Establishing a Receptor Binding Assay for Ciguatoxins: Challenges, Assay Performance and Application.

Ciguatera, a global issue, lacks adequate capacity for ciguatoxin analysis in most affected countries. The Caribbean region, known for its endemic ciguatera and being home to a majority of the global small island developing states, particularly needs established methods for ciguatoxin detection in seafood and the environment. The radioligand receptor binding assay (r-RBA) is among the in vitro bioassays currently used for ciguatoxin analysis; however, similarly to the other chemical-based or bioassays that have been developed, it faces challenges due to limited standards and interlaboratory comparisons. This work presents a single laboratory validation of an r-RBA developed in a Cuban laboratory while characterizing the performance of the liquid scintillation counter instrument as a key external parameter. The results obtained show the assay is precise, accurate and robust, confirming its potential as a routine screening method for the detection and quantification of ciguatoxins. The new method will aid in identifying high-risk ciguatoxic fish in Cuba and the Caribbean region, supporting monitoring and scientific management of ciguatera and the development of early warning systems to enhance food safety and food security, and promote fair trade fisheries.

Chemodiversity of Brevetoxins and Other Potentially Toxic Metabolites Produced by Karenia spp. and Their Metabolic Products in Marine Organisms.

In recent decades, more than 130 potentially toxic metabolites originating from dinoflagellate species belonging to the genus Karenia or metabolized by marine organisms have been described. These metabolites include the well-known and large group of brevetoxins (BTXs), responsible for foodborne neurotoxic shellfish poisoning (NSP) and airborne respiratory symptoms in humans. Karenia spp. also produce brevenal, brevisamide and metabolites belonging to the hemi-brevetoxin, brevisin, tamulamide, gymnocin, gymnodimine, brevisulcenal and brevisulcatic acid groups. In this review, we summarize the available knowledge in the literature since 1977 on these various identified metabolites, whether they are produced directly by the producer organisms or biotransformed in marine organisms. Their structures and physicochemical properties are presented and discussed. Among future avenues of research, we highlight the need for more toxin occurrence data with analytical techniques, which can specifically determine the analogs present in samples. New metabolites have yet to be fully described, especially the groups of metabolites discovered in the last two decades (e.g tamulamides). Lastly, this work clarifies the different nomenclatures used in the literature and should help to harmonize practices in the future.

Guidance Level for Brevetoxins in French Shellfish.

Brevetoxins (BTXs) are marine biotoxins responsible for neurotoxic shellfish poisoning (NSP) after ingestion of contaminated shellfish. NSP is characterized by neurological, gastrointestinal and/or cardiovascular symptoms. The main known producer of BTXs is the dinoflagellate Karenia brevis, but other microalgae are also suspected to synthesize BTX-like compounds. BTXs are currently not regulated in France and in Europe. In November 2018, they have been detected for the first time in France in mussels from a lagoon in the Corsica Island (Mediterranean Sea), as part of the network for monitoring the emergence of marine biotoxins in shellfish. To prevent health risks associated with the consumption of shellfish contaminated with BTXs in France, a working group was set up by the French Agency for Food, Environmental and Occupational Health & Safety (Anses). One of the aims of this working group was to propose a guidance level for the presence of BTXs in shellfish. Toxicological data were too limited to derive an acute oral reference dose (ARfD). Based on human case reports, we identified two lowest-observed-adverse-effect levels (LOAELs). A guidance level of 180 µg BTX-3 eq./kg shellfish meat is proposed, considering a protective default portion size of 400 g shellfish meat.

The Latin America and Caribbean HAB status report based on OBIS and HAEDAT maps and databases.

Harmful Algae Blooms (HAB) have been documented for at least fifty years in Latin America and the Caribbean (LAC), however, their impacts at social, ecological and economic levels are still little known. To contribute to the impact assessment of HABs in LAC region, the available information in HAEDAT, OBIS, CAREC, and CARPHA databases, and scientific literature was analyzed. This historical analysis allows identification of the main syndromes and causal organisms. Considering the existence of two regional working groups of the Intergovernmental Oceanographic Commission (IOC): Algas Nocivas del Caribe (ANCA) and Floraciones Algales Nocivas en Sudamérica (FANSA), representing Central American/Caribbean and South American countries, respectively, the analysis is presented both globally and subregional. For the FANSA region, the HAEDAT data base listed 249 records from 1970 to 2019, with a total of 1432 human intoxications, including 37 fatalities. The majority of these events comprised Paralytic Shellfish Toxins (49%), Diarrhetic Shellfish Toxins (34%), Cyanotoxins (12%) and 6 % other toxins. The total number of harmful taxa in the OBIS database includes 79 species distributed over 25 genera. The most commonly reported species are Alexandrium catenella/tamarense, Gymnodinium catenatum and the Dinophysis acuminata complex. Two new species Prorocentrum caipirignum Fraga, Menezes and Nascimento and Alexandrium fragae Branco and Menezes were newly described from Brazilian waters. In the ANCA region, HAEDAT listed 131 records from 1956 to 2018. The main problems are PSP and Ciguatera and common HAB taxa are Gambierdiscus, Gymnodinium, Pyrodinium, Alexandrium and Dinophysis. The most reported HAB forming species are Gymnodinium catenatum, Pyrodinium bahamense and Gambierdiscus spp. In recent years Margalefidinium polykrikoides blooms have become frequent, causing fish and invertebrates massive mortalities and impacts on touristic activities. In the LAC region, the greatest economic losses were produced by ichthyotoxic massive events causing salmon deaths associated to Pseudochattonella verruculosa and Alexandrium catenella in Chile and tuna deaths related to Tripos furca and Chattonella spp. in the Mexican Pacific. In the last decade, several studies in LAC have linked HAB events with local mesoscale oceanographic and atmospheric phenomena. Trends analyzed up to 2019 are related to the increasing awareness about presence of toxic species, the geographical expansion of already known species, the detection of new toxins for the region, and HAB events duration and/or impacts.

Ciguatoxin Occurrence in Food-Web Components of a Cuban Coral Reef Ecosystem: Risk-Assessment Implications.

In Cuba, ciguatera poisoning associated with fish consumption is the most commonly occurring non-bacterial seafood-borne illness. Risk management through fish market regulation has existed in Cuba for decades and consists of bans on selected species above a certain weight; however, the actual occurrence of ciguatoxins (CTXs) in seafood has never been verified. From this food safety risk management perspective, a study site locally known to be at risk for ciguatera was selected. Analysis of the epiphytic dinoflagellate community identified the microalga Gambierdiscus. Gambierdiscus species included six of the seven species known to be present in Cuba (G. caribaeus, G. belizeanus, G. carpenteri, G. carolinianus, G. silvae, and F. ruetzleri). CTX-like activity in invertebrates, herbivorous and carnivorous fishes were analyzed with a radioligand receptor-binding assay and, for selected samples, with the N2A cell cytotoxicity assay. CTX activity was found in 80% of the organisms sampled, with toxin values ranging from 2 to 8 ng CTX3C equivalents g-1 tissue. Data analysis further confirmed CTXs trophic magnification. This study constitutes the first finding of CTX-like activity in marine organisms in Cuba and in herbivorous fish in the Caribbean. Elucidating the structure-activity relationship and toxicology of CTX from the Caribbean is needed before conclusions may be drawn about risk exposure in Cuba and the wider Caribbean.

Morphology, toxicity and molecular characterization of Gambierdiscus spp. towards risk assessment of ciguatera in south central Cuba.

Ciguatera poisoning is caused by the consumption of reef fish or shellfish that have accumulated ciguatoxins, neurotoxins produced by benthic dinoflagellates of the genera Gambierdiscus or Fukuyoa. Although ciguatera constitutes the primary cause of seafood intoxication in Cuba, very little information is available on the occurrence of ciguatoxins in the marine food web and the causative benthic dinoflagellate species. This study conducted on the south-central coast of Cuba reports the occurrence of Gambierdiscus and Fukuyoa genera and the associated benthic genera Ostreopsis and Prorocentrum. Gambierdiscus/Fukuyoa cells were present at low to moderate abundances depending on the site and month of sampling. This genus was notably higher on Dictyotaceae than on other macrophytes. PCR analysis of field-collected samples revealed the presence of six different Gambierdiscus and one Fukuyoa species, including G. caribaeus, G. carolinianus, G. carpenteri, G. belizeanus, F. ruetzleri, G. silvae, and Gambierdiscus sp. ribotype 2. Only Gambierdiscus excentricus was absent from the eight Gambierdiscus/Fukuyoa species known in the wider Caribbean region. Eleven clonal cultures were established and confirmed by PCR and SEM as being either G. carolinianus or G. caribaeus. Toxin production in each isolate was assessed by a radioligand receptor binding assay and found to be below the assay quantification limit. These novel findings augment the knowledge of the ciguatoxin-source dinoflagellates that are present in Cuba, however further studies are needed to better understand the correlation between their abundance, species-specific toxin production in the environment, and the risk for fish contamination, in order to develop better informed ciguatera risk management strategies.

Insights into the Biosynthesis of Cyclic Guanidine Alkaloids from Crambeidae Marine Sponges.

Among the outstanding chemical diversity found in marine sponges, cyclic guanidine alkaloids, present in species of the family Crambeidae, are particularly attractive, not only because of their unique chemical features, but also due to a broad range of biological activities. Despite a growing interest in these natural products as therapeutic agents, their metabolic pathway has not been experimentally investigated. Ex situ feeding experiments using radiolabeled precursors performed on the Mediterranean sponge Crambe crambe suggest arginine and fatty acids as precursors in the metabolic pathway of crambescins. A subsequent bio-inspired approach supported the change of paradigm in the metabolic pathway of cyclic guanidine alkaloids. A large part of the chemical diversity of this family would therefore originate from a tethered Biginelli-like reaction between C-2/C-3 activated fatty acids and a central guanidinylated pyrrolinium.

A radioligand receptor binding assay for ciguatoxin monitoring in environmental samples: Method development and determination of quality control criteria.

Ciguatoxins are algal toxins responsible for tens of thousands of human intoxications yearly, both in tropical and subtropical endemic regions as well as worldwide through fish exportation. Previously developed methods for biotoxin surveillance in the environment and seafood include analytical methods and in vivo and in vitro bioassays. The radioligand receptor binding assay (r-RBA) is among the in vitro methodologies currently used for the detection and quantification of marine biotoxins. For the ciguatoxin group, the r-RBA has been widely used as a means to characterize the mode of action and as detection method in various biological matrices. Yet, screening methods have not been standardized, and the details of the ciguatoxin-specific r-RBA are not well-documented, which limit interlaboratory comparison and progress toward method validation. This work presents the development of an optimized r-RBA for ciguatoxins and provides guidance on its use and quality control checks for analysis of environmental samples. We focus on the analysis of critical parameters involved in determining assay acceptability. Calculation of toxin concentrations in fish samples is illustrated with four examples. Thus, this paper provides the detailed information required for a full validation of the r-RBA, a necessary step toward the development and implementation of a regulatory monitoring programme for ciguatoxins in seafood products using the r-RBA.

A study of the influence of brevetoxin exposure on trace element bioaccumulation in the blue mussel Mytilus edulis.

Marine organisms are exposed to and affected by a multitude of chemicals present in seawater and can accumulate in their tissues a wide range of contaminants as well as natural biotoxins associated with harmful algal blooms (HABs). Trace elements and biotoxins may modify physiological functions in exposed organisms, and studies have been conducted to better understand their respective kinetics and effects in marine species. Despite the increasing concern of concurrent toxic HABs and pollution events due to anthropogenic pressures and global change, very little information is available on their combined effects. Chemical interactions between biotoxins and trace elements have been reported, and exposure to certain biotoxins is known to modify ion transport pathways, suggesting that biotoxins have the potential to alter trace element uptake. Using specific and sensitive radiotracer techniques (radioligand receptor binding assay and γ-spectrometry), this laboratory study examined the influence of pre-exposure to the brevetoxins (PbTxs)-producing microalgae Karenia brevis on the bioaccumulation of selected non-essential (Cd) and essential (Co, Mn and Zn) trace elements in the blue mussel Mytilus edulis. PbTxs are a group of neurotoxins known to accumulate in bivalves but also to have lethal effects on a number of marine organisms including fish and mammals. We found that, over 23 days exposure to the radiotracers, the bioaccumulation of the dissolved essential trace elements Co, Mn and Zn in M. edulis was not significantly affected by pre-exposure to toxic K. brevis. In contrast, the uptake rate constant ku of Cd was significantly higher in the pre-exposed group (p < 0.05), likely caused by a decrease in mussel clearance rates after K. brevis exposure. These results suggest that the effects of algal toxin exposure on bioaccumulation of trace elements in mussels may be trace element-dependent.

Experimental evidence of dietary ciguatoxin accumulation in an herbivorous coral reef fish.

Ciguatoxins (CTXs) are potent algal toxins that cause widespread ciguatera poisoning and are found ubiquitously in coral reef food webs. Here we developed an environmentally-relevant, experimental model of CTX trophic transfer involving dietary exposure of herbivorous fish to the CTX-producing microalgae Gambierdiscus polynesiensis. Juvenile Naso brevirostris were fed a gel-food embedded with microalgae for 16 weeks (89 cells g-1 fish daily, 0.4 µg CTX3C equiv kg-1 fish). CTXs in muscle tissue were detectable after 2 weeks at levels above the threshold for human intoxication (1.2 ±â€¯0.2 µg CTX3C equiv kg-1). Although tissue CTX concentrations stabilized after 8 weeks (∼3 ±â€¯0.5 µg CTX3C equiv kg-1), muscle toxin burden (total µg CTX in muscle tissue) continued to increase linearly through the end of the experiment (16 weeks). Toxin accumulation was therefore continuous, yet masked by somatic growth dilution. The observed CTX concentrations, accumulation rates, and general absence of behavioural signs of intoxication are consistent with field observations and indicate that this method of dietary exposure may be used to develop predictive models of tissue-specific CTX uptake, metabolism and depuration. Results also imply that slow-growing fish may accumulate higher CTX flesh concentrations than fast-growing fish, which has important implications for global seafood safety.

The role of marine biotoxins on the trophic transfer of Mn and Zn in fish.

Essential nutrients are critical for physiological processes of organisms. In fish, they are obtained primarily from the diet, and their transfer and accumulation are known to be impacted by environmental variables such as water temperature, pH and salinity, as well as by diet composition and matrices. Yet, prey items consumed by fish may also contain toxic compounds such as marine toxins associated with harmful algae. These biotoxins have the potential to affect essential trace element assimilation in fish through chemical interactions such as the formation of trace element-toxin complexes or by affecting general fish physiology as in the modification of ion-specific transport pathways. We assessed the influence of dietary exposure to brevetoxins (PbTxs), ichthyotoxic neurotoxins produced by the dinoflagellate Karenia brevis, on trophic transfer of two essential trace elements, Mn and Zn, in a fish model. Using ecologically relevant concentrations of PbTxs and trace elements in controlled laboratory conditions, juvenile turbots Scophthalmus maximus were given food containing PbTxs before or at the same time as a feeding with radiotracers of the chosen essential elements (54Mn and 65Zn). Treatments included simultaneous exposure (PbTxs + 54Mn + 65Zn) in a single-feeding, 3-week daily pre-exposure to dietary PbTx followed by a single feeding with 54Mn and 65Zn, and a control (54Mn and 65Zn only). After a 21-day depuration period, turbot tissue brevetoxin levels were quantified and assimilation efficiencies of 54Mn and 65Zn were assessed. PbTxs were found in turbot tissues in each exposure treatment, demonstrating dietary trophic transfer of these toxins; yet, no differences in assimilation efficiencies of Mn or Zn were found between treatments or the control (p > 0.05). These results indicate that, in our experimental conditions, PbTx exposure does not significantly affect the trophic transfer of Mn and Zn in fish.

Speciation of americium in seawater and accumulation in the marine sponge Aplysina cavernicola.

The fate of radionuclides in the environment is a cause of great concern for modern society, seen especially in 2011 after the Fukushima accident. Among the environmental compartments, seawater covers most of the earth's surface and may be directly or indirectly impacted. The interaction between radionuclides and the marine compartment is therefore essential for better understanding the transfer mechanisms from the hydrosphere to the biosphere. This information allows for the evaluation of the impact on humans via our interaction with the biotope that has been largely undocumented up to now. In this report, we attempt to make a link between the speciation of heavy elements in natural seawater and their uptake by a model marine organism. More specifically, because the interaction of actinides with marine invertebrates has been poorly studied, the accumulation in a representative member of the Mediterranean coralligenous habitat, the sponge Aplysina cavernicola, was investigated and its uptake curve exposed to a radiotracer (241)Am was estimated using a high-purity Ge gamma spectrometer. But in order to go beyond the phenomenological accumulation rate, the speciation of americium(III) in seawater must be assessed. The speciation of (241)Am (and natural europium as its chemically stable surrogate) in seawater was determined using a combination of different techniques: Time-Resolved Laser-Induced Fluorescence (TRLIF), Extended X-ray Absorption Fine Structure (EXAFS) at the LIII edge, Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy and Scanning Electron Microscopy (SEM) and the resulting data were compared with the speciation modeling. In seawater, the americium(III) complex (as well as the corresponding europium complex, although with conformational differences) was identified as a ternary sodium biscarbonato complex, whose formula can be tentatively written as NaAm(CO3)2·nH2O. It is therefore this chemical form of americium that is accumulated by the sponge A. cavernicola.

A roadmap for hazard monitoring and risk assessment of marine biotoxins on the basis of chemical and biological test systems.

Aquatic food accounts for over 40% of global animal food products, and the potential contamination with toxins of algal origin--marine biotoxins--poses a health threat for consumers. The gold standards to assess toxins in aquatic food have traditionally been in vivo methods, i.e., the mouse as well as the rat bioassay. Besides ethical concerns, there is also a need for more reliable test methods because of low inter-species comparability, high intra-species variability, the high number of false positive and negative results as well as questionable extrapolation of quantitative risk to humans. For this reason, a transatlantic group of experts in the field of marine biotoxins was convened from academia and regulatory safety authorities to discuss future approaches to marine biotoxin testing. In this report they provide a background on the toxin classes, on their chemical characterization, the epidemiology, on risk assessment and management, as well as on their assumed mode of action. Most importantly, physiological functional assays such as in vitro bioassays and also analytical techniques, e.g., liquid chromatography coupled mass spectrometry (LC-MS), as substitutes for the rodent bioassay are reviewed. This forms the basis for recommendations on methodologies for hazard monitoring and risk assessment, establishment of causality of intoxications in human cases, a roadmap for research and development of human-relevant functional assays, as well as new approaches for a consumer directed safety concept.

Linking ciguatera poisoning to spatial ecology of fish: a novel approach to examining the distribution of biotoxin levels in the great barracuda by combining non-lethal blood sampling and biotelemetry.

Ciguatera in humans is typically caused by the consumption of reef fish that have accumulated Ciguatoxins (CTXs) in their flesh. Over a six month period, we captured 38 wild adult great barracuda (Sphyraena barracuda), a species commonly associated with ciguatera in The Bahamas. We sampled three tissues (i.e., muscle, liver, and blood) and analysed them for the presence of ciguatoxins using a functional in vitro N2A bioassay. Detectable concentrations of ciguatoxins found in the three tissue types ranged from 2.51 to 211.74pg C-CTX-1 equivalents/g. Blood and liver toxin concentrations were positively correlated (ρ=0.86, P=0.003), indicating that, for the first time, blood sampling provides a non-lethal method of detecting ciguatoxin in wild fish. Non-lethal blood sampling also presents opportunities to couple this approach with biotelemetry and biologging techniques that enable the study of fish distribution and movement. To demonstrate the potential for linking ciguatoxin occurrence with barracuda spatial ecology, we also present a proof-of-concept case study where blood samples were obtained from 20 fish before releasing them with acoustic transmitters and tracking them in the coastal waters using a fixed acoustic telemetry array covering 44km(2). Fish that tested positive for CTX may have smaller home ranges than non-toxic fish (median distance travelled, U=2.21, P=0.03). Results presented from this study may help identify high risk areas and source-sink dynamics of toxins, potentially reducing the incidence and human health risk of ciguatera fish poisoning. Moreover, development of the non-lethal sampling approach and measurement of ciguatera from blood provide future opportunities to understand the mechanistic relationship between toxins and the spatial ecology of a broad range of marine fish species.