fish from any particular region limits the usefulness of

such analyzes for fish poisoning confirmation, much less

the assessment of exposure thresholds. Information

derived from the study of ciguatera disease outbreaks has

improved clinical recognition and timely treatment of this

disease. Where implicated meal remnants are available

such studies are equally important for the differentiation of

ciguatoxin profiles in fish from one region to the next, the

determination of toxicity thresholds in humans, and the

development of regulatory policy to protect consumers.

In the Pacific Lehane and Lewis (2000) noted that mild

outbreaks of ciguatera occur after exposures of 1 ng P-CTX1/kg body weight. They assumed consumption of 500 g

fish/meal to derive a threshold ciguatoxin contamination

rate in fish of 0.1 ppb P-CTX-1 equivalent toxicity using the

mouse bioassay. Applying a 10X safety factor to address

individual human risk factors, uncertainty in the amount of

fish consumed, and uncertainty in assay accuracy, the

authors suggested that a ‘‘safe’’ fish would contain no more

that 0.01 ppb P-CTX-1 equivalent toxicity (see also Lehane,

2000). Similarly, in the Caribbean Vernoux and Lewis

(1997) estimated a threshold contamination rate of

1.0 ppb C-CTX-1 equivalent toxicity using the mouse

bioassay, and no more than 0.1 ppb C-CTX-1 for safe fish

after a 10X safety factor is applied. A review of exposure

levels in 109 outbreaks of ciguatera in the United States

from 1998–2008 was consistent with the suggested

adverse effects thresholds and ‘‘safe’’ contamination rates

for C-CTX-1 and P-CTX-1 equivalent toxicity in fish (Dickey

et al., 2008; Dickey, 2008). These estimates were derived

from analyses of implicated ‘‘meal remnant’’ fish tissues

using a tiered protocol of in vitro neuroblast cell assay to

measure toxicity and LC-MS/MS to confirm the molecular

presence of ciguatoxins in fish tissues. The aforementioned

studies may serve as the basis for industry and consumer

advisory levels of 0.10 ppb C-CTX-1 equivalent toxicity in

fish from the tropical Atlantic, Gulf of Mexico, Caribbean,

and 0.01 ppb P-CTX-1 equivalent toxicity in fish from

Pacific regions.

8. Summary

Establishment of industry and consumer guidance for

controlling the ciguatera hazard and prevent illness has

been constrained by many factors. Much work remains

before risk assessments and effective management plans

can be undertaken. Interdisciplinary science has made

progress in developing the critical information required

for ciguatera risk analysis. The principal ciguatera toxins

found in fish have been identified and are traceable

through the food web to a single genus of dinoflagellate,

Gambierdiscus spp.; the toxins produced by Gambierdiscus spp. are assimilated and metabolized through trophic

transfer in many species of food fish; and the burden of

toxins accumulated by fish that induce ciguatera fish

poisoning among consumers can be measured. Significant progress in ciguatera research has been made in

recent years but many questions pertaining to environmental and public health risk analysis remain to be

addressed.

Conflict of interest

The authors declare that there are no conflicts of

interest.

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