A Review of Cyclic Imines in Shellfish: Worldwide Occurrence, Toxicity and Assessment of the Risk to Consumers.

Cyclic imines are a class of lipophilic shellfish toxins comprising gymnodimines, spirolides, pinnatoxins, portimines, pteriatoxins, prorocentrolides, spiro-prorocentrimine, symbiomines and kabirimine. They are structurally diverse, but all share an imine moiety as part of a bicyclic ring system. These compounds are produced by marine microalgal species and are characterized by the rapid death that they induce when injected into mice. Cyclic imines have been detected in a range of shellfish species collected from all over the world, which raises the question as to whether they present a food safety risk. The European Food Safety Authority (EFSA) considers them to be an emerging food safety issue, and in this review, the risk posed by these toxins to shellfish consumers is assessed by collating all available occurrence and toxicity data. Except for pinnatoxins, the risk posed to human health by the cyclic imines appears low, although this is based on only a limited dataset. For pinnatoxins, two different health-based guidance values have been proposed at which the concentration should not be exceeded in shellfish (268 and 23 µg PnTX/kg shellfish flesh), with the discrepancy caused by the application of different uncertainty factors. Pinnatoxins have been recorded globally in multiple shellfish species at concentrations of up to 54 times higher than the lower guidance figure. Despite this observation, pinnatoxins have not been associated with recorded human illness, so it appears that the lower guidance value may be conservative. However, there is insufficient data to generate a more robust guidance value, so additional occurrence data and toxicity information are needed.

Targeted Metabolite Fingerprints of Thirteen Gambierdiscus, Five Coolia and Two Fukuyoa Species.

The genus Gambierdiscus produces an array of bioactive hydrophilic and lipophilic secondary metabolites that range in mode of action and toxicity. In this study, the metabolite fingerprint was mapped for thirteen Gambierdiscus, five Coolia and two Fukuyoa species (34 isolates) by assessing the production of 56 characterised secondary metabolites. Gambierdiscus polynesiensis was the only species to produce Pacific-ciguatoxin-3B (P-CTX3B), P-CTX3C, iso-P-CTX3B/C, P-CTX4A, P-CTX4B and iso-P-CTX4A/B. G. australes produced maitotoxin-1 (MTX-1) and MTX-5, G. cheloniae produced MTX-6 and G. honu produced MTX-7. Ubiquitous production of 44-methylgambierone was observed amongst all the Gambierdiscus isolates, with nine species also producing gambierone. Additional gambierone analogues, including anhydrogambierone (tentatively described herein), were also detected in all Gambierdiscus species, two Coolia and two Fukuyoa species. Gambieroxide was detected in G. lewisii and G. pacificus and gambieric acid A was detected in ten Gambierdiscus species, with G. australes (CAWD381) being the only isolate to produce gambieric acids A-D. This study has demonstrated that the isolates tested to date produce the known CTXs or MTXs, but not both, and highlighted several species that produced 'unknown' compounds displaying characteristics of cyclic polyethers, which will be the focus of future compound discovery efforts.

Structural Characterization of Maitotoxins Produced by Toxic Gambierdiscus Species.

Identifying compounds responsible for the observed toxicity of the Gambierdiscus species is a critical step to ascertaining whether they contribute to ciguatera poisoning. Macroalgae samples were collected during research expeditions to Rarotonga (Cook Islands) and North Meyer Island (Kermadec Islands), from which two new Gambierdiscus species were characterized, G. cheloniae CAWD232 and G. honu CAWD242. Previous chemical and toxicological investigations of these species demonstrated that they did not produce the routinely monitored Pacific ciguatoxins nor maitotoxin-1 (MTX-1), yet were highly toxic to mice via intraperitoneal (i.p.) injection. Bioassay-guided fractionation of methanolic extracts, incorporating wet chemistry and chromatographic techniques, was used to isolate two new MTX analogs; MTX-6 from G. cheloniae CAWD232 and MTX-7 from G. honu CAWD242. Structural characterization of the new MTX analogs used a combination of analytical chemistry techniques, including LC-MS, LC-MS/MS, HR-MS, oxidative cleavage and reduction, and NMR spectroscopy. A substantial portion of the MTX-7 structure was elucidated, and (to a lesser extent) that of MTX-6. Key differences from MTX-1 included monosulfation, additional hydroxyl groups, an extra double bond, and in the case of MTX-7, an additional methyl group. To date, this is the most extensive structural characterization performed on an MTX analog since the complete structure of MTX-1 was published in 1993. MTX-7 was extremely toxic to mice via i.p. injection (LD50 of 0.235 µg/kg), although no toxicity was observed at the highest dose rate via oral administration (155.8 µg/kg). Future research is required to investigate the bioaccumulation and likely biotransformation of the MTX analogs in the marine food web.

Cyclic Imine Pinnatoxin G is Cytotoxic to Cancer Cell Lines via Nicotinic Acetylcholine Receptor-Driven Classical Apoptosis.

Pinnatoxin G is a cyclic imine neurotoxin produced by dinoflagellates that has been reported in shellfish. Like other members of the pinnatoxin family, it has been shown to have its effects via antagonism of the nicotinic acetylcholine receptors, with preferential binding to the α7 subunit often upregulated in cancer. Because increased activity of α7 nicotinic acetylcholine receptors contributes to increased growth and resistance to apoptosis, the effect of pinnatoxin G on cancer cell viability was tested. In a panel of six cancer cell lines, all cell types lost viability, but HT29 colon cancer and LN18 and U373 glioma cell lines were more sensitive than MDA-MB-231 breast cancer cells, PC3 prostate cancer cells, and U87 glioma cells, correlating with expression levels of α7, α4, and α9 nicotinic acetylcholine receptors. Some loss of cell viability could be attributed to cell cycle arrest, but significant levels of classical apoptosis were found, characterized by caspase activity, phosphatidylserine exposure, mitochondrial membrane permeability, and fragmented DNA. Intracellular Ca2+ levels also dropped immediately upon pinnatoxin G treatment, which may relate to antagonism of nicotinic acetylcholine receptor-mediated Ca2+ inflow. In conclusion, pinnatoxin G can decrease cancer cell viability, with both cytostatic and cytotoxic effects.

Transcriptomic investigation into polyketide toxin synthesis in Ostreopsis (Dinophyceae) species.

In marine ecosystems, dinoflagellates can become highly abundant and even dominant at times, despite their comparatively slow growth. Their ecological success may be related to their production of complex toxic polyketide compounds. Ostreopsis species produce potent palytoxin-like compounds (PLTX), which are associated with human skin and eye irritations, and illnesses through the consumption of contaminated seafood. To investigate the genetic basis of PLTX-like compounds, we sequenced and annotated transcriptomes from two PLTX-producing Ostreopsis species; O. cf. ovata, O. cf. siamensis, one non-PLTX producing species, O. rhodesae and compared them to a close phylogenetic relative and non-PLTX producer, Coolia malayensis. We found no clear differences in the presence or diversity of ketosynthase and ketoreductase transcripts between PLTX producing and non-producing Ostreopsis and Coolia species, as both groups contained >90 and > 10 phylogenetically diverse ketosynthase and ketoreductase transcripts, respectively. We report for the first-time type I single-, multi-domain polyketide synthases (PKSs) and hybrid non-ribosomal peptide synthase/PKS transcripts from all species. The long multi-modular PKSs were insufficient by themselves to synthesize the large complex polyether backbone of PLTX-like compounds. This implies that numerous PKS domains, including both single and multi-, work together on the biosynthesis of PLTX-like and other related polyketide compounds.

Antifouling activity of portimine, select semisynthetic analogues, and other microalga-derived spirocyclic imines.

A range of natural products from marine invertebrates, bacteria and fungi have been assessed as leads for nature-inspired antifouling (AF) biocides, but little attention has been paid to microalgal-derived compounds. This study assessed the AF activity of the spirocyclic imine portimine (1), which is produced by the benthic mat-forming dinoflagellate Vulcanodinium rugosum. Portimine displayed potent AF activity in a panel of four macrofouling bioassays (EC50 0.06-62.5 ng ml-1), and this activity was distinct from that of the related compounds gymnodimine-A (2), 13-desmethyl spirolide C (3), and pinnatoxin-F (4). The proposed mechanism of action for portimine is induction of apoptosis, based on the observation that portimine inhibited macrofouling organisms at developmental stages known to involve apoptotic processes. Semisynthetic modification of select portions of the portimine molecule was subsequently undertaken. Observed changes in bioactivity of the resulting semisynthetic analogues of portimine were consistent with portimine's unprecedented 5-membered imine ring structure playing a central role in its AF activity.

Toxicology of Gambierdiscus spp. (Dinophyceae) from Tropical and Temperate Australian Waters.

Ciguatera Fish Poisoning (CFP) is a human illness caused by the consumption of marine fish contaminated with ciguatoxins (CTX) and possibly maitotoxins (MTX), produced by species from the benthic dinoflagellate genus Gambierdiscus. Here, we describe the identity and toxicology of Gambierdiscus spp. isolated from the tropical and temperate waters of eastern Australia. Based on newly cultured strains, we found that four Gambierdiscus species were present at the tropical location, including G. carpenteri, G. lapillus and two others which were not genetically identical to other currently described species within the genus, and may represent new species. Only G. carpenteri was identified from the temperate location. Using LC-MS/MS analysis we did not find any characterized microalgal CTXs (P-CTX-3B, P-CTX-3C, P-CTX-4A and P-CTX-4B) or MTX-1; however, putative maitotoxin-3 (MTX-3) was detected in all species except for the temperate population of G. carpenteri. Using the Ca2+ influx SH-SY5Y cell Fluorescent Imaging Plate Reader (FLIPR) bioassay we found CTX-like activity in extracts of the unidentified Gambierdiscus strains and trace level activity in strains of G. lapillus. While no detectable CTX-like activity was observed in tropical or temperate strains of G. carpenteri, all species showed strong maitotoxin-like activity. This study, which represents the most comprehensive analyses of the toxicology of Gambierdiscus strains isolated from Australia to date, suggests that CFP in this region may be caused by currently undescribed ciguatoxins and maitotoxins.

Recent Trends in Marine Phycotoxins from Australian Coastal Waters.

Phycotoxins, which are produced by harmful microalgae and bioaccumulate in the  marine food web, are of growing concern for Australia. These harmful algae pose a threat to  ecosystem and human health, as well as constraining the progress of aquaculture, one of the fastest  growing food sectors in the world. With better monitoring, advanced analytical skills and an  increase in microalgal expertise, many phycotoxins have been identified in Australian coastal  waters in recent years. The most concerning of these toxins are ciguatoxin, paralytic shellfish  toxins, okadaic acid and domoic acid, with palytoxin and karlotoxin increasing in significance. The  potential for tetrodotoxin, maitotoxin and palytoxin to contaminate seafood is also of concern,  warranting future investigation. The largest and most significant toxic bloom in Tasmania in 2012  resulted in an estimated total economic loss of~AUD$23M, indicating that there is an imperative to  improve  toxin  and  organism  detection  methods,  clarify  the  toxin  profiles  of  species  of  phytoplankton and carry out both intra- and inter-species toxicity comparisons. Future work also  includes the application of rapid, real-time molecular assays for the detection of harmful species  and toxin genes. This information, in conjunction with a better understanding of the life histories  and  ecology  of  harmful  bloom  species,  may  lead  to  more  appropriate  management  of  environmental, health and economic resources.

Ciguatoxins and Maitotoxins in Extracts of Sixteen Gambierdiscus Isolates and One Fukuyoa Isolate from the South Pacific and Their Toxicity to Mice by Intraperitoneal and Oral Administration.

Ciguatoxins (CTXs), and possibly maitotoxins (MTXs), are responsible for Ciguatera Fish Poisoning, an important health problem for consumers of reef fish (such as inhabitants of islands in the South Pacific Ocean). The habitational range of the Gambierdiscus species is expanding, and new species are being discovered. In order to provide information on the potential health risk of the Gambierdiscus species, and one Fukuyoa species (found in the Cook Islands, the Kermadec Islands, mainland New Zealand, and New South Wales, Australia), 17 microalgae isolates were collected from these areas. Unialgal cultures were grown and extracts of the culture isolates were analysed for CTXs and MTXs by liquid chromatography tandem mass spectrometry (LC-MS/MS), and their toxicity to mice was determined by intraperitoneal and oral administration. An isolate of G. carpenteri contained neither CTXs nor MTXs, while 15 other isolates (including G. australes, G. cheloniae, G. pacificus, G.honu, and F. paulensis) contained only MTX-1 and/or MTX-3. An isolate of G. polynesiensis contained both CTXs and MTX-3. All the extracts were toxic to mice by intraperitoneal injection, but those containing only MTX-1 and/or -3 were much less toxic by oral administration. The extract of G. polynesiensis was highly toxic by both routes of administration.

The Epiphytic Genus Gambierdiscus (Dinophyceae) in the Kermadec Islands and Zealandia Regions of the Southwestern Pacific and the Associated Risk of Ciguatera Fish Poisoning.

Species in the genus Gambierdiscus produce ciguatoxins (CTXs) and/or maitotoxins (MTXs), which may cause ciguatera fish poisoning (CFP) in humans if contaminated fish are consumed. Species of Gambierdiscus have previously been isolated from macroalgae at Rangitahua (Raoul Island and North Meyer Islands, northern Kermadec Islands), and the opportunity was taken to sample for Gambierdiscus at the more southerly Macauley Island during an expedition in 2016. Gambierdiscus cells were isolated, cultured, and DNA extracted and sequenced to determine the species present. Bulk cultures were tested for CTXs and MTXs by liquid chromatography-mass spectrometry (LC-MS/MS). The species isolated were G. australes, which produced MTX-1 (ranging from 3 to 36 pg/cell), and G. polynesiensis, which produced neither MTX-1 nor, unusually, any known CTXs. Isolates of both species produced putative MTX-3. The risk of fish, particularly herbivorous fish, causing CFP in the Zealandia and Kermadec Islands region is real, although in mainland New Zealand the risk is currently low. Both Gambierdiscus and Fukuyoa have been recorded in the sub-tropical northern region of New Zealand, and so the risk may increase with warming seas and shift in the distribution of Gambierdiscus species.

Myeloperoxidase-dependent lipid peroxidation promotes the oxidative modification of cytosolic proteins in phagocytic neutrophils.

Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification, we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A 4-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NADPH oxidase and myeloperoxidase activity and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. The addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The post-translational modification of neutrophil proteins will influence the functioning and fate of these immune cells in the period following phagocytic activation, and provides a marker of neutrophil activation during infection and inflammation.

The marine cytotoxin portimine is a potent and selective inducer of apoptosis.

Portimine is a recently discovered member of a class of marine micro-algal toxins called cyclic imines. In dramatic contrast to related compounds in this toxin class, portimine has very low acute toxicity to mice but is highly cytotoxic to cultured cells. In this study we show that portimine kills human Jurkat T-lymphoma cells and mouse embryonic fibroblasts (MEFs), with LC50 values of 6 and 2.5 nM respectively. Treated cells displayed rapid caspase activation and phosphatidylserine exposure, indicative of apoptotic cell death. Jurkat cells overexpressing the anti-apoptotic protein Bcl-2 or Bax/Bak knockout MEFs were completely protected from portimine. This protection was apparent even at high concentrations of portimine, with no evidence of necrotic cell death, indicating that portimine is a selective chemical inducer of apoptosis. Treatment of the Bcl-2-overexpressing cells with both portimine and the Bcl-2 inhibitor ABT-737 proved a powerful combination, causing >90 % death. We conclude that portimine is one of the most potent naturally derived inducers of apoptosis to be discovered, and it displays strong selectivity for the induction of apoptotic pathways.

Clinical diagnosis and chemical confirmation of ciguatera fish poisoning in New South Wales, Australia.

Ciguatera fish poisoning is common in tropical and sub-tropical areas and larger fish (> 10 kg) are more susceptible to toxin accumulation with age. Although the coastal climate of northern New South Wales is considered sub-tropical, prior to 2014 there has only been 1 documented outbreak of ciguatera fish poisoning from fish caught in the region. During February and March 2014, 2 outbreaks of ciguatera fish poisoning involved 4 and 9 individuals, respectively, both following consumption of Spanish mackerel from northern New South Wales coastal waters (Evans Head and Scotts Head). Affected individuals suffered a combination of gastrointestinal and neurological symptoms requiring hospital treatment. At least 1 individual was symptomatic up to 7 months later. Liquid chromatography-tandem mass spectrometry detected the compound Pacific ciguatoxin-1B at levels up to 1.0 µg kg(-1) in fish tissue from both outbreaks. During April 2015, another outbreak of ciguatera fish poisoning was reported in 4 individuals. The fish implicated in the outbreak was caught further south than the 2014 outbreaks (South West Rocks). Fish tissue was unavailable for analysis; however, symptoms were consistent with ciguatera fish poisoning. To our knowledge, these cases are the southernmost confirmed sources of ciguatera fish poisoning in Australia. Educational outreach to the fishing community, in particular recreational fishers was undertaken after the Evans Head outbreak. This highlighted the outbreak, species of fish involved and the range of symptoms associated with ciguatera fish poisoning. Further assessment of the potential for ciguatoxins to occur in previously unaffected locations need to be considered in terms of food safety.

Polyketide synthesis genes associated with toxin production in two species of Gambierdiscus (Dinophyceae).

BACKGROUND: Marine microbial protists, in particular, dinoflagellates, produce polyketide toxins with ecosystem-wide and human health impacts. Species of Gambierdiscus produce the polyether ladder compounds ciguatoxins and maitotoxins, which can lead to ciguatera fish poisoning, a serious human illness associated with reef fish consumption. Genes associated with the biosynthesis of polyether ladder compounds are yet to be elucidated, however, stable isotope feeding studies of such compounds consistently support their polyketide origin indicating that polyketide synthases are involved in their biosynthesis. RESULTS: Here, we report the toxicity, genome size, gene content and transcriptome of Gambierdiscus australes and G. belizeanus. G. australes produced maitotoxin-1 and maitotoxin-3, while G. belizeanus produced maitotoxin-3, for which cell extracts were toxic to mice by IP injection (LD50 = 3.8 mg kg(-1)). The gene catalogues comprised 83,353 and 84,870 unique contigs, with genome sizes of 32.5 ± 3.7 Gbp and 35 ± 0.88 Gbp, respectively, and are amongst the most comprehensive yet reported from a dinoflagellate. We found three hundred and six genes involved in polyketide biosynthesis, including one hundred and ninety-two ketoacyl synthase transcripts, which formed five unique phylogenetic clusters. CONCLUSIONS: Two clusters were unique to these maitotoxin-producing dinoflagellate species, suggesting that they may be associated with maitotoxin biosynthesis. This work represents a significant step forward in our understanding of the genetic basis of polyketide production in dinoflagellates, in particular, species responsible for ciguatera fish poisoning.

Measurement of Estradiol in Human Serum by LC-MS/MS Using a Novel Estrogen-Specific Derivatization Reagent.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described that employs a novel derivatization reagent for the measurement of serum estradiol (E2), with simultaneous analysis of underivatized testosterone (T) and dihydrotestosterone (DHT). The main advantage of the new derivatization reagent 1,2-dimethylimidazole-5-sulfonyl chloride is its analyte-specific fragmentation that enables monitoring of confirmatory mass transitions with high sensitivity. The reaction mixture can be analyzed without additional purification steps using a 9.5 min gradient run, and sensitive detection is achieved with a triple quadrupole mass spectrometer using atmospheric pressure photoionization. Method validation was performed with human serum samples, including a comparison with a standard LC-MS/MS method using 120 samples from a clinical study, and analysis of certified E2 serum reference materials BCR-576, BCR-577, and BCR-578. The lower limits of quantification for E2, T, and DHT were 0.5 pg/mL, 25 pg/mL, and 0.10 ng/mL, respectively, from a 200-µL sample. Validation results indicated good accuracy and agreement with established, conventional LC-MS/MS assays, demonstrating suitability for analysis of samples containing E2 in the low pg/mL range, such as serum from men, children, and postmenopausal women.

Warm temperature acclimation impacts metabolism of paralytic shellfish toxins from Alexandrium minutum in commercial oysters.

Species of Alexandrium produce potent neurotoxins termed paralytic shellfish toxins and are expanding their ranges worldwide, concurrent with increases in sea surface temperature. The metabolism of molluscs is temperature dependent, and increases in ocean temperature may influence both the abundance and distribution of Alexandrium and the dynamics of toxin uptake and depuration in shellfish. Here, we conducted a large-scale study of the effect of temperature on the uptake and depuration of paralytic shellfish toxins in three commercial oysters (Saccostrea glomerata and diploid and triploid Crassostrea gigas, n = 252 per species/ploidy level). Oysters were acclimated to two constant temperatures, reflecting current and predicted climate scenarios (22 and 27 °C), and fed a diet including the paralytic shellfish toxin-producing species Alexandrium minutum. While the oysters fed on A. minutum in similar quantities, concentrations of the toxin analogue GTX1,4 were significantly lower in warm-acclimated S. glomerata and diploid C. gigas after 12 days. Following exposure to A. minutum, toxicity of triploid C. gigas was not affected by temperature. Generally, detoxification rates were reduced in warm-acclimated oysters. The routine metabolism of the oysters was not affected by the toxins, but a significant effect was found at a cellular level in diploid C. gigas. The increasing incidences of Alexandrium blooms worldwide are a challenge for shellfish food safety regulation. Our findings indicate that rising ocean temperatures may reduce paralytic shellfish toxin accumulation in two of the three oyster types; however, they may persist for longer periods in oyster tissue.

Single-Laboratory Validation of a Multitoxin Ultra-Performance LC-Hydrophilic Interaction LC-MS/MS Method for Quantitation of Paralytic Shellfish Toxins in Bivalve Shellfish.

A single-laboratory validation study was conducted for the hydrophilic interaction-LC-MS/MS analysis of paralytic shellfish toxins (PSTs) in bivalve shellfish. The method was developed as an alternative to the precolumn oxidation AOAC 2005.06 and postcolumn oxidation AOAC 2011.02 LC with fluorescence detection methods, receptor binding assay AOAC 2011.27, as well as the mouse bioassay AOAC 959.08. PSTs assessed were saxitoxin, neosaxitoxin, deoxydecarbamoylsaxitoxin, decarbamoylsaxitoxin, decarbamoylneosaxitoxin, gonyautoxins 1-6, decarbamoylgonyautoxins 2-3, and N-sulfocarbamoyl gonyautoxins 2&3. The method also included the determination of decarbamoylgonyautoxins 1&4, N-sulfocarbamoyl gonyautoxins 1&4, and M toxins. Twelve commercially produced bivalve species from both New Zealand and the United Kingdom were assessed, including mussels, oysters, scallops, and clams. Validation studies demonstrated acceptable method performance characteristics for specificity, linearity, recovery, repeatability, and within-laboratory reproducibility. LOD and LOQ were significantly improved in comparison to current fluorescence-based detection methods, and the method was shown to be rugged. The method performed well in comparison to AOAC 2005.06, with evidence obtained from both comparative analysis of 1141 PST-contaminated samples and successful participation in proficiency testing schemes. The method is suitable for use in regulatory testing and will be submitted for an AOAC collaborative study.

The Effect of Experimental Protocol on the Toxicity of Saxitoxin in Mice.

Regulatory limits for toxins in shellfish are required to ensure the health of consumers. However, these limits also impact the profitability of shellfish industries making it critical that they are fit for purpose. Since human toxicity data is rarely available, the setting of regulatory limits is dependent on animal data which can then be extrapolated for use in the assessment of human risk. The dependence on animal data to keep humans safe means that it is critical that the toxicity data used is robust and of high quality. Worldwide, the protocols used in toxicity testing are varied, making it hard to compare results and adding confusion over which results better reflect the true toxicity. In this study, we look at the effect of mouse gender, i.p. dose volume, mouse body weight and feeding protocols (both acute and sub-acute) on the toxicity of saxitoxin. This allowed the effect of different variables used in toxicity testing to be understood and showed that the feeding protocol used in both acute and sub-acute studies greatly influenced the toxicity of saxitoxin in mice. Therefore, the adoption of a standard protocol for the testing of shellfish toxins is recommended.

A Sub-Acute Dosing Study of Saxitoxin and Tetrodotoxin Mixtures in Mice Suggests That the Current Paralytic Shellfish Toxin Regulatory Limit Is Fit for Purpose.

Paralytic shellfish poisoning is a worldwide problem induced by shellfish contaminated with paralytic shellfish toxins. To protect human health, a regulatory limit for these toxins in shellfish flesh has been adopted by many countries. In a recent study, mice were dosed with saxitoxin and tetrodotoxin mixtures daily for 28 days showing toxicity at low concentrations, which appeared to be at odds with other work. To further investigate this reported toxicity, we dosed groups of mice with saxitoxin and tetrodotoxin mixtures daily for 21 days. In contrast to the previous study, no effects on mouse bodyweight, food consumption, heart rate, blood pressure, grip strength, blood chemistry or hematology were observed. Furthermore, no histological findings were associated with dosing in this trial. The dose rates in this study were 2.6, 3.8 and 4.9 times greater, respectively, than the highest dose of the previous study. As rapid mortality in three out of five mice was observed in the previous study, the deaths are likely to be due to the methodology used rather than the shellfish toxins. To convert animal data to that used in a human risk assessment, a 100-fold safety factor is required. After applying this safety factor, the dose rates used in the current study were 3.5, 5.0 and 6.5 times greater, respectively, than the acute reference dose for each toxin type set by the European Union. Furthermore, it has previously been proposed that tetrodotoxin be included in the paralytic shellfish poisoning suite of toxins. If this were done, the highest dose rate used in this study would be 13 times the acute reference dose. This study suggests that the previous 28-day trial was flawed and that the current paralytic shellfish toxin regulatory limit is fit for purpose. An additional study, feeding mice a diet laced with the test compounds at higher concentrations than those of the current experiment, would be required to comment on whether the current paralytic shellfish toxin regulatory limit should be modified.

Diversity and distribution of benthic dinoflagellates in Tonga include the potentially harmful genera Gambierdiscus and Fukuyoa.

Benthic dinoflagellates that can cause illness, such as ciguatera poisoning (CP), are prevalent around the Pacific but are poorly described in many locations. This study represents the first ecological assessment of benthic harmful algae species in the Kingdom of Tonga, a country where CP occurs regularly. Surveys were conducted in June 2016 in the Tongatapu island group, and in June 2017 across three island groups: Ha'apai, Vava'u, and Tongatapu. Shallow subtidal coastal habitats were investigated by measuring water quality parameters and conducting quadrat surveys. Microalgae samples were collected using either macrophyte collection or the artificial substrate method. Benthic dinoflagellates (Gambierdiscus and/or Fukuyoa, Ostreopsis, and Prorocentrum) were counted using light microscopy, followed by molecular analyses (real-time PCR in 2016 and high throughput sequencing (metabarcoding) in 2017) to identify Gambierdiscus and Fukuyoa to species level. Six species were detected from the Tongatapu island group in 2016 (G. australes, G. carpenteri, G. honu, G. pacificus, F. paulensis, and F. ruetzleri) using real-time PCR. Using the metabarcoding approach in 2017, a total of eight species (G. australes, G. carpenteri, G. honu, G. pacificus, G. cheloniae, G. lewisii, G. polynesiensis, and F. yasumotoi) were detected. Species were detected in mixed assemblages of up to six species, with G. pacificus and G. carpenteri being the most frequently observed. Ha'apai had the highest diversity with eight species detected, which identifies this area as a Gambierdiscus diversity 'hotspot'. Vava'u and Tongatapu had three and six species found respectively. Gambierdiscus polynesiensis, a described ciguatoxin producer and proposed causative agent of CP was found only in Ha'apai and Vava'u in 2017, but not in Tongatapu in either year. Ostreopsis spp. and Prorocentrum spp. were also frequently observed, with Prorocentrum most abundant at the majority of sites. In 2016, the highest number of Gambierdiscus and/or Fukuyoa cells were observed on seagrass (Halodule uninervis) from Sopu, Tongatapu. In 2017, the highest numbers of Gambierdiscus and/or Fukuyoa from artificial substrate samples were recorded in the Halimeda dominant habitat at Neiafu Tahi, Vava'u, a low energy site. This raised the question of the effect of wave motion or currents on abundance measurements from artificial substrates. Differences in detection were noticed between macrophytes and artificial substrates, with higher numbers of species found on artificial substrates. This study provides a baseline of benthic dinoflagellate distributions and diversity for Tonga that may be used for future studies and the development of monitoring programmes.