Ciguatoxins In Fish Research Articles

3-Epimers of Caribbean ciguatoxins in fish and algae

Ciguatera poisoning (CP) is endemic to several subtropical and tropical regions and is caused by the consumption of fish contaminated with ciguatoxins (CTXs). The recent discovery of Caribbean CTXs (C-CTXs) in Gambierdiscus spp. isolated from the Caribbean resulted in the identification of a precursor analogue, C-CTX5, that is reduced into C-CTX1. C-CTX5 has two reducible sites, a ketone at C-3 and hemiketal at C-56. Chemical reductions of C-CTX5 into C-CTX3/4 resulted in two peaks in the LC−HRMS chromatograms with a ratio that differed markedly from that observed in fish extracts and the reduction of C-CTX1 isolated from fish. Reduction of C-CTX5 should have produced four diastereoisomers of C-CTX3/4, prompting a more detailed study of the reduction products. LC–HRMS with a slow gradient was used to separate and detect the four stereoisomers of C-CTX3/4, and to determine the distribution of these analogues in naturally contaminated fish tissues and following chemical reduction of isolated analogues. The results showed that in naturally contaminated fish tissues C-CTX1/2 is a mixture of two diastereoisomers at C-3 and that C-CTX3/4 is a mixture of two pairs of diastereoisomers at C-3 and C-56. The data suggests that there is variability in the enzymatic reduction at C-3 and C-56 of C-CTXs in reef fish, leading to variations in the ratios of the four stereoisomers. Based on these findings, a naming convention for C-CTXs is proposed which aligns with that used for Pacific CTX congeners and will aid in the identification of the structure and stereochemistry of the different CTX analogues.

Evaluation of relative potency of calibrated ciguatoxin congeners by near-infrared fluorescent receptor binding and neuroblastoma cell-based assays

Ciguatera fish poisoning (CFP) is a foodborne illness affecting > 50,000 people worldwide annually. It is caused by eating marine invertebrates and fish that have accumulated ciguatoxins (CTXs). Recently, the risk of CFP to human health, the local economy, and fishery resources have increased; therefore, detection methods are urgently needed. Functional assays for detecting ciguatoxins in fish include receptor binding (RBA) and neuroblastoma cell-based assay (N2a assay), which can detect all CTX congeners. In this study, we made these assays easier to use. For RBA, an assay was developed using a novel near-infrared fluorescent ligand, PREX710-BTX, to save valuable CTXs. In the N2a assay, a 1-day assay was developed with the same detection performance as the conventional 2-day assay. Additionally, in these assays, we used calibrated CTX standards from the Pacific determined by quantitative NMR for the first time to compare the relative potency of congeners, which differed significantly among previous studies. In the RBA, there was almost no difference in the binding affinity among congeners, showing that the differences in side chains, stereochemistry, and backbone structure of CTXs did not affect the binding affinity. However, this result did not correlate with the toxic equivalency factors (TEFs) based on acute toxicity in mice. In contrast, the N2a assay showed a good correlation with TEFs based on acute toxicity in mice, except for CTX3C. These findings, obtained with calibrated toxin standards, provide important insights into evaluating the total toxicity of CTXs using functional assays.

Ciguatoxin-like toxicity distribution in flesh of amberjack (Seriola spp.) and dusky grouper (Epinephelus marginatus)

Ciguatoxins (CTXs) are marine neurotoxins that cause ciguatera poisoning (CP), mainly through the consumption of fish. The distribution of CTXs in fish is known to be unequal. Studies have shown that viscera accumulate more toxins than muscle, but little has been conducted on toxicity distribution in the flesh, which is the main edible part of fish, and the caudal muscle is also most commonly targeted for the monitoring of CTXs in the Canary Islands. At present, whether this sample is representative of the toxicity of an individual is undisclosed. This study aims to assess the distribution of CTXs in fish, considering different muscle samples, the liver, and gonads. To this end, tissues from four amberjacks (Seriola spp.) and four dusky groupers (Epinephelus marginatus), over 16.5 kg and captured in the Canary Islands, were analyzed by neuroblastoma-2a cell-based assay. Flesh samples were collected from the extraocular region (EM), head (HM), and different areas from the fillet (A-D). In the amberjack, the EM was the most toxic muscle (1.510 CTX1B Eq·g−1), followed by far for the caudal section of the fillet (D) (0.906 CTX1B Eq·g−1). In the dusky grouper flesh samples, D and EM showed the highest toxicity (0.279 and 0.273 CTX1B Eq·g−1). In both species, HM was one of the least toxic samples (0.421 and 0.166 CTX1B Eq·g−1). The liver stood out for its high CTX concentration (3.643 and 2.718 CTX1B Eq·g−1), as were the gonads (1.620 and 0.992 CTX1B Eq·g−1). According to these results, the caudal muscle next to the tail is a reliable part for use in determining the toxicity of fish flesh to guarantee its safe consumption. Additionally, the analysis of the liver and gonads could provide further information on doubtful specimens, and be used for CTX monitoring in areas with an unknown prevalence of ciguatera.

Cyclodextrin polymer clean-up method for the detection of ciguatoxins in fish with cell-based assays

Ciguatoxins (CTXs) are marine toxins produced by microalgae of the genera Gambierdiscus and Fukuyoa, which are transferred through the food webs, reaching humans and causing a poisoning known as ciguatera. The cell-based assay (CBA) is commonly used for their detection because of its high sensitivity and the provided toxicological information. However, matrix effects may interfere in the CBA. In this work, γ-cyclodextrin-hexamethylene diisocyanate (γ-CD-HDI), γ-cyclodextrin-epichlorohydrin (γ-CD-EPI) and γ-CD-EPI conjugated to magnetic beads (γ-CD-EPI-MB) have been evaluated as clean-up materials for fish flesh extracts containing CTXs. The best results were achieved with γ-CD-HDI in column format, which showed a CTX1B recovery of 42% and 32% for Variola louti and Seriola dumerili, respectively, and allowed exposing cells to at least 400 mg/mL of fish flesh. This clean-up strategy provides at least 4.6 and 3.0-fold higher sensitivities to the assay for V.louti and S.dumerili, respectively, improving the reliability of CTX quantification.

A smartphone-controlled amperometric immunosensor for the detection of Pacific ciguatoxins in fish

Ciguatoxins (CTXs) are marine neurotoxins produced by microalgae of the genera Gambierdiscus and Fukuyoa. CTXs may reach humans through food webs and cause ciguatera fish poisoning (CFP). An immunosensor for the detection of Pacific CTXs in fish was developed using multiwalled carbon nanotube (MWCNT)-modified carbon electrodes and a smartphone-controlled potentiostat. The biosensor attained a limit of detection (LOD) and a limit of quantification (LOQ) of 6 and 27 pg/mL of CTX1B, respectively, which were 0.001 and 0.005 μg/kg in fish flesh. In the analysis of fish samples from Japan and Fiji, excellent correlations were found with sandwich enzyme-linked immunosorbent assays (ELISAs), a cell-based assay (CBA) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Stability of at least 3 months at −20 °C was predicted. In just over 2 h, the biosensor provides reliable, accurate and precise Pacific CTX contents in fish extracts, being suitable for monitoring and research programs.

Ciguatoxin-Producing Dinoflagellate Gambierdiscus in the Beibu Gulf: First Report of Toxic Gambierdiscus in Chinese Waters.

Ciguatera poisoning is mainly caused by the consumption of reef fish that have accumulated ciguatoxins (CTXs) produced by the benthic dinoflagellates Gambierdiscus and Fukuyoa. China has a long history of problems with ciguatera, but research on ciguatera causative organisms is very limited, especially in the Beibu Gulf, where coral reefs have been degraded significantly and CTXs in reef fish have exceeded food safety guidelines. Here, five strains of Gambierdiscus spp. were collected from Weizhou Island, a ciguatera hotspot in the Beibu Gulf, and identified by light and scanning electron microscopy and phylogenetic analyses based on large and small subunit rDNA sequences. Strains showed typical morphological characteristics of Gambierdiscus caribaeus, exhibiting a smooth thecal surface, rectangular-shaped 2′, almost symmetric 4″, and a large and broad posterior intercalary plate. They clustered in the phylogenetic tree with G. caribaeus from other locations. Therefore, these five strains belonged to G. caribaeus, a globally distributed Gambierdiscus species. Toxicity was determined through the mouse neuroblastoma assay and ranged from 0 to 5.40 fg CTX3C eq cell−1. The low level of toxicity of G. caribaeus in Weizhou Island, with CTX-contaminated fish above the regulatory level in the previous study, suggests that the long-term presence of low toxicity G. caribaeus might lead to the bioaccumulation of CTXs in fish, which can reach dangerous CTX levels. Alternatively, other highly-toxic, non-sampled strains could be present in these waters. This is the first report on toxic Gambierdiscus from the Beibu Gulf and Chinese waters and will provide a basis for further research determining effective strategies for ciguatera management in the area.

In Vitro Glucuronidation of Caribbean Ciguatoxins in Fish: First Report of Conjugative Ciguatoxin Metabolites.

Ciguatoxins (CTX) are potent marine neurotoxins, which can bioaccumulate in seafood, causing a severe and prevalent human illness known as ciguatera poisoning (CP). Despite the worldwide impact of ciguatera, effective disease management is hindered by a lack of knowledge regarding the movement and biotransformation of CTX congeners in marine food webs, particularly in the Caribbean and Western Atlantic. In this study we investigated the hepatic biotransformation of C-CTX across several fish and mammalian species through a series of in vitro metabolism assays focused on phase I (CYP P450; functionalization) and phase II (UGT; conjugation) reactions. Using liquid chromatography high-resolution mass spectrometry to explore potential C-CTX metabolites, we observed two glucuronide products of C-CTX-1/-2 and provided additional evidence from high-resolution tandem mass spectrometry to support their identification. Chemical reduction experiments confirmed that the metabolites were comprised of four distinct glucuronide products with the sugar attached at two separate sites on C-CTX-1/-2 and excluded the C-56 hydroxyl group as the conjugation site. Glucuronidation is a novel biotransformation pathway not yet reported for CTX or other related polyether phycotoxins, yet its occurrence across all fish species tested suggests that it could be a prevalent and important detoxification mechanism in marine organisms. The absence of glucuronidation observed in this study for both rat and human microsomes suggests that alternate biotransformation pathways may be dominant in higher vertebrates.

Development and Validation of an LC-MS/MS Method for the Ultra-Trace Analysis of Pacific Ciguatoxins in Fish.

BackgroundCiguatera fish poisoning (CFP) poses a serious threat to both public health and the use of aquatic resources from the various warm‐water regions of the world. Hence, a process for the efficient determination of the relevant toxins is required.ObjectiveWe sought to develop and validate the first LC-MS/MS method to quantify the major toxins prevalent in fish from the Pacific Ocean.MethodToxins were extracted from fish flesh (2 g) using a methanol–water mixture (9:1, v/v). The extract was heated at 80°C, and low-polarity lipids were eliminated using hexane, initially from the basic solution and later from the acidic solution. The cleanup was performed using solid-phase extraction, Florisil, silica, reversed-phase C18, and primary secondary amine columns. A validation study was conducted by spiking fish flesh with two representative toxins having different skeletal structures and polarities and was calibrated by NMR (qNMR) spectroscopy.ResultsThe validation parameters for the ciguatera toxins CTX1B and CTX3C at spiked levels of 0.1 µg/kg were as follows: repeatabilities of 2.3–3.5% and 3.2–5.3%; intermediate precisions of 6.3–9.8% and 6.0–7.4%; recoveries of 80–107% and 95–120%, respectively. The lowest detection levels were 0.004 µg/kg for CTX1B, 0.005 µg/kg for 51-hydroxyCTX3C, and 0.009 µg/kg for CTX3C.ConclusionsThe described method practically clears the international action level of 0.01 µg/kg CTX1B equivalents set by the U.S. Food and Drug Administration and the European Food Safety Authority and satisfies the global standards set by Codex and AOAC INTERNATIONAL.HighlightsA validation study for an LC-MS/MS method for ciguatoxin detection was completed for the first time using calibrated toxin standards.

LC–MS/MS Analysis of Ciguatoxins Revealing the Regional and Species Distinction of Fish in the Tropical Western Pacific

Ciguatera fish poisoning (CFP) is one of the most frequently reported seafood poisoning diseases. It is endemic to the tropical region and occurs most commonly in the regions around the Pacific Ocean, Indian Ocean, and Caribbean Sea. The principal toxins causing CFP are ciguatoxins (CTXs). In the Pacific region, more than 20 analogs of CTXs have been identified to date. Based on their skeletal structures, they are classified into CTX1B-type and CTX3C-type toxins. We have previously reported species-specific and regional-specific toxin profiles. In this study, the levels and profiles of CTXs in fish present in the tropical western Pacific regions were analyzed using the liquid chromatography–tandem mass spectrometry (LC–MS/MS) technique. Forty-two fish specimens, belonging to the categories of snappers, groupers, Spanish mackerel, and moray eel, were purchased from various places such as Fiji, the Philippines, Thailand, and Taiwan. Only the fish captured from Fijian coastal waters contained detectable amounts of CTXs. The toxin levels in the fish species found along the coastal regions of the Viti Levu Island, the main island in Fiji, and the toxin profiles were significantly different from those of the fish species present in other coastal regions. The toxin levels and profiles varied among the different fish samples collected from different coastal areas. Based on the toxin levels and toxin profiles, the coast was demarcated into three zones. In Zone-1, which covers the northern coast of the main island and the regions of the Malake Island and Korovau, CTXs in fish were below the detection level. In Zone-2, CTX3C-type toxins were present in low levels in the fish. CTX1B-type and CTX3C-type toxins co-occurred in the fish present in Zone-3. The toxin profiles may have reflected the variation in Gambierdiscus spp.

Evidence for the Range Expansion of Ciguatera in French Polynesia: A Revisit of the 2009 Mass-Poisoning Outbreak in Rapa Island (Australes Archipelago).

Ciguatera poisoning (CP) results from the consumption of seafood contaminated with ciguatoxins (CTXs). This disease is highly prevalent in French Polynesia with several well-identified hotspots. Rapa Island, the southernmost inhabited island in the country, was reportedly free of CP until 2007. This study describes the integrated approach used to investigate the etiology of a fatal mass-poisoning outbreak that occurred in Rapa in 2009. Symptoms reported in patients were evocative of ciguatera. Several Gambierdiscus field samples collected from benthic assemblages tested positive by the receptor binding assay (RBA). Additionally, the toxicity screening of ≈250 fish by RBA indicated ≈78% of fish could contain CTXs. The presence of CTXs in fish was confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The potential link between climate change and this range expansion of ciguatera to a subtropical locale of French Polynesia was also examined based on the analysis of temperature time-series data. Results are indicative of a global warming trend in Rapa area. A five-fold reduction in incidence rates was observed between 2009 and 2012, which was due in part to self-regulating behavior among individuals (avoidance of particular fish species and areas). Such observations underscore the prominent role played by community outreach in ciguatera risk management.

Advances in Detecting Ciguatoxins in Fish.

Ciguatera fish poisoning (CFP) is currently the most common marine biotoxin food poisoning worldwide, associated with human consumption of circumtropical fish and marine invertebrates that are contaminated with ciguatoxins. Ciguatoxins are very potent sodium-channel activator neurotoxins, that pose risks to human health at very low concentrations (>0.01 ng per g of fish flesh in the case of the most potent Pacific ciguatoxin). Symptoms of CFP are nonspecific and intoxication in humans is often misdiagnosed. Presently, there is no medically approved treatment of ciguatera. Therefore, to mitigate the risks of CFP, reliable detection of ciguatoxins prior to consumption of fish tissue is acutely needed, which requires application of highly sensitive and quantitative analytical tests. During the last century a number of methods have been developed to identify and quantify the concentration of ciguatoxins, including in vivo animal assays, cell-based assays, receptor binding assays, antibody-based immunoassays, electrochemical methods, and analytical techniques based on coupling of liquid chromatography with mass spectrometry. Development of these methods, their various advantages and limitations, as well as future challenges are discussed in this review.

Caribbean Ciguatoxin-1 stability under strongly acidic conditions: Characterisation of a new C-CTX1 methoxy congener

ABSTRACTThe recent emergence of ciguatera in the eastern Atlantic, particularly in the Canary Islands (Spain) and Madeira (Portugal) prompted the development and implementation of liquid chromatography tandem–mass spectrometry (LC/MS-MS) methods for the detection of ciguatoxins in fish. The complexity of fish tissue matrices, low concentrations of ciguatoxins in hazardous fish, and the scarcity of ciguatoxin standards present challenging issues for successful implementation of routine ciguatoxin analysis. A laboratory reference material of Caribbean Ciguatoxin-1 (C-CTX1), which was previously confirmed in fish responsible for ciguatera outbreaks in the Canary Islands, was used to assess the toxin’s stability under strongly acidic conditions and solvent systems commonly used in LC-MS/MS. It was observed that strongly acidic conditions caused the transformation of C-CTX1 to a C56 methoxy congener, C-CTX1-Me. C-CTX1 was structurally characterised by LC-MS/MS and fragmentation pathways are presented showing the same fragmentation pattern as C-CTX1-Me. These results suggest that the use of strongly acidic conditions during sample pretreatment for C-CTX analysis, might produce significant artefacts, and risks failing to detect the presence of C-CTX1.

Ciguatera-Causing Dinoflagellate Gambierdiscus spp. (Dinophyceae) in a Subtropical Region of North Atlantic Ocean (Canary Islands): Morphological Characterization and Biogeography.

Dinoflagellates belonging to the genus Gambierdiscus produce ciguatoxins (CTXs), which are metabolized in fish to more toxic forms and subsequently cause ciguatera fish poisoning (CFP) in humans. Five species of Gambierdiscus have been described from the Canary Islands, where CTXs in fish have been reported since 2004. Here we present new data on the distribution of Gambierdiscus species in the Canary archipelago and specifically from two islands, La Palma and La Gomera, where the genus had not been previously reported. Gambierdiscus spp. concentrations were low, with maxima of 88 and 29 cells·g−1 wet weight in samples from La Gomera and La Palma, respectively. Molecular analysis (LSUrRNA gene sequences) revealed differences in the species distribution between the two islands: only G. excentricus was detected at La Palma whereas four species, G. australes, G. caribaeus, G. carolinianus, and G. excentricus, were identified from La Gomera. Morphometric analyses of cultured cells of the five Canary Islands species and of field specimens from La Gomera included cell size and a characterization of three thecal arrangement traits: (1) the shape of the 2′ plate, (2) the position of Po in the anterior suture of the 2′ plate, and (3) the length–width relationship of the 2″″ plate. Despite the wide morphological variability within the culture and field samples, the use of two or more variables allowed the discrimination of two species in the La Gomera samples: G. cf. excentricus and G. cf. silvae. A comparison of the molecular data with the morphologically based classification demonstrated important coincidences, such as the dominance of G. excentricus, but also differences in the species composition of Gambierdiscus, as G. caribaeus was detected in the study area only by using molecular methods.

Ciguatoxin prevalence in 4 commercial fish species along an oceanic exposure gradient in the US Virgin Islands.

Ciguatera fish poisoning is a seafood-toxin illness resulting from consumption of fish contaminated with ciguatoxins. Managing ciguatera fish poisoning is complex. It is made easier, however, by local fishers from endemic areas reporting regional predictability for local fish species' ciguatera fish poisoning risk, which the present study then tested. We investigated the prevalence of ciguatoxins in 4 commonly marketed and consumed species (Balistes vetula, Haemulon plumierii, Ocyurus chrysurus, and Epinephelus guttatus) across an oceanic gradient (north, south, east, and west) from the US Virgin Islands. Fish muscle extracts were analyzed for Caribbean ciguatoxins using an in vitro mouse neuroblastoma (N2a) cytotoxicity assay and confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fish collected from the north location had 0 fish with detectable ciguatoxins; this site also had the greatest wave energy. Caribbean ciguatoxins in fish ranged from 0.01 to 0.11, 0.004 to 0.10, and 0.005 to 0.18 ng Caribbean ciguatoxin-1 eq/g, from the west, east, and south respectively. Ciguatoxin-like activity was detectable by the N2a assay in 40, 41, 50, and 70% of H. plumierii, O. chrysurus, B. vetula, and E. guttatus, respectively. Of the fish collected, 4% had Caribbean ciguatoxin levels exceeding the US Food and Drug Administration guidance of 0.1 ng Caribbean ciguatoxin-1 eq/g fish. These findings concurred with spatial ciguatera fish poisoning prevalence information provided by local fishers in the US Virgin Islands and demonstrate how partnerships between researchers and fishers can aid the improvement of science-based ciguatera fish poisoning management. Environ Toxicol Chem 2018;39:1852-1863. Published 2018 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

An Updated Review of Ciguatera Fish Poisoning: Clinical, Epidemiological, Environmental, and Public Health Management.

Ciguatera Fish Poisoning (CFP) is the most frequently reported seafood-toxin illness in the world. It causes substantial human health, social, and economic impacts. The illness produces a complex array of gastrointestinal, neurological and neuropsychological, and cardiovascular symptoms, which may last days, weeks, or months. This paper is a general review of CFP including the human health effects of exposure to ciguatoxins (CTXs), diagnosis, human pathophysiology of CFP, treatment, detection of CTXs in fish, epidemiology of the illness, global dimensions, prevention, future directions, and recommendations for clinicians and patients. It updates and expands upon the previous review of CFP published by Friedman et al. (2008) and addresses new insights and relevant emerging global themes such as climate and environmental change, international market issues, and socioeconomic impacts of CFP. It also provides a proposed universal case definition for CFP designed to account for the variability in symptom presentation across different geographic regions. Information that is important but unchanged since the previous review has been reiterated. This article is intended for a broad audience, including resource and fishery managers, commercial and recreational fishers, public health officials, medical professionals, and other interested parties.

Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®- PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®- PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample throughput, and is well-suited for routine CTX monitoring programs.

Toxicological Perspective on Climate Change: Aquatic Toxins.

In recent years, our group and several others have been describing the presence of new, not previously reported, toxins of high toxicity in vectors that may reach the human food chain. These include tetrodotoxin in gastropods in the South of Europe, ciguatoxin in fish in the South of Spain, palytoxin in mussels in the Mediterranean Sea, pinnatoxin all over Europe, and okadaic acid in the south of the U.S. There seem to be new marine toxins appearing in areas that are heavy producers of seafood, and this is a cause of concern as most of these new toxins are not included in current legislation and monitoring programs. Along with the new toxins, new chemical analogues are being reported. The same phenomenom is being recorded in freshwater toxins, such as the wide appearance of cylindrospermopsin and the large worldwide increase of microcystin. The problem that this phenomenon, which may be linked to climate warming, poses for toxicologists is very important not only because there is a lack of chronic studies and an incomplete comprehension of the mechanism driving the production of these toxins but also because the lack of a legal framework for them allows many of these toxins to reach the market. In some cases, it is very difficult to control these toxins because there are not enough standards available, they are not always certified, and there is an insufficient understanding of the toxic equivalency factors of the different analogues in each group. All of these factors have been revealed and grouped through the massive increase in the use of LC-MS as a monitoring tool, legally demanded, creating more toxicological problems.

Simultaneous quantification of Pacific ciguatoxins in fish blood using liquid chromatography–tandem mass spectrometry

Ciguatera fish poisoning (CFP) is a food intoxication caused by exposure to ciguatoxins (CTXs) in coral reef fish. Rapid analytical methods have been developed recently to quantify Pacific-CTX-1 (P-CTX-1) in fish muscle, but it is destructive and can cause harm to valuable live coral reef fish. Also fish muscle extract was complex making CTX quantification challenging. Not only P-CTX-1, but also P-CTX-2 and P-CTX-3 could be present in fish, contributing to ciguatoxicity. Therefore, an analytical method for simultaneous quantification of P-CTX-1, P-CTX-2, and P-CTX-3 in whole blood of marketed coral reef fish using sonication, solid-phase extraction (SPE), and liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. The optimized method gave acceptable recoveries of P-CTXs (74-103 %) in fish blood. Matrix effects (6-26 %) in blood extracts were found to be significantly reduced compared with those in muscle extracts (suppressed by 34-75 % as reported in other studies), thereby minimizing potential for false negative results. The target P-CTXs were detectable in whole blood from four coral reef fish species collected in a CFP-endemic region. Similar trends in total P-CTX levels and patterns of P-CTX composition profiles in blood and muscle of these fish were observed, suggesting a relationship between blood and muscle levels of P-CTXs. This optimized method provides an essential tool for studies of P-CTX pharmacokinetics and pharmacodynamics in fish, which are needed for establishing the use of fish blood as a reliable sample for the assessment and control of CFP.

First Toxin Profile of Ciguateric Fish in Madeira Arquipelago (Europe)

Ciguatera fish poisoning (CFP) is a human foodborne intoxication caused by ingestion of tropical fishes contaminated with the potent polyether toxins known as ciguatoxins (CTXs). These toxins are issued from Gambierdiscus species of dinoflagellates. Herbivorous fish accumulate these toxins in their musculature and viscera after ingesting dinoflagellates. Epidemiological studies showed that CFP has been present in areas between 35 degrees North and 35 degrees South latitude, mainly, Indo-pacific and Caribbean areas, but not in waters closed to European and African continent. In the present paper, a specimen of Seriola dumerili weighing 70 kg and a smaller Seriola fasciata specimen, captured in waters belonging to Selvagens Islands (Madeira Arquipelago), were analyzed. Fishes from this genus were implicated in previous suspected ciguatera poisoning outbreaks in the Portuguese Madeira Arquipelago in the North Atlantic Ocean. Analysis was performed by two approaches, a functional method using cerebellar granule cells and by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) method. The study was carried out in one portion of the tail muscle of Seriola fasciata and five parts of the body of Seriola dumerili (tail muscle, head, ventral muscle, mid muscle, and liver). The functional method consisted in the modification of the inward sodium current in cerebellar granule cells and the chemical method was a high resolution chromatography, which allowed elucidating the toxin profile in the samples. In addition, UPLC-MS technique was optimized and used for detecting and quantifying CTXs for the first time. After fish extraction and clean up, the chromatograms revealed the presence of CTX-1B at 1111.6 m/z, CTX-3C at 1023.5 m/z, a CTX analogue at 1040.6 m/z, and a CTX from the Caribbean or Indic waters at 1141.6 m/z. Therefore, the results obtained in the present paper for both methods confirm, for the first time, the presence of CTX in fish from Madeira Arquipelago.

Characteristic profiles of Ciguatera toxins in different strains of Gambierdiscus spp.

Ciguatera fish poisoning characterizes the intoxication caused by consumption of fish from tropical and subtropical areas, which have accumulated ciguatoxins (CTXs). The observed pattern of ciguatoxins in fish highly depends on the marine region and the causative organisms. It is evident that differences exist between ciguatoxins produced by certain strains of the dinoflagellate Gambierdiscus toxicus and other Gambierdiscus spp. In this context cultured strains purchased from the Provasoli–Guillard National Center for Culture of Marine Phytoplankton (CCMP) and strains from Vietnam were analyzed. Besides, lyophilized samples of several Gambierdiscus spp. from the National Oceanic and Atmospheric Administration (NOAA), USA and lyophilized samples of G. toxicus from Vietnam were analyzed. The latter has been cultured at different salinities. We observed differences between the toxin ratios of the analogues in the strain from Vietnam depending on the salinity. The CTX profiles of the Vietnamese samples were compared with cultures of Gambierdiscus spp. from CCMP and the National Oceanic and Atmospheric Administration (NOAA) resulting in an overview of toxins in cultures from different regions. Hence, it was obvious that the strain from Vietnam forms a characteristic CTX profile which is not directly comparable to CTX pattern observed in other tropical marine regions.