Contribution to the risk characterization of ciguatoxins: LOAEL estimated from eight ciguatera fish poisoning events in Guadeloupe (French West Indies)

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Contribution to the risk characterization of ciguatoxins: LOAEL estimated from eight ciguatera fish poisoning events in Guadeloupe (French West Indies) Virginie Hossen a, Lucia Soliño b, Patricia Leroy a, Eric David c, Pierre Velge d, Sylviane Dragacci a,n, Sophie Krys a, Harold Flores Quintana e, Jorge Diogène b a Université Paris-Est, ANSES-Laboratory for Food Safety, National Reference Laboratory for the Control of Marine biotoxins, 14 rue Pierre et Marie Curie, 94701 Maisons-Alfort, France b Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Ctra. Poble Nou km 5.5, Sant Carles de la Rapita, Spain c Ministry of Agriculture, Direction de l’Alimentation de l’Agriculture et de la Forêt de Guadeloupe, Abymes, France d Ministry of Agriculture, General Directorate for Food, Paris, France e U.S. Food and Drug Administration (FDA), Division of Seafood Science and Technology, Gulf Coast Seafood Laboratory, 1 Iberville Drive, Dauphin Island, AL 36528, USA

art ic l e i nf o

a b s t r a c t

Article history: Received 18 March 2015 Received in revised form 30 July 2015 Accepted 14 September 2015

From 2010 to 2012, 35 ciguatera fish poisoning (CFP) events involving 87 individuals who consumed locally-caught fish were reported in Guadeloupe (French West Indies). For 12 of these events, the presence of ciguatoxins (CTXs) was indicated in meal remnants and in uncooked fish by the mouse bioassay (MBA). Caribbean ciguatoxins (C-CTXs) were confirmed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. Using a cell-based assay (CBA), and the only available standard Pacific ciguatoxin-1 (P-CTX-1), the lowest toxins level detected in fish samples causing CFP was 0.022 mg P-CTX1 equivalent (eq.)·kg
1 fish. Epidemiological and consumption data were compiled for most of the individuals afflicted, and complete data for establishing the lowest observable adverse effects level (LOAEL) were obtained from 8 CFP events involving 21 individuals. Based on toxin intakes, the LOAEL was estimated at 4.2 ng P-CTX-1 eq./individual corresponding to 48.4 pg P-CTX-1 eq. kg
1 body weight (bw). Although based on limited data, these results are consistent with the conclusions of the European Food Safety Authority (EFSA) opinion which indicates that a level of 0.01 mg P-CTX-1 eq. kg
1 fish, regardless of source, should not exert effects in sensitive individuals when consuming a single meal. The calculated LOAEL is also consistent with the U.S. Food and Drug Administration guidance levels for CTXs (0.1 mg CCTX-1 eq. kg
1 and 0.01 mg P-CTX-1 eq. kg
1 fish). & 2015 Elsevier Inc. All rights reserved.

Keywords: Ciguatera fish poisoning Ciguatoxins Mouse bioassay Neuro-2A cell-based assay LOAEL Guadeloupe

1. Introduction Ciguatera fish poisoning (CFP) is the most common food-borne illness related to the consumption of tropical and subtropical reef fish. The causative agents are toxins belonging to the ciguatoxins (CTXs) group (Bagnis et al., 1979; Lehane and Lewis, 2000; Yasumoto, 2005). CTXs result from the bioaccumulation and metabolism of precursor toxins along the fish food webs. Precursor toxins named gambiertoxins, are produced by benthic dinoflagellates of the genus Gambierdiscus whose distribution includes tropical and n Correspondence to: ANSES-Laboratory for Food Safety, National Reference Laboratory for the Control of Marine biotoxins, 14 rue Pierre et Marie Curie, 94701 Maisons-Alfort, France. E-mail address: [email protected] (S. Dragacci).

subtropical coral reef areas (Yasumoto et al., 1977; Litaker et al., 2009). These benthic microalgae grow on substrates like sediments and coral, and can be grazed upon by fish, and their toxins can move along food webs reaching top predators (e.g. in the Caribbean Sea: grouper, barracuda, snapper and jack). Symptoms of CFP include common gastrointestinal disturbances such as nausea, vomiting, and diarrhea. Rashes and neurological disorders like paresthesia, and in particular a temperature-related dysesthesia, are very typical symptoms of this disease. Some of these symptoms can last for weeks or months (Bagnis et al., 1979; Ruff and Lewis, 1994). Cardiovascular and respiratory disorders may also be triggered by the intoxication. At the cellular level, it has been showed that CTXs activate the sodium ion channels, causing Na þ influx and K þ efflux, cell membrane excitability, and cell disruption (Lewis et al., 2000; Hidalgo et al., 2002).

http://dx.doi.org/10.1016/j.envres.2015.09.014 0013-9351/& 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Hossen, V., et al., Contribution to the risk characterization of ciguatoxins: LOAEL estimated from eight ciguatera fish poisoning events in.... Environ. Res. (2015), http://dx.doi.org/10.1016/j.envres.2015.09.014i

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Most countries concerned by CFP have set up regulations or recommendations. European Union (EU) Regulation no. 854/2004 states that checks should be implemented to ensure that fishery products which may contain toxins including CTXs are not placed on the market (European Union Commission, 2004). However, there are no EU regulatory limits, nor reference method, for CTXs in fish because the lack of data does not allow a complete risk assessment to be performed (Caillaud et al., 2010). France is paying special attention to CFP when considering its overseas territories, Polynesia and New Caledonia (Pacific Ocean), Reunion Island (Indian Ocean), and Guadeloupe and Martinique (French West Indies). Preventive actions to manage the risk linked to CFP are based on the establishment of local regulations that include a list of highly susceptible ciguatoxic fish species that cannot be put on the market. For instance, in Guadeloupe, the prefectural decree no. 1249 (Prefectural Decree, 2002) indicates several fish species such as barracudas, kingfishes, snappers and yellowtails that are not allowed for sale and consumption. In some cases, the human clinical diagnosis of CFP has been confirmed by analysis of CTXs in fish meal remnants or remaining portions of the same fish from which the meal was prepared (Friedman et al., 2008; Lehane and Lewis, 2000; Dickey, 2008; Abraham et al., 2012; Hossen et al., 2013). Nevertheless, full documentation of CFP cases including fish consumption and toxicity levels, remain scarce as emphasized by Pottier et al. (2001), and as more recently described in the InVS and CIRE Antilles Guyane report (2013). Therefore toxin intakes and exposure estimates are often missing in CFP reports. In July 2006, the European Commission requested the European Food Safety Authority (EFSA) to release a scientific opinion to assess the current regulatory limits in the EU with regard to human health and analytical methods for marine biotoxins (EFSA, 2010). The acute reference dose (ARfD) represents the amount of a substance that can be ingested in an acute duration (24 h or less) without appreciable health risk. When sufficient relevant human data are available, the lowest observable adverse effects level (LOAEL) can be calculated, and the ARfD can be derived by applying adequate uncertainty factors. For CTXs, EFSA reported that “due to the very limited quantitative data issued from experimental animal studies and from observed human intoxications, the panel concluded that the establishment of an oral ARfD was not possible”. It was nevertheless indicated that “a concentration of 0.01 mg P-CTX-1 eq. kg
1of fish is expected not to exert effects in sensitive individuals”. It should be noted that the concentration is based on equivalent of “P-CTX-1” standard. In several published works, C-CTX-1 was demonstrated as being ten-fold less potent than P-CTX-1 in mice by i.p. administration (Vernoux and Lewis, 1997; Lewis et al., 1999; Caillaud et al., 2010). Taking this into consideration and other reports (Dickey, 2008; Dickey et al., 2008; Dickey and Plakas, 2010), the U.S. Food and Drug Administration has established guidance levels of 0.1 mg C-CTX-1 eq. kg
1 and 0.01 mg P-CTX-1 eq. kg
1 fish, for Caribbean and Pacific CTXs, respectively (U.S. Food and Drug Administration, 2011). Additional data, especially quantitative determination of CTXs in fish samples, will help better characterize the risk of CFP for consumers. Two tools are most commonly implemented to evaluate CTXs content in fish tissues according to toxicological response: the mouse bioassay (MBA) as a qualitative test (Yasumoto et al., 1984; Vernoux, 1994) and the Neuro-2A cell-based assay (Neuro-2A CBA) as a quantitative test (Manger et al., 1995; Caillaud et al., 2012) that allows estimation of CTXs levels (toxicity equivalents) using a CTX standard calibration curve. The Neuro-2A CBA was identified by the EFSA as suitable method to estimate contents of CTXs in fish, and this was considered relevant to establish the LOAEL (EFSA, 2010). Analytical methods based on liquid chromatography coupled to mass spectrometry are promising methods for CTXs

identification and quantification and can be used to confirm the presence of CTXs in fish (Lewis et al., 2009). From 2010 to 2012, 35 CFP events involving 87 individuals who had consumed locally-caught fish were reported in Guadeloupe (French West Indies). In 12 of those cases, meal remnants and uncooked fish portions associated with illnesses were available and CTXs contamination was presumed by the MBA. When sufficient remnants were available, in 10 of the 12 cases, the CTXs content was estimated by the Neuro-2A CBA. In two of those cases, samples were submitted for LC–MS/MS analysis, where C-CTX-1 congener was confirmed. In this study, we describe epidemiological data and information of CTXs content in food remnants, whenever available, from 8 among 35 CFP events. These data sets, comprising 21 individuals, collected through the outbreak reporting system, were used to estimate a dose–response relationship and to establish a LOAEL.

2. Materials and methods 2.1. Ethics statement Patients’ medical records were retrospectively reviewed, and all data collected were de-identified in standardized forms according to procedures of the Commission Nationale de l’Informatique et des Libertés (the French Information Protection Commission). 2.2. Investigation of CFP events The CFP events recorded in Guadeloupe (French West Indies) from 2010 to 2012 were identified through the French reporting system for food poisoning outbreaks (Hossen et al., 2011). In the West Indies islands, CFP has been a concern for health authorities for many years (InVS and CIRE Antilles Guyane report, 2013). Since 2010, to address the risk assessment needs for CTXs, the local veterinary services of Guadeloupe have strengthened their investigation of CFP to obtain more detailed data on fish; this include portion sizes of fish ingested and, where available, meal remnants in order to analyze for CTXs. When a CFP event was alerted to the authorities, epidemiological data were recorded anonymously using a questionnaire including the number of cases, onset dates, symptom identification, symptom severity, and recovery time. Whenever possible, the portion of fish consumed and personal information (sex, age and weight) of the affected persons were also collected. 2.3. Analysis of uncooked fish and meal remnants Samples of uncooked fish and meal remnants were frozen and were sent for analysis to the French National Reference Laboratory for marine biotoxins (ANSES, Maisons-Alfort, France). Eleven samples were portions of the fish consumed whereas one (event 1) was a fish of the same species caught at the same time. Four out of the eleven samples (events 2–5) were uncooked fish portions from which meals implicated in illness were prepared. The other samples (events 6-12) were collected as meal remnants. Details on the preparation of fish meals (e.g. grilled, boiled or simply macerated into dressing) were not provided. The toxicity level of samples was estimated by MBA, and when sufficient quantity of sample remained, aliquots were forwarded to IRTA, Sant Carles de la Rapita, Spain, for the estimation of CTXs contents by the Neuro2A CBA. CTX confirmation was performed by LC–MS/MS at FDA’s Gulf Coast Seafood Laboratory, Dauphin Island, Alabama, USA.

Please cite this article as: Hossen, V., et al., Contribution to the risk characterization of ciguatoxins: LOAEL estimated from eight ciguatera fish poisoning events in.... Environ. Res. (2015), http://dx.doi.org/10.1016/j.envres.2015.09.014i

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2.4. Mouse bioassay (MBA) The MBA (i.p. administration in mice) was conducted according to the national chart on the ethics of animal experimentation jointly edited by the French Ministry of Research and the French Ministry of Agriculture (agreement no. 10/06/08-07B). The MBA was carried out according to Vernoux (1994) with some modifications as described hereafter. All solvents were of analytical grade (Fisher Scientific, France). Briefly, a 50 g fish muscle tissue sample was minced and extracted twice with methanol (2  100 mL), the methanolic extract was defatted with n-hexane (170 mL) and partitioned twice with diethyl ether (2  340 mL). The combined diethyl ether fractions were dried by rotary evaporator (Büchi R-134, Fisher Bioblock scientific, France). The resulting residues were dissolved in 80% of methanol (2 mL), and defatted again with n-hexane (2 mL). The methanolic extracts were finally dried down under a gentle stream of nitrogen gas at 40 °C (Reacti-Vap, Pierce Biotechnology, France). The residues were suspended in 2 mL of 1%-Tween-60 (Acros Organics) and injected to mice intraperitoneally (0.04 mL g
1 of body weight). For each extract, two male Swiss albino mice of 18
22 g (Charles River Laboratory, France) were required. Mice were allowed free access to food and tap water throughout the experimental period. The death time and/or the 24 h loss of weight were carefully recorded. The death of one mouse or two mice qualifies the fish sample as “not edible”, and the final result was reported as “positive”. If not dead, a body weight loss of more than 5% for at least one mouse qualifies the fish sample as “in the limit of edibility”, and the result was also recorded as “positive”. If the mice have gained or lost less than 5% of their weight, the fish sample was considered as “edible” and the result was then reported as “negative”. 2.5. Neuro-2A cell-based assay (Neuro-2A CBA) 2.5.1. Extraction and purification of CTXs from fish samples Fish flesh was extracted and purified according to the protocol described by Lewis (2003). All the solvents were of HPLC grade (Merck, Darmstadt, Germany). Briefly, a 10 g portion of fish flesh was cooked (70 °C, 10 min) and extracted twice with acetone by homogenization (UltraTurrax, 17,500  g), centrifugation (10 min at 3000  g) (Jouan MR23i, Saint Herblain, France) and filtration through a 0.45 μm nylon filter. Acetone extracts were pooled and dried on a rotary evaporator (Büchi R-200, Flawil, Switzerland). The dried extract was dissolved in 5 mL methanol:water (9:1, v:v) and partitioned twice with 5 mL n-hexane. The methanol:water fractions were dried, dissolved in 5 mL ethanol:water (1:3, v-v) and partitioned twice with 5 mL diethyl ether. Ether fractions were pooled, dried, dissolved in 4 mL of methanol and stored at
20 °C until analysis. 2.5.2. Neuro-2A CBA cell maintenance and assay Neuro-2A cells (ATCC, CCL131) were maintained in 10% fetal bovine serum (FBS)-RPMI medium (Sigma-Aldrich, USA) at 37 °C in a 5% CO2 humid atmosphere (Binder, Tuttlingen, Germany). The day before the assays, cells were seeded in a 96-well microplate in 5% FBS-RPMI medium at a density of 35,000 cells per well. Cells were incubated in the same temperature and atmospheric conditions as described for cell maintenance. After a 24 h-period of incubation, the Neuro-2A cells were exposed to increasing concentrations of fish extract or to P-CTX-1 standard. Reference P-CTX-1 standard was provided by R.J. Lewis (The Queensland University, Australia) and is presently used at IRTA as a reference toxin for the routine evaluation of CTXs in fish regardless of their origin. It is also in line with EFSA which references to P-CTX-1 to express the toxicity level of samples. Other CTX reference

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materials, including C-CTXs, are currently not commercially available. For assay purposes, P-CTX-1 standard solution or fish extracts were evaporated (TurboVap, Caliper, Hopkinton, USA), then reconstituted in culture medium and added to each well in presence or absence of 0.1 mM of ouabain (Sigma-Aldrich, USA) and 0.01 mM of veratridine (Sigma-Aldrich, USA). All assays were performed in triplicate. To minimize matrix effects, the highest concentration of tissue equivalent (TE) in the assay was 100 mg TE mL
1. The detection limit of the assay was 0.0096 pg P-CTX-1 eq. mg TE
1. Sensitivity of the Neuro-2A cells to the presence of CTX-compounds was calibrated each day of the experiment with a standard solution of P-CTX-1. After a 24 h-period of exposure to fish extracts or to P-CTX-1 standard solutions, cell viability was assessed by using the colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] assay. Absorbance was read at 570 nm with an automated multi-well scanning spectrophotometer (Biotek, Synergy HT, Winooski, Vermont, USA) and results were analyzed with the software Prism 4 (GraphPad, San Diego, California, USA). Viability of cells was expressed in relation to the viability of the corresponding cell control with (O/V þ) or without (O/V
) ouabain/ veratridine treatment, and the 50% effect concentration (IC50O/V þ ) was calculated after a sigmoid regression curve with variable Hill slope. Responses producing less than 80% cell viability in relation to the control (100%) were considered as toxic. Results were expressed in mg P-CTX-1 eq. kg
1 of fish flesh weight (FW). Jack and blackfin snapper samples from Guadeloupe that were found negative for CTXs by the MBA were used as negative controls. 2.5.3. Estimation of the lowest observed adverse effect level (LOAEL) The LOAEL corresponds to the lowest toxin intake that exhibits symptoms in human. Toxin intakes were obtained by multiplying the toxin contents as determined by the Neuro-2A CBA by the estimated weight of the ingested fish portion. The LOAEL can be expressed in quantity of toxins per person or, if the weight of the person is known, it can be expressed in quantity of toxins per kg of body weight. The LOAEL was deduced from 21 people and expressed in pg P-CTX-1 eq. per individual. For the 17 individuals for whom the body weight was available, the LOAEL was expressed in pg P-CTX-1 eq. kg
1 body weight. 2.6. Liquid chromatography–mass spectrometry (LC–MS/MS) 2.6.1. Sample clean-up For LC–MS/MS analysis, the methanolic Neuro-2 CBA extracts were cleaned up by solid phase extraction (SPE). The SPE cartridge (500 mg NH2 SPE, Agilent Bond Elut, CA, USA) was conditioned with 3 mL of chloroform. Two mL of methanolic sample extract (5 g TE) were dried at 65 °C under nitrogen gas. The resulting residue was re-dissolved in 100 mL of chloroform and passed through a previously conditioned SPE cartridge. The sample container was rinsed three times with 100 mL of chloroform and applied to the cartridge. The SPE cartridge was washed with 3 mL of chloroform and CTXs were eluted with 3 mL of chloroform:isopropanol (2:1) by gravity. The eluate was collected and dried at 65 °C under nitrogen gas. The resulting residue was reconstituted with 100 mL of methanol before LC–MS/MS analysis. 2.6.2. LC–MS/MS analysis The LC–MS/MS method used for the confirmatory analysis of C-CTX was based on previous FDA studies (Dickey, 2008; Abraham et al., 2012; Robertson et al., 2014). The LC–MS system consisted of Agilent 1260 series HPLC system (Agilent Technologies, Palo Alto, CA, USA (couple to a 4000 QTrap) equipped with a Turbo Spray ion

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source (Applied Biosystems, Inc., Foster city, CA, USA). Analyte separation was achieved on a Kinetex C8 column (75  2.1 mm, 2.6 mm particle size) equipped with a Phenomenex KrudKatcher Ultra HPLC in-line filter (Phenomenex, Torrance, CA, USA) both maintained at 40 °C. Mobile phase was water (A) and 95:5 acetonitrile:water (B) both with 5 mM ammonium formate and the flow rate was set to 0.3 mL/min. Ten microliter of sample was injected onto the column. The column gradient program started with 10% B for 1 min, linear gradient to 95% B in 1.5 min, held at 95% B for 3.5 min, and returned to 10% B in 0.2 min. The column was re-equilibrated with 10% B for 4.3 min prior to the next run. The mass spectrometer with Turbo Ion-Spray ionization was operated in positive ion mode using multiple reaction monitoring (MRM), with unit resolution set for Q1 and low resolution for Q3, and a dwell time of 175 ms. The main source parameters were as follows: source temperature, 400 °C; ion spray voltage, 5.5 kV; curtain gas, 20 psi; ion source gas 1 and 2, both set to 60 psi; and, collision gas, medium setting. Declustering, entrance, and cell exit potentials were set at 75 V, 10 V, 15 V, respectively, and collision energy at 35 eV for all ion transitions. Data analysis was performed

with Analyst software, version 1.6.1 (Applied Biosystems, Inc., Ontario, Canada). C-CTX-1 standard, obtained from the FDA, was used for structural confirmation of this congener in the samples.

3. Results 3.1. Epidemiological data and consumption data During the period from 2010 to 2012, 35 events involving a total of 87 individuals showing CFP symptoms were traced through the official reporting system in Guadeloupe. All the reported events were related to the consumption of fish caught in Guadeloupe waters. However, a thorough investigation was possible for only 12 events involving 41 illnesses (Table 1). Table 1 shows the identification number of each event, the sex and age of each individual listed anonymously, their weight when available, symptoms and their onset, and the fish species and estimated portions ingested. According to these data, the diagnosis of CFP appears to be unequivocal as regards to the polymorphic early-

Table 1 Epidemiological and consumption data (sex, age, and weight of individuals, symptoms and their onsets, implicated fish species, and portion ingested) for the 12 CFP events (n¼41 individuals) recorded in Guadeloupe (French West Indies) over 2010–2012. Symptomsa

Event

Individuals

no.

Sex (M or F)

Age (year)

Weight (in kg)

1

M F M F M F M F unkn. unkn. M M M F F unkn. unkn. unkn. M M F M F M F M M M M F unkn. unkn. unkn. unkn. unkn. unkn. unkn. unkn. unkn. F M

71 67 55 50 45 40 60 60 14 12 67 42 36 62 39 15 9 7 63 36 40 28 47 41 37 16 13 3 52 58 4 4 4 4 4 4 4 4 4 67 67

94 52 70 unkn.c 80 45 75 75 unkn. unkn. 85 101 75 128 70 unkn. unkn. unkn. 64 54 72 89 85 80 60 64 38 13 87 57 unkn. unkn. unkn. unkn. unkn. unkn. unkn. unkn. unkn. 67 83

2 3 4

5

6

7 8

9 10

11 12

50 50 50 50 50 50 50 50 50

Symptoms onset

Fish species

(h) AC, D, N, DYS, PRU AC, N, DYS, PRU DYS, PAR, GW DYS, PAR, GW AC, D AC, D AC, D, SD AC, D, SD unkn. unkn. D, V, DYS, PAR, SD D, V, DYS, PAR, SD D, V, DYS, PAR, SD D, V, DYS, PAR, SD D, V, DYS, PAR, SD SD, GW unkn. unkn. D, V, DYS, PAR D, V, DYS, PAR D, V, DYS, PAR AC, D, PAR, SD, GW AC, D, PAR, SD, GW AC, D, PAR, DYS, V AC, D, PAR, DYS, V AC, D, PAR, DYS, V AC, D, PAR, DYS, V AC, D, PAR, DYS, V N, V, PAR, GW N, V, PAR, GW AC, D, N, DYS, PAR, PRU, AC, D, N, DYS, PAR, PRU, AC, D, N, DYS, PAR, PRU, AC, D, N, DYS, PAR, PRU, AC, V, DYS, GW AC, V, DYS, GW AC, V, DYS, GW AC, V, DYS, GW AC, V, DYS, GW AC, PRU unkn.

GW GW GW GW

9.5 9.5 unkn. unkn. 6 6 6 6 unkn. unkn. 2 2 2 2 2 2 unkn. unkn. 7 7 7 3.5 3.5 unkn. unkn. unkn. unkn. unkn. unkn. unkn. 3 3 3 3 9 9 9 9 9 4.5 unkn.

Fish portion (g)

Horse eye jack (Caranx latus) Grey snapper (Lutjanus griseus) Dog snapper (Lutjanus jocu) Dog snapper (Lutjanus jocu)

Yellow fish grouper (Mycteroperca venenosa)

Blackfin snapper (Lutjanus buccanella)

Snapper (Lutjanus sp) Blackfin snapper (Lutjanus buccanella)

Snapper (Lutjanus sp) Snapper (Lutjanus sp)

Jack (Caranx sp) Yellow jack (Caranx bartholomaei)

250 400b unkn. unkn. 200 100 unkn.b unkn.b unkn. unkn. 150 150 150 150 150 unkn. unkn. unkn. 150 150 150 350 50 (fish head) 400b 300b 400b 200b 100b 100 200 150 150 150 150 unkn. unkn. unkn. unkn. unkn. 300 50 (fish head)

a description of symptoms: AC (abdominal cramps), D (diarrhea), DYS (dysesthesia), GW (general weakness), N (nausea), PAR (paraesthesia), PRU (pruritis), SD (skin disorders), V (vomiting). b in two meals. c unkn.: unknown.

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appearing symptoms. In addition, the fish species consumed correspond to those known as potentially ciguatoxic. The age of individuals, when reported, ranged from 3 to 71 years. The health examinations disclosed a variety of symptoms typically associated with CFP, in particular dysesthesias (72%, paradoxical thermal sensation) and paresthesis (64%, especially tingling of hands and feet). Diarrhoea, i.e. at least three liquid stools in a day, was reported in most of the cases (67%) accompanied with abdominal cramps (64%). Other reported symptoms were vomiting (56%), general weakness (44%), nausea (22%), skin disorders especially swelling (28%), and itching (19%). The onset of symptoms ranged from 2 to 9.5 h after consuming the fish. The estimated weights of fish portions ingested ranged from 50 to 400 g, but often the largest portions were eaten in two meals within a day. Approximately 50% of ill individuals consumed fish portions between 100 and 200 g. All the fish species involved in the events belonged to genera for which harvesting and consumption are forbidden by local regulation: Lutjanus (8 cases), Caranx (3 cases), and Mycteroperca (1 case). 3.2. Toxicity levels of CTXs in remnants Table 2 provides the levels of toxicity detected by the MBA in fish samples and their CTXs contents as estimated by the Neuro2A CBA. Among the samples that were found positive according to the MBA, 4 were qualified as “in the limit of edibility” as mice did not die but lost more than 5% of their weight in 24 h (events 1–3, 5). The Neuro-2A CBA proved to be suitable for the determination of P-CTX-1 eq. in samples in the range of concentrations tested (Fig. 1). Samples from events 8 and 12 were not analyzed for their CTXs contents because there were no remaining fish after the MBA (Table 2). The CTXs contents ranged from 0.0220 (event 1) to 0.4708 mg P-CTX-1 eq. kg
1 of fish (event 10). No obvious correlation was found when qualifying samples as “not edible” or “in the limit of edibility”, with respect to the contents of P-CTX-1 eq. as measured by the Neuro-2A CBA. For events 4, 6–7 and 9-12

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(samples not edible according to the MBA), the CTXs contents ranged ten-fold from 0.0421 (event 9) to 0.4708 (event 10) mg P-CTX-1 eq. kg
1 of fish. For events 1–3 and 5, where the MBA indicates that the samples are “in the limit of edibility”, the measured CTXs contents by the Neuro-2A CBA ranged from 0.0220 (event 1) to 0.1714 mg P-CTX-1 eq. kg
1 of fish (event 5). 3.3. Estimation of the toxin intakes and the LOAEL Where data for fish portion ingested and CTX contents were available, the toxin intakes that triggered CFP symptoms were calculated (Table 3). This information was available for 21 individuals involved in events 1, 3, 5–7, 9-11. Among them, body weight was available for 17 people. These data allowed the derivation of a LOAEL expressed either as per individual or per kg bw. The toxin intakes ranged from 4.2 to 70.6 ng P-CTX-1 eq./individual. In terms of consumption expressed per body weight, intakes ranged approximately ten-fold from 48.4 to 429.4 pg PCTX-1 eq. kg
1 bw. Fig. 2 illustrates toxin intakes expressed in pg P-CTX-1 eq. kg
1 bw and ranked in ascending order. The LOAEL of 48.4 pg P-CTX-1 eq. kg
1 bw, was determined from a 52 years old male of 87 kg having consumed a 100 g portion of contaminated snapper containing 0.0421 mg P-CTX-1 eq. kg
1 of fish (event 9). 3.4. LC–MS/MS confirmation of C-CTXs A direct analysis of the Neuro-2A CBA diethyl ether extracts was not possible without the SPE clean-up due to a high signal suppression from the matrix. The extracts cleaned up by SPE, were re-submitted for LC–MS/MS analysis and provided structural confirmation of C-CTX-1. The data were acquired by MRM where the precursor/product ion transitions were m/z 1123.6 41087.6 and 1123.64 1069.6. Samples exhibiting the highest activity for CTXs by Neuro-2A CBA with sufficient remaining sample were submitted for confirmatory analysis. C-CTX-1 was structurally confirmed in two samples by comparison of retention time and ion transitions relative to the C-CTX-1 reference standard (Fig. 3).

Table 2 Determination of toxicity level of the implicated fish samples by the mouse bioassay (MBA) and their ciguatoxin content by the Neuro-2A cell-based assay (Neuro-2A CBA). The MBA toxicity level of samples is expressed as positive (not edible or in the limit of edibility) depending on the response of the tested mice (2 mice per test). The ciguatoxin contents are expressed in mg P-CTX-1 eq. kg
1 of fish. Event no. Implicated fish species

Analysis of fish samples by MBA

Analysis of fish samples by Neuro-2A CBA

Level of toxicity

Response of mice tested (2 mice /test)

(limit of

Weight losses: 15% and 17%

expressed in mg of P-CTX-1 eq. kg
1 of fish (FW) 0.0220

(limit of

Weight losses: 18% and 20%

0.0424

(limit of

Weight losses: 14% and 19%

0.0759

(not edible) (limit of

2/2 mice dead at 5 h25 and 22 h30 Weight losses: 15% and 19%

0.1108 0.1714

(not edible)

1/2 mouse dead in the 23–24 h interval Weight loss for the surviving mouse: 15% 1/2 mouse dead in the 2–19 h interval Weight loss for the surviving mouse: 15% 1/2 mouse dead in the 21–23 h interval Weight loss for the surviving mouse: 20% 1/2 mouse dead in the 2–18 h interval Weight loss for the surviving mouse: 14% 2/2 mice dead in the 2–18 h interval 2/2 mice dead in the 1–2 h interval 1/2 mouse dead in the 1–2 h interval

0.1058

1

Horse eye jack (Caranx latus)

2

Grey snapper (Lutjanus griseus)

3

Dog snapper (Lutjanus jocu)

4 5 6

Dog snapper (Lutjanus jocu) Yellow fish grouper (Mycteroperca venenosa) Blackfin snapper (Lutjanus buccanella)

POSITIVE edibility) POSITIVE edibility) POSITIVE edibility) POSITIVE POSITIVE edibility) POSITIVE

7

Snapper (Lutjanus sp)

POSITIVE (not edible)

8

Blackfin snapper (Lutjanus buccanella)

POSITIVE (not edible)

9

Snapper (Lutjanus sp)

POSITIVE (not edible)

10 11 12

Snapper (Lutjanus sp) Jack (Caranx sp) Yellow jack (Caranx bartholomaei)

POSITIVE (not edible) POSITIVE (not edible) POSITIVE (not edible)

0.1092

Not analyzed

0.0421

0.4708 0.0548 Not analyzed

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Fig. 1. Evaluation of cytotoxicity of fish extracts and P-CTX-1 standard by Neuro-2A cells: (a) P-CTX-1 standard dose–response curve; (b) negative control of a jack fish showing no toxicity in the absence (OV
) or presence (OV þ) of ouabaine and veratridine and (c) dose–response curve of Lutjanus sp. extract from event 10. Fish extract concentration is expressed in mg/mL of tissue equivalents.

4. Discussion Guadeloupe belongs to the French West Indies and is located in the Caribbean Sea. These islands are part of the Archipelago of Antilles, a CFP-endemic area. The estimated number of individual CFP cases which occur each year in the tropics ranges from 10,000 to 50,000. However, CFP incidence significantly varies according to the geographic endemic areas. For instance, the CFP incidence is greater in South Florida and the Bahamas, and from Puerto Rico to the North of Guadeloupe, than in Martinique (Friedman et al., 2008). In the InVS and CIRE Antilles Guyane report (2013), epidemiological data from Guadeloupe and Martinique surveys showed that the CFP incidence remains at a rather low level in the French West Indies compared to other endemic areas such as the French Polynesia. The authors mentioned 13 cases a year for the period of 2004–2008, and 6–8 cases a year for 2009–2011 for Guadeloupe, whereas for Martinique a mean of 9 cases/year was recorded for 1997–2007 and around 15 cases/year since 2007 to the present. Nevertheless, the authors (InVS and CIRE Antilles Guyane report, 2013) highlighted an increase of CFP in 2012, in Guadeloupe, involving 71 individuals. However they indicated that further data would be needed to determine whether this increase might be considered as a single event or linked to the ecological

change of the marine environment. CFP has been known for centuries in the Guadeloupe area, and coastal residents and fishermen are generally aware of the risk resulting from the consumption of coral reef fish. Despite this traditional knowledge, CFP events occur almost every year. In 8 out of the 12 studied CFP cases (events 1–6, 8, 12), the fish species were reported as Caranx latus, Lutjanus jocu, Mycteroperca venenosa, and Lutjanus buccanella. In the 4 other cases (events 7, 9– 11), fish were identified only by their genus: Lutjanus sp. (n ¼3) and Caranx sp. (n ¼ 1). These are all carnivorous fish commonly involved in CFP. In the review by Pottier et al. (2001), the authors mentioned that no herbivorous fish were reported in CFP events in the Caribbean, in contrast to other seas, particularly in regions of the Pacific areas. Several other reports (Lehane and Lewis, 2000; Caillaud et al., 2010; InVS and CIRE Antilles Guyane report, 2013) have mentioned that omnivorous and herbivorous fish, such as parrotfish and surgeonfish, can contain CTXs in their flesh but would probably be less concentrated than those in carnivorous fish. More information is needed on fish feeding habits and habitats, as well as differences in the bioaccumulation or biotransformation of CTXs (InVS and CIRE Antilles Guyane report, 2013). Local regulation prohibits the sale and consumption of all the fish genera/species reported in the study, as they are known to be more likely contaminated by CTXs in Guadeloupe waters. The origin of fish supply is often informal. However, the occurrence of such CFP events illustrates that local people may underestimate the risk associated to the potentially-ciguatoxic fish species. As illustrated in our study, collection of full epidemiological and consumption data is difficult to achieve, even with very responsive official control services and good coordination between local authorities. Out of 35 CFP events reported in the last three years, only 12 events (41 individuals) were described in detail including patient descriptions, clinical symptoms, and information on the fish consumed. Among them, 8 events (21 afflicted individuals) were fully documented for the weight of the fish portion consumed. This information is crucial for calculating toxin intakes and cohort study LOAEL. There are few published data on the effect of heating or cooking on the chemical stability of CTXs in fish flesh, including on the possible leaching of toxins out of the cooked samples which may lead to improper estimation of toxicity. In studies with horse eye jack and barracuda (Pottier et al., 2001; Abraham et al., 2012), the results indicated that there was little effect of heating and/or cooking on the toxicity. In particular, Pottier et al. (2001), found no differences in toxicity in cooked and uncooked fish samples by MBA. They also found that heating samples to 120 °C did not affect the toxicity level of fish extracts. In fish samples from the Caribbean, Abraham et al. (2012) found similar cytotoxicity and toxin profiles in cooked and uncooked portions of the same fish, and that C-CTX-1 was a major contributor of composite toxicity. When describing their gastrointestinal and neurological disorders, patients did not mention any significant predominance between them. Dysesthesia which includes the feeling of hot and cold temperature reversibility, known as being typical of CFP, was described as lasting longer than gastrointestinal symptoms. In the Caribbean area, gastrointestinal symptoms are mainly seen in the acute phase, and they are rapidly followed by neurological symptoms (Dickey and Plakas, 2010). In our study, no obvious correlation was found between the severity of the symptoms and the corresponding ingested quantity of fish or toxins. Such variation in the clinical syndrome and severity of the disease has been reported, and has been linked to human factors such as individual patient variation, ethnic group, prior exposure to CTXs, combined action of several toxins, physical and dietary behaviors, and subjective descriptions by patients (Bagnis et al., 1979; Lehane and Lewis, 2000; Dickey and Plakas, 2010). It is also worth noting that

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Table 3 Estimation of toxin intakes for 21 individuals from events 1, 3, part of 5, 6–7, 9-11 where relevant details were available for LOAEL estimation (ciguatera fish poisoning, Guadeloupe, French West Indies, 2010–2012). Event no. Individual’s age (year) Individual’s weight (kg) Fish ingested (g) CTXsa contents (mg P-CTX-1 eq. kg
1 of fish

Toxin intakes

ng P-CTX-1 eq./ individual 5.5 8.8

pg P-CTX-1 eq./ kg bwb 58.5 169.2

1

71 67

94 52

250 400

0.0220

3

45 40

80 45

200 100

0.0759

15.2 7.6

189.8 168.7

5

62 39 67 42 36

128 70 85 101 75

150 150 150 150 150

0.1714

25.7 25.7 25.7 25.7 25.7

200.9 367.3 302.5 254.6 342.8

6

63 36 40

64 54 72

150 150 150

0.1058

15.9 15.9 15.9

248.0 293.9 220.4

7

28 47

89 85

350 50

0.1092

38.2 5.5

429.4 64.2

9

58 52

57 87

200 100

0.0421

8.4 4.2

147.7 48.4

10

450 450 450 450 67

unkn. unkn. unkn. unkn. 67

150 150 150 150 300

0.4708

70.6 70.6 70.6 70.6 16.4

245.4

11 a b

0.0548

CTXs: Ciguatoxins; the contents were determined by the Neuro-2A CBA. bw: body weight.

Fig. 2. Toxin intakes in a series of CFP events in Guadeloupe, 2010–2012, and the estimation of the LOAEL. Toxin intakes are ranked from lowest to highest estimated values expressed in pg P-CTX-1 eq./kg bw from the 17 individual cases fully documented.

in this study, two individuals got ill after eating only the head of the fish corresponding to a small flesh quantity (events 7 and 12, Table 1). This is in accordance with the fact that the head and viscera have been reported to contain the highest toxin levels

(Dickey and Plakas, 2010;, Hossen et al., 2013). For event 2 where Lutjanus griseus was involved, the two ill people exhibited typical CFP symptoms such as dysesthesia, paresthesia and general weakness. The implicated fish analyzed by the MBA was found at the limit of edibility and estimation of its CTXs content by the Neuro-2A CBA showed a rather low but not insignificant level of 0.0424 mg P-CTX-1 eq. kg
1 of fish. Indeed, L. griseus even though not registered among the “forbidden” species in the local official list, belongs to a genus where other species are often involved in CFP. In this study, 10 fish samples (events 1–7 and 9–11) were found positive with MBA and their CTXs contents were determined by the Neuro-2A CBA. The most cytotoxic samples (from two events) were submitted for LC–MS/MS analysis and Caribbean ciguatoxin congener C-CTX1 was structurally confirmed. There was a clear relationship between the clinical diagnosis, the ciguatoxic potential of the genus/ species, the toxic symptoms and CTXs content in the fish samples. Moreover, it should be emphasized that 3 out of the 7 samples recorded as “in limit of edibility” by MBA were implicated in illnesses. The lowest concentration of toxins in fish responsible for intoxication in our study was found at 0.0220 mg P-CTX-1 eq. kg
1 of fish. These values are in agreement with the conclusions of the European Food Safety Authority (EFSA, 2010) indicating that a content of 0.01 mg P-CTX-1 eq. kg
1 fish is expected to exert no effect in sensitive individuals. Even if not very sensitive (Wong et al., 2008), the MBA has demonstrated its usefulness to screen for toxic samples. However, the Neuro-2A CBA displays a suitable detection capability for assessing composite toxicity, even if it does not provide any information on the toxin profile, but combined

Please cite this article as: Hossen, V., et al., Contribution to the risk characterization of ciguatoxins: LOAEL estimated from eight ciguatera fish poisoning events in.... Environ. Res. (2015), http://dx.doi.org/10.1016/j.envres.2015.09.014i

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Fig. 3. Extracted ion chromatogram of C-CTX-1 standard (top) and a representative fish extract of Lutjanus sp. from event 10 (bottom), showing two precursor/product confirmatory ion transitions.

with LC–MS/MS analysis, toxin profile can be obtained and CFP may be confirmed. The present study contributes to the risk assessment of CTXs in fish by providing information on the toxin intakes that have produced typical symptoms of CFP. While a limited set of data was available, a LOAEL of 4.2 ng eq. P-CTX-1/ individual was calculated, corresponding to 48.4 pg P-CTX1 eq. kg
1 bw. As for events 1 and 7, the toxin intakes of 58.5 and 64.2 pg P-CTX-1 eq. kg
1 bw, respectively, which triggered CFP symptoms, are slightly above the calculated LOAEL in this report. It should be emphasized that uncertainty is associated with calculation of LOAEL values, because of various potential sources; however, calculation of uncertainty is not always quantifiable (EFSA, 2010). In our set of data, the main source of uncertainty that was identified lies in consumption data, such as fish portion and details on preparation of fish meals. These uncertainties should be considered with respect to the calculated LOAEL, and emphasizes the need for similar studies in the risk assessment process, as recommended by EFSA (2010). However, to our knowledge, this is the first time that a LOAEL for CTXs was derived from human case reports involving epidemiological, consumption, and analytical data sets.

Funding sources The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/20072013) under the ECsafeSEAFOOD project (Grant agreement no. 311820). URV-IRTA-Banco de Santander also has delivered a PhD. Grant to Lucia Soliño, one of the co-authors.

Acknowledgments The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/20072013) under the ECsafeSEAFOOD project (Grant agreement no. 311820). We would like to acknowledge URV-IRTA-Banco de Santander PhD. grant to Lucia Soliño.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.envres.2015.09.014.

Please cite this article as: Hossen, V., et al., Contribution to the risk characterization of ciguatoxins: LOAEL estimated from eight ciguatera fish poisoning events in.... Environ. Res. (2015), http://dx.doi.org/10.1016/j.envres.2015.09.014i

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