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FAO and WHO. 2020. Report of the Expert Meeting on Ciguatera Poisoning. Rome, 19–23 November 2018. Food Safety and Quality No. 9. Rome. https://doi.org/10.4060/ca8817en.

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CONTENTS

Tables and figures

Contributors

Abbreviations and acronyms

Declarations of interest

Executive summary

CHAPTER 1
INTRODUCTION

1.1 Background

1.2 Objectives

1.3 Meeting approach

CHAPTER 2
OCCURRENCE OF CAUSATIVE ORGANISMS AND CTXs

2.1 Occurrence of Gambierdiscus species

2.2 Distribution of CP causative organisms

2.3 Methods for species identification

2.3.1 Polymerase chain reaction (PCR)

2.3.2 Fluorescence in situ hybridization (FISH) probes

2.3.3 Restriction fragment length polymorphism (RFLP) typing

2.3.4 High-throughput sequencing (HTS) metabarcoding

2.4 Sampling strategies

2.5 Gambierdiscus abundance

2.6 Toxicity of CP causative organisms

2.6.1 Detection of CTXs in Gambierdiscus and Fukuyoa and in the environment

2.7 Occurrence of CTXs in seafood

CHAPTER 3
CHEMISTRY AND BIOSYNTHETIC PATHWAYS

3.1 Chemistry

3.1.1 Structures

3.1.2 Chemical characteristics

3.1.3 Biosynthetic pathways

3.2 Ciguatoxin classification

CHAPTER 4
DETECTION METHODOLOGIES FOR CTXs

4.1 Screening assays for ciguatoxins

4.1.1 In vitro assays

4.1.1.1 N2A-MTT assay for ciguatoxins

4.1.1.2 Receptor binding assay (RBA) for ciguatoxins

4.1.1.3 Immunological assays

4.1.2 In vivo assays

4.2 Structure-based confirmatory methods

4.3 Concluding remarks

CHAPTER 5
HAZARD IDENTIFICATION

5.1 Absorption, distribution, metabolism and excretion (ADME)

5.1.1 Animal data

5.2 Toxicological studies

5.2.1 Acute toxicity

5.2.2 Short-term studies

5.2.3 Medium-term studies

5.2.4 Long-term studies

5.2.5 Repeated versus acute toxicity

5.2.6 Conclusion from toxicological studies in rodents

5.3 Mode of action

CHAPTER 6
HUMAN DATA

6.1 ADME

6.2 Clinical features and toxicity

6.2.1 Acute symptoms

6.2.2 Chronic symptoms

6.2.3 Acute recurrence/exacerbation of ciguatera symptoms

6.2.4 Lethality

6.3 Epidemiology

6.4 Treatments

CHAPTER 7
EXPOSURE ASSESSMENT

7.1 Introduction

7.2 Detection frequency of CTXs and levels of contamination

7.3 Consumption

7.3.1 Mean estimates of fish consumption

7.3.2 High percentile estimates of fish consumption

7.4 Ciguatoxin doses causing Ciguatera poisoning

7.5 Dietary exposure to ciguatoxins

CHAPTER 8
CONCLUSIONS

8.1 Risk management considerations

REFERENCES

ANNEX
CP FLOW DIAGRAM, AND RESEARCH PROJECTS

Research projects

Ciguatools

Eurocigua

AlertoxNet

TABLES

1. Biological activity reported in Gambierdiscus and Fukuyoa species

2. Relative ciguatoxin-like toxicities of Gambierdiscus and Fukuyoa species reported to date

3. Minimum fish weight to require CTX analysis in the Canary Islands, Spain

4. Fish and marine invertebrates reported to bioaccumulate CTXs and their locations

5. Classification of CTXs and abbreviation synonyms

6. Gambierdiscus metabolites other than CTXs

7. Available screening assays and confirmation methods

8. Acute toxicity and relative potency of CTXs

9. Common chronic symptoms of ciguatera poisoning.

10. Factors contributing to the recurrence or exacerbation of symptoms

11. Ciguatera as a global health problem

12. Highest incidence rates per 10 000 population in selected locations

13. Poisoning cases with causative fish and toxins

14. Summary of studies on the ciguatoxin content of marine fish

15. Scenario-based estimation of acute dietary CTX exposure

16. Marine species in specific regions that are known to exhibit high site fidelity

FIGURES

1. Gambierdiscus: G. belizeanus (A), G. australes (B), G. caribaeus (C)

2. Gambierdiscus and Fukuyoa occurrence in the Caribbean and adjacent seas

3. Gambierdiscus and Fukuyoa occurrence in Macaronesia, Europe and the Near East

4. Gambierdiscus and Fukuyoa occurrence in the Pacific Ocean, N=162

5. Global Gambierdiscus and Fukuyoa occurrence from published records, 2009-2018

6. Mean Gambierdiscus abundances from 46 studies

7. Ciguatoxin toxin profiles found in the microalga Gambierdiscus polynesiensis, in the herbivorous fish Chlorurus microrhinos (ex. Scarus gibbus) and in the carnivorous fish Gymnothorax javanicus

8. Ciguatoxin classification

9. Gambierdiscus metabolites other than CTXs

10. Example of reported acute symptoms of ciguatera poisoning

11. Distribution of doses of CTX1B equivalents associated with CP cases in Guadeloupe

A1. Flow of CP responses and needs

CONTRIBUTORS

EXPERTS

Ann Abraham, Division of Seafood Science and Technology, United States Food and Drug Administration

Nathalie Arnich, Risk Assessment Department, French Agency for Food, Environmental and Occupational Health and Safety (ANSES), France

Mireille Chinain, Institut Louis Malardé, Laboratoire de Recherche sur les Biotoxines Marines, French Polynesia

Peter Cressey, Institute of Environmental Science and Research Limited, New Zealand

Marie-Yasmine Dechraoui Bottein, Radioecology Laboratory of the IAEA Environment Laboratories, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Monaco

Jorge Diogène Fadini, IRTA, Marine and Continental Waters Program, Spain

Miriam Friedemann, German Federal Institute for Risk Assessment, Department Exposure, Centre for Documentation and Assessment of Poisonings, Germany

Ana Gago-Martínez, Universidad de Vigo, Department of Analytical and Food Chemistry, Spain

Clémence Gatti, Institut Louis Malardé, Laboratoire de Recherche sur les Biotoxines Marines, French Polynesia

Arjen Gerssen, RIKILT Wageningen University and Research, Netherlands

Matthew O. Gribble, Department of Environmental Health, Department of Epidemiology, Emory University Rollins School of Public Health, United States of America

Tim Harwood, Cawthron Institute, New Zealand

Philipp Hess, Ifremer, Atlantic Center, Laboratoire Phycotoxines, France

James M Hungerford, Applied Technology Center, Pacific Northwest Laboratory, Office of Regulatory Affairs, United States Food and Drug Administration, USA

Iddya Karunasagar, Department of International Relations, NITTE University, India

Raphaёle Le Garrec, Univ Brest, LIEN, F-29200 Brest, France

Naomasa Oshiro, Marine Biotoxin Laboratory, Division of Biomedical Food Research, National Institute of Health Sciences, Japan

Alison Robertson, Marine Ecotoxicology Laboratory, Department of Marine Sciences, University of South Alabama and Dauphin Island Sea Laboratory, United States of America

Patricia Tester, Ocean Tester, United States of America

Martin van den Berg, Institute for Risk Assessment Sciences, Utrecht University, Netherlands

Jean-Paul Vernoux, Normandy University, UNICAEN, ABTE Research Team, France

OBSERVERS

Henrik Enevoldsen, Intergovernmental Oceanographic Commission of UNESCO, IOC Science and Communication Centre on Harmful Algae, Marine Biological Section, University of Copenhagen

SECRETARIAT

Verna Carolissen, Codex Secretariat, Joint FAO/WHO Food Standards Programme, Food and Agriculture Organization of the United Nations

Gracia Brisco, Codex Secretariat, Joint FAO/WHO Food Standards Programme, Food and Agriculture Organization of the United Nations

Vittorio Fattori, FAO Secretariat, Food Safety and Quality Unit, Agriculture and Consumer Protection Department, Food and Agriculture Organization of the United Nations

Markus Lipp, FAO Secretariat, Food Safety and Quality Unit, Agriculture and Consumer Protection Department, Food and Agriculture Organization of the United Nations

Esther Garrido Gamarro, FAO Secretariat, Fisheries and Aquaculture Department, Food and Agriculture Organization of the United Nations

Angelika Tritscher, WHO Joint Secretary, Department of Food Safety and Zoonoses, World Health Organization

Lisa Scheuermann, WHO Joint Secretary, Department of Food Safety and Zoonoses, World Health Organization

Luis M. Botana, Department Pharmacology, Faculty of Veterinary Medicine, Campus Lugo, University of Santiago de Compostela, Spain

ABBREVIATIONS AND ACRONYMS

ADME absorption, distribution, metabolism and excretion

AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

C-CTX Caribbean ciguatoxin

CBA cell bioassay

CDC Centers for Disease Control and Prevention

CP ciguatera poisoning

CIFOCOss FAO/WHO Chronic Individual Food Consumption database

CTX ciguatoxin

EFSA European Food Safety Authority

ELISA enzyme-linked immunosorbent assay

FAO Food and Agricultural Organization of the United Nations

FDA Food and Drug Administration (the United States of America)

FISH fluorescence in situ hybridization

GABA gamma amino butyric acid

GEADE Global Estimate of Acute Dietary Exposure

HRMS high-resolution mass spectrometry

HTS high-throughput sequencing

I-CTX Indian Ocean ciguatoxin

IAEA International Atomic Energy Agency

ICR Institute of Cancer Research

IESTI, International Estimate of Short-term Intake

InsP3 inositol triphosphate

ip intraperitoneal

iv intravenous

JECFA Joint FAO/WHO Expert Committee on Food Additives

LC-MS liquid chromatography coupled with mass spectrometry

LD lethal dose

LOAEL lowest observed adverse effect level

LOD limit of detection

LOQ limit of quantitation

MBA mouse bioassay

MTT 3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyltetrazolium bromide

MTX maitotoxin

MU mouse unit

N2A (N2a) mouse neuroblastoma assay

NMDA N-methyl-D-aspartic acid

NOAA National Oceanic and Atmospheric Administration

NOAEL non-observable adverse effect level

P-CTX Ciguatoxins (CTX) initially isolated from biota from the pacific region

PbTx brevetoxin

PCR polymerase chain reaction

PICTs Pacific island countries and territories

PKS polyketide synthase

PSS Poison Severity Score

qPCR quantitative polymerase chain reaction

RASFF Rapid Alert System for Food and Feed

RBA receptor binding assay

RFLP restriction fragment length polymorphism

SEM scanning electronic microscopy

SP substance P

SPATT solid phase adsorption toxin tracking

STX saxitoxin

TEF toxicity equivalency factor

TRPA1 transient receptor potential ankyrin 1

TTX tetrodotoxin

UHPLC ultra-high-pressure liquid chromatography

Vd volume of distribution

VGSC voltage-gated sodium channel

VGPC Voltage-gated potassium channel

WHO World Health Organization

DECLARATIONS OF INTEREST

All participants in the Joint FAO/WHO Expert Meeting on Ciguatera Poisoning completed a declaration of interest form in advance of the meeting. In relation to the subject of this Expert Meeting, the following declarations were made: (i) Ann Abraham, James M. Hungerford and Clémence Mahana declared having paid employment; (ii) Mireille Chinain, Ana Gago-Martínez, David Timothy Harwood, Philipp Hess, Iddya Karunasagar and Clémence Mahana declared having received or anticipating research support; (iii) Marie-Yasmine Dechraoui Bottein, Jorge Diogene, Ana Gago-Martinez, David Timothy Harwood and Clémence Mahana reported participating in an expert committee or scientific advisory group; (iv) Peter Cressey and Jorge Diogene have provided expert opinion or testimony as part of a regulatory, legislative, judicial, or other governmental process; and (v) David Timothy Harwood indicated knowledge of a technology that could be impacted by the outcome of the meeting.

Following the FAO Guidance Document for Declaration of Interests, the declarations noted above were assessed as to the extent to which each interest could be reasonably expected to affect and exercise influence on the experts’ judgement. The declared interests of Ann Abraham, Mireille Chinain, Peter Cressey, Marie-Yasmine Dechraoui Bottein, Jorge Diogene, Ana Gago, David Timothy Harwood, Philipp Hess, James M. Hungerford, Iddya Karunasagar and Clémence Mahana were considered unlikely to impair their objectivity or have a significant influence on the impartiality, neutrality and integrity of the work. Meeting participation by these individuals was neither reasonably expected to create unfair competitive advantages, nor were the meeting outcomes reasonably foreseen to affect the individuals’ declared interests. Neither FAO nor WHO received any public comments in response to the online posting of the names and brief biographies of the individuals considered for participation in the Expert Meeting. The interests of all participants were disclosed to all attendees at the beginning of the Expert Meeting.

EXECUTIVE SUMMARY

Ciguatera poisoning (CP) is reported in historical documents of the sixteenth century. The first report of the organism Gambierdiscus (originally referred to as Goniodoma sp.) dates from October 1948, in Cabo Verde. Today, the term ciguatera identifies poisoning caused by the ingestion of certain reef fish and shellfish from tropical and subtropical regions, especially the South Pacific Ocean, Indian Ocean and the Caribbean Sea. Through the food chain, these fish and shellfish have accumulated certain lipid-soluble toxins (ciguatoxins [CTXs]) that are produced by dinoflagellates of the genera Gambierdiscus and Fukuyoa. Ciguatera is a worldwide problem that is expanding due, among other reasons, to climate change. In general, CP can be regarded as the most significant non-bacterial poisoning associated with fish consumption worldwide. A typical sign of the poisoning is cold allodynia, and there are more than 175 gastrointestinal, cardiovascular and neurological symptoms. It is unclear whether the toxins cause harm to those herbivorous or carnivorous fish that take them through the food chain.

In 2016, at the Thirty-Second Session of the Codex Committee on Fisheries and Fishery Products, the Pacific Nations raised CP as an issue that is increasingly affecting the tropical and subtropical regions of the Pacific Ocean, Indian Ocean and Caribbean Sea between the latitudes of 35°N and 35°S. Indeed, it was noted that, due to climate change, the frequency of storms and hurricanes is increasing, as is the sea surface temperature, which affects the distribution and proliferation of CTXs and makes the occurrence of CP less predictable. The issue of CP was raised at the Eleventh Session of the Codex Committee on Contaminants in Food. The Committee agreed to request scientific advice from FAO/WHO to enable the development of appropriate risk management options, in particular: full evaluation of known CTXs (toxicological assessment and exposure assessment), including geographic distribution and rate of illness, congeners, and methods of detection; and guidance for the development of risk management options.

There are now 16 described Gambierdiscus species: G. australes, G. balechii, G. belizeanus, G. caribaeus, G. cheloniae, G. carpenteri, G. carolinianus, G. excentricus, G. pacificus, G. polynesiensis, G. scabrosus, G. toxicus, G. silvae, G. lapillus, G. honu and G. jejuensis. Recently, two globular species Gambierdiscus have been reclassified as Fukuyoa (F. yasumotoi and F. ruetzleri), and a new species described (F. paulensis). Both F. ruetzleri and F. paulensis produce toxins. Optimum growth takes place between 26.5 °C and 31.1 °C, with thermal limits from 15–21 °C to 31–34 °C, and salinities from 24.7 g/litre to 35 g/litre with light irradiances below 231 μmol photons per square metre per second. A variety of techniques can be considered for the identification of species at any given site, including optical microscopy as a screening tool, and scanning electronic microscopy (SEM) and/or molecular techniques (sequencing, PCR, RFLP, FISH probes, etc.) as confirmation tools.

Gambierdiscus cells are distributed in a very patchy manner; coefficients of variation among adjacent samples range from 50 percent to > 150 percent. The frequency distributions of average cell densities are similar in both the Atlantic and Pacific. Ten percent of the abundance estimates are between 1 000 cells/g wet weight algae and 10 000 cells/g wet weight algae, with 5 percent exceeding 100 000 cells/g wet weight algae.

More than 425 species of fish have been linked to ciguatera events. Coral reef fishes contribute to the expansion of CP intoxications. Reef fish known to potentially accumulate these toxins are: barracuda (Sphyraenidae), amberjack (Seriola), grouper (Serranidae), snapper (Lutjanidae), po’ou (Labridae spp.), jack (Carangidae spp.), trevally (Caranx spp.), wrasse (Labridae spp.), surgeon fish (Acanthuridae spp.), moray eel (Muraenidae spp.), roi (Cephalopholis spp.), and parrotfish (Scaridae spp.). A large variety of marine invertebrates including urchins, gastropods, bivalves and echinoderms have also been reported to contain CTXs, but their implication in CP is far less important than fish. Due to world trade, and consumption of imported fish there are poisoning reports in many geographic areas, such as Canada, Germany (e.g. Hamburg), the Paris area (France) and California, New York, Rhode Island and Vermont (the United States of America), and also CP has been reported after returning to their countries by patients having consumed ciguateric fish in endemic areas. Global warming is facilitating the expansion of Gambierdiscus, but there are bodies of water that are warm enough to depress their growth. Several reports indicate the presence of Gambierdiscus in new areas (Brazil, Morocco or Thailand), but there is no solid link yet to this being caused by climate change. Another subject not yet clarified is how the change in pH and carbon dioxide (CO2) levels and in sea surface temperatures may affect toxin production, as the growth of microalgae is influenced by these parameters. The approach followed by many countries is to impose fish size restrictions as a ciguatera risk management action, but the toxicity of some species is associated to seasonal variations and for most species, there is no proven correlation between the toxicity of fish and their size/weight.

Ciguatoxins (CTXs) are a class of large polyether ladder-like lipid-soluble compounds that are thermostable and resistant to mild pH changes; they contain 13–14 fused rings. Representative backbone structures of CTXs identified to date are represented by CTX4A, CTX3C and C-CTX1. The diastereoisomers 52-epi-54-deoxyCTX1B and 54-deoxyCTX1B (also known as CTX2 and CTX3, respectively) represent less oxidized forms of CTX1B. The only difference between CTX1B, 52-epi-54-deoxyCTX1B and 54-deoxyCTX1B involves modification at one end of the CTX. The backbone structure of CTX3C toxins differs from CTX4A group on the E-ring (i.e. an eight-membered ring in CTX3C and a seven-membered ring in CTX4A) and by the absence of an aliphatic side chain on the A-ring. Several toxic Caribbean CTX analogues have been identified and isolated from fish. The major Caribbean toxin is C-CTX1 and its 56-epimer C-CTX2. C-CTX2 was found as a minor analogue in fish. The C-CTX backbone shares characteristics with CTX4A and CTX3C but does not possess the aliphatic side chain on the A-ring and it contains an additional ring on the right wing of the molecule. Additional Caribbean CTXs have been reported in several studies but have yet to be structurally elucidated. An Indian Ocean group has also been described, and although masses have been reported in the literature, no molecular structures have been determined to date. The most toxic analogue described to date is CTX1B, which is stable at 100 °C, 1 N NaOH, and in sunlight for 1 h, but loses toxicity in 1 N HCl after 10 min. Ciguatoxins are odourless, tasteless, heat stable and present at very low (typically < ppb) levels in contaminated seafood, making them difficult to detect without advanced detection methods. The toxic potency of CTXs has been shown to increase as they become more oxidized. Analogues isolated from the Pacific are currently thought to be the most potent and have been well characterized. Some CTXs are metabolites generated through the process of enzyme-mediated biotransformation in invertebrates and fish. Experimental CTX1B oral and intraperitoneal dosing studies in mice have confirmed the rapid absorption capacity of CTXs, and their ability to cross the blood–brain barrier. There is a similar compartmentalization of toxins across tissues into liver, spleen, brain, muscle, gonads, fat and bone. A remarkable feature of Gambierdiscus is its unique biochemical machinery, responsible for the production of multiple structurally complex polyether toxins, including CTXs, gambierol, gambierone, gambieroxide, gambieric acids and maitotoxins. Three families of CTXs have commonly been classified according to their geographical location, i.e. Pacific CTXs, Caribbean CTXs (C-CTXs) and Indian Ocean CTXs (I-CTXs). However, it is now possible and appropriate to classify CTXs based on the known chemical structures. The structural characteristics of CTXs actually allow further classification into two separate groups based on their chemical structure, i.e. CTX3C vs CTX4A backbones and their derivatives. The Caribbean CTXs represent a third group. To date, only two Caribbean CTXs have been structurally elucidated (C-CTX1 and C-CTX2). A classification into five groups has been suggested, and this report uses this classification: CTX3C, CTX4A, C-CTX, I-CTX (the existence of this group is still speculative as the structure elucidation is pending), and other Gambierdiscus metabolites.

None of the methods described has been reported to have undergone single- or multi-laboratory validation. While some laboratories have produced reference materials and quantified standards on a small scale, recent data have most consistently reported CTX3C equivalents, owing to commercial availability. Several assays for the screening of fish samples for CTXs have been described, based on in vitro assays (N2A-MTT assay, immunoassays, receptor binding assay [RBA]) and in vivo (mouse) bioassays. These in vitro assays all have high throughput capacity due to the 96-well plate formats used, which allow parallel measurements at the respective endpoints of each assay. However, reliable quantification of the bioactivities and/or toxicity of CTXs in seafood extracts requires a certified reference and, where possible, a matrix-matched reference. Liquid chromatography coupled with mass spectrometry (LC-MS) is a suitable approach for the identification and confirmation of CTXs and related compounds in a range of matrices.

Information on fish consumption, particularly in countries with high rates of CP, is fragmentary. The estimated doses eliciting CP are in the range 48.4–429 pg/kg bw CTX1B equivalents. The minimum eliciting dose of 48.4 pg/kg bw CTX1B equivalents provides an estimate of a lowest observed adverse effect level (LOAEL) for CP in humans. Both the United States Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) have proposed a fish CTX concentration of 0.01 μg CTX1B/kg fish flesh as being unlikely to elicit symptoms of CP. This concentration is just below the lowest concentrations seen in fish samples associated with CP cases (0.02 μg CTX1B equivalents/kg fish flesh). As documented by mouse ip LD_50, variability in the potencies exists between some different analogues of CTXs. On the basis of LD_50 estimates in mice, oral CTX1B (also known as CTX1) (0.22 μg/kg bw) was similar in potency to ip CTX1B (0.25 μg/kg bw). CTX1B and CTX4C have a cumulative effect on the cardiac tissue at a dose of 0.1 μg/kg bw for 15 days. A medium term low dose CTX1B exposure impairs spatial learning and reference memory in rats. Repeated exposures of rats (every 3 days for 8 weeks) to a low dose of CTX1B (0.065 μg/kg bw) after an initial high dose (0.26 μg/kg bw) leads to the development of anxiety-like behaviour learning and memory deficits, and decision-making impairment. Neurotoxic effects in rats are similar to symptoms reported in humans. After about 18 weeks, mice treated with 0.1 μg/kg bw show hypertrophy and histological changes in the heart. No effects are observed in mice treated with 0.05 μg/kg bw. A non-observable adverse effect level (NOAEL) for heart toxicity is 0.05 μg/kg bw (after 40 weeks, once a week). None of these studies were considered suitable to establish a health-based guidance value (acute or chronic). Human data allowed identification of a LOAEL of 50 pg CTX1B/kg bw after actute exposure.

The main target of CTXs is the voltage-gated sodium channel (VGSC, Nav), causing hyperexcitability of the nerve membrane, eliciting spontaneous and repetitive action potentials by interacting with receptor-site 5 of the alpha subunit pore of the VGSC. Ciguatoxins show affinity for all the VGSC isoforms (Nav 1.1–1.9), with differences in potency between the different toxin types. CTX binding to the VGSC causes a shift in voltage dependence of Na+ conductance to more negative membrane potentials, allowing an increase in Na+ influx and spontaneous action potential firing. The overall effect is an increase in excitability. This action, especially in peripheral nerves where binding of CTXs to Nav is long-lasting, explains most of the effects of the group.

Data on CTX toxicokinetics in humans are very limited. Many ciguatera cases express central nervous system symptoms, suggesting that CTXs can enter the brain. Ciguatoxins have been measured in blood several hours after ingestion. However, toxins may not persist long in blood as they are undetectable in serum, plasma or urine 90 hours after poisoning. Ciguatoxins have been detected in human liver in an autopsy of a lethal case six days after fish consumption. Case reports describing symptoms among infants of ciguatera-affected mothers suggest that women may eliminate toxins via breast milk, and that toxins may be resorbed through breast milk. Transplacental toxin transfer is possible. The consumption of ciguatoxic fish is followed by the onset within 48 hours of specific, incident neurological symptoms: cold allodynia (which may be considered as nearly pathognomonic), paraesthesia, dysaesthesia, pruritus, myalgia, arthralgia and/or dizziness.

Unspecific symptoms such as severe fatigue and any kind of pain (e.g. myalgia, arthralgia and dentalgia) are very common. More than 175 different symptoms have been reported to date. Chronic ciguatera symptoms are those that persist beyond three months after the initial poisoning, and concern at least 20 percent of ciguatera-affected persons. Ciguatera poisoning may have neurological, psychiatric and/or general symptoms that can persist for months or years after the initial poisoning. There is no specific treatment. The fatality rate has been estimated as < 0.5 percent, but in some contexts may exceed 10 percent. Death due to CTX exposure often follows cardiovascular and/or complications of the central nervous system. It might be preventable by avoiding consumption of fish heads, liver and viscera, or possibly through better clinical management practices. If, as suspected, only 10–20 percent of actual intoxications are formally reported to the authorities, the problem of CP poisoning is much larger than official figures show.

Although there are many gaps in the available information about CP, there are some needs that require urgent attention regarding both risk management and research. The main needs for risk management are for the definition of clear protocols to avoid the risk of consuming toxic fish, mainly by local people and tourists, but also consumers purchasing imported fish from certain areas. This includes a well-defined information and outreach programme, and a clear identification of the geographic distribution of fish and causative organisms. The main research needs refer to detection methods, both screening and analytical, and the need to have a stable supply programme of analytical standards.

CHAPTER 1

INTRODUCTION

1.1 BACKGROUND

The first historical event involving a ciguatera poisoning was reported in 1521, it affected several captains of the Spanish army in the Gulf of Guinea and led to the death of Juan Sebastian Elcano and others (Urdaneta, 1580; Baeza, 2009). Captain Cook’s crew was also poisoned in 1786, as described by the ship’s surgeon (Pearn, 1994). In 1787, Parra described the neurological symptoms after ingestion of a local gastropod (Livona sp.) in the Antilles, calling the poisoning “siguatera” [cited in Lee, 1980]). The first report about the toxin-producing organism Gambierdiscus (originally referred as Goniodoma sp.) dates from October 1948, in Cabo Verde (Sousa e Silva, 1956). In 1955, Martyr described (Gudger, 1930; Holmes, Brust and Lewis, 2014) Ciguatera poisoning (CP) as the term used for an poisoning originally named in Cuba after the ingestion of Turbo pica, a marine snail called cigua by local people (Guzmán-Pérez and Park, 2000; Gudger, 1930).

Today, the term ciguatera identifies an poisoning syndrome caused by the ingestion of certain reef fish and shellfish from tropical and subtropical regions, especially South Pacific, Indian Ocean and the Caribbean. These fish and shellfish have accumulated certain toxins (ciguatoxins [CTXs]) through the food chain. These lipid-soluble toxins are produced by dinoflagellates of the genus Gambierdiscus and Fukuyoa (Vlamis and Katikou, 2014). Ciguatera is a worldwide problem, in some countries it is considered a globally neglected tropical disease that is expanding due, among other reasons, to climate change (Gingold, Strickland and Hess, 2014; Rhodes et al., 2014a; Hallegraef, 2015). Mild cases of palytoxin poisoning have been wrongfully identified as ciguatera (Lewis and Holmes, 1993), but in general CP can be regarded as the most significant non-bacterial poisoning associated with fish consumption worldwide (Lewis, Molgó and Adams, 2000). A typical sign of the poisoning is cold allodynia, and there are more than 175 gastrointestinal, cardiovascular and neurological symptoms (Gatti, Oelher and Legrand, 2008). It is unclear whether the toxins cause harm to those herbivorous or carnivorous fish that take them through the food chain, but there is evidence that they may also affect fish nerves (Flowers, Capra and Cameron, 1992).

More than 425 species of fish have been linked to ciguatera events (Perez-Arellano et al., 2005). Coral reef fishes are a premium sea product with global distribution, which contributes to the expansion of CP intoxications. Reef fish commonly involved in ciguatera are: barracuda (Sphyraenidae), amberjack (Seriola), grouper (Serranidae), snapper (Lutjanidae), po’ou (Labridae spp.), jack (Carangidae spp.), trevally (Caranx spp.), wrasse (Labridae spp.), surgeon fish (Acanthuridae spp.), moray eel (Muraenidae spp.), roi (Cephalopholis spp.), and parrotfish (Scaridae spp.) (FDA, 2011). Gambierdiscus are different from other open-water dinoflagellates in that they flourish in calm and protected locations, such as coral reefs or atolls, where water movement is low. For this reason, destruction or alteration of the reefs has also been associated with an increase in CP events, as the spread of Gambierdiscus allows a wider dissemination to the food chain (Rongo and van Woesik, 2013; Bagnis, 1994; Dickey, 2008).

1.2 OBJECTIVES

In 2015, FAO organized an interagency meeting to discuss CP as an increasing food safety threat. At the meeting, a plan of action was defined and the need for international-level guidance was identified.

At the Thirty-second Session of the Codex Committee on Fisheries and Fishery Products (2016), the Pacific Nations raised CP as an issue that increasingly affects the tropical and subtropical regions of the Pacific Ocean, Indian Ocean, and Caribbean Sea, between the latitudes 35°N and 35°S. Indeed, it was noted that, due to climate change, the frequency of storms and hurricanes is increasing, as is the sea surface temperature, which affects the distribution and proliferation of CTXs and makes the occurrence of CP less predictable.

In addition to climate change, globalization of trade might also contribute to the spread of CTXs. As such, further guidance might be needed for those countries that have not previously considered CTXs in their risk management programmes.

The matter of CP was raised at the Eleventh Session of the Codex Committee on Contaminants in Food. The Committee agreed to request scientific advice from FAO/WHO to enable the development of appropriate risk management options. In particular, the requested scientific advice of FAO/WHO entails:

> full evaluation of known CTXs (toxicological assessment and exposure assessment), including geographic distribution and rate of illness; congeners; and methods of detection;

> based on this, guidance for the development of risk management options.

1.3 MEETING APPROACH

The Expert Meeting was jointly organized by FAO and WHO. Both organizations posted a public call for data and a call for experts on their respective websites, and distributed the calls widely to all Codex contact points and elsewhere.

Applications to the call for experts were screened for relevant experiences, and the most suitable experts were identified considering the need for a gender-balanced representation of all regions. Selected experts were submitted to a process of evaluation of potential conflict of interest as per applicable FAO and WHO policies. No significant interests have been identified by the experts attending the meeting. All data received were made available to the experts and informed the discussions during the meeting and this report (main text and annex).

CHAPTER 2

OCCURRENCE OF CAUSATIVE ORGANISMS AND CTXs

2.1 OCCURRENCE OF GAMBIERDISCUS SPECIES

Ciguatera poisoning is a tropical disease, endemic between latitudes 35°N and 35°S, but the occurrence of ciguatoxic fish is global due to the international shipment of fish and other seafood products (Dickey and Plakas, 2010). The link between toxins produced by a benthic microorganism and CP was conjectured to be through grazing herbivorous fish to carnivorous fish and was proposed in 1958 (Randall, 1958). However, the causative organism, a dinoflagellate in the genus Gambierdiscus, was not described until 1977 by Yasumoto and colleagues (Adachi and Fukuyo, 1979) after having material collected previously by Bagnis and colleagues in the Gambier Islands, French Polynesia. Due to the lack of understanding of the ecology and taxonomy of Gambierdiscus at the time, the organism was named Diplopsalis (Dickey, 2008). Gambierdiscus toxicus (Adachi and Fukuyo, 1979; Bagnis, et al., 1980; Vlamis and Katikou, 2014) was the only species in the genus for almost 20 years; hence, the early literature is rife with erroneous reports of G. toxicus (Figure 1). There are now 16 described Gambierdiscus species: G. australes, G. balechii, G. belizeanus, G. caribaeus, G. cheloniae, G. carpenteri, G. carolinianus, G. excentricus, G. pacificus, G. polynesiensis, G. scabrosus, G. toxicus, G. silvae, G. lapillus, G. honu and G. jejuensis (Jang, Jeong and Yoo, 2018; Kibler et al., 2012; Fraga et al., 2016; Pisapia et al., 2017a; Smith et al., 2016). Recently, two globular species of Gambierdiscus have been reclassified as Fukuyoa (F. yasumotoi and F. ruetzleri) and a new species described (F. paulensis) (Gomez et al., 2015). Both F. ruetzleri and F. paulensis produce toxins (Laza-Martinez et al., 2016; Rhodes and Smith, 2018; Leung et al., 2018).

Gambierdiscus is an epiphytic benthic (bottom-dwelling) dinoflagellate, and toxic cells are the first step in contaminated marine food webs. There is poor understanding about the ecological or environmental factors that affect the production of toxins by Gambierdiscus. A study of eight species shows that maximum growth takes place between 26.5 °C and 31.1 °C, with thermal limits from 15–21 °C to 31.34 °C. Salinities range from 24.7 g/litre to 35 g/litre for maximum growth with sufficient light at irradiances generally below 231 μmol photons per square metre per second (Kibler et al., 2012). Each species has unique combinations of optimal temperature, salinity and light conditions (Kibler et al., 2012). Gambierdiscus grow slowly compared with many other dinoflagellates, and it is estimated that 1–5 months are necessary for significant increases in cell abundance to occur. At exponential growth, the division rate for G. polynesiensis is 0.13 ± 0.03 divisions/day (Chinain, et al., 2010a). The fastest rate of division was reported in a Hawaiian strain of G. toxicus, 0.55 div/day (Chinain et al., 2010a). There is no clear correlation between Gambierdiscus cell density and toxicity, depending on the species. Lag times of up to 17 months have been reported between a Gambierdiscus bloom and transfer of toxins in the food web leading to elevated CP incidences (Clausing et al., 2016). Correlation between water temperature and bloom occurrence in Hawaii, the United States of America, was reported after water temperature increased from 25.4 °C to 26.5 °C (Parsons, Settlemier and Bienfang, 2010), suggesting blooms may develop above a certain temperature threshold. However, this relationship weakens when temperatures exceed 31 °C. To understand how increasing temperatures may affect the distribution of Gambierdiscus and Fukuyoa species in the Caribbean, one study (Kibler et al., 2015) used temperature vs growth data in combination with projected water temperatures at six representative sites to predict how the growth rates of six Gambierdiscus species may change as oceans warm in the twenty-first century.

They predicted:

> northward progression of species currently restricted to lower latitude environments;

> increased abundance of species already present in from subtropical to temperate areas;

> reduced occurrence of some species in the Caribbean Sea as temperatures exceed their upper thermal growth limits.

Temperature increases are not the only changes that will accompany global warming. Sea-level rise, regional increases in precipitation, and nutrient input and habitat (substrate) alterations will also follow. Each of these factors will affect habitat suitability and, in turn, the distribution and abundance of Gambierdiscus and Fukuyoa species (Yong et al., 2018).

2.2 DISTRIBUTION OF CP CAUSATIVE ORGANISMS

Species in the genera Gambierdiscus and Fukuyoa have a pan-tropical distribution and the genus is reported throughout the Caribbean Sea (Figure 2), the North East Atlantic and the Mediterranean Sea (Figure 3), and Hawaii (the United States of America), French Polynesia, Australia, Japan, Southeast Asia, the Pacific Ocean (Figure 4). In 2012 and 2014, two new Gambierdiscus species were described from the Canary Islands (Spain) and both are toxic (see Section 2.6.1, Table 2). This genus has been found in the Mediterranean Sea, the Gulf of Mexico, and in the Atlantic Ocean off the coast of North Carolina (the United States of America) (34.7°N) (Villareal et al., 2007; Aligizaki and Nikolaidis, 2008; Litaker et al., 2009; Fraga and Rodriguez, 2014; Fraga et al., 2011; Fraga et al., 2016; Rodriguez et al., 2017). Gambierdiscus has also been observed on the coast of Angola (Isabel Rangel, personal communication), and CTXs have been detected in fish from Cameroon (Bienfang, Oben and DeFelice, 2008), but the entire coast of Africa remains mostly unexplored in this regard. Until 2009, establishing the distribution of various Gambierdiscus species was difficult because the taxonomy of the genus was poorly defined. Studies simply reported counts as Gambierdiscus “toxicus” or Gambierdiscus sp. (Tester et al., 2008; Litaker et al., 2009). A revision of the genus in 2009, using both morphological and genetic methods, made it possible to unambiguously identify Gambierdiscus species. As with the global distribution (Figure 5) of most micro-organisms, it is possible with additional sampling that species now known as endemic to either the Atlantic or Pacific may eventually be found in both regions.

The different Gambierdiscus species have been identified on most tropical and subtropical coasts (Figures 2–5):

> G. toxicus has been reported in French Polynesia (Chinain, Faust and Pauillac, 1999), Mexican Caribbean (Hernández-Becerril and Amazán, 2004), New Caledonia, Réunion, Indian Ocean (Chinain, Faust and Pauillac, 1999), Viet Nam (Roeder et al., 2010), and Malaysian Borneo (Leaw, Lim and Tan, 2011).

> G. belizeanus has been reported in Kiribati (Xu et al., 2014), Saudi Arabia (Red Sea), Belize (Faust, 1995), Mexican Caribbean (Hernández-Becerril and Amazán, 2004), Malaysia, Florida (the United States of America) (Litaker et al., 2009), Pakistan (Munir, Siddiqui and Morton, 2011), Queensland (Australia) (Kohli, Farrell and Murray, 2015), and Saint Barthélemy – Caribbean (Litaker et al., 2010).

> F. yasumotoi has been reported in Kuwait and the Gulf of Aqaba (Jordan) (Saburova, Polikarpov and Al-yamani, 2013), New Zealand (Rhodes et al., 2014b), Singapore (Holmes, 1998), Japan (Nishimura et al., 2013), Mexican Caribbean (Hernández-Becerril and Amazán, 2004), Queensland (Australia) (Kohli, Farrell and Murray, 2015), and Nha Trang – Viet Nam (Kohli, Farrell and Murray, 2015).

> G. australes has been reported in French Polynesia (Chinain, Faust and Pauillac, 1999), Japan (Nishimura et al., 2013), Cook Islands (Rhodes et al., 2010), Hawaii (the United States of America) (Litaker et al., 2009; Pisapia et al., 2017a), Pakistan (Munir, Siddiqui and Morton, 2011), and the Kermadec Islands (New Zealand) and Zealandia regions of the Southwest Pacific (Rhodes et al., 2017a; Rhodes et al., 2017b).

> G. silvae (this species was formerly known as G. ribotype 1) has been reported in the Canary Islands (Spain) (Rodriguez et al., 2017; Pisapia et al., 2017a), and Belize – Caribbean (Litaker et al., 2010) and Japan (Nishimura et al., 2013).

> G. pacificus has been reported in French Polynesia (Chinain, Faust and Pauillac, 1999), the Marshall Islands and the Society Islands (French Polynesia) – Micronesia (Litaker et al., 2010), Kota Kinabalu and Sipadan Island, Malaysia (Kohli, Farrell and Murray, 2015), and Nha Trang – Viet Nam (Roeder et al., 2010).

> G. polynesiensis has been reported in French Polynesia (Chinain, Faust and Pauillac, 1999), Pakistan (Munir, Siddiqui and Morton, 2011), and Nha Trang – Viet Nam (Roeder et al., 2010).

> G. caribaeus has been reported in Florida (the United States of America), Belize – Caribbean, Tahiti (French Polynesia), Palau, Hawaii (the United States of America) (Litaker et al., 2009), Flower Gardens – Gulf of Mexico, Ocho Rios – Jamaica (Holland et al., 2013), the Bahamas, Grand Cayman Island, Tol-truk (Micronesia [Federated States of]) (Litaker et al., 2010), Hainan Island (China), Jeju Island (the Republic of Korea) (Jeong et al., 2012), and the Canary Islands (Spain) (Rodriguez et al., 2017).

> G. carolinianus has been reported in North Carolina (the United States of America), Atlantic Ocean (Litaker et al., 2009), Bermuda, Mexico (Litaker et al., 2010), Puerto Rico, Flower Gardens – Gulf of Mexico, Ocho Rios – Jamaica, Crete (Greece) (Holland et al., 2013), and the Canary Islands (Spain) (Rodriguez et al., 2017).

> G. carpenteri has been reported in Australia (Kohli et al., 2014b), Kiribati, Belize, Guam, Fiji (Litaker et al., 2009), Hawaii (the United States of America) (Litaker et al., 2010), Dry Tortugas – Florida (the United States of America), Flower Gardens – Gulf of Mexico, and Ocho Rios – Jamaica (Holland et al., 2013).

> G. scabrosus (formerly known as Gambierdiscus sp. type 1) has been reported in Japan (Nishimura et al., 2014), and Viet Nam (Pisapia et al., 2017a; Nishimura et al., 2014).

> G. balechii (formerly known as Gambierdiscus sp. type 6) has been reported in Kiribati (Dai et al., 2017), and Manado, Celebes Sea, Indonesia (Pisapia et al., 2017a; Fraga et al., 2016).

> F. ruetzleri has been reported in North Carolina (the United States of America), and Belize – Caribbean (Litaker et al., 2009).

> G. honu has been reported in Rarotonga (Cook Islands) and in North Meyer Island, and the Kermadec Islands and South West Pacific (Rhodes et al., 2017a).

> G. cheloniae has been reported in Rarotonga (Cook Islands) (Smith et al., 2016).

> G. excentricus has been reported in the Canary Islands (Spain) (Fraga et al., 2011; Rodriguez et al., 2017).

> G. lapillus has been reported in the Great Barrier Reef (Australia) (Kretzschmar et al., 2017).

> G. jejuensis (formerly known as Gambierdiscus sp. type 2) has been reported from Jeju Island (the Republic of Korea) (Jang, Jeong and Yoo, 2018).

> Gambierdiscus ribotype 2 has been reported in Belize – Caribbean, Martinique – Caribbean (Litaker et al., 2010), and Puerto Rico (Holland et al., 2013).

> Gambierdiscus jejuensis has been reported in the Republic of Korea (Jang, Jeong and Yoo, 2018), and Japan (Nishimura et al., 2013).

> Gambierdiscus sp. ribotype 3 has been reported in the Canary Islands (Spain) (Rodriguez et al., 2017) and Japan (Nishimura et al., 2013).

> Gambierdiscus has also been reported in many other areas, although the species were not identified, such as Cyprus, Rhodes (Greece), Saronic Gulf (Greece), French West Indies, Cuba, Veracuz, Costa Rica, Brazil, Angola, and Cameroon (Kohli, Farrell and Murray, 2015). Moreover, some species were thought to be endemic to certain zones, such as: G. australes and G. pacificus (Pacific area); G. belizeanus, G. caribaeus, G. ruetzleri, G. carpenteri and G. carolinianus (Atlantic and Pacific); and G. yasumotoi (Pacific) (Kohli, Farrell and Murray, 2015). Nevertheless, this might be due to lack of extensive searches, as G. australes has recently been found in the Canary Islands (Spain) (Rodriguez et al., 2017).

2.3 METHODS FOR SPECIES IDENTIFICATION

A variety of techniques can be considered for the identification of species at any given site, including: optical microscopy as a screening tool, and then scanning electronic microscopy (SEM) and/or molecular techniques (sequencing, polymerase chain reaction [PCR], restriction fragment length polymorphism [RFLP], fluorescence in situ hybridization [FISH] probes, etc.) as confirmation tools. A combination of SEM and molecular techniques should be considered where new species are to be described.

2.3.1 POLYMERASE CHAIN REACTION (PCR)

Polymerase chain reaction (PCR) assays targeting unique sequences found within the SSU, ITS, and D1/D3 LUS ribosomal domains are available for most Gambierdiscus species to help detect described species or identify new ones. These assays are cost-effective, produce rapid, accurate measurements of relative cell concentrations, and can be adapted to survey large numbers of environmental samples.

Semi-quantitative PCR assays are available for: G. belizeanus, G. caribaeus, G. carpenteri, G. carolinianus, G. ruetzleri and Gambierdiscus ribotype 2 (Vandersea et al., 2012); G. polynesiensis, G. toxicus, G. pacificus and G. australes (Darius et al., 2018a). In addition, PCR assays have been published for: G. lapillus (Kretzschmar et al., 2017), G. scabrosus, Fukuyoa cf. yasumotoi (Nishimura et al., 2016); and G. excentricus and G. silvae (Litaker, Tester and Vandersea, 2019).

2.3.2 FLUORESCENCE IN SITU HYBRIDIZATION (FISH) PROBES

In situ hybridization with rRNA-targeted fluorescently labelled oligonucleotides has been reported to be a reasonable and rapid method for the detection, identification and enumeration of species (Pitz, 2016). Species-specific FISH probes can be used in combination with epifluorescence microscopy to obtain data on the abundance and diversity of Gambierdiscus species in field samples. Different species have probes of different wavelengths; thus, the use of multiple filter sets on the epifluorescence microscope allows species-specific abundance counts of up to six species in any given sample. As this technology develops, additional species and automated through-flow cytometry may be possible.

2.3.3 RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) TYPING

This technique can be used either as a screening method prior to the selection of species and strains for further study, or in combination with other methods of community diversity profiling (e.g. quantitative polymerase chain reaction [qPCR]) (Lyu et al., 2017). It is based on the comparison of DNA-extract profiles obtained from different strains/species of Gambierdiscus and Fukuyoa following their digestion using appropriate restriction enzymes. The RFLP digestion products are further separated and analysed by electrophoresis on agarose gel.

2.3.4 HIGH-THROUGHPUT SEQUENCING (HTS) METABARCODING

High-throughput sequencing (HTS) metabarcoding uses universal PCR primers to mass-amplify specific gene sequences from environmental samples and enables the characterization of all species or specific taxa present in the sample (Smith et al., 2017). This approach allows greater resolution of microbial community composition than do traditional morphological and molecular methodologies. The use of HTS metabarcoding for characterizing microbial communities is rapidly increasing due to the adaptability of the methods and a continual lowering of cost per sample (Lallias et al., 2015).

2.4 SAMPLING STRATEGIES

Several sampling methods to confirm the presence of Gambierdiscus species are available (Steidinger and Castillo, 2018). For example, sampling of natural substrates such as representative macrophytes (e.g. turf algae, Dictyota spp. and Halimeda spp.) or seagrass (Thalassia spp. and Halophila spp.) in a region, or the in situ deployment of artificial supports (e.g. window screens or PVC tiles) for a given period (e.g. 24 h or 30 d). The use of either macroalgal samples and/or artificial substrates in combination with qPCR techniques can be useful for providing information on the diversity and relative abundance of the different species present in a given location (Tan et al., 2013; Tester et al., 2013; Tester et al., 2014; Darius et al., 2018b). Care should be taken in using artificial substrates in high-energy sites as these are less reflective of the natural community diversity and abundance under these conditions (Smith et al., 2017). However, the absence of toxigenic species does not necessarily reflect low risk. Due to the potential patchiness of Gambierdiscus, both macroalgal and artificial substrate collections should employ a robust sampling design that covers both spatial and temporal scales. When monitoring for the occurrence of Gambierdiscus and Fukuyoa species and cell abundance, long-term data series should be considered, if possible, to address the issue of seasonality as both the abundance and species compositions are known to vary over time. The sampling design should consider patchy distributions and include preliminary range finding exercises to determine the patch sizes (spatial scales) to capture inherent environmental variability with statistical rigour and the possibility of substrate preference.

2.5 GAMBIERDISCUS ABUNDANCE

Gambierdiscus cells are distributed in a very patchy manner, even over small distances (Ballantine et al., 1985; Ballantine, Tosteson and Bardales, 1988; Lobel, Anderson and Durand-Clement, 1988). Typically, coefficients of variation among adjacent samples range from 50 percent to > 150 percent. Even with this high variation, average Gambierdiscus abundance data from 46 published studies were used to estimate density distributions for the Atlantic and Pacific Oceans (Litaker et al., 2010) (Figure 6). The frequency distributions of average cell densities were similar in both the Atlantic and Pacific. Eighty-five percent of the abundance estimates were < 1 000 cells/g wet weight algae. About 10 percent of the abundance estimates were between 1 000 cells/g wet weight algae and 10 000 cells/g wet weight algae. Estimates exceeding 100 000 cells/g wet weight algae were fewer than 5 percent of the total. The only estimate that exceeded 1 000 000 cells/g wet weight algae was from the Pacific. It is likely that the highest 10 percent of the densities represent localized epibenthic blooms of Gambierdiscus (Nakajima, Oshima and Yasumoto, 1981; Withers, 1981; Darius et al., 2007) including a recent bloom in the Canary Islands (Spain) with 104 cells/g of G. caribaeus (Soler-Onís et al., 2016).

Collecting, counting (Tester et al., 2014) and identification methods for benthic microplankton are well documented (Steidinger and Castillo, 2018).

Early reports highlighted increased abundance of Gambierdiscus sp. following disturbance events and anthropogenic impacts in reef ecosystems (Rongo and van Woesik, 2013; Bagnis, 1994), including hurricanes, salinity fluxes due to heavy rain, rise in sea surface temperature, earthquakes, bomb testing, shipwrecks, pollution, sedimentation, and destructive fishing practices (Randall, 1958). Native Fijians documented that a 200 m section of reef became toxic after macroalgae coverage was destroyed on the coral following a severe hurricane in 1929. Fresh Creek, Andros Island, Bahamas, experienced several years of fishing closures following a series of ciguatera outbreaks after a severe storm in 1908 (Randall, 1958). Several outbreaks of ciguatera in the Pacific and Atlantic following hurricanes have also been described (de Sylva, 1994). In the Pacific Ocean, military activity, dredging of shipping channels, and waterside construction have all been implicated with increased ciguatera outbreaks, although this could be attributed to population increases and the improved reporting of CP (Ruff, 1989). Other regions have suffered fish poisoning events related to environmental pollution. Mariel Bay, Cuba, exceeded quality standards for safe human contact making it the most polluted waters of Cuba and macroalgae blanketed the coral reefs (Morrison et al., 2008). The bay also experienced the highest rates of CP in Cuba, with 70 percent of nationwide cases occurring there from 1993 to 2002.

2.6 TOXICITY OF CP CAUSATIVE ORGANISMS

2.6.1 DETECTION OF CTXs IN GAMBIERDISCUS AND FUKUYOA AND IN THE ENVIRONMENT

The evaluation of CTX production in Gambierdiscus and Fukuyoa can be approached by establishing cultures from field isolates in the laboratory and evaluating the toxins produced (Table 1). It is also important to characterize the toxicogenic potency of natural populations. Where possible, direct sampling of Gambierdiscus and Fukuyoa blooms for further evaluation of toxin content should also be conducted in order to characterize both the toxin concentration and profile in wild cells. Generally, a known amount of Gambierdiscus or Fukuyoa cells are obtained through filtration or centrifugation of cultures or directly from field samples. Harvested cells are extracted using chemical methods to evaluate CTXs. Care should be taken to eliminate crossover between CTXs and maitotoxins (MTXs) (and related compounds) as both can invoke toxicity depending on the methodology used. Toxin cell quota can be calculated when the CTX content or toxicity is standardized to a known cell number. Very few studies have characterized the complete toxin profiles as many CTXs have not yet been structurally elucidated. This is an area of need that is somewhat hampered by the slow growth rates of toxigenic Gambierdiscus in culture, resulting in insufficient material for isolation and purification. Identification of previously elucidated CTXs such as the CTX 1B and 3 groups (see Section 3) in Gambierdiscus isolates is more straightforward and can be conducted by LC-MS/MS methods (see Section 4) but the lack of available standards for all elucidated congeners makes confirmation and quantification challenging.

The monitoring of toxins produced by Gambierdiscus and Fukuyoa in the water column with the use of passive adsorption approaches (e.g. solid phase adsorption toxin tracking [SPATT]) is worth considering. SPATT consists of the immersion in water of small devices holding resins within a planktonic mesh. Generally, SPATT bags are immersed for 3–10 days. Several groups of toxins including CTXs adsorb to the resin (e.g. HP20 resin). This approach has been implemented in the laboratory to detect and quantify CTXs in the medium of Gambierdiscus cultures (Fraga et al., 2011) and also in the environment within ciguatera endemic areas (Roué et al., 2016). The approach is a complementary strategy to the evaluation of CTXs in microalgal populations. SPATT may be useful as an early warning tool to detect CTXs in areas where Gambierdiscus or Fukuyoa may have not yet been reported or where molecular identification was not available. In addition, extraction of adsorbed toxins from SPATT bags reduces matrix effects that may interfere in the consequent analytical evaluation of CTXs.

This approach can provide complementary information on the presence not only of CTXs but also of other toxin classes adsorbing to the resin in a given site, as other toxin-producing benthic toxigenic species such as Ostreopsis and Prorocentrum are often present in high abundance in benthic assemblages of ciguateric biotopes.

In addition to the evaluation of CTXs, one may consider, for scientific purposes, identifying other toxins (MTXs1-4) and bioactive compounds not proved to be directly implicated in ciguatera (gambieric acid, gambierol, gambierone, etc.) produced by Gambierdiscus and Fukuyoa (Table 2).

When assessing the occurrence of microalgae involved in the production of CTXs, both Gambierdiscus and Fukuyoa have to be addressed. Whenever possible, data about Gambierdiscus and Fukuyoa cell abundance, species diversity and their respective toxin profiles should be documented, as these parameters can significantly impact the toxin profiles in surrounding fish and help explain variations in fish toxicity observed at a regional/local scale (Figures 2–5 and Section 3.2 Ciguatoxin classification).

2.7 OCCURRENCE OF CTXs IN SEAFOOD

More than 425 species of fish have been linked to ciguatera events (Perez-Arellano et al., 2005). Coral reef fishes are a premium sea product with global distribution, which contributes to the expansion of CP intoxications. Reef fish known to potentially accumulate these toxins are: barracuda (Sphyraenidae), amberjack (Seriola), grouper (Serranidae), snapper (Lutjanidae), po’ou (Chielinus rodochrous), jack (Carangidae spp.), trevally (Caranx spp.), wrasse (Labridae spp.), surgeon fish (Acanthuridae spp.), moray eel (Muraenidae spp.), roi (Cephalopholis spp.), and parrotfish (Scaridae spp.) (FDA, 2011) (see Tables 3 and 4, and Figure 7). A large variety of marine invertebrates including urchins, gastropods, bivalves, echinoderms, etc. have also been reported to contain CTXs and present an additional source of poisoning to seafood consumers.

Most of the tropical fish are territorial. Hence, those areas with toxicity usually remain toxic, while the areas without toxicity usually remain safe. This knowledge is used by local people of Pacific and Caribbean islands as a precautionary measure to avoid CP. Nevertheless, it is common that they have chronic levels of CTX that at a certain point reach a threshold resulting in disease (Dickey, 2008). Gambierdiscus has been also reported as free-swimming cell in the water column (Price et al., 2016), which further complicates a potential monitoring plan.

Due to international trade, there are reports of poisoning in many geographical areas without the risk of indigenous ciguatera (such as: Canada, California, New York, Rhode Island and Vermont in the United States of America (Graber et al., 2013); Hamburg and elsewhere in Germany (Schlaich et al., 2012; Gestal-Otero, 2014); and the Paris area in France, and also from patients returning to their countries after travelling. A report of CP poisoning after consumption of a farm-cultured salmon shows the complexity of the problem (Ebesu, Nagai and Hokama, 1994). It is also a matter of concern that the terminology as well as the denominations used for commercial fish change depending on the country.

The recent appearance of significant amounts of CTXs in the Canary Islands (Spain) (Boada et al., 2010; Perez-Arellano et al., 2005) and Madeira (Portugal) (Otero et al., 2010) suggest that CTXs are expanding their ecological niches, probably as a consequence of global warming (Kohli, Farrell and Murray, 2015). It has been reported that in the Caribbean Sea and the West Indies, where optimal growth of Gambierdiscus is above 29 °C, the number of days with sea surface temperatures above 29 °C has doubled, from 44 days to 86 days, in the last 30 years (Tester et al., 2010). Nevertheless, although global warming is facilitating the growth and expansion of Gambierdiscus, there are bodies of water that are warm enough to depress their growth, and this is a factor to take into account in understanding the role of global warming in ciguatera expansion (Llewellyn, 2010). Although several reports indicate the presence of Gambierdiscus in new areas (Brazil [Nascimento et al., 2012], Morocco [Ennaffah and Chaira, 2015] and Thailand [Tawong et al., 2015]), there is no solid link yet to this being caused by climate change (Botana, 2016; Silva et al., 2015). Another subject not yet clarified is how the change in pH and CO2 levels and in sea surface temperatures may affect toxin production, as these parameters influence the growth of microalgae (Schippers, Lürling and Scheffer, 2004; Hinga, 1992).

The poisoning of a fisher in Madeira (Portugal) was reported to be caused by Seriola, and the components showed a mixture of toxins identified as Caribbean or Indian and Pacific, namely CTX1B, CTX3C, and Caribbean or Indian Ocean analogues of 1 141.6 m/z (Otero et al., 2010). The concentrations reported were about 50 ng/g fish tissue. In the Canary Islands (Spain), several Gambierdiscus species have been identified (G. silvae and G. australes) (Fraga and Rodriguez, 2014), and the nine intoxications reported to date were associated to Caribbean C-CTX1 (Boada et al., 2010; Perez-Arellano et al., 2005; Nunez et al., 2012). In the Mediterranean Sea, there are reports of Gambierdiscus in several macroalgae (Padina pavonica, Corallina elongate, Jania spp. and Cystoseira spp.) in Crete (Greece) (Aligizaki and Nikolaidis, 2008), and although CP poisoning has never been reported in this region, ciguateric species have been proposed in the eastern Mediterranean (Chevaldonné, 1990; Bentur and Spanier, 2007).

The fact that CTXs enter the food chain from herbivores (both invertebrates and fish) to end up in carnivorous fish allows the accumulation of these toxins in hundreds of marine species (Yasumoto, 2001). The toxin profile in fish flesh, observed during a 20-year interval, was reported to be species-dependent – CTX1B and two deoxy congeners were found in snappers and groupers, CTX4A and CTX4B in spotted knifejaw (Oplegnathus punctatus) from Okinawan (Japan) waters, CTX3C-type toxins in spotted knifejaw from Miyazaki (Japan), and CTX1B-type and CTX3C-type toxins in red snapper (Lutjanus bohar) and amberjack (Seriola dumerili) (Yogi et al., 2011; Yogi et al., 2014). A possible explanation of species-specific toxin profiles was attributed to genetic differences in CTX bio-oxidation by the liver enzymatic complex CYP3A4. These changes in toxin profiles are the result of the bio-oxidative transformations that CTXs undergo as they move up the food chain, leading to more oxidized and also more toxic congeners (Lewis and Holmes, 1993).

For example, one study (Ikehara et al., 2017) has shown that CTX1B, -2 and -3 actually derive from the enzymatic oxidation of CTX4A and -4B, whereas the biotransformation of CTX3C leads to CTX2A1 (see Figure 7).

This illustrates the complexity of a potential monitoring process, as depending on the species, the analytical challenge is different.

One issue that has been identified as a potential problem for CP prevention is the mislabelling of fish species, either unintentionally or by fraudulent initiatives. This may lead to ciguateric species that replace others being put on the market (Stewart et al., 2009). Moreover, the identification of ciguateric fish and their catch location is often difficult, and this adds to the difficulty of establishing the real toxic levels that did cause the poisoning, due to analytical limitations (Farrell et al., 2017).

The approach followed by many countries is to impose fish size restrictions as a ciguatera risk management action (Clausing, Chinain and Dechraoui Bottein, 2016). As an example, the Sydney Fish Market (Market, 2013) is to warn of possible ciguatoxic fish that may be sold (surgeonfish [all Acanthuridae members], flowery rockcod [Epinephelus fuscoguttatus], yellowtail kingfish and samsonfish [Seriola spp.], narrow-barred Spanish mackerel [Scomberomorus commerson], coral trout [Plectropomus spp. and Variola spp.) and parrot fish [all Scaridae members]), and to forbid others (Chinamanfish [Symphorus nematophorus], tripletail Maori wrasse [Cheilinus trilobatus] and humphead Maori wrasse [Cheilinus undulatus], red bass [Lutjanus bohar], paddletail [Lutjanus gibbus], giant moray [Gymnothorax javanicus]) obtained in the regions of Kiribati, certain Queensland areas, Marshall Islands, Northern Australian Territory waters (Bremer Island, Bonner Rocks, Miles Island, Cape Arnhem, etc), and Fijian waters. For high-risk species, a maximum size is allowed, with a range that varies from, for example, 3 kg for coral rockcod (Cephalopholis spp.) to 10 kg for mackerel (Scomberomorus spp.).

The approach taken in the Canary Islands (Spain), is to check for the presence of CTXs in fishes of certain species above a threshold weight, and always excluding viscera (Table 3).

The method for routine control is the neuroblastoma assay (Canarias, 2017; Caillaud et al., 2012) (see Section 4).

The toxicity of some species is associated to seasonal variations. Sphyraena barracuda was reported to increase in toxicity in winter and autumn (60–70 percent toxic fish), while toxic fish is only 10 percent in summer and December (Tosteson, Ballantine and Durst, 1988). This observation, taken in Puerto Rico, might be associated to changes in the toxicity of fish prey.

Some countries count on reporting systems. In the United States of America, the reporting of cases should be made to the surveillance system of Centers for Disease Control and Prevention (CDC; www.cdc.gov/nors/index.html). In the European Union, the Rapid Alert System for Food and Feed (RASFF; https://ec.europa.eu/food/safety/rasff_en) provides alerts to food safety agencies and consumers. In French Polynesia, the Institut Louis Malardé (www.ilm.pf) provides information and issues alerts about CP.

CHAPTER 3

CHEMISTRY AND BIOSYNTHETIC PATHWAYS

3.1 CHEMISTRY

3.1.1 STRUCTURES

Ciguatoxins (CTXs) are a class of large polyether ladder-like structures that contain 13–14 fused rings (Nicolaou, Frederick and Aversa, 2008). Representative backbone structures of CTXs identified so far are represented by CTX4A, CTX3C and C-CTX1.

After the initial description of “ciguatoxin” in 1967 (Scheuer et al., 1967), the structure of “CTX” (= CTX1B) and its analogue (CTX4A) were reported in 1989 following significant isolation and purification efforts from moray eel and Gambierdiscus cultures, respectively (Murata et al., 1989). The diastereoisomers 52-epi-54-deoxyCTX1B and 54-deoxyCTX1B (CTX2 and CTX3, respectively) were subsequently described, and these represent less oxidized forms of CTX1B (Lewis et al., 1991). The only difference between CTX1B, 52-epi-54-deoxyCTX1B and 54-deoxyCTX1B involves modification at one end of the CTX structure with no hydroxyl group on the M-ring, and this manifests in modest differences in potency (Lewis et al., 1991). The structure of CTX3C was first elucidated in 1993 after extraction from a culture of what was at that time described as G. toxicus and is now thought to have been a strain of G. polynesiensis. Subsequent structures of the oxidized forms of the toxin were determined in 1998 from fish (Satake et al., 1998). The backbone structure of CTX3C toxins differs from the CTX4A group on the E-ring (i.e. an eight-membered ring in CTX3C and a seven-membered ring in CTX4A) and absence of an aliphatic side chain on the A-ring (Satake et al., 1998; Satake et al., 1996; Satake, Murata and Yasumoto, 1993b). Several toxic Caribbean CTX analogues have been identified and isolated from fish for structural characterization (Crouch et al., 1995). The structure of the major Caribbean toxin C-CTX1 and its 56-epimer C-CTX2 were first described by Lewis and Vernoux in 1997 and 1998 (Vernoux and Lewis, 1997; Lewis, Vernoux and Brereton, 1998). C-CTX2 was found as a minor analogue in fish, rearranging to C-CTX1 in solution. The C-CTX backbone shares characteristics with CTX4A and CTX3C but does not possess the aliphatic side chain on the A-ring and contains an additional ring on the right wing of the molecule. Additional Caribbean CTXs have been reported in several studies but are yet to be structurally elucidated (Pottier et al., 2002b). An Indian Ocean group has also been described (Hamilton et al., 2002b), and although masses have been reported in the literature (Diogene et al., 2017), no molecular structures have been determined to date. Yasumoto and Satake (1996) provide a concise summary of the chemistry and aetiology of CTXs and other related toxins that could be associated with ciguatera poisoning.

3.1.2 CHEMICAL CHARACTERISTICS

Ciguatoxins are lipid-soluble compounds that are thermostable and resistant to mild pH changes (Guzmán-Pérez and Park, 2000). The most toxic analogue described to date is CTX1B, which is stable at 100 °C, 1 N NaOH, and in sunlight for 1 h, but loses toxicity in 1 N HCl after 10 min (Guzmán-Pérez and Park, 2000; Nukina, Koyanagi and Scheuer, 1984). Ciguatoxins are odourless, tasteless, heat stable and present at very low (typically < ppb) levels in contaminated seafood, making them difficult to detect without advanced detection methods (see Section 4 for more details on detection methods). The toxic potency of CTXs has been shown to increase as they become more oxidized (Lewis et al., 1991). Analogues originally isolated from the Pacific are currently thought to be the most potent and have been well characterized. Some CTXs are metabolites generated through the process of enzyme-mediated biotransformation in invertebrates and fish. For example, one study has demonstrated the oxidation of algal toxins CTX4A, CTX4B and CTX3C in vitro to the analogues found in fish using human liver CYP enzymes and fish liver microsomes (S9 fractions) (Ikehara et al., 2017). However, the low yields reported using this approach make large-scale production of standards impractical at present. Biotransformation contributes to the large number and structural diversity of CTXs observed in fish, with more than 30 analogues reported to date (Yasumoto et al., 2000; Vernoux and Lewis, 1997; Hamilton et al., 2002b; Diogene et al., 2017). Ciguatoxins are lipophilic, a property recognized in early studies (1950–1960), based on bioassay-guided separation of components in toxic fish (Hashimoto, 1956; Banner et al., 1960). Ciguatoxin lipophilicity has been estimated from the partition coefficient of CTX1B between ethyl acetate and HEPES-buffered Ringer solution in a 1:1 ratio at 37 °C (Lewis, Hoy and Sellin, 1993). As the ratio was determined to be > 2.0, it was confirmed that CTX1B was a lipophilic compound and suggested that it could cross cellular membranes (Lewis, Hoy and Sellin, 1993). Experimental CTX1B oral and intraperitoneal (ip) dosing studies in mice confirmed the rapid absorption capacity of CTXs, and ability to cross the blood–brain barrier as evidenced by CTX1B levels in post-mortem brain tissue (Bottein, Wang and Ramsdell, 2011). Reports in wild-caught fish contaminated with CTXs showed similar compartmentalization of toxins across tissues into liver, spleen, brain, muscle, gonads, fat and bone (Vernoux et al., 1985a). While these studies confirm the lipophilic nature of CTX1B, no detailed studies of partitioning coefficients or cellular transport mechanisms have been conducted on this CTX analogue to date, largely due to the lack of available standards. The polarity of other CTXs has been estimated based on the order of elution (i.e. retention time) when using reversed-phase chromatography (Satake et al., 1998; Yasumoto et al., 2000; Hessel, Halstead and Peckham, 1960; Scheuer et al., 1967; Hashimoto, 1956). It would be advantageous to explore quantitative structure activity relationship modelling from known and newly elucidated structures as they become available in order to help predict the absorption of CTXs in humans and aquatic organisms, particularly fish.

3.1.3 BIOSYNTHETIC PATHWAYS

A remarkable feature of Gambierdiscus is its unique biochemical machinery, responsible for the production of multiple structurally complex polyether toxins, including CTXs, gambierol, gambierone, gambieroxide, gambieric acids and maitotoxins. Different Gambierdiscus spp. produce varying amounts of toxin, generate different toxin profiles, and strain differences are also observed. For example, a recent toxicity study of various Gambierdiscus species, and strains, showed that they all produce MTX-like activity, in the range 1.5–86 pg MTX eq/cell, and CTX-like activity in the range 0.6–50 fg CTX3C eq/cells (Pisapia et al., 2017a). In addition, a strain of G. excentricus isolated from the Canary Islands (Spain) was reported to produce 1 426 fg CTX3C eq/cell (Pisapia et al., 2017a), and a strain of G. polynesiensis from the South Pacific produced 18.2 pg/cell (Rhodes et al., 2016). A comprehensive table of Gambierdiscus toxicity can be found in Section 2.6 Toxicity of CP causative organisms. In the Pacific, the link between algal CTX production and those observed in fish has been established (Ikehara et al., 2017). However, more evidence is needed before this information can be used to predict the presence of CTXs in seafood from a specific region.

The size of dinoflagellate genomes is enormous, with more than 100 chromosomes and up to 80-fold the human haploid genome (Lin, 2011), and this large genome is suspected to be one of the causes of their slow growth. Dinoflagellates possess some of the largest genomes known from eukaryotes, from 1.85 Gbp to 112 Gbp (LaJeunesse et al., 2005). Some of the toxins they produce are among the largest non-polymeric compounds reported in nature. For example, MTX is produced by several Gambierdiscus spp. and has a molecular weight of 3 422 Da, containing 32 rings, with 99 elements of stereochemistry. It is the largest and most toxic natural product characterized to date (Murata et al., 1993; Nicolaou, Frederick and Aversa, 2008). Therefore, Gambierdiscus has the genetic and biochemical machinery for synthesis of extremely large and complex natural compounds.

The genes involved in Gambierdiscus toxin production are not known, and application of molecular methods to detect toxic species is therefore not currently possible. The enzymes needed for construction of these complex molecules are thought to be extender unit polyketide synthases (PKSs), which are one of the most important and diverse class of enzymes occurring in plants, bacteria and fungi. They are a large multimodular enzymatic group that participate in the synthesis of many natural compounds (Barrios-Gonzalez and Miranda, 2010), and are classified into three types, according to their domain organization: (i) Type I PKS are large multifunctional enzymes with modular or iterative activity, where multiple catalytic sites exist on a single polypeptide; (ii) Type II PKSs are organized in complexes of smaller monofunctional enzymes with iterative activity, where each gene encodes a single catalytic enzyme; and (iii) Type III PKSs are similar to Type II but smaller. To date, transcriptome data are available for four species of Gambierdiscus: G. polynesiensis, G. belizeanus, G. australes and G. excentricus (Pawlowiez, et al., 2014; Kohli et al., 2017; Kohli et al., 2015); and PKS genes-related sequence data show similarity to type I PKSs, as reported in other dinoflagellates (Van Dolah et al., 2017). The biosynthesis of MTX by PKS would require multiple steps that include polyepoxidation, cyclization and sulfonation (Kohli et al., 2015). Some species, such as G. australes, produce MTX and not CTX (Rhodes et al., 2014a). Several studies report low CTX production in cultures (Holmes, Lewis and Gillespie, 1990; Murata et al., 1990), and it has been shown that environmental manipulation can supress toxin production (Morton et al., 1993). Nevertheless, each strain has its own unique toxic profile, which is dependent on the optimal growth conditions. G. polynesiensis produces similar amounts of MTX and CTX in culture, but as the cells age, CTX becomes dominant (Chinain et al., 2010a). Some investigators have reported that Gambierdiscus cultures require an acclimation time to produce toxins, between 16 weeks and 52 weeks (Chinain et al., 2010a). The rate of toxin production is inverse to the rate of growth. Hence, maximum toxin levels correspond to a stationary phase, which also suggests that CTXs are secondary metabolites (Sperr and Doucette, 1996). Further research is required to determine the toxin profile of the different Gambierdiscus spp., and to understand why toxin production changes when in culture and how this process is modulated.

3.2 CIGUATOXIN CLASSIFICATION

Three families of CTXs have commonly been classified according to their geographical location, i.e. Pacific (P-CTXs), Caribbean (C-CTXs) and Indian Ocean (I-CTXs). Thanks to advances in structural elucidation, it is now possible and appropriate to classify CTXs based on the known chemical structures. The structural characteristics of P-CTXs actually allow further classification into two separate groups based on their chemical structure, i.e. CTX3C vs CTX4A backbones and their derivatives. The Caribbean CTXs represent a third group. To date, only two Caribbean CTXs have been structurally elucidated (C-CTX1 and C-CTX2) (Lewis, Vernoux and Brereton, 1998; Vernoux and Lewis, 1997). Several further compounds have been identified as potential C-CTXs; however, there is little evidence concerning their structure or relative toxicity (Pottier et al., 2002b, 2002a). Figure 8 shows the backbone structures and associated analogues of these three groups. An Indian Ocean group has also been referred to; however, their structures have not been elucidated (Hamilton et al., 2002a; Hamilton et al., 2002b). More research is needed to isolate the toxins observed in the Indian Ocean region in order to determine their structural characteristics and potency, and whether they are related to CTXs found in other regions. Tables 5 and 6 shows the classification and abbreviation synonyms of CTXs.

While the biotransformation of algal toxins into metabolites through the food web, and in particular fish, has been investigated for CTX3C and CTX4A analogues (Yasumoto, 2001; Ikehara et al., 2017), no such information is available for the C-CTX group or Indian Ocean CTXs at this time.

There are other known bioactive compounds produced by Gambierdiscus spp. that potentially have relevance to human health, including maitotoxin 1–4, gambierone, gambieroxide, gambierol and gambieric acids; structures of these compounds are shown in Figure 9. While for some compounds (e.g. MTX1) a different mode of action has been determined, the toxicity mechanisms remain to be ascertained for the other compounds. There is no clear evidence that these compounds accumulate in fish flesh to significant levels, while they have been consistently found at higher levels in visceral tissues (Kohli et al., 2014a; Diogene et al., 2017). However, in processing environments, it cannot be excluded that cross-contamination of fish flesh could occur from poorly handled visceral tissues. In many areas of the world, invertebrates and fish are eaten whole, and for a variety of fish species many organs are targeted for direct consumption (e.g. liver, gonads and roe) and eaten on a regular basis. Thus, the contribution of these other bioactive compounds in the human health risk associated with ciguatera cannot be discounted.

CHAPTER 4

DETECTION METHODOLOGIES FOR CTXs

Due to their high potency, their complex structures, trace occurrence, and the scarce availability of analytical standards, detecting CTXs in seafood and algae presents many analytical challenges (Suzuki et al., 2017; Vilarino et al., 2018). Proposed analytical strategies for detecting CTXs can be divided into screening and confirmation methods. To date, none of the methods described has been reported to have undergone single- or multi-laboratory validation. Table 7 provides an overview of the existing published methods along with the advantages and disadvantages of each strategy. While some laboratories have produced reference materials and quantified standards on a small scale, recent data have most consistently reported CTX3C equivalents due to commercial availability. While not commercially available, some quantified reference materials are available through a variety of laboratories.

4.1 SCREENING ASSAYS FOR CIGUATOXINS

Several assays for the screening of fish samples for CTXs have been described, based on in vitro assays, in vivo (mouse) bioassays, and immunoassays. These assays all have high-throughput capacity (apart from the mouse bioassay), which allow parallel measurements at the respective endpoints of each assay. However, reliable quantification of the bioactivities and/or toxicity of CTXs in seafood extracts requires a full dilution series (6–8 concentrations) of a certified reference and, where possible, matrix-matched reference.

4.1.1 IN VITRO ASSAYS

The in vitro assays for CTXs make use of selective binding to site 5 (Catterall, Trainer and Baden, 1992; Trainer et al., 1993) of the voltage-gated sodium channel (VGSC). Although several in vitro assays have been proposed, the most widely used in screening are the N2A-MTT assay and receptor binding assays. (RBA).

4.1.1.1 N2A-MTT assay for ciguatoxins

Development of cell assays for marine toxins was stimulated by early work by Kogure et al. (1988) using mouse neuroblastoma (N2a) cells to detect marine toxins active at VGSCs. Their use of a morphological endpoint limited the assay’s utility, and the assay was directed at saxitoxins (STXs) and tetrodotoxins (TTXs) rather than CTXs. The neuroblastoma N2A assay (N2A-MTT) proposed by Manger et al. (Manger et al., 1993; Manger et al., 1995) is based on detection of mitochondrial dehydrogenase activity using MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium]) to detect viability colorimetrically. Since its introduction by Mossman (Mosmann, 1983), cell biologists have used MTT across many different cell lines for the evaluation of cytotoxicity. However, direct cytotoxicity is not readily observed with CTXs or other sodium channel active toxins, and this is how the N2A-MTT assay differs. A key aspect of the assay that has been optimized for CTXs, brevetoxins and site 5 sodium channel toxins is the synergistic effect between site 2 and site 5 toxins of VGSCs described by Catterall and Risk (1981). Cells are treated with veratridine, which targets site 2 of the sodium channel, and blockade of the sodium-potassium ATPase by ouabain forces sodium accumulation and cell death. Site 5 toxins such as the CTXs and brevetoxins are detected with very high-sensitivity due to the site 2 – site 5 synergism. The N2A-based methods require only low quantities of CTX calibrants (25–50 pg CTX1B or C-CTX1 is sufficient for an eight-point curve) and samples (1–2 g). Measuring the absorbance at 570 nm is used for detecting mitochondrial metabolism of MTT and omission of veratridine and ouabain in (-O/V) control experiments reveals any (non-specific) cell death not caused by site 5 toxins. The -O/V control experiments, performed in parallel to the assay as full dose-response curves, are crucial to data interpretation and success. Other important parameters are: pre-treatment of Neuro-2a (N2a) cells obtained from culture collections (e.g. American Tissue Type Collection, and Sigma); plating consistency (i.e. standardized number of cells seeded per assay well); stable growth rates in maintenance cultures (which can be obtained by cell enumeration during passaging and tracking following propagation from cryopreservation); among other factors. These considerations will be important parameters for future harmonization.

As with all analytical methods for CTXs, their use for commercial trading would require a validation that has not been performed to date due to the absence of certified reference materials. There are other drawbacks. As activity-based assays, N2A assays do not supply structure information but simply respond to site 5 activity of any component within the sample extract. Many investigators have improved the specificity of CBA-N2a by optimizing different concentrations of OV treatment for the specific detection of either VGSC activators (brevetoxins [PbTxs] and CTXs) or blockers (STXs and TTXs), as they have opposite mode of action (Canete and Diogene, 2008).

The N2A-MTT can be sensitive to matrix effects such as protein and lipid content and other sample matrix components (e.g. phytosterols, and free fatty acids). Refinements of the method have addressed matrix effects and included sample conditioning and cleanup steps. Although maximum sensitivity can require a 24 h incubation, the assay can be performed more rapidly following sample matrix conditioning. These requirements vary depending on fish matrix lipid content. Interest in the N2A-MTT assay remains high, and the need for standardization of the assay has been asserted (Caillaud et al., 2012).

Related methods using voltage-sensitive fluorescent dyes for high throughput have also been reported using neuroblastomas (Louzao et al., 2004) or synaptosomes (David et al., 2003). This latter approach does not require use of ouabain or MTT as it is based on cell membrane depolarization rather than cell death. This approach is promising as depolarization can be observed in minutes versus the overnight incubations typically used for the viability end point of the N2A-MTT assays. Flow cytometric detection (Manger et al., 2014) allows selection of the most responsive cells using electronic gating. Assays based on voltage-sensitive dyes (including N2A-Flow) have not been optimized or practised by other laboratories to the same extent as the N2A-MTT format, with the latter used extensively and refined over the past 25 years.

4.1.1.2 Receptor binding assay (RBA) for ciguatoxins

Following study of an RBA for brevetoxins by Poli et al. (Poli, Mende and Baden, 1986) and Sharkey et al. (1987) the first RBA for CTXs was demonstrated by Lombet et al. (Lombet, Bidard and Lazdunski, 1987). The first application to an outbreak of ciguatera was by Poli et al. (1997). This radioreceptor assay using rat brain synaptosomes, is based on the competition for the receptor between a brevetoxin and CTXs, has since been refined (Diaz-Asencio et al., 2018). The assay allows CTX quantitation by means of using tritiated brevetoxin-3 or tritiated brevetoxin-9 (Pawlowiez et al., 2013; Gaboriau et al., 2014; Darius et al., 2013; Chinain et al., 2010b; Poli, Mende and Baden, 1986; Dechraoui et al., 1999; Darius et al., 2007). The Radioligand RBA for CTXs is operational in several countries impacted by ciguatera, however the need to use radiolabelled compounds is a drawback to its use. A comparison of RBA and N2a assay has shown that N2a was 12-fold more sensitive for ciguatoxins, whereas RBA was 3–24-fold more sensitive for brevetoxins (Dechraoui et al., 2005). A similar approach with brevetoxin-2 conjugated with fluorescent labels (Texas Red-PbTx-2, 6-TAMRA-PbTx-2, and BODIPY®-PbTx-2) has also been proposed for detection of PbTxs and applied for CTXs and Caribbean fish by Hardison et al. (Hardison et al., 2016) with a performance equivalent to the radioligand RBA assay (McCall et al., 2014) and semi-quantitative limit of detection of 0.075 μg/kg CTX3C equivalent (0.0975 μg/kg C-CTX1). Recently, improvements have been presented concerning chemiluminescent probes (Yasumoto, 2018). Preliminary results using chemiluminescent acridinium ABTX probes have shown superior sensitivity compared with tritium and fluorescent probes. However, more research on this probe is needed before making final conclusions.

The method based on the RBA with the fluorescent probe is part of the programme of the National Oceanic and Atmospheric Administration (NOAA) for detection of CTXs, and it is currently being used to screen invasive lionfish in the Caribbean (https://coastalscience.noaa.gov/news/habs/noaa-improves-monitoring-ciguatera-fish). The fluorescent radioreceptor assay has been announced to be a commercially available kit by SeaTox Research, Inc. The International Atomic Energy Agency (IAEA) has been providing support for the establishment of the radioligand RBA. It is used by the Caribbean regional network for early warning (Cuellar-Martinez et al., 2018).

4.1.1.3 Immunological assays

Although attempts have been made to develop immunoassays kits for CTXs, until recently none has been reported in the literature with sufficient performance to meet the FDA advisory levels of 0.01 ppb for Pacific CTXs (as CTX1B equivalents). Campora et al. reported an enzyme-linked immunosorbent assay (ELISA) with a detection limit of 0.078 ng/ml CTX1 in crude fish extract (Campora et al., 2008). This level is already at least ten times the advisory level even before accounting for dilution during extraction. However, they concluded that their ELISA was not suitable for commercial purposes. More recent synthetic haptens have provided a route to improved immunoassays that have resulted in a highly sensitive, direct sandwich ELISA format (Tsumuraya, Fujii and Hirama, 2014). ELISAs based on the multiple antibodies developed from these fragments now allow detection of the Pacific CTXs below 0.01 ppb CTX1B, and allow the simultaneous detection of multiple Pacific CTXs (CTX1B, CTX3C, 51-hydroxy CTX3C, and 54-deoxyCTX1B) (Tsumuraya, Fujii and Hirama, 2014). An ELISA kit for the simultaneous detection of CTX1B and 54-deoxy CTX1B is now commercially available (FUJIFILM Wako Chemicals, Japan) (Tsumuraya et al., 2018). Users of the assay should keep in mind that these assays are specific for the Pacific congeners and do not cross-react with Caribbean CTXs.

It should be noted that a commercial immunoassay distributed as Cigua-Check was once available, and is still in circulation in some regions, but it had significant rates of false positives and false negatives, so cannot be recommended (Lehane and Lewis, 2000; Bienfang, DeFelice and Dowling, 2011).

4.1.2 IN VIVO ASSAYS

The mouse bioassay (MBA) was used for years until in vitro and analytical methods started to replace it (FAO, 2004). In the United States of America, it was recommended in 1994 that this bioassay be replaced for regulatory analyses by in vitro assays (Dickey, 2008). The method was described by Banner et al. (Banner et al., 1960) and later improved (Lewis and Sellin, 1993; Lewis, 1995). It is based on an acetone extraction and partitioning into hexane, ethanol-water and diethyl ether. The extraction method is designed to eliminate the interference of maitotoxin. A nitrogen-dried ether extract is injected in 0.5 ml 1–5 percent Tween 60/0.9 percent saline solution to two mice. The observation time is 24 h (continuous observation for the first 2 h), with animals of standard weight (18–21 g) housed at 23 ± 2 °C. The lethality is expressed in mouse units (MU); an MU is the intraperitoneal LD_50 at 24 h, equivalent to 5 ng (CTX1B), 48 ng (52-epi-54-deoxyCTX), 18 ng (CTX3C) or 80 ng for CTX4B (Guzmán-Pérez and Park, 2000; Lewis, Hoy and McGiffin, 1992; FAO, 2004). The LD_50 is 0.25 μg CTX1B/kg bw and 3.7 μg C-CTX1/kg bw (Lewis et al., 1991; Vernoux and Lewis, 1997). The relationship between MU and time of death is log MU = 2.3 log (1 + T-1) (T being the time to death in hours). The observable signs of toxicity are diverse, such as inactivity, piloerection, jumping, cyanosis, diarrhoea, weight loss, hypersalivation, lacrimation and dyspnoea with gasping (Vernoux, 1994; Caillaud et al., 2010). Because CTXs induce hypothermia in mice, rectal body temperature may be measured, as the drop in temperature is of about 5 °C after 60 min (Lewis and Sellin, 1993; Lewis and Hoy, 1993). The bioassay was further improved by including a Florisil solid-phase extraction cleanup to further eliminate lipid contaminants (Wong et al., 2009) – the recovery improves up to 96.7 percent, while the standard procedure provides a 49–77 percent recovery (Lewis and Sellin, 1993).

The need for a dose response curve, required for the calibration and calculation of MU, makes this method difficult to implement on a routine base, given the limited commercial supply of CTXs. Although the symptoms could identify the toxin group (Vernoux, 1994), the bioassay does not inform about the toxin causing the effect. Therefore, its lack of selectivity is a problem, along with the ethical and legal acceptability that animal experiments poses in many countries (European Communities, 1986).

Several other methods described, not currently used, have been proposed as alternatives to the bioassay, such as contraction of guinea pig atria (Miyahara, Akau and Yasumoto, 1979), brine shrimp (Granade, Cheng and Doorenbos, 1976), cat (Hashimoto, 1956), mosquito (Bagnis, et al., 1985b) or Diptera larvae (Labrousse and Matile, 1996) assays. Chicken bioassay was also used as a feeding test, and its significant symptoms included hypersalivation along with an important drop in rectal temperature and weight losses, as in mice. Accumulation of toxins was proved: repeated administration, once a day, of toxic extracts at a subsymptomatic level, induced lethality. For the same cumulative dosage, lethality decreased to zero through repeated doses given once a day during a week. However, when a single sublethal dosage eliciting the lowest response 48 h after oral feeding was given, the subsequent feeding at the same dose level given to the same chicken had to be retarded by at least 7 days in order to have no lethal effect. This showed that detoxification is low in chickens (Vernoux et al., 1985b, Vernoux and Lahlou, 1986; Pottier and Vernoux, 2003).

4.2 STRUCTURE-BASED CONFIRMATORY METHODS

Liquid chromatography coupled with mass spectrometry (LC-MS) is a suitable approach for the identification and confirmation of CTXs, and related compounds, in a range of matrices. All liquid chromatographic methods use a serial analysis format that ultimately limits throughput in screening applications, in contrast to most of the assays described above. However, this approach allows accurate quantitation of individual CTX analogues, provided availability of analytical standards. All approaches described here involve the use of liquid chromatography coupled with electrospray ionization mass spectrometry, either in low-resolution tandem mass spectrometry (MS/MS) or high-resolution (HRMS) modes. Key limitations for the implementation and validation of these techniques are the lack of pure toxin standards and tissue reference materials. While most laboratories employ these methods for confirmatory purposes, they cannot be used for screening, especially in the absence of toxin standards for many analogues.

The first attempt to identify CTXs and MTXs using ion spray was by Lewis et al. in 1994 (Lewis, et al., 1994; Lewis and Jones, 1997). Due to the low concentrations of CTXs in fish that are relevant to human health (Moreiras, Leao and Gago-Martinez, 2018), it is necessary to significantly concentrate them from crude extracts. Sample preparation and the removal of matrix co-extractives haves also been shown to be very important for the performance of LCMS-based methods (as reviewed in Harwood, Murray and Boundy, 2017). As CTXs are lipophilic, it is important to eliminate potential interfering compounds (e.g. lipids) from the sample, to improve the efficiency of the extraction and to minimize matrix effects during MS analysis. Further refinements of the instrumentation have improved the detection and quantitation of CTXs in the past 25 years (Moreiras, Leao and Gago-Martinez, 2018; Otero et al., 2010; Dickey, 2008; Lewis, Yang and Jones, 2009; Yogi et al., 2014; Pottier and Vernoux, 2003). This has included the use of new generation LC-MS systems allowing faster separation (ultra-high-pressure liquid chromatography [UHPLC]), improved sensitivity (lower detection limits) and a greater level of specificity (through use of HRMS systems). These represent the main advantages of this approach. A key challenge associated with LC-MS methods is extensive sample cleanup, resulting in low recoveries that may compromise the ability to meet the guidance levels established by the Food and Drug Administration of the United States of America (FDA) (0.01 μg CTX1B eq/kg; 0.1 μg C-CTX1 eq/kg).

Given the large number of CTX analogues, the need for standards represents one of the most critical aspects of CTX research and control. The only CTX standard currently available commercially is CTX3C. The lack of calibrants also limits the ability to perform the required validation. Recent improvements include: the detection of CTXs using low- or high-resolution mass spectrometry (Sibat et al., 2018a); the systematic assessment of parameters for MS optimization (Moreiras, Leao and Gago-Martinez, 2018); and a method combining MTXs and CTXs within the same analysis (Murray et al., 2018). This has allowed the profiling of toxins in microalgal and environmental samples and seafood (fish, bivalves, sea urchins, starfish and gastropods). It has also been successfully applied to confirm the presence of CTXs and profiles in fish from the Pacific and Caribbean areas (Estevez et al., 2019; Yogi et al., 2011; Yogi et al., 2014; Abraham et al., 2012a) and the enzymatic biotransformation in fish (Ikehara et al., 2017). Recent implementations on LC-MS/MS also allowed the confirmation of the presence of C-CTX1 in fish from Atlantic coasts of Spain and Portugal (Estevez et al., 2019; Costa et al., 2018). A new hydroxyl metabolite of C-CTX1 was also confirmed in samples from the Canary Islands (Spain). However, this remains to be elucidated and confirmed. Further implementation on the LC-MS/MS methods for Caribbean CTX analogues described by Pottier et al. (Pottier et al., 2003; Pottier, Vernoux and Lewis, 2001; Pottier et al., 2002b) allowed investigators to conclude that the CTX profiles of CTX implicated fish from the Canary Islands (Spain) and Madeira (Portugal) were similar to those observed in Sphyraena barracuda and Caranx latus from the French West Indies.

4.3 CONCLUDING REMARKS

Quantitative LC-MS methods and in vitro bioassays need to be validated internationally based global standards and guidelines. Such validation will ensure harmonization and method comparability. At this stage, formal validation is not possible due to the lack of certified calibrants and matrix reference materials. Method comparability is also needed in order to give confidence in the testing methods used for analysis of CTXs. To achieve comparability between LC-MS (analogue-specific method) and screening assays (e.g. N2A or RBA), it is necessary that all toxicologically relevant analogues be accurately quantified and have reliable toxicity equivalency factors (TEFs). Several recent publications have shown a correlation between screening methods and LC-MS. Nevertheless, more data are required in order to strengthen the comparability of the various methods employed.

As toxin calibrants are scarce, a double-tiered approach is recommended; first, using a screening method, followed by a further interrogation of positive samples with a confirmatory method such as LC-MS/MS. The United States of America has defined advisory toxin levels for consideration in monitoring and control (FDA, 2011). The system utilizes screening based on semi-quantitative in vitro mouse neuroblastoma cell assay (N2A-MTT) and later toxin confirmation by LC/MS-MS (Dickey and Plakas, 2010). The composite toxicity of the sample is determined by N2A-MTT assay, and samples positive by N2A are analysed by LC-MS/MS for confirming the presence of CTX analogues. A similar approach has been proposed in Australia (Stewart et al., 2009). One of the main challenges for CTX detection in a surveillance system is the low amount of toxin in fish that, combined with matrix effects and number of analogues present in a single sample, make the analysis very complex (Friedman et al., 2017). For this reason, it is also important to have procedures for the rapid identification of CTX in the blood and liver of patients (Hamilton et al., 2010; Bottein Dechraoui et al., 2005; Bottein Dechraoui, Wang and Ramsdell, 2007). Although there has been some progress, given the high potency of the toxins and the large number of analogues, this is still a problem to be resolved (Matta et al., 2002).

Even more powerful than using the methods separately is the combination of N2A-MTT assay and mass spectrometry. Ciguatoxin profiles can be studied in detail by performing fractionation of samples by LC and performing N2A-MTT assay (Manger et al., 1993) to provide cytotoxicity profiles (Dechraoui et al., 2005; Abraham et al., 2012b). In this way, detailed structural confirmation can be combined with detection of toxin bioactivity.

CHAPTER 5

HAZARD IDENTIFICATION

5.1 ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION (ADME)

5.1.1 ANIMAL DATA

Available data on absorption, distribution, metabolism and excretion (ADME) are scarce, mainly limited to rats and mouse models, and to one purified CTX analogue, one of the most toxic, the CTX1B (also known as CTX or CTX1) found in the Pacific region. Two major studies from Dechraoui Bottein et al. (Bottein, Wang and Ramsdell, 2011) and Ledreux and Ramsdell (Ledreux and Ramsdell, 2013) performed in rats using CTX1B (> 90 percent purity), LD_50 0.25 μg/kg bw in mouse (Lewis, et al., 1991) yielded reliable data on CTX toxicokinetics. Due to the lack of 14C-radiolabelled CTXs and of sufficiently sensitive analytical methods, toxin levels in blood, excreta and tissues were determined using in vitro bioassays such as the N2a neuroblastoma cell-based assay. Sprague Dawley rats were exposed by ip or oral (gavage) route to a high dose of 0.26 μg CTX1B/kg bw or by intravenous (iv) route to 0.13 μg CTX1B/kg bw (Bottein, Wang and Ramsdell, 2011; Ledreux and Ramsdell, 2013).

Results indicated a rapid absorption following ip and oral exposure (Tmax values of 25 min and 2 h, respectively; alpha half-lives of 1.15 h and 2.61 h, respectively) and by combining ip and oral blood data study with the iv study, results suggested a “bioavailability” (of the parent CTX but also bioactive metabolites, given the bioassay used) of 75 percent and 39 percent, respectively. The Cmax after ip exposure was found to be 3.3-fold higher than the Cmax following administration of the same dose via oral route (6.40 ± 0.43 and 1.95 ± 0.15 pg/ml, respectively). However the areas under the blood concentration–time curve were not statistically different (282.22 ± 106.63 and 144.78 ± 52.45 pg/ml, respectively) indicating a similar toxin bioavailability regardless of the route of administration.

The volume of distribution (Vd) was comparable between ip and oral routes (77 litres/kg and 78 litres/kg, respectively) but lower for the iv route (35 litres/kg). Considering that no first-pass metabolism is taking place after iv administration, this Vd could represent that of the parent CTX (i.e. the CTX1B), whereas ip and oral route Vd would also include distribution of its potential metabolites. The very large Vd, especially for ip and oral routes, is indicative of additional extravascular distribution. Indeed, CTX1B was still detected in muscle, liver and brain tissues four days after administration (as measured by CBA at the end of the study), with 96 percent of the recovered total CTX activity found in muscle and 2–3 percent in the other tissues, regardless of the administration route. This is further evidenced by the biphasic elimination phase of the time course of CTX in blood following oral, ip and iv administration.

The Vd, which exceeds the total body water volume of the rat (~0.7 litres/kg), indicates a larger distribution of CTXs than in aqueous compartments. As CTXs are not soluble in very non-polar solvents such as hexane (Amzil, Vernoux and Pottier, 2001), they are unlikely to be stored in fat (neutral lipids) of adipose tissue. Rather, CTXs are expected to bind to tissue proteins, primarily to Nav channels (Au et al., 2016) but possibly also to muscular proteins as reported in fish (Hahn, Capra and Walsh, 1992), and/or to blood serum lipoproteins as shown for brevetoxins (Woofter, Spiess and Ramsdell, 2005).

Regarding CTX metabolism, toxin analysis in liver by CBA at termination of the ip and oral experiment revealed presence of CTX1B with two additional less polar metabolites (Bottein, Wang and Ramsdell, 2011). Ciguatoxin metabolization is further evidenced by the induction of gene expression of numerous cytochrome and glutathione S-transferases in mouse liver, 4 h post ip exposure to the same CTX1B dose (Morey et al., 2008), indicative of a complex biotransformation through both phase I and II enzymes. The induction of the expression of many members of the solute carrier family in the liver argues for a CTXs hepatobiliary excretion. In rat faeces, three main bioactive less-polar metabolites were found 2 days (oral route) or 3 days (ip route) after CTX1B exposure (Bottein, Wang and Ramsdell, 2011), thus confirming the remaining activity previously reported in blood 3 days after ip exposure to the same dose (Bottein-Dechraoui et al., 2008). Biotransformation in mammals to more polar metabolites may be a strategy to favour elimination of CTXs. However, it could lead to a toxification of the compounds as it occurs in fish.

The beta half-lives following oral, ip and iv administration were 81.82 h, 112.27 h and 35.5 h, respectively. The elimination of CTXs occurred predominantly via faeces (hepatobiliary route), and in lower quantities into the urine, regardless of the administration route.

CTX1B was undetectable at day 7 after a single administration by gavage at 0.26 μg/kg bw (Zhang et al., 2013). The excitatory neurotoxic actions on ACC neurons after acute single-dosage CTX exposure in rats could be recovered after 7 days of exposure.

In another rat study (Wang et al., 2017), where Sprague Dawley rats were exposed to CTX1B (purity > 95 percent) by gavage at an initial dose of 0.26 μg/kg bw, followed by administration of a fourth of this dose (0.065 μg/kg bw) every 72 h for 8 weeks, CTX1B concentration in blood samples ranged from 0.006 μg/litre to 0.015 μg/litre at 24 h, 48 h and 72 h after the final exposure to the toxin at week 8, and no difference was detected among these three time points (F = 2.72, p > 0.05, n = 4 rats for each time point). Higher concentrations were detected in the brain samples of rats after repetitive administration of CTX1B (0.034 ± 0.010, 0.030 ± 0.009 and 0.035 ± 0.005 μg/kg brain sample at 24 h, 48 h and 72 h, respectively). No difference was detected among these three time points (F = 0.11, p > 0.05, n = 4 rats for each time point). In this experiment, a low dose of CTX1B was administrated every 72 h; thus the toxin level at 72 h was the baseline concentration for the next toxin administration. These data suggest that accumulative quantities of the toxin are present in the brain and blood of rats as a result of chronic low-dose CTX1B treatment.

In mice, no quantitative assessment of the bioavailability was found. However, LD_50 studies conducted (ip and oral) using semi purified toxins (> 85 percent) on male Institute of Cancer Research (ICR) mice (four-weeks-old), suggest a very high oral uptake as identical LD0 values and almost similar signs of intoxications were reported for the two routes of administration (Ito, Yasumoto and Terao, 1996; Lewis and Hoy, 1993; Lewis, Hoy and Sellin, 1993; Lehane and Lewis, 2000; Terao et al., 1991). Diarrhoea was only caused by CTX when ip administered; with a dose of 4/5 MU (10.4 ng/28 g). Diarrhoea started within 10 min after administration and lasted until 30 min, associated with accelerated mucus secretion and peristalsis in the colon. It is likely that CTX given by oral route was absorbed and metabolized in a slightly different manner from that of ip route, and therefore did not cause diarrhoea. Another study, ip or orally administering CTX1B or a mixture CTX-m (CTX1B, 52-epi-54-deoxyCTX1B and 54-deoxyCTX1B) found no apparent differences in signs between CTX1B and CTX-m. However, signs induced by oral administration, including hind-limb paralysis, swollen tongue, delayed effects and the absence of diarrhoea, do not occur following ip administration (Lewis, Hoy and Sellin, 1993). Such differences were not evident in previous studies on the effects of orally administered CTX (Ogura, Nara and Yoshida, 1968; Terao et al., 1991).

In conclusion, CTXs are efficiently absorbed and rapidly distributed to the tissues after ingestion in laboratory animal models.

5.2 TOXICOLOGICAL STUDIES

5.2.1 ACUTE TOXICITY

Species highly sensitive to CP have been described and used efficiently to assess ciguateric fish by feeding or gavage test: cat (Hessel, Halstead and Peckham, 1960; Lewis and Endean, 1983); mongoose (Banner et al., 1960; Hamilton et al., 2002b); and chicks (Kosaki and Stephens, 1968; Vernoux et al., 1985b). Cat, which is very sensitive, was also used to study CTX mode of action (Legrand, Galonnier and Bagnis, 1982).

In the laboratory, mouse bioassay was used to monitor extracted CTXs and to assess their LD_50 (Vernoux, 1994), by either oral or ip route. Ciguatoxins, regardless of the route of administration, induce a set of signs, which, as a whole, hallmarks acute ciguatoxicity: loss of activity, piloerection, hypothermia, profuse diarrhoea, hypersalivation, lachrymation cyanosis (especially of the penis during transient pre-erection state), motricity disorders, dyspnea with gasping, possible hind-limb paralysis, and death due to respiratory distress. Surviving mice recover in hours, except from a significant weight loss induced by sublethal doses, whose recovery takes a few days. As shown by studies performed with pure CTXs from the Pacific (Lewis et al., 1993; Lewis and Hoy, 1993), Caribbean (Vernoux and Lewis, 1997; Lewis, Vernoux and Brereton, 1998) and Indian Ocean (Hamilton et al., 2002a; Hamilton et al., 2002b) areas, this typical symptomatology in mice is common to all tested CTX analogues, regardless of the geographic origin. Clinical signs in mouse induced by pure congeners isolated from microalgae or carnivorous fish are also similar (Terao et al., 1991; Lewis, 1995). Ciguateric extracts obtained from accepted extraction procedures induced similar effects in mice (Vernoux and Talha, 1989; Lewis and Sellin, 1993) to those from purified substances. Limited data suggest that effects observed are similar following ip and oral administration (Terao et al., 1991; Lewis and Hoy, 1993).

As documented by mouse ip LD_50 data (see Table 8), variability in the potencies exists between some different analogues of CTXs (Nicholson and Lewis, 2006; Solino and Costa, 2018). However, many data are lacking for a number of identified congeners. On the basis of LD_50 estimates in mice, oral CTX1 (a.k.a. CTX1B) (0.22 μg/kg) was similar in potency to ip CTX1B (0.25 μg/kg [Lewis, et al., 1991]). This publication proposes the use of CTX1B (also known as CTX1) as a worldwide reference CTX due to its commercial availability.

An original study by Terao et al. (Terao et al., 1991) reported both clinical signs and histopathological changes observed in mice from 10 min to 24 h following ip injection or gavage of 0.7 μg/kg bw of pure (> 99 percent purity) CTX1B or CTX4C (i.e. above LD_50, which is 0.25 μg/kg bw). Clinical signs included laborious movements, lumbar muscle contraction, defecation followed by severe watery diarrhoea then severe dyspnea, cyanosis and death within 24 h in 70 percent of the mice, whereas paw paralysis and/or penis erection occurred in the survived mice. Macroscopical examination of the organs of deceased mice showed dilated heart, lung oedema and congestion of all the other organs. Microscopical examination indicated heart, lung, medulla of adrenal glands, autonomic nerves and penis as target organs. Histopathological changes included mainly necrotizing and swollen cardiac muscle cells, and plasma effusion in the heart and lung, and swollen neuromuscular synapses in the small intestine. There were no significant differences in clinical signs or histopathological symptoms induced by the two toxins and the two administration routes. Atropine pre-treatment prevented diarrhoea, indicating that it was caused by actions on the autonomic nerve system in the intestinal wall. However, atropine did not prevent cardiac injuries.

In a second study conducted by the same team, a single ip sublethal dose of 0.1 μg/kg bw of CTX1B or CTX4C caused no discernible morphological changes in macroscopic, microscopic and at even ultrastructural level in hearts of mice (Terao, Ito and Yasumoto, 1992).

Symptomatology in rats was reported (Zhang et al., 2013) for acute toxicity obtained by gavage of pure (> 95 percent) CTX1B at a single dose of a sublethal dosage of 0.26 μg/kg bw given to adult male Sprague–Dawley rats (250–350 g) . It induced decreased body weight, reduced food consumption, and severe visceral pain after 5 h exposure was observed, mimicking the clinical gastrointestinal symptoms noted in patients with acute ciguatera poisoning, especially visceral allodynia and hyperalgesia. Neurotoxicity was also observed (Asthana et al., 2018).

5.2.2 SHORT-TERM STUDIES

In four-week-old male C57BL/6 mice, a second ip administration of a sublethal dose of 0.26 μg/kg bw of purified CTX1B, (CTX1, provided by R.J. Lewis) three days after an initial ip dose of 0.26 μg/kg bw prolonged the hypothermic response, and enhanced the reduced motor activity and the antinociceptive effect (by the tail flick assay) seen after the first administration. In addition, 30 percent (n=2 over 6) of the mice died within 7 h after the second injection whereas no deaths were observed after the first injection at the same dose. The second exposure also caused a twofold greater body weight loss after 7 h. The CTX1B concentration in blood measured by N2a 1h post-exposure was higher after the second injection compared with the first injection (16.6 ± 1.0 pg/ml vs 9.0 ± 1.0 pg/ml, respectively) (Bottein-Dechraoui et al., 2008).

Repeated ip and oral administrations of purified CTX1B and CTX4C (99 percent pure) to four-week-old male ICR mice at a dose of 0.1 μg/kg bw for 15 days resulted in marked swelling of cardiac cells and endothelial lining cells of blood capillaries in the heart. Single doses caused no discernible pathological changes. After 15 doses, both ventricles were dilated at necropsy; the rest of the bodies were normal. Damage to the capillaries was followed by prominent effusion of serum and erythrocytes into the interstitial spaces of the myocardium. Swelling of the endothelial lining cells of capillaries caused narrowing of the lumen and accumulation of blood platelets in capillaries, which resulted in multiple single-cell necrosis of cardiac muscle cells. According to the authors, the ultrastructural changes of the heart were similar to those observed after a single dose of 0.7 μg/kg bw.

About one month after the final administration, myocytes and capillaries appeared to be normal. Effusion in the interstitial spaces resulted in formation of bundles of dense collagen, which persisted for 14 months. Up to the fifth month, considerable hypertrophy of cardiac muscle cells was also noted. The mean weight of the hearts of CTX-treated mice at 14 months (280 ± 36 mg) was significantly heavier than that of controls (207 ± 15 mg), (P < 0.05).

No differences in clinical signs or histopathology between CTX1B and CTX4C were observed.

This study shows that CTX1B and CTX4C have a cumulative effect on cardiac tissue. After 15 injections of CTX1B or CTX4C at a dose of 0.1 μg/kg bw, marked swelling of the myocardia and the endothelial lining cells of blood capillaries was observed, whereas there were no discernible changes after a single administration of the same dose (Terao, Ito and Yasumoto, 1992).

5.2.3 MEDIUM-TERM STUDIES

Adult Sprague Dawley male rats (280–300 g) received purified CTX1B (purity ≥ 95 percent) dissolved in saline 1 percent Tween 60 by gavage at an initial dose of 0.26 μg/kg bw followed by administration of one-quarter of this dose (0.065 μg/kg bw) every 72 h for 8 weeks. This protocol was designed to mimic exposure in endemic regions. The author selected the dose of 0.065 μg/kg bw because it did not affect the average food intake per unit body weight nor the average body weight increase. The control group was treated with saline containing 1 percent Tween 60 of the same dosage and at the same time points as the CTX1B group. Body weight and food intake were monitored and reported every week. Behavioural tests were performed between week 5 and week 8 (open field test, elevated plus maze, Morris water maze, and rat gambling task). Rats were sacrificed at different time intervals (24 h, 48 h and 72 h) after the final exposure at week 8. Blood and brain were collected and analysed by Neuro-2a assay to quantify CTX with CTX1B standard (in-house standard, based on CTX1B provided by R.J. Lewis).

In the open field test, no difference between the treated and control rats was observed for the locomotor function (total distance moved and mean speed). Spontaneous exploratory activity in CTX1B-treated rats was suppressed compared with control rats, as indicated by a reduced time spent in the centre area (23.95 ± 2.43 s vs 13.31 ± 2.26 s; p < 0.01), and a decreased number of entries into the centre area (10.25 ± 0.91 s vs 7.50 ± 1.24 s; p < 0.05). These results suggest that chronic low CTX1B exposure leads to increased anxiety-like behaviour in rats.

Results in the elevated plus maze test confirmed the absence of effects on locomotor function, and increased anxiety-like behaviour in rats, expressed as the decreased time spent in the open arms and the lower number of entries into the open arms.

In the Morris water maze, CTX1B rats showed longer escape latency than control rats during the training process (p < 0.05), suggesting spatial learning ability was affected in these rats. Moreover, in the probe test, chronic CTX1B rats showed significantly shorter duration in the target quadrant (39.57 ± 3.46 s vs 23.46 ± 3.01 s; p < 0.01) and shorter duration in the platform region (2.76 ± 0.51 s vs 1.40 ± 0.34 s; p < 0.05) compared with control rats. These results suggest that chronic low dose CTX1B exposure impaired spatial learning and reference memory in rats.

In the rats gambling task, the proportion of good decision makers decreased in the CTX1B group (65.0 percent vs 41.2 percent), and the proportion of poor decision makers increased (15.0 percent vs 41.2 percent) compared with controls. Similar results in the proportion of undecided rats were detected between the two groups (20.0 percent vs 17.6 percent). The difference in the proportions of the three types of decision-making behaviours between the two groups was significant (Mann-Whitney U test, U = 102.5; p < 0.05). The mean food reward obtained during the task by CTX rats was significantly lower than controls (159.6 ± 18.3 vs 109.3 ± 12.0; p < 0.05). These data indicate that rats developed a decision-making deficit after chronic low dose CTX1B treatment (Wang et al., 2017).

In conclusion, after repeated exposures of rats (every 3 days for 8 weeks) to low dose of CTX1B (0.065 μg/kg bw) after an initial high dose (0.26 μg/kg bw), this study observed the development of anxiety-like behaviour (by open field test and elevated plus maze test), learning and memory deficits (by the Morris water maze) and decision-making impairment (by the rats gambling task).

This study showed neurotoxic effects in rats relevant with symptoms reported in humans. However, only one dose of CTX1 was tested, so it is not possible to identify a lowest observed adverse effect level (LOAEL) or a non-observable adverse effect level (NOAEL).

5.2.4 LONG-TERM STUDIES

Terao et al. (1994) conducted a long-term toxicity of CTX1B in mice focused on heart effects (Terao et al., 1994). CTX1B isolated and purified (purity and composition not known) from contaminated fish (Micronesia, Okinawa [Japan]) were given orally by intubation into male ICR mice (4 weeks of age) once a week at a dose of 0.1 μg/kg bw (n=20) for 25 weeks or 0.05 μg/kg bw (n=15) for 40 weeks. A control group of five mice received saline once a week for 40 weeks. Two mice from the group treated with CTX1B at 0.1 μg/kg bw were sacrificed 5 h after administration at the 12th, 14th, 18th and 23rd week from the beginning of the treatment. Two mice from the group treated with CTX1B at 0.05 μg/kg bw were sacrificed 5 h after administration at the 18th and 40th week from the beginning of the treatment.

Until about ten weeks, the mice showed no abnormal clinical symptoms. After about 18 weeks, mice treated with 0.1 μg/kg bw showed marked hypertrophy of the heart. Histopathological analysis showed swelling and rupture of the endothelium of the capillaries and widening caused by exudation or collagen fibres in the interstitial space. Occasionally, degenerated or swollen mitochondria were prominent in the myocardium. Accumulations of platelets in the capillaries were frequently observed. No effects were observed in mice treated with 0.05 μg/kg bw, even ultrastructural changes at 40 weeks.

This study provides a NOAEL for heart toxicity of 0.05 μg/kg bw (after 40 weeks, once a week).

5.2.5 REPEATED VERSUS ACUTE TOXICITY

Cardiotoxicity in mice was studied after a single dose of purified CTX1B of 0.1 ug/kg bw and a similar daily dose during 15 days. While no cardiotoxicity was observed after a single dose, severe morphological effects on the heart were observed after the multiple dose exposure, indicating an accumulative process for this endpoint.

A single dose of 0.7 ug/kg of purified CTX1B caused similar cardiotoxic symptoms as observed after multiple daily administrations during the 15 day period.

Thus, cardiotoxicity is similar for a single high dose versus multiple lower daily dosages (Terao, Ito and Yasumoto, 1992).

A long-term study for 25 weeks and 40 weeks with oral administration of 0.1 and 0.05 ug/kg once a week also observed similar cumulative cardiotoxic effects for the mice with the highest dose level. No cardiotoxicity was observed for the low dose group over a period of 40 weeks (Terao, Ito and Yasumoto, 1992).

Two single ip dosages of 0.26 ug/kg of purified CTX1B were given to mice, and bodyweight, thermoregulation, motor activity and tail flicking were studied. These effects were more pronounced after the second dose, but from a qualitative point of view similar effects could be found after a single or double dose (Bottein-Dechraoui et al., 2008).

In rats, an initial dose of 0.26 ng/kg of purified CTX1B follow by a dose of 0.065 ug/kg every three days during an eight-week period was used to study neurobehavioural effect. This study showed that after an initial high dose of CTX1B followed by a long-term study, neurobehavioural deficits could be observed. These effects included anxiety-like behaviour, and learning and memory deficits. The study indicates that chronic low-level exposure to CTXs after a clinically relevant poisoning period may cause neurobehavioural deficits (Wang et al., 2017; Bottein-Dechraoui et al., 2008).

Overall, these studies indicate that, in mice and rats, subchronic exposure may lead to similar toxic effects, e.g. cardiotoxicity, as found with a single higher dose level. Cardiotoxicity was the only endpoint studied in both acute and repeated toxicity studies (by the same team). However, similar symptoms are not reported in human.

5.2.6 CONCLUSION FROM TOXICOLOGICAL STUDIES IN RODENTS

There are very few oral studies in mice or rats, and only using purified CTX1B or CTX4C. None is suitable to establish a health-based guidance value (acute or chronic).

In a limited study in mice, a single oral dose of 0.1 μg/kg bw of purified CTX1B caused no clinical or histopathological changes, whereas an oral dose of 0.7 μg/kg bw caused severe toxicity in the heart, adrenal gland and penis. Repeated oral doses of 0.1 μg/kg bw also caused severe toxicity (Terao, Ito and Yasumoto, 1992; Terao, et al., 1994). However, the experts of this Expert Meeting highlighted that the quantification of the doses in the Terao study is probably not accurate. In addition, CTX4C is an old terminology, and in the absence of chemical information in the paper, it is not possible to identify this compound. Repeated low oral dose of purified CTX1B (0.065 μg/kg bw) after an initial high dose (0.26 μg/kg bw) caused anxiety-like behaviour, learning and memory deficits, and decision-making impairment in rats (Wang et al., 2017).

5.3 MODE OF ACTION

The main molecular target of ciguatoxins is the voltage-gated sodium channel (VGSC, Nav) (Bidard et al., 1984), to which they bind to the “receptor-site 5” of the alpha subunit. This binding site is shared with brevetoxins (Gawley et al. 1992; Dechraoui et al. 1999), although with lower affinity. The receptor-site 5 is located in the region of interaction of certain residues of segments S5 of domain IV and S6 of domains I (Trainer et al., 1993). The binding of CTXs leads to a shift in the voltage dependence of the channel activation to more negative membrane potentials (hyperpolarizing shift in the thresholds of activation) and an increase in the recovery rate from inactivation. The overall effect is a persistent activation of Nav (i.e. Na+ influx) at the resting membrane potential and, as a consequence in excitable cells, CTXs cause an increase in the membrane excitability, eliciting depolarization and even spontaneous and repetitive actions potentials at the resting potential (Bidard et al. 1984; Benoit et al. 1986; Benoit and Legrand 1994; Strachan et al. 1999; Hogg et al. 2002; Ghiaroni et al. 2006). This action, especially in peripheral nerves where binding of CTXs to Nav is long-lasting (Strachan et al. 1999; Au et al. 2016), explains most of the effects of the group. CTXs show affinity for all the VGSC alpha subunit isoforms (Nav 1.1 to 1.9; Inserra et al. 2017), with difference in potency between the different congeners of CTXs.

The membrane hyperexcitability induced by CTXs is primarily due to the Nav activation but also to a blockade of voltage-gated potassium channels (VGPCs, Kv), although the potency of this effect varies according to congeners and study models. In frog myelinated axons, CTX4B reduced potassium currents (Benoit and Legrand 1994) and was shown to be more effective in blocking Kv than Nav and, compared to CTX1B, 4 times more potent at blocking Kv but 50 fold less potent at activating Nav (Schlumberger et al., 2010a). Low nanomolar concentrations of CTX1B partially blocked potassium currents in cultured primary rat (mammalian) skeletal muscle cells (Hidalgo et al. 2002). In rat sensory neurons, CTX1B blocking effect on Kv was shown to involve the delayed-rectified and A-type potassium channels (Birinyi-Strachan, Gunning, et al., 2005b). CTX3C failed to inhibit potassium currents in taste cells (Ghiaroni et al. 2006), whereas it inhibited them more potently than 51-hydroxy-CTX3C and CTX1B in cerebellar granule cells (Pérez et al. 2011).

Na+ influx induced by CTXs elicits several secondary cell events, including the water entry in nervous structures leading to swelling. CTXs cause the volume increase of frog myelinated axons (nodes of Ranvier) and motor nerve terminals (Benoit et al. 1996; Mattei et al. 1997, 1999a, b). The CTX1B-induced nodal swelling of frog myelinated axons was shown to be the combined result of the Na+ influx subsequent to the direct Nav activation by the toxins and a K+ efflux subsequent to the depolarization-induced opening of Kv channels (Mattei et al. 2014). An opening of calcium-activated potassium channels following the activation of the nitric oxide-cGMP pathway was involved in the swelling of frog erythrocytes induced by CTXs (Sauviat et al. 2006).

Another consequence triggered by the increased Nav gating and conductance is an increase in cytosolic calcium concentration shown in several neuronal or myocardial models, by mechanisms involving either the reverse mode of the Na+-Ca2+ exchanger (Molgó et al. 1993a; Gaudry-Talarmain et al. 1996) or the inositol triphosphate (InsP3)-dependent mobilization of internal Ca2+ stores (Molgó et al. 1993b; Hidalgo et al. 2002). Consequently, calcium-dependent responses, such as exocytosis of neurotransmitter and neuromodulators, are induced: acetylcholine release from motor nerve terminals, cholinergic models (Molgo et al. 1990, 1992) and intestine vagal innervation (Lewis and Endean 1984; Tatsumi et al. 1985; Lewis and Hoy 1993), noradrenaline release from sympathetic innervation of the smooth muscle of artery (Brock et al. 1995, 1997), heart (Lewis et al. 1992; Lewis and Hoy 1993) or the vas deferens (Ohizumi et al. 1981; Lewis and Endean 1984; Tatsumi et al. 1985), GABA release from cortical neurons (Martin et al. 2015a) and release of the neuropeptides CGRP (calcitonin gene-related peptide) and substance P (SP) release from sensory neurons (Zimmermann et al. 2013; Le Garrec et al. 2016; Touska et al. 2017).

In mice following intraplantar administration of CTX, the induced cold allodynia was demonstrated to involve the activation of the Nav1.8 isotype in CGRP-positive sensory neurons that triggers a calcium influx through the cold-sensitive Transient Receptor Potential Ankyrin 1 (TRPA1) cation channel. CTX1B does not directly activate TRPA1 but sensitizes it to cold, leading to cold allodynia (Vetter et al., 2012). The neuropeptide CGRP is a marker of peptidergic sensory neurons that, together with SP, mediates neurogenic inflammation, pain and also itch (Andoh et al. 1998; McCoy et al. 2012), which is a typical symptom of CFP. The human intracutaneous injection of (sub)nanomolar concentrations of CTX elicited the release of enough CGRP to induce a lasting axon reflex flare, i.e. a neurogenic skin inflammation sign, which was accompanied by localized pruritus, burning pain and cold allodynia (Zimmermann et al., 2013).

Another secondary effect of CTX following Nav activation is gene expression modulation (Rubiolo et al. 2018), including upregulation of the immediate early genes Arc and Egr and downregulation of glutamate NMDA and AMPA receptors in cortical neurons (Martin et al. 2015b). The inducible nitric oxide synthase was also upregulated in macrophages (Kumar-Roine et al., 2008).

Regarding water-soluble compounds produced by Gambierdiscus spp., which can be present in digestive viscera (see CTX classification), the mode of action of MTX is by inserting in the plasma membrane to increase massive influx of calcium (Reyes et al., 2014). Gambierol potently blocked Kv channels in several models, whereas it had mild or no effect on Nav channels (Inoue et al. 2003; Ghiaroni et al. 2005, 2006; Louzao et al. 2006; Cuypers et al. 2008; Schlumberger et al. 2010b; Cao et al. 2014). Gambieric acid and gambierone are mild inhibitors of the Nav channels (Inoue et al., 2003; Rodriguez et al., 2015).

CHAPTER 6

HUMAN DATA

6.1 ADME

Data on CTX toxicokinetics in humans are very limited. Aside from one experiment showing that recombinant human CYP3A4 enzyme can oxidize CTX4A and CTX4B to CTX1B, 54-deoxyCTX1B, and 52-epi-54-deoxyCTX1B (Ikehara et al., 2017), toxicokinetic findings are deduced from clinical observations. As systemic symptoms may appear soon (in some cases in less than one hour) after toxin ingestion (Friedemann, 2016; Bagnis, 1968; Chateau-Degat et al., 2007), it appears that systemic distribution in humans may occur quickly after ingestion. Ciguatoxin resorption via mucosa appears possible, based on a case report of systemic effects in a female after vaginal sexual intercourse with a ciguatera-affected male (Lange, Lipkin and Yang, 1989; Geller, Olson and Senecal, 1991; Ting, Brown and Pearn, 1998) and another case report of systemic effects in a male after sexual intercourse with a female (Hevia Pumariega and Hernández Mullings, 2008). Other reports of symptoms among sexual partners of ciguatera cases further support mucosal elimination (Lange, Lipkin and Yang, 1989; Geller, Olson and Senecal, 1991). Many ciguatera cases express central nervous system symptoms (Allsop et al., 1986; Friedman et al., 2017; Gatti, Oelher and Legrand, 2008), suggesting that CTXs can enter the brain. Ciguatoxins have been measured in blood several hours after ingestion – i.e. CTX1B and CTX3B/3C measured by mass spectrometry hours after consumption of red snapper (Lutjanus campechanus) (Mendoza et al., 2013). However, toxins may not persist long in blood. In a case series of four tourists who had eaten the same barracuda in Cuba and developed cold allodynia (Butera et al., 2000), CTXs were undetectable in serum, plasma or urine 90 h after poisoning using HPLC or the cell-based, synaptosome receptor binding assay. Ciguatoxins were detected in human liver in an autopsy of a lethal case six days after fish consumption (Hamilton et al., 2010), indicating that CTXs may persist in tissues after exposure. Case reports describing symptoms among infants of ciguatera-affected mothers suggest that women may eliminate toxins via breast milk, and that toxins may be resorbed through breast milk (Bagnis and Legrand, 1987; Blythe and de Sylva, 1990). However, the importance of this mode of toxin elimination may vary across individuals. In another case report describing a mother who presented with ciguatera-related symptoms, CTXs were detected in fish remnants but not in breast milk, and the breastfed infant remained healthy (CDC, 2009). Case reports of apparent toxicity among foetuses of ciguatera-affected women suggest that transplacental toxin transfer is possible (Rivera-Alsina et al., 1991; Pearn et al., 1982). Other reports of apparently uncomplicated pregnancies of ciguatera-affected women suggest heterogeneity in the extent of transplacental toxin transfer (Fenner et al., 1997; Rivera-Alsina et al., 1991; Geller, Olson and Senecal, 1991; Senecal and Osterloh, 1991).

6.2 CLINICAL FEATURES AND TOXICITY

Drawing on the case definition provided in a recent epidemiological review (Friedman et al., 2017), which adapted definitions provided by the CDC Yellow Book, the European Food Safety Authority (EFSA) Framework Agreement, and the FDA Bad Bug Book, a possible case definition for ciguatera may be defined (as described in Box 1): the consumption of ciguatoxic fish followed by the onset within 48 hours of specific, incident neurological symptoms: cold allodynia (which may be considered as nearly pathognomonic), paraesthesia, dysaesthesia, pruritus, myalgia, arthralgia, and/or dizziness.

6.2.1 ACUTE SYMPTOMS

Ciguatera poisoning, although defined by its neurological symptoms, is characterized by a combination of non-specific symptoms including possible gastrointestinal, neurological, cardiovascular and other systemic manifestations (Friedman et al., 2017). General, unspecific symptoms such as severe fatigue and any kind of pain (e.g. myalgia, arthralgia and dentalgia) are very common. More than 175 different symptoms have been reported to date (Sims, 1987) (see Figure 10).

The organ systems most commonly affected during the acute phase of the poisoning are (Bagnis, Kuberski and Laugier, 1979; Palafox et al., 1988; Pearn, 2001; Dickey and Plakas, 2010; Friedman, et al., 2017):

> the gastrointestinal tract, e.g. nausea, vomiting, abdominal pain, diarrhoea;

> the cardiovascular system, e.g. bradycardia, hypotension (Geller, Olson and Senecal, 1991);

> the peripheral nervous system, e.g. paraesthesia (tingling of the mouth and digits, numbness, pruritus), dysaesthesia (cold allodynia), hyporeflexia, dysphagia (Bagnis et al., 1977; Friedman et al., 2007; Oehler et al., 2009).

> the central nervous system, e.g. headache, dizziness, anxiety, tremor, ataxia, changes of behaviour (Bagnis et al., 1977; Friedman et al., 2007).

> the autonomic nervous system, e.g. disturbances of thermoregulation, hypersalivation, mydriasis, meiosis, altered sense of smell, laryngeal spasm.

> the arthro-muscular system, e.g. muscle weakness, myalgia, arthralgia.

The most characteristic manifestations of ciguatera, found in all patients, are neurological symptoms. Paraesthesia of the extremities and other body surfaces generally appears within 12 h postprandial, may begin perioral a few minutes after ingestion of marine food products (Bagnis, 1968; Chateau-Degat et al., 2007; Friedemann, 2016), and can last days, month or even years (Friedemann, 2016). Paraesthesia causes great discomfort in ciguatera poisoning patients and may worsen symptoms of anxiety or depression (Friedemann, 2016).

Ciguatoxins may act on the central thermoregulation centre in the hypothalamus (Peng et al., 1995). Hypothermia is observed during the first stage of the disease in many human patients, consistent with symptomatology described in animals (Gatti, Oelher and Legrand, 2008; Boucaud-Maitre et al., 2018). Shivering and chills are often present in the acute phase (Boucaud-Maitre et al., 2018; Gatti, Oelher and Legrand, 2008; Gatti et al., 2018; Mattei et al., 2014).

Cold allodynia is nearly pathognomonic for ciguatera (Mattei et al., 2014). This modification of thermal sensation (dysaesthesia) makes victims feel an unpleasant or painful “burning cold” sensation, tingling or electric discharges (Bagnis, 1968; Palafox and Buenconsejo-Lum, 2001; Pearn, 2001) as consequence of any contact with cold fluids, cold surfaces (e.g. bathroom floors, door handles of buildings or vehicles), or cold wind. It has been suggested that this pathological thermal sensation is mediated by bilateral responses in the medial insula, medial cingulate cortex, secondary somatosensory cortex, frontal areas, and cerebellum (Eisenblatter et al., 2017). Vetter et al. presented TRPA1-dependent Ca2+ influx in response to mild cooling as responsible for the development of pain and cold allodynia (Vetter, Zimmermann and Lewis, 2014).

Unlike the gastrointestinal symptoms of ciguatera (e.g. nausea, vomiting, diarrhoea and abdominal pain), which may appear within 12 h and last for up to 1 or 2 days, or the cardiovascular symptoms (e.g. bradycardia and hypotension) that remain for 3–4 days (Butera et al., 2000), neurological manifestations can last for weeks, months or even years (Friedman, et al., 2007; Friedman, et al., 2017).

Psychiatric symptoms have also been reported, such as depression, anxiety, and memory disturbances (Pearn, 2001; Arena et al., 2004; Dickey, 2008).

Symptoms in the urogenital tract (pain, paraesthesia, dyspermia, dyspareunia) sometimes occur and are often primarily misdiagnosed (Lange, Lipkin and Yang, 1989; Pearn, 1989; Müller and Majer, 2018).

Worldwide occurrence of gastrointestinal, neurological, and cardiovascular ciguatera symptoms:

Gastrointestinal, neurological and cardiovascular symptoms can all occur after consumption of contaminated seafood from any of the major areas where ciguatera is endemic (i.e. Pacific Ocean, Indian Ocean, and the Caribbean) (Friedman, et al., 2017).

Ciguatera poisoning severity criteria:

The Poison Severity Score (PSS) is a standardized inventory for grading the severity of acute poisonings in adults and children (Persson et al., 1998), and has been recommended as a guideline for the evaluation of poisonings regardless of causative agent by WHO since 2011 (WHO, 2011). According to the PSS, poisoning cases presenting with an acute bradycardia (< 40 BPM) should be considered severe, consistent with past ciguatera studies where authors suggested using cardiovascular symptoms as a ciguatera poisoning severity indicator (Morris et al., 1982; Katz, Terrell-Perica and Sasaki, 1993; Chateau-Degat et al., 2007; Friedemann, 2016). Other symptoms of ciguatera poisoning that should be considered as severe include: intense abdominal pain (Friedemann, 2016; Chan, 2016), extreme agitation, areflexia, generalized seizures (Chan, 2016), paralysis (Chan, 2016; Oehler et al., 2009), ophthalmoplegia (Chan, 2016; Bagnis, 1979), decreased function or paralysis of respiratory tract muscles (Chan, 2016). Often, deep coma precedes death (Chan, 2016; Oehler et al., 2009; Bagnis, 1979).

6.2.2 CHRONIC SYMPTOMS

Chronic ciguatera symptoms are those that persist beyond three months after the initial poisoning, in accordance with the definition of chronic pain (Treede et al., 2015), and concern at least 20 percent of ciguatera-affected persons (Pearn, 1996; Palafox and Buenconsejo-Lum, 2001; Friedemann, 2016). Gatti et al. reported persistent symptoms in 100 percent, i.e. 9 persons, 6 months after a single exposure linked to the consumption of the gastropod Tectus niloticus in French Polynesia (Gatti et al., 2018), where high amounts of CTXs were detected in Tectus niloticus samples collected in the same area shortly after this outbreak (Darius et al., 2018a).

Ciguatera poisoning may have neurological, psychiatric and/or general symptoms that can persist months or years after the initial poisoning (Blythe et al., 1992; Baumann, Bourrat and Pauillac, 2010; Friedemann, 2016; Gatti et al., 2018) (Table 9). Smoking has been suggested as a potential risk factor for ciguatera symptom persistence (Chateau-Degat et al., 2007).

The mechanism underlying the long persistence of symptoms in ciguatera poisoning patients is not confirmed, but it has been suggested that one of the possible mechanisms could be that CTXs may be stored in deep tissue and occasionally released into the blood stream following lipid metabolism activation (Nicholson and Lewis, 2006). Another hypothesis is that immune dysregulation could be involved (Ryan, Wu and Shoemaker, 2015; Shoemaker, House and Ryan, 2010).

6.2.3 ACUTE RECURRENCE/EXACERBATION OF CIGUATERA SYMPTOMS

The acute exacerbation of specific symptoms in chronic courses of ciguatera poisoning is characterized by the transient recurrence of neurological and possibly other systemic symptoms, following the consumption of certain foods, exposure to certain environmental factors, or enactment of specific behaviours (see Table 10) (Bagnis, Kuberski and Laugier, 1979; Gillespie et al., 1986; Lange, Snyder and Fudala, 1992; Fleming and Blythe, 1997; Lewis, 2001; Vigneau et al., 2008; Gatti et al., 2018; Friedman et al., 2017). Ciguatera-specific symptoms may reappear even in cured patients when they are exposed to certain triggers, e.g. to ciguatoxic and non-toxic fish consumption (Pottier, Vernoux and Lewis, 2001).

6.2.4 LETHALITY

Although ciguatera poisoning usually has a benign prognosis, there have been reports of lethal cases in all endemic regions (Rabenjarison et al., 2016; Chan, 2016; Friedman, et al., 2017). The ciguatera poisoning case fatality rate has been estimated as < 0.5 percent (Bagnis, Kuberski and Laugier, 1979; Allsop et al., 1986), but in some contexts may exceed 10 percent (Bagnis 1970; Rabenjarison et al., 2016). Death due to CTX exposure often follows cardiovascular and/or complications of the central nervous system. It might be preventable by avoiding consumption of fish heads, liver and viscera or possibly through better clinical management practices (Hamilton et al., 2010; Rabenjarison et al., 2016).

6.3 EPIDEMIOLOGY

Ciguatera poisoning is a global health problem. Although CP is endemic to countries from intertropical area around the globe, “imported cases” are reported in countries far from the tropics through tourists being poisoned during travel in ciguatera-endemic regions and/or poisoning cases with imported toxic fishes from ciguatera endemic regions (Table 11). Population-specific estimates of incidence rates have been published for some locations, from 0.002/10 000 persons in Kakeroma Island, Japan (Chan, 2015b) to 280/10 000 persons in Marquesas, French Polynesia (Chateau-Degat et al., 2007). However, epidemiological understanding of the global burden of disease is extremely limited, in part due to systematic under-reporting (Begier et al., 2006; Tester et al., 2010; Skinner et al., 2011). Recent initiatives to consolidate global monitoring of harmful algal events, such as ciguatera poisonings, have been implemented, for example, as the Harmful Algae Information System operated by the Intergovernmental Oceanographic Commission (http://haedat.iode.org).

Inferences regarding the likely impacts of climate change on the global burden of ciguatera poisoning are limited by the dearth of high-quality quantitative data on the historical burden of ciguatera poisoning. A retrospective analysis of telephone calls to the United States Poison Control System identified direct associations between major storms and upticks in ciguatera reports about 18 months later and suggested that there could be dramatic increases in the ciguatera burden if the temporal trends continue (Gingold, Strickland and Hess, 2014). These results coincide with observations in the Caribbean, where intervals of 1–2 years between the occurrence of severe storms and upsurges in ciguatera incidence or toxicity of fish were reported (Pottier, Vernoux and Lewis, 2001). However, there have not been consistent time trends identified in 30-year time trends analysis of reported cases in the Marshall Islands (Radke et al., 2013). It has been suggested that the evolution of the ciguatera burden under climate change may be more nuanced than a simple increase with rising temperature, as ciguatera-causing algae may have preferred environmental conditions (Llewellyn, 2010).

The European Rapid Alert System for Food and Feed (RASFF) has issued 8 alerts for ciguatoxins in the past 5 years. In all eight cases the notifying country was France (https://webgate.ec.europa.eu/rasff-window/portal), and the poisoning includes; snapper fillets (27 July 2017) and chilled kingfish (22 August 2016) from India; chilled great barracuda from Senegal (22 August 2016); frozen whole barracuda from India (19 August 2016); wild-caught fish from Sri Lanka (27 January 2015); frozen red snapper from Viet Nam (17 March 2017) and India (14 July 2016 and 16 November 2012).

Outbreaks of CP have also been reported from tropical or subtropical regions, such as Australia (Gillespie et al., 1986), China, Hong Kong SAR (Chan, 2014; Wong et al., 2008; Wong et al., 2005), French Polynesia (Gaboriau et al., 2014; Chinain et al., 2010b), Okinawa, Japan (Chan, 2015a; Oshiro et al., 2010), and the United States of America (Barrett, et al., 2017).

Because CP is associated with tropical marine environments between latitudes 35°N and 35°S, most of the intoxications are located in these areas, although, as mentioned above, international trade is increasing the poisoning cases of non-native countries. In the Atlantic, it is common in Florida (the United States of America), the Bahamas, throughout the Caribbean (Boucaud-Maitre et al., 2018), particularly in Cuba, the Dominican Republic, Haiti, Puerto Rico, and the Leeward Islands, including the Virgin Islands. In the Pacific, it occurs in Australia, Cook Islands, Fiji, Hawaii (the United States of America), French Polynesia, the Marshall Islands, New Caledonia, the Philippines, Samoa, Tonga, and Vanuatu. In the Indian Ocean, it appears in Comoros, Chagos Islands (British Indian Ocean Territory), India, Madagascar, Maldives, Mauritius, Réunion, Seychelles and Sri Lanka (Gestal-Otero, 2014).

In the Pacific region there are about 3 400–4 700 ciguateric intoxications per year, which most probably represent only 10–20 percent of the real number of cases (Gestal-Otero, 2014). It has been reported that a classical CP case presented to 36 clinicians in southern Florida (the United States of America) (an endemic area), and 68 percent identified the syndrome, but only half of them were aware of the obligation to report the case to health authorities (Friedman et al., 2017). Tables 12 and 13 show incidences in certain locations with high values.

If, as suspected, only 10–20 percent of actual intoxications are formally reported to the authorities, the problem of CP poisoning is much larger than official figures show (Friedman et al., 2017). In Florida (the United States of America), an outbreak is considered one single case (Klekamp, Bodager and Matthews, 2015), while CDC requires at least two cases (CDC, 2000). The number of reported cases is also related to the awareness of clinicians to report it (for example, in Florida [the United States of America], in many cases doctors are not aware of the need to report it [Friedman et al., 2017]), and with the actual identification of the poisoning, as the symptoms are in many cases misguiding and lead to incorrect diagnosis (Friedman et al., 2017). It is estimated that 50 000–500 000 poisonings per year may be occurring (Fleming et al., 1998). In Puerto Rico and United States Virgin Islands alone, it has been estimated that 20 000–40 000 illnesses per year may occur (Tosteson, 1995). It has been estimated that the annual incidence is about 10 percent of local Pacific island populations (Lewis and Ruff, 1993). The prevalence of the poisoning in endemic areas covers a range from 0.1 percent of the population in continental lands (Queensland [Australia] or Florida [the United States of America]) to more than 50 percent in small South Pacific or Caribbean islands (Dickey and Plakas, 2010; Skinner et al., 2011). One of the most affected countries is Tuvalu, with a prevalence of 240 cases per 10 000 population, while in the United States of America it is 50–70 cases per 10 000 population (Gestal-Otero, 2014). There were 39 677 reported cases in 17 Pacific Island Countries and Territories (PICTs), with an annual incidence of 194 cases per 100 000 population from 1998 to 2008. This, compared with the 104 cases per 100 000 population from 1973 to 1983, means a 60 percent increase (Skinner et al., 2011). Using the conservative estimate that the official reported CP accounts for 20 percent of actual incidence, the real overall incidence for the region would be 970 cases per 100 000 population for 1998–2008.

In the United States of America, from 1998 to 2015, out of the 260 000 people intoxicated by fish in 857 outbreaks, 227 outbreaks were caused by CTX, especially linked to grouper fish (Barrett et al., 2017). In Florida (the United States of America) alone, there were 137 cases from 2012 to 2014 (Klekamp, Bodager and Matthews, 2015). In China, Hong Kong SAR, the annual incidence of CP of 10.2 cases per million people from 1989 to 2008 was caused by groupers (replacing snappers as the main cause) (Chan, 2015a, 2014). In French Polynesia, the prevalence between 1992 and 2001 was of 35.6 cases per 10 000 population, but the incidence decreased to 14.5 cases per 10 000 in 2008 by improving the public health system (Château-Degat et al., 2009). A risk assessment evolution in French Polynesia concluded that the locations known by local people as safe would actually be safe, with low Gambierdiscus populations (Darius et al., 2007), but those fish with sizes thought to be safe were actually toxic (Gaboriau et al., 2014). In Cook Islands, annual CP incidence varied from 204 cases per 10 000 population to 1 058 cases per 10 000 population between 1994 and 2010 (Rongo and van Woesik, 2013, 2011). The prevalence of poisoning in New Caledonia is in general low, 10 cases per 10 000 population, and doctors rarely report food-borne intoxications. It is a common situation that CP is often underdiagnosed and under-reported, with only 2–10 percent of cases reported to health authorities (Friedman et al., 2017). Estimates of the annual incidence of CP in Oceania have ranged from 0.5 cases per 10 000 population in Hawaii (the United states of America) to 5 850 cases per 10 000 population in French Polynesia.

Ciguatera poisoning had never been reported on the coast of West Africa until a 2004 outbreak in the Canary Islands (Spain), which was followed by two additional outbreaks in 2008-2009. These cases were linked to consumed lesser amberjack (Seriola rivoliana) captured in local waters. The Canary Islands had been considered a non-endemic region for CP (Perez-Arellano et al., 2005). After these incidents several species of Gambierdiscus were identified in the zone (Rodriguez et al., 2017). The patient showed all the typical symptoms of CP. In 2008, public health authorities of the Canary Islands (Spain) investigated a second outbreak of CP involving 20–30 patients that showed symptoms a few hours after eating lesser amberjack, similar to the 2004 outbreak. The amberjack involved in this event were captured off the north coast of the Canary Islands (Spain), near the Selvagens Islands (Portugal), another area reported to have caused intoxications (Otero et al., 2010). In 2009, a third outbreak occurred in the same location, affecting 10–40 individuals who had eaten lesser amberjack bought from a supermarket, and as in former incidents, the fish had been captured north of the Canary Islands (Spain), near the Selvagens Islands (Portugal).

In other supposedly non-endemic areas, such as the eastern Mediterranean, there have been confirmed reports of intoxications from rabbit fish (Siganus sp.) caught in Haifa Bay (Israel) (Raikhlin-Eisenkraft and Bentur, 2002). Barracuda (Sphyraena barracuda) and snapper (Lutjanus sp.) from Cameroon have also caused intoxications (Bienfang, Oben and DeFelice, 2008).

In recent years, there has also been an increase in inquiries for CP patients who have spent their holidays in tropical regions, particularly in the Pacific islands and the northern regions of Australia, and in the Caribbean.

Outside the Canary Islands (Spain), the frequency of intoxications in Spain is very low, mostly from tourists returning from Cuba or the Dominican Republic (Herrero-Martínez et al., 2011). In 2011, a case was described (Herrero-Martínez et al., 2011) of a 44-year-old woman after returning from Santo Domingo (the Dominican Republic) after consumption of boiled silk snapper (L. vivanus).

In Europe, intoxications are a consequence of international trade. The episode in Hamburg (Germany) in 2009 was caused by fish (bigeye trevally [Caranx sexfasciatus] and red grouper [Cephalopholis miniata]) caught in the Caribbean (Schlaich et al., 2012). This poisoning affected 14 sailors who had consumed frozen fish, and symptoms persisted for 14 days. Further outbreaks occurred in Germany between 2012 and 2017 caused by snappers imported from India, Indonesia and Viet Nam (Friedemann 2019).

A recent report of a large single poisoning was reported in Mangalore, India, in 2016. This case affected more than 200 people working in a seafood processing unit, with typical symptoms of abdominal pain, vomiting, weakness and tingling sensations (Karunasagar et al., 2018). Each individual has consumed 1–6 pieces of cooked heads of large red snapper (Lutjanus bohar). Gastrointestinal symptoms started after 4–5 h, and after 12 h all were sick, with neurological symptoms. While 25 percent of these cases were mild, the rest were severe, and 10 percent experienced cardiovascular symptoms (hypotension, and sinus bradycardia) that were responsible for a longer hospitalization. Analysis suggested Caribbean and Indian Ocean CTXs, in amounts equivalent to CTX3C of 1.1–2.6 ng/g.

6.4 TREATMENTS

No therapy has demonstrated efficacy for treating acute or chronic manifestations of CP. Supportive medical management of CP relies on symptom-managing medications, such as antispasmodic, antiemetic, antidiarrheic, cardioactive and antidepressant drugs, as well as chronotropic pressors, rehydration, calcium gluconate, antihistaminics, analgesics, calcium channel blockers, and/or B1, B6, B12 vitamins (Berlin, King and Blythe, 1992; Lewis, 2001; Kumar-Roiné et al., 2010; Friedman et al., 2017).

The use of mannitol at an early stage of the poisoning has been suggested to reduce Schwann cell oedema and to act as a scavenger of free radicals (Birinyi-Strachan et al., 2005a, Palafox, 1992; Palafox et al., 1988). Infusion of 10 ml/kg of 20 percent solution, infused slowly for up to 45 min, within 48 h post-poisoning, has been shown to treat neurological symptoms in well-rehydrated patients (Pearn, 2001; Bagnis et al., 1992; Mullins and Hoffman, 2017).

The effectiveness of pregabalin and gabapentin for the alleviation of pain and allodynia has not been demonstrated in randomized trials (Friedman et al., 2017).

In the Indo-Pacific and West Indies endemic regions, the use of 90 different traditional herbal remedies such as Heliotropium foertherianum, Euphorbia hirta, Rosmarinus spp., Vitex spp. as well as castor oil and carapat oil have been reported (Kumar-Roiné et al., 2011), but none of them has been clinically evaluated (Pottier, Vernoux and Lewis, 2001; Friedman, et al., 2017).

CHAPTER 7

EXPOSURE ASSESSMENT

7.1 INTRODUCTION

While there is evidence to suggest that individuals may experience multiple instances of CP over time and may exhibit chronic adverse health effects, there is little evidence that chronic exposure to CTXs at subacute poisoning levels is associated with distinct adverse health effects. Consequently, assessment of dietary exposure to CTXs is primarily concerned with acute (single event or single day) exposures.

7.2 DETECTION FREQUENCY OF CTXs AND LEVELS OF CONTAMINATION

Table 14 summarizes information on concentrations of CTXs in samples of fish associated with CP cases (suspect) and from monitoring studies (random). These concentrations were derived using a range of activity- and structure-based analytical methods, with concentrations expressed as equivalents of a range of different CTX. Consequently, concentration values are not necessarily comparable from study to study. Only recently, with the advance of analytical methods, has this information become more complete. Nevertheless, the lack of CTX standards is still a major limitation to understand the nature of the intoxications.

While some of the studies in Table 14 are classified as random, all information currently available on CTXs or ciguatoxicity in fish are from studies that are targeted to some extent. A number of studies relate to samples of fish implicated as the cause of cases of CP. A smaller number of prospective monitoring studies are available, but often focus on particular risk fish species or risk environments.

Table 14 contains summary information from studies that were considered to contain sufficient detail to be of potential use for exposure assessment.

7.3 CONSUMPTION

Information on fish consumption, particularly in countries with high rates of CP, is fragmentary.

7.3.1 MEAN ESTIMATES OF FISH CONSUMPTION

Analysis of food balance sheets confirms that the countries with the highest per capita pelagic fish availability for consumption levels are mainly island nations. The highest per capita pelagic fish availability was for the Maldives (163 kg/year or 446 g/day), followed by Kiribati (142 g/day), Iceland (120 g/day) and Samoa (92 g/day). It should be noted that these are population mean availability figures and are not generally useful for assessing acute dietary exposure.

An analysis of CP cases in Guadeloupe included information on the portion size consumed for 25 cases of people who consumed fish fillet and two cases of people who consumed fish head (Hossen et al., 2015). The mean fillet serving size was 204 g (range 100–400 g), while for the two cases who had consumed fish head the serving size was 50 g in both cases. On a body weight basis, the mean fillet serving size was 3.2 g/kg bw (range 1.1–7.7 g/kg bw) for cases where both serving size and body weight information was available (n = 21) and 0.59 g/kg bw for the two cases who consumed fish head. The minimum serving size for fish fillet associated with CP was 100 g.

The FAO/WHO Chronic Individual Food Consumption database (CIFOCOss) is based on studies that surveyed at least two consumption days. While these data are not strictly appropriate for acute dietary exposure assessment, the highest individual mean consumption is 202 g/day (3.1 g/kg bw).

The EFSA Comprehensive European Food Consumption Database (EFSA, 2018) contained food consumption information from 60 surveys carried out in 25 European countries. Mean single-day estimates for fish (meat) consumption by adults are in the range of from 45 g/day (Denmark) to 220 g/day (Hungary). However, as Hungary is a land-locked country, the higher estimate is likely to include a substantial proportion of freshwater fish species. The highest single-day mean fish consumptions for maritime countries were 215 g/day (Romania) and 212 g/day (Croatia). The mean of the various European mean estimates was 139 g/day. Estimated single-day mean fish consumption by children was lower than for adults; in the range of from 33 g/day (Denmark) to 130 g/day (Austria). The mean of all mean estimates was 81 g/day.

7.3.2 HIGH PERCENTILE ESTIMATES OF FISH CONSUMPTION

Data submitted to the current consultation by Food Standards Australia New Zealand, for the Australian population, include large portion sizes (97.5th percentile of consumers only) for a range of seafood species. Large portion sizes range from 15 g (anchovies) to 490 g (snapper).

The current model for the International Estimate of Short-term Intake (IESTI) includes an even higher large-portion size for marine fish of 1 040 g (WHO). The large portion database, used by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) for the Global Estimate of Acute Dietary Exposure (GEADE) for veterinary drugs includes a 97.5th 1-day fish consumption figure of 2 000 g, based on data from Slovakia. However, little information is available to assess the reliability of this very high consumption figure.

The EFSA Comprehensive European Food Consumption Database reported 97.5th percentile single-day estimates for fish consumption by adults as being in the range of from 183 g/day (Denmark) to 717 g/day (Poland). However, due to the number of respondents in the latter survey, the 97.5th percentile was not considered to be a reliable high percentile. The highest reliable 97.5th percentile single-day fish consumption estimate was 558 g/day (Croatia). Estimated single-day 97.5th percentile fish consumption by children was in the range of from 120 g/day (Germany) to 340 g/day (Italy). The highest reliable 97.5th percentile was 296 g/day (Spain).

As mentioned above, CIFOCOss is based on studies that surveyed at least two consumption days. While these data are not strictly appropriate for acute dietary exposure assessment, the highest consumer 97.5th percentile consumption figure for marine fish is 655 g (9.98 g/kg bw) for the Brazilian population.

Based on these data, four “reference” fish consumption levels were selected: 100 g/day (mean; non-major fish-consuming nations); 200 g/day (mean; major fish-consuming nations); 500 g/day (97.5th percentile; major fish-consuming nations); and 1 000 g/day (highest reliable single-day consumption estimate), for scenario assessment.

7.4 CIGUATOXIN DOSES CAUSING CIGUATERA POISONING

The study by Hossen et al. (Hossen, et al., 2015) appears to be unique in the literature in reporting estimated doses of CTX associated with cases of CP, rather than only the concentration of toxins or toxicity in the implicated fish (Figure 11). Data were available for 17 cases for which the consumption amount, the CTX concentration (determined by N2a, as CTX1B equivalents) and the consumer body weight were available. The estimated doses eliciting CP were in the range 48.4–429 pg/kg bw CTX1B equivalents, with a mean of 221 pg/kg bw CTX1B equivalents and a median of 220 pg/kg bw CTX1B equivalents. The distribution of doses can be well represented by a normal distribution (Figure 11, mean 220 pg/kg bw CTX1B equivalents, standard deviation 108 pg/kg bw CTX1B equivalents). The minimum eliciting dose of 48.4 pg/kg bw CTX1B equivalents provides an estimate of a LOAEL for CP in humans.

It should be noted that, although results were expressed in terms of CTX1B, this was due to availability of that CTX and the actual CTXs present were Caribbean CTXs, which was confirmed by LC-MS/MS.

This study was carried out in a CP endemic area, and it is possible that the cases analysed were chronically exposed to CTX in addition to the exposure quantified from the suspected fish meal.

Lehane and Lewis (Lehane and Lewis, 2000) estimated a minimum dose likely to cause mild CP from the minimum concentration of toxin detected in fish associated with CP outbreaks (0.1 μg/kg CTX1B) and assuming a 500 g consumption level and a 50 kg body weight. However, the estimated exposure dose (1 ng/kg bw CTX1B equivalents or 1 000 pg/kg bw CTX1B equivalents) is higher than any of the doses estimated in the study by Hossen et al. (Hossen et al., 2015). A number of factors could contribute to this apparent large difference in estimates of LOAEL, including the sensitivity of the analytical method used.

Yasumoto et al. (Yasumoto, Raj and Bagnis, 1984) reported that illness in adults could result from oral intake of as little as 0.1 μg of CTX. Assuming a 60 kg body weight, this would equate to an exposure dose of 1.7 ng/kg bw or 1 700 pg/kg bw.

Both the FDA and EFSA have proposed a fish CTX concentration of 0.01 μg/kg CTX1B as being unlikely to elicit symptoms of CP. This concentration is just below the lowest concentrations seen in fish samples associated with CP cases (0.02 μg/kg CTX1B equivalents) (Farrell et al., 2017; Hossen et al., 2015).

7.5 DIETARY EXPOSURE TO CIGUATOXINS

Given the targeted nature of all of the studies summarized in Table 13 and the diversity of analytical methods and bases for analytical results, the available concentration data do not provide a suitable basis for nationally or internationally applicable acute dietary exposure assessment. Instead, a scenario-driven approach to dietary CTX exposure has been adopted. Table 15 provides estimates of acute dietary exposure based on four fish-serving sizes (100 g, 200 g, 500 g and 1 000 g) and four concentrations of CTX in fish. The particular CTX has not been specified; this is so that these scenarios can be fitted to a variety of situation.

Concentrations of ciguatoxicity in fish suspected to have caused CP as low as 0.02 μg/kg CTX1B equivalents have been reported (Farrell et al., 2017; Hossen et al., 2015). The minimum doses associated with CP cases from these two studies were determined using different analytical methods (LC-MS/MS and N2a, respectively). Depending on the rate of consumption of the fish, this level of toxin may equate to a dietary exposure of 33–340 pg/kg bw CTX1B equivalents for serving sizes of 100–1 000 g.

There is no evidence to suggest that the susceptibility of children to the effects of CTX is any different from that of adults. However, children consume food at a higher rate than adults in relation to their body weight, and this may increase their risk of CP from consumption of CTX-contaminated fish, compared with adults consuming flesh from the same fish.

CHAPTER 8

CONCLUSIONS

Ciguatera poisoning can be caused by consumption of several marine organisms, mainly fish, which have accumulated CTXs via the food chain in tropical waters. Abundance and distribution of CTX-producing organisms (Gambierdiscus and Fukuyoa species) can be modulated by multiple factors. These include eutrophication of water through excess nutrient input, damage to coral reefs or water temperature change, which may result in specific favouring environmental conditions in regions that had no prior history of Gambierdiscus growth. Taking into consideration the circumtropical distribution of ciguatera, international trade and export of potentially toxic fish, and tourist travel to endemic areas, ciguatera poisoning has become a global problem.

While, due to the existing data gaps, it was not possible to carry out and complete a full risk assessment, the Expert Meeting did outline the following considerations.

CTX classification

Taking into account the current knowledge of structural features as well as existing nomenclature of CTXs, the Expert Meeting suggested and used a classification of five groups: CTX3C, CTX4A, C-CTX, I-CTX (the existence of this group is still speculative as the structure elucidation is pending), and other Gambierdiscus metabolites.

Analytical challenges

Regarding the analytical determination of CTXs, the Expert Meeting identified the following major constraints: (i) there is no certified CTX standard; (ii) there is no reference material; (iii) there is no reference chemical protocol for CTX extraction in a biological matrix validated internationally; and (iv) there is no single- or multi- interlaboratory validation for any method, either LC-MS/MS, screening methods or bioassay. The current lack of reference materials makes it impossible to completely validate the analytical methods and, therefore, extremely difficult to compare analytical results between methods and/or laboratories. Moreover, all current screening methods/bioassays have been only incompletely validated. To date, LC-MS/MS is the only known confirmatory method, but it has also been only incompletely validated. According to current best analytical practices, the recommendation is to use screening methods prior to the use of LC-MS/MS. Among all the methods examined (LC-MS/MS, N2a, RBA, MBA and ELISA), the detection methodology with the lowest limit of detection (LOD) is the neuroblastoma assay (N2a). The LOD of this assay is 0.01 μg/kg fish flesh when optimized; however, this LOD is not always achieved.

While researchers have made some progress using purified toxins that are being made available, investment of significant resources is required in order to develop commercial standards and ultimately certified reference materials for the major toxin forms. For this reason, criteria for such purified compounds should include sufficiently high purity, characterization of impurities, and broad and continued availability.

Purified toxin standards are required for at least the following: (i) of the group of CTX4A analogues, CTX4A, CTX4B, CTX1B (these three are critical), 52-epi-54-deoxyCTX1B and 54-deoxyCTX1B, M-seco-CTX4A/B; (ii) of the group of CTX3C analogues, CTX3C (critical), 49-epiCTX3C, 51-hydroxyCTX3C, 2-hydroxyCTX3C, 2,3-dihydroCTX3C, M-seco-CTX3C; and (iii) of the group of C-CTX analogues, C-CTX1 (critical), C-CTX2. Moreover, Indian Ocean CTXs are poorly understood, and research is required in order to determine their algal source, chemical structures, and toxicity. The high fatality rate associated with shark poisoning in the Indian Ocean region makes further investigation of the structure, toxicity and prevalence of these toxins a high priority.

The Expert Meeting also noted that, due to the lack of information on the genes responsible for CTX production by Gambierdiscus, it is not possible at present to use targeted molecular approaches to identify toxigenic species. Research on this topic is needed as it remains an important step to elucidate the specific genetic pathways responsible for toxin production by Gambierdiscus spp.

Hazard identification and ciguatoxin toxicity

Animal data show that CTXs are: (i) efficiently absorbed and distributed in the body after ingestion in laboratory animal models; and (ii) eliminated mainly through faeces in animals. Most pure CTX ADME animal studies involve Pacific CTXs and CTX1B in particular, and all have used activity-based detection methods to quantify toxins in tissues. On the other hand, the available human data show that: (i) ingested CTX is rapidly systemically distributed and mucosal absorption appears possible; (ii) CTX4A and B are metabolized in vitro by CYP3A4; and (iii) CTXs may cross the placenta and enter breast milk.

The toxicological studies performed to date show that, with regard to acute toxicity: (i) all tested CTX analogues from all regions are acutely toxic and induce common effects in mice; (ii) with the accepted extraction procedures, CTX extracts induced similar effects in mice to those from purified and synthesized substances; (iii) the limited data suggest that the effects observed are similar following ip and oral administration, and based on limited ip LD50 data the potencies of the different analogues within and between the different classes appears to vary; and (iv) the method used for extraction of the CTX (i.e. the first solvent) has to be taken into consideration. The acetone method is designed to eliminate interference with MTX, which is acetone-insoluble, and should be used.

The results obtained with repeated dose studies show that there are very few oral studies in mice or rats, only using purified CTX1B (and in one study, purified CTX4C). Respective information is lacking for the other CTX compounds. The available studies include two short-term studies in mice, one medium-term study in rats (focused on neurological effects), and one long-term study in mice (focused on heart toxicity). None of these studies is suitable to establish a health-based guidance value (acute or chronic).

With regard to the mode of action, it is well accepted that the shared primary molecular mechanism of CTXs is the binding and the activation of the VGSC. This leads to an increase in membrane excitability, as well as to secondary cellular effects that are the basis of most human symptoms. In addition, CTXs block the VGPC, and this effect also contributes to increased membrane excitability. The potencies of CTXs in affecting these two channels vary according to CTX congeners.

The available information about TEFs suggests that the toxicity of all CTXs may not be covered by the in vitro bioassays and that in vitro bioassays have not yet been sufficiently validated for use in risk assessment. Based on the existing data, MBA is the only current in vivo assay that allows relative effect potencies to be determined; however, due to limited data from oral in vivo studies, it has not been possible to derive TEFs.

Clinical features and epidemiology of Ciguatera poisoning

The clinical features of CP can be classified into acute and chronic symptoms. Acute symptoms are observed in almost all organ systems and are qualitatively observed in all geographical CP regions. While it is unclear whether the frequency of all symptoms between various CP regions differs, fatalities have been reported in all regions. With regard to chronic symptoms, they are characterized by the presence of neurological, neuropsychiatric and systemic symptoms beyond three months after the initial poisoning. At least 20 percent of affected persons show chronic symptoms (and the prevalence can reach 100 percent after a single marine invertebrate CTX exposure), and a transient recurrence or exacerbation of neuromuscular or other systemic symptoms may appear due to certain food-based, behavioural or other external triggering factors. This phenomenon can be present from the early days of the individual poisoning and can last for months or years, and decrease in intensity and frequency over time.

The epidemiological data suggest that CP is vastly under-reported, limiting the understanding of its epidemiology. Considering the scale of affected countries, consolidated data on the global burden of CP are needed. It should be noted that there are no specific treatments for CP; hence, symptomatic treatments are being used. Mannitol is commonly administered quickly after the onset of neurological symptoms to manage CP. However, its efficacy in treating CP remains controversial. Traditional remedies have been reported; however, their efficacy remains to be demonstrated in clinical trials.

Exposure assessment

Available data do not provide a suitable basis for nationally or internationally applicable dietary exposure assessment. To date, none of the available CTX monitoring studies is suitable for exposure assessment. There is only one publication available providing CTX dose information using N2a assay, allowing the estimation of the exposure dose in cases and identification of a LOAEL in humans. For risk assessment, the acute exposure to CTX is the most relevant. However, the data on which the assessment is based are from endemic areas where multiple exposures cannot be excluded. It is important to highlight the fact that the animal and human data available are insufficient to derive an acute reference dose. Moreover, while human data allowed identification of a LOAEL of 50 pg/kg bw CTX1B (other studies report a higher LOAEL), the observed uncertainties do not allow the derivation of an acute reference dose. The lowest concentration measured in fish associated with symptoms in humans was determined at about 0.02 μg/kg CTX1B-equiv in fish flesh from the Caribbean. Depending on the rate of consumption of the fish, this level of toxin may equate to a dietary exposure of 33–340 pg/kg bw CTX1B equivalents for serving sizes of 100–1 000g. Children consume food at a higher rate than adults in relation to their body weight and this may increase their risk of CP from consumption of CTX-contaminated fish, compared with adults consuming flesh from the same fish.

8.1 RISK MANAGEMENT CONSIDERATIONS

Considering the currently existing scientific gaps, it was not possible to complete a full risk assessment. However, the Expert Meeting noted that, by drawing on existing knowledge as well as traditional practices from CP endemic areas, some risk management considerations could be given to inform regional interventions.

The Expert Meeting noted that effective and integrated risk management options would require definition of toxin profiles in each region, both in algal strains and in seafood to define risk evaluation protocols. Recognizing the strong influence of the regional and local circumstances on the occurrence of the organisms as well as the production of the toxins, any conclusions should be considered as of local or regional significance only, and care must be taken when transferring these to other areas. While the link between algal CTXs and those observed in fish has been established in the Pacific, evidence from the Caribbean and the Indian Ocean is still needed and warrants ongoing investigation.

The following considerations can provide assistance in the identification and management of CP.

Establishing and/or strengthening surveillance programmes

Causative organisms should be defined, if possible, in the areas where more-toxic species have been identified. It might also be useful to develop hazard maps for each region. The Expert Meeting identified the following combination of causative organisms and regions that may be useful in informing the establishment of surveillance programmes:

> G. polynesiensis (currently only known from the Pacific);

> G. scabrosus (currently found in Japan and Okinawa [Japan]);

> G. excentricus (currently known from the Atlantic and Caribbean);

> G. silvae (currently known from the Atlantic and Caribbean);

> G. australes (currently known from the Pacific, Atlantic and Mediterranean);

Currently, it cannot be excluded that other species may be significant contributors to the occurrence of CTXs. It would also be important to consider long-term studies for a comprehensive description of population dynamics of Gambierdiscus and Fukuyoa, in order to help address seasonality variations.

Sampling

It is necessary to establish a careful sampling design for Gambierdiscus and Fukuyoa to address identification of species, quantification of populations and eventually identification and quantification of toxins in natural populations. The sampling of the causative organism and sentinel fish species needs to consider the relatively low density of benthic organisms in the water and the temporal and spatial dispersions of Gambierdiscus and Fukuyoa as well as of fish species.

The sampling of migratory fish species may benefit from a better understanding of migratory patterns, and sampling plans may also be informed by the traditional knowledge of indigenous fishers. During the sampling step, it should be endured that enough samples are collected to make further analysis using SEM and qPCR techniques, which require different preservation methods.

The use of passive sampling should be considered for an extensive overview of toxins in water, including those toxins not directly involved in CP. This allows for further differential diagnosis in poisoning events – in particular, the co-exposure to multiple toxins is to be suspected in the presence of atypical poisoning cases.

Monitoring ciguatera transmission in the food web

Depending on the region of interest and habitat, sentinel invertebrate or fish species can be selected for biomonitoring. Because fishing habits differ based on sociocultural, historical, biodiversity and regional factors, a first step would include a survey of fish across multiple trophic levels (herbivore, mesopredator [including invertivores] and predator). The Expert Meeting identified a combination of marine species in specific regions that are known to exhibit high site fidelity (i.e. resident species that stay within a small area for their whole life) (Table 16).

Definition of key target species

There is large regional variation in the occurrence of CTXs in fish in general, including within the same species. There is inconclusive evidence that size or weight of fish is an indicator for the risk of CP in every region. If possible, the marine species found to contain CTXs and commonly consumed by humans should be defined by region. Epidemiology surveys can also aid in selection of target species that have been associated with illness, although caution needs to be taken to ensure correct species identification. Selection of species identified in such surveys should start with species that have high site fidelity. Moreover, there should be a set of audit/inspection schemes that provide suitably protective measures to allow trade from endemic areas (imported fish). In addition, it should also be noted that, while changes in the occurrence of CTX in migratory species have been observed, it is unclear to what extent they can be traced back to anthropogenic factors. Therefore, it is extremely difficult to predict the risk of CP associated to migratory fish species. Traceability is an important issue in the identification of fish species, and, therefore, accurate seafood product labelling should also be improved.

Good practices

There is evidence that removing the viscera, liver and head of the fish prior to consumption will lower the risk of CP. In areas with a known high prevalence of CTXs in seafood, managers should consider the development of guidance that includes avoidance of visceral organs, roe (fish eggs), and carcasses (e.g. fish heads, eyes and bones), as these tissues can contain high levels of CTXs and other bioactive compounds that may pose a significant health risk. Moreover, good practices should provide guidance on the disposal of ciguatoxic fish, fish head and viscera.

Awareness raising and communication

Awareness raising is an important part of the risk management options, including the need for risk communication to be tailored to local situations with regard to the possible limitation of consumption and trade of specific marine species. Public outreach programmes need to inform consumers about the potential risks posed by consuming fish (including head and viscera) as well as invertebrates.

The increase in occurrence of ciguatera poisoning in certain regions suggests that surveillance, public outreach/education and further research are required also in areas where this is becoming an emerging issue.

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ANNEX

CP FLOW DIAGRAM, AND RESEARCH PROJECTS

RESEARCH PROJECTS

CIGUATOOLS

This project, already finished, was funded by the Research Executive Agency (FP7/2007-2013) under Grant Agreement 311765 between three academic partners and Cifga, a European ISO 17034 company for the production of certified marine toxins, to develop immunoassay-based methods and obtain certified standards. The result of the project was the availability of gambierone (Rodriguez et al., 2015) as potential certified calibration for ciguatoxin (CTX) analysis by LC-MS/MS. The antibody research part was not successful. A high-resolution mass spectrometry (HRMS) method was developed (Silva et al., 2015).

EUROCIGUA

The European Food Safety Authority (EFSA) is partner with several Member States’ organizations in a multiannual project Risk Characterization of Ciguatera Food Poisoning (CP) in Europe (http://www.aecosan.msssi.gob.es/AECOSAN/web/ciguatera/home/aecosan_home_ciguatera.htm).

The project is cofunded by and 14 European organizations. The main objectives of the project are:

> The estimation of the incidence of ciguatera in Europe and the determination of the epidemiological characteristics of cases.

> The assessment of the presence of ciguatoxin in Gambierdiscus and in fish in Europe.

> The development and validation of methods for the detection, quantification and confirmation of the presence of ciguatoxin-contaminated specimens.

To accomplish the first objective, a surveillance protocol for CP in the European Union was created. The protocol includes: the definitions for ciguatera cases and outbreaks; the recommended public health measures for CP; and two specific questionnaires for ciguatera cases and outbreaks. Based on these questionnaires, a database for collecting the data was created. A list of possible data sources (at country and European level) for ciguatera cases and outbreaks was elaborated (http://www.aecosan.msssi.gob.es/AECOSAN/web/ciguatera/subseccion/documents_and_publications.htm).

Sampling and culturing of Gambierdiscus and sampling of fish in Europe is ongoing, together with the evaluation of the presence of CTX. The development, optimization and validation of LC-MS/MS and HRMS for identification and confirmation of CTX is also ongoing.

ALERTOXNET

This European Interreg project is designed to implement a network of emerging toxin warnings and characterization. The network includes 14 partners and, starting in October 2017, will run for three years.

In Spain, at the national level, there is a project with Cifga specifically devoted to the development of reference standards for early, reliable and specific detection of CTXs (Reference EMP-TU-2016-4878). The project started in September and will run for three years.

The GlobalHAB programme, funded by the Scientific Committee on Oceanic Research (NSF) and UNESCO Intergovernmental Oceanographic Commission, aims to promote coordinated research on harmful algal blooms (HABs), including CP. GlobalHAB focuses on the human health impacts of marine microalgae-produced toxins, ecophysiology and oceanic processes that modulate HAB dynamics, and ecology and epidemiology of HAB illnesses (Berdalet et al., 2017). As another example, a Global Burden of Disease CP study (e.g. http://www.healthdata.org/gbd) would allow for a better understanding of the extent and economic implications of CP, and changes in international CP distribution. The One Health approach (http://onehealthinitiative.com/) seeks cooperation among environmental and health disciplines in order to improve CP diagnosis, treatment and reporting worldwide.

1 The nomenclature of for the different toxins in Figure 7 might differ from the one used throughout the document.