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Ciguatoxins (CTXs), potent neurotoxins produced by dinoflagellates of the genera Gambierdiscus and Fukuyoa, accumulate in commonly consumed fish species, causing human ciguatera poisoning. Field collections of Pacific reef fish reveal that consumed CTXs undergo oxidative biotransformations, resulting in numerous, often toxified analogs. Following our study showing rapid CTX accumulation in flesh of an herbivorous fish, we used the same laboratory model to examine the tissue distribution and metabolization of Pacific CTXs following long-term dietary exposure. Naso brevirostris consumed cells of Gambierdiscus polynesiensis in a gel food matrix over 16 weeks at a constant dose rate of 0.36 ng CTX3C equiv g−1 fish d−1. CTX toxicity determination of fish tissues showed CTX activity in all tissues of exposed fish (eight tissues plus the carcass), with the highest concentrations in the spleen. Muscle tissue retained the largest proportion of CTXs, with 44% of the total tissue burden. Moreover, relative to our previous study, we found that larger fish with slower growth rates assimilated a higher proportion of ingested toxin in their flesh (13% vs. 2%). Analysis of muscle extracts revealed the presence of CTX3C and CTX3B as well as a biotransformed product showing the m/z transitions of 2,3-dihydroxyCTX3C. This is the first experimental evidence of oxidative transformation of an algal CTX in a model consumer and known vector of CTX into the fish food web. These findings that the flesh intended for human consumption carries the majority of the toxin load, and that growth rates can influence the relationship between exposure and accumulation, have significant implications in risk assessment and the development of regulatory measures aimed at ensuring seafood safety. [ABSTRACT FROM AUTHOR]

Algal toxins pose a serious threat to human and coastal ecosystem health, even if their potential impacts are poorly documented in New Caledonia (NC). In this survey, bivalves and seawater (concentrated through passive samplers) from bays surrounding Noumea, NC, collected during the warm and cold seasons were analyzed for algal toxins using a multi-toxin screening approach. Several groups of marine microalgal toxins were detected for the first time in NC. Okadaic acid (OA), azaspiracid-2 (AZA2), pectenotoxin-2 (PTX2), pinnatoxin-G (PnTX-G), and homo-yessotoxin (homo-YTX) were detected in seawater at higher levels during the summer. A more diversified toxin profile was found in shellfish with brevetoxin-3 (BTX3), gymnodimine-A (GYM-A), and 13-desmethyl spirolide-C (SPX1), being confirmed in addition to the five toxin groups also found in seawater. Diarrhetic and neurotoxic toxins did not exceed regulatory limits, but PnTX-G was present at up to the limit of the threshold recommended by the French Food Safety Authority (ANSES, 23 μg kg−1). In the present study, internationally regulated toxins of the AZA-, BTX-, and OA-groups by the Codex Alimentarius were detected in addition to five emerging toxin groups, indicating that algal toxins pose a potential risk for the consumers in NC or shellfish export. [ABSTRACT FROM AUTHOR]

Gymnodimines (GYMs) are a group of fast-acting phycotoxins and their toxicological effects on human beings are still unclear due to the lack of sufficiently well-characterized large quantities of purified toxins for toxicology studies. In this study, a certified reference material (CRM) of GYM-A was prepared from the dinoflagellate Karenia selliformis, followed by multi-step chromatography separation and purification. Subsequently, the stability of GYM-A in methanolic media was evaluated at different temperature (−20, 4, and 20 °C) and pH (3, 5, and 7) conditions for 8 months, and the conversion products of GYM-A were explored by liquid chromatography–high resolution mass spectrometry (LC-HRMS). The results show that the stability of GYM-A decreased with increasing temperature and pH values. The GYM-A was stable during storage at −20 °C regardless of pH, but it decreased rapidly (81.8% ± 9.3%) at 20 °C in pH 7 solution after 8 months. Moreover, the concentrations of GYM-A did not significantly change at all temperatures in solutions with pH 3 (p > 0.05). It is recommended that GYM-A should be stored at low temperature (≤−20 °C) and pH (≤3) conditions for long-term storage in aqueous methanolic media. In addition, two conversion products of GYM-A, tentatively named as GYM-K (m/z 540) and GYM-L (m/z 524), were identified in the samples stored at high levels of pH and temperature. Based on the LC-HRMS data, the hypothetical chemical structures of both converting derivatives were proposed. A useful strategy for long-term storage of GYM-A CRM in aqueous methanolic media was suggested and two hypothesized conversion products of GYM-A were discovered in this study. [ABSTRACT FROM AUTHOR]

One of the greatest challenges in the design of shape-memory elements (mostly binary Nickel-Titanium wires) is to ensure that the required travel (stroke) is achieved, as this is subject to variation due to various influencing factors. One way of predicting the stroke is to use a suitable energy model. In the past, for example, a model was developed by Oelschläger with which the stroke can be calculated on the basis of the electrical energy. However, so far no model takes into account the change of the phase transformation temperature. In this study, the model of Oelschläger is extended and verified to consider the degradation behavior over the whole lifetime. For this purpose, fatigue tests of 52 wires (2 different load scenarios) were performed. Based on these tests and the application of statistical methods (distribution models, goodnes-of-fit tests etc.), a target model was developed for each load scenario, which is used to verify the extended energy model. The energy model was applied to wires of both load scenarios to simulate the stroke progression. The verification of the extended simulation model shows that it is possible to simulate the longterm behavior of the stroke for one of the two load scenarios. The second load scenario shows deviations between the target model and the simulation, which is due to problems in the area of measurement equipment, convection, and temperature distribution in the wire. Nevertheless, a decisive modeling approach could be developed, which can be used to consider the long-term behavior of the phase transformation temperature of wires in simulations. [ABSTRACT FROM AUTHOR]

Gambierdiscus and Fukuyoa dinoflagellates produce a suite of secondary metabolites, including ciguatoxins (CTXs), which bioaccumulate and are further biotransformed in fish and marine invertebrates, causing ciguatera poisoning when consumed by humans. This study is the first to compare the performance of the fluorescent receptor binding assay (fRBA), neuroblastoma cell-based assay (CBA-N2a), and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the quantitative estimation of CTX contents in 30 samples, obtained from four French Polynesian strains of Gambierdiscus polynesiensis. fRBA was applied to Gambierdiscus matrix for the first time, and several parameters of the fRBA protocol were refined. Following liquid/liquid partitioning to separate CTXs from other algal compounds, the variability of CTX contents was estimated using these three methods in three independent experiments. All three assays were significantly correlated with each other, with the highest correlation coefficient (r2 = 0.841) found between fRBA and LC-MS/MS. The CBA-N2a was more sensitive than LC-MS/MS and fRBA, with all assays showing good repeatability. The combined use of fRBA and/or CBA-N2a for screening purposes and LC-MS/MS for confirmation purposes allows for efficient CTX evaluation in Gambierdiscus. These findings, which support future collaborative studies for the inter-laboratory validation of CTX detection methods, will help improve ciguatera risk assessment and management. [ABSTRACT FROM AUTHOR]

Ciguatera fish poisoning (CFP) is a foodborne disease caused by the consumption of seafood (fish and marine invertebrates) contaminated with ciguatoxins (CTXs) produced by dinoflagellates in the genus Gambierdiscus. The report of a CFP-like mass-poisoning outbreak following the consumption of Tectus niloticus (Tegulidae, Gastropod) from Anaho Bay on Nuku Hiva Island (Marquesas archipelago, French Polynesia) prompted field investigations to assess the presence of CTXs in T. niloticus. Samples were collected from Anaho Bay, 1, 6 and 28 months after this poisoning outbreak, as well as in Taiohae and Taipivai bays. Toxicity analysis using the neuroblastoma cell-based assay (CBA-N2a) detected the presence of CTXs only in Anaho Bay T. niloticus samples. This is consistent with qPCR results on window screen samples indicating the presence of Gambierdiscus communities dominated by the species G. polynesiensis in Anaho Bay. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed that P-CTX-3B was the major congener, followed by P-CTX-3C, P-CTX-4A and P-CTX-4B in toxic samples. Between July 2014 and November 2016, toxin content in T. niloticus progressively decreased, but was consistently above the safety limit recommended for human consumption. This study confirms for the first time T. niloticus as a novel vector of CFP in French Polynesia. [ABSTRACT FROM AUTHOR]

Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, G. excentricus, is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island G. excentricus strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca2+ flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to pinpoint potential MTX analogs. Subsequent non-targeted HRMS analysis permitted the identification of a novel MTX analog, maitotoxin-4 (MTX4, accurate mono-isotopic mass of 3292.4860 Da, as free acid form) in the most toxic fractions. HRMS/MS spectra of MTX4 as well as of MTX are presented. In addition, crude methanolic extracts of five other strains of G. excentricus and 37 other strains representing one Fukuyoa species and ten species, one ribotype and one undetermined strain/species of Gambierdiscus were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS). This targeted analysis indicated the original maitotoxin (MTX) was only present in one strain (G. australes S080911_1). Putative maitotoxin-2 (p-MTX2) and maitotoxin-3 (p-MTX3) were identified in several other species, but confirmation was not possible because of the lack of reference material. Maitotoxin-4 was detected in all seven strains of G. excentricus examined, independently of their origin (Brazil, Canary Islands and Caribbean), and not detected in any other species. MTX4 may therefore serve as a biomarker for the highly toxic G. excentricus in the Atlantic area. [ABSTRACT FROM AUTHOR]

Vulcanodinium rugosum, a recently described species, produces pinnatoxins. The IFR-VRU-01 strain, isolated from a French Mediterranean lagoon in 2010 and identified as the causative dinoflagellate contaminating mussels in the Ingril Lagoon (French Mediterranean) with pinnatoxin-G, was grown in an enriched natural seawater medium. We tested the effect of temperature and salinity on growth, pinnatoxin-G production and chlorophyll a levels of this dinoflagellate. These factors were tested in combinations of five temperatures (15, 20, 25, 30 and 35 °C) and five salinities (20, 25, 30, 35 and 40) at an irradiance of 100 µmol photon m–2 s–1. V. rugosum can grow at temperatures and salinities ranging from 20 °C to 30 °C and 20 to 40, respectively. The optimal combination for growth (0.39 ± 0.11 d–1) was a temperature of 25 °C and a salinity of 40. Results suggest that V. rugosum is euryhaline and thermophile which could explain why this dinoflagellate develops in situ only from June to September. V. rugosum growth rate and pinnatoxin-G production were highest at temperatures ranging between 25 and 30 °C. This suggests that the dinoflagellate may give rise to extensive blooms in the coming decades caused by the climate change-related increases in temperature expected in the Mediterranean coasts. [ABSTRACT FROM AUTHOR]

Vulcanodinium rugosum, a recently described dinoflagellate species producing a potent neurotoxin (pinnatoxin G), has been identified in French Mediterranean lagoons and was responsible for recurrent episodes of shellfish toxicity detected by mouse bioassay. Until now, the biology and physiology of V. rugosum have not been fully investigated. We studied the growth characteristics and toxicity of a V. rugosum strain (IFR-VRU-01), isolated in the Ingril lagoon in June 2009 (North-Western French Mediterranean Sea). It was cultivated in Enriched Natural Sea Water (ENSW) with organic (urea) and inorganic (ammonium and nitrate) nitrogen, at a temperature of 25 °C and irradiance of 100 μmol/m2·s-1. Results showed that ammonium was assimilated by cells more rapidly than nitrate and urea. V. rugosum is thus an osmotrophic species using urea. Consequently, this nitrogen form could contribute to the growth of this dinoflagellate species in the natural environment. There was no significant difference (Anova, p = 0.856) between the growth rate of V. rugosum cultivated with ammonium (0.28 ± 0.11 day-1), urea (0.26 ± 0.08 day-1) and nitrate (0.24 ± 0.01 day-1). However, the production of chlorophyll a and pinnatoxin G was significantly lower with urea as a nitrogen source (Anova, p < 0.027), suggesting that nutritional conditions prevailing at the moment of the bloom could determine the cellular toxicity of V. rugosum and therefore the toxicity measured in contaminated mollusks. The relatively low growth rate (≤0.28 day-1) and the capacity of this species to continuously produce temporary cysts could explain why cell densities of this species in the water column are typically low (≤20,000 cells/L). [ABSTRACT FROM AUTHOR]

ß-N-methylamino-L-alanine (BMAA) is a neurotoxic non-protein amino acid suggested to be involved in neurodegenerative diseases. It was reported to be produced by cyanobacteria, but also found in edible aquatic organisms, thus raising concern of a widespread human exposure. However, the chemical analysis of BMAA and its isomers are controversial, mainly due to the lack of selectivity of the analytical methods. Using factorial design, we have optimized the chromatographic separation of underivatized analogues by a hydrophilic interaction chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) method. A combination of an effective solid phase extraction (SPE) clean-up, appropriate chromatographic resolution and the use of specific mass spectral transitions allowed for the development of a highly selective and sensitive analytical procedure to identify and quantify BMAA and its isomers (in both free and total form) in cyanobacteria and mollusk matrices (LOQ of 0.225 and 0.15 |ig/g dry weight, respectively). Ten species of cyanobacteria (six are reported to be BMAA producers) were screened with this method, and neither free nor bound BMAA could be found, while both free and bound DAB were present in almost all samples. Mussels and oysters collected in 2009 in the Thau Lagoon, France, were also screened, and bound BMAA and its two isomers, DAB and AEG, were observed in all samples (from 0.6 to 14.4 μg/g DW), while only several samples contained quantifiable free BMAA. [ABSTRACT FROM AUTHOR]

Ostreopsis cf. ovata produces palytoxin analogues including ovatoxins (OVTXs) and a putative palytoxin (p-PLTX), which can accumulate in marine organisms and may possibly lead to food intoxication. However, purified ovatoxins are not widely available and their toxicities are still unknown. The aim of this study was to improve understanding of the ecophysiology of Ostreopsis cf. ovata and its toxin production as well as to optimize the purification process for ovatoxin. During Ostreopsis blooms in 2011 and 2012 in Villefranche-sur-Mer (France, NW Mediterranean Sea), microalgae epiphytic cells and marine organisms were collected and analyzed both by LC-MS/MS and hemolysis assay. Results obtained with these two methods were comparable, suggesting ovatoxins have hemolytic properties. An average of 223 µg·kg-1 of palytoxin equivalent of whole flesh was found, thus exceeding the threshold of 30 µg·kg-1 in shellfish recommended by the European Food Safety Authority (EFSA). Ostreopsis cells showed the same toxin profile both in situ and in laboratory culture, with ovatoxin-a (OVTX-a) being the most abundant analogue (~50%), followed by OVTX-b (~15%), p-PLTX (12%), OVTX-d (8%), OVTX-c (5%) and OVTX-e (4%). Ostreopsis cf. ovata produced up to 2 g of biomass per L of culture, with a maximum concentration of 300 pg PLTX equivalent cell-1. Thus, an approximate amount of 10 mg of PLTX-group toxins may be produced with 10 L of this strain. Toxin extracts obtained from collected biomass were purified using different techniques such as liquid-liquid partition or size exclusion. Among these methods, open-column chromatography with Sephadex LH20 phase yielded the best results with a cleanup efficiency of 93% and recovery of about 85%, representing an increase of toxin percentage by 13 fold. Hence, this purification step should be incorporated into future isolation exercises. [ABSTRACT FROM AUTHOR]

Pinnatoxin G (PnTX-G) is a marine toxin belonging to the class of cyclic imines and produced by the dinoflagellate Vulcanodinium rugosum. In spite of its strong toxicity to mice, leading to the classification of pinnatoxins into the class of "fast-acting toxins", its hazard for human health has never been demonstrated. In this study, crude extracts of V. rugosum exhibited significant cytotoxicity against Neuro2A and KB cells. IC50 values of 0.38 μg mL-1 and 0.19 μg mL-1were estimated on Neuro2A cells after only 24 h of incubation and on KB cells after 72 h of incubation, respectively. In the case of Caco-2 cells 48 h after exposure, the crude extract of V. rugosum induced cell cycle arrest accompanied by a dramatic increase in double strand DNA breaks, although only 40% cytotoxicity was observed at the highest concentration tested (5 μg mL-1). However, PnTX-G was not a potent cytotoxic compound as no reduction of the cell viability was observed on the different cell lines. Moreover, no effects on the cell cycle or DNA damage were observed following treatment of undifferentiated Caco-2 cells with PnTX-G. The crude extract of V. rugosum was thus partially purified using liquid-liquid partitioning and SPE clean-up. In vitro assays revealed strong activity of some fractions containing no PnTX-G. The crude extract and the most potent fraction were evaluated using full scan and tandem high resolution mass spectrometry. The dereplication revealed the presence of a major compound that could be putatively annotated as nakijiquinone A, N-carboxy-methyl-smenospongine or stachybotrin A, using the MarinLit™ database. Further investigations will be necessary to confirm the identity of the compounds responsible for the cytotoxicity and genotoxicity of the extracts of V. rugosum. [ABSTRACT FROM AUTHOR]

Ciguatera poisoning is caused by the ingestion of fish or shellfish contaminated with ciguatoxins produced by dinoflagellate species belonging to the genera Gambierdiscus and Fukuyoa. Unlike in the Pacific region, the species producing ciguatoxins in the Atlantic Ocean have yet to be definitely identified, though some ciguatoxins responsible for ciguatera have been reported from fish. Previous studies investigating the ciguatoxin-like toxicity of Atlantic Gambierdiscus species using Neuro2a cell-based assay identified G. excentricus as a potential toxin producer. To more rigorously characterize the toxin profile produced by this species, a purified extract from 124 million cells was prepared and partial characterization by high-resolution mass spectrometry was performed. The analysis revealed two new analogs of the polyether gambierone: sulfo-gambierone and dihydro-sulfo-gambierone. Algal ciguatoxins were not identified. The very low ciguatoxin-like toxicity of the two new analogs obtained by the Neuro2a cell-based assay suggests they are not responsible for the relatively high toxicity previously observed when using fractionated G. excentricus extracts, and are unlikely the cause of ciguatera in the region. These compounds, however, can be useful as biomarkers of the presence of G. excentricus due to their sensitive detection by mass spectrometry. [ABSTRACT FROM AUTHOR]

Tetrodotoxins (TTXs) are potentially lethal paralytic toxins that have been identified in European shellfish over recent years. Risk assessment has suggested comparatively low levels (44 µg TTX-equivalent/kg) but stresses the lack of data on occurrence. Both bacteria and dinoflagellates were suggested as possible biogenic sources, either from an endogenous or exogenous origin. We thus investigated TTXs in (i) 98 shellfish samples and (ii) 122 bacterial strains, isolated from French environments. We optimized a method based on mass spectrometry, using a single extraction step followed by ultrafiltration without Solid Phase Extraction and matrix-matched calibration for both shellfish and bacterial matrix. Limits of detection and quantification were 6.3 and 12.5 µg/kg for shellfish and 5.0 and 10 µg/kg for bacterial matrix, respectively. Even though bacterial matrix resulted in signal enhancement, no TTX analog was detected in any strain. Bivalves (either Crassostrea gigas or Ruditapes philippinarum) were surveyed in six French production areas over 2.5–3 month periods (2018–2019). Concentrations of TTX ranged from 'not detected' to a maximum of 32 µg/kg (Bay of Brest, 17 June 2019), with events lasting 2 weeks at maximum. While these results are in line with previous studies, they provide new data of TTX occurrence and confirm that the link between bacteria, bivalves and TTX is complex. [ABSTRACT FROM AUTHOR]

Ciguatera poisoning (CP) cases linked to the consumption of deep-water fish occurred in 2003 in the Gambier Islands (French Polynesia). In 2004, on the request of two local fishermen, the presence of ciguatoxins (CTXs) was examined in part of their fish catches, i.e., 22 specimens representing five deep-water fish species. Using the radioactive receptor binding assay (rRBA) and mouse bioassay (MBA), significant CTX levels were detected in seven deep-water specimens in Lutjanidae, Serranidae, and Bramidae families. Following additional purification steps on the remaining liposoluble fractions for 13 of these samples (kept at −20 °C), these latter were reanalyzed in 2018 with improved protocols of the neuroblastoma cell-based assay (CBA-N2a) and liquid chromatography tandem mass spectrometry (LC–MS/MS). Using the CBA-N2a, the highest CTX-like content found in a specimen of Eumegistus illustris (Bramidae) was 2.94 ± 0.27 µg CTX1B eq. kg−1. Its toxin profile consisted of 52-epi-54-deoxyCTX1B, CTX1B, and 54-deoxyCTX1B, as assessed by LC–MS/MS. This is the first study demonstrating that deep-water fish are potential ciguatera vectors and highlighting the importance of a systematic monitoring of CTXs in all exploited fish species, especially in ciguatera hotspots, including deep-water fish, which constitute a significant portion of the commercial deep-sea fisheries in many Asian–Pacific countries. [ABSTRACT FROM AUTHOR]

Dinoflagellate species of the genera Gambierdiscus and Fukuyoa are known to produce ciguatera poisoning-associated toxic compounds, such as ciguatoxins, or other toxins, such as maitotoxins. However, many species and strains remain poorly characterized in areas where they were recently identified, such as the western Mediterranean Sea. In previous studies carried out by our research group, a G. australes strain from the Balearic Islands (Mediterranean Sea) presenting MTX-like activity was characterized by LC-MS/MS and LC-HRMS detecting 44-methyl gambierone and gambieric acids C and D. However, MTX1, which is typically found in some G. australes strains from the Pacific Ocean, was not detected. Therefore, this study focuses on the identification of the compound responsible for the MTX-like toxicity in this strain. The G. australes strain was characterized not only using LC-MS instruments but also N2a-guided HPLC fractionation. Following this approach, several toxic compounds were identified in three fractions by LC-MS/MS and HRMS. A novel MTX analogue, named MTX5, was identified in the most toxic fraction, and 44-methyl gambierone and gambieric acids C and D contributed to the toxicity observed in other fractions of this strain. Thus, G. australes from the Mediterranean Sea produces MTX5 instead of MTX1 in contrast to some strains of the same species from the Pacific Ocean. No CTX precursors were detected, reinforcing the complexity of the identification of CTXs precursors in these regions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Whereas (technical) standards often affect society as a whole, they are mostly developed by men. In the context of the United Nations' Sustainable Development Goal 5 (gender equality), this article motivates research on the gender gap in standardization, focusing in a first step on the under-representation of women in science, technology, engineering, mathematics and leadership positions as one possible cause. A novel data set of more than 8000 organizations that develop formal standards and 28,000 affiliated experts (10.5% female) confirms that women are descriptively under-represented. A logistic regression shows that organizations' size, industry, and geographical location are significant factors that are associated with representation by female standardizers. Standard-development for construction, mechanical and electrical engineering is especially male-dominated, while the east of Germany shows more female representation than the west. The presented empirical evidence of female under-representation suggests a need for standard-setting organizations to expand their focus from considering gender in standards documents to actively promoting female participation in their committees. It further adds to the debate on stakeholder representation in standardization and its legitimacy as a co-regulative system in the EU. [ABSTRACT FROM AUTHOR]

The circular economy (CE) represents an environmentally and sustainability-focused economic paradigm that has gained momentum in recent years. Innovation ecosystems are the evolving interconnected sets of actors, activities, artefacts, and institutions who are vital to the innovative performances of single actors or actor groups consisting largely of firms in the products and services sector. To develop sustainable CE ecosystems, participating firms need to involve the consumers and users in their innovation processes. The automotive industry is to a large extent an industry in which incorporating customer requirements in product development is critical to success. In addition, growing expectations and growing awareness of environmental issues drive the industry to develop environmentally friendly products. However, CE solutions and, specifically, sustainable tyres have not yet been given due consideration. Likewise, the specific preferences of the end-users of sustainability-focused cars such as electric vehicles (EVs) and users of biofuels are unknown in the CE context so far. Based on the current state of research, this article addresses an important, unexplored topic of product circularity. Being the first article on consumer interests and active contributions to CE automotive products, it also extends the first articles on CE software products. A survey of 168 traditional car owners (no EV/biofuels users), 29 users of biofuels, and 40 EV affine consumers was conducted in Germany to create an empirical foundation for the specification of CE configuration software for sustainable automotive products, particularly sustainable tyres. The results show different preferences among these user groups, but also the importance of other characteristics not captured by the distinction by car ownership. In particular, the perception of climate change and the use of test reports or rating portals were variables that had significant influence on configuration preferences. [ABSTRACT FROM AUTHOR]

Coolia is a genus of marine benthic dinoflagellates which is widely distributed in tropical and temperate zones. Toxicity has been reported in selected Coolia species, although the identity of causative compounds is still controversial. In this study, we investigated the taxonomical and toxicological aspects of Coolia species from Brazil. Since light- and electron microscopy-based morphology was not enough to distinguish small-celled species, ITS and LSU D1-D3 phylogenetic analyses were used for species definition. Cultures of Coolia palmyrensis and Coolia santacroce were established from samples collected along the northeastern Brazilian coast, the first record of both species in South Atlantic waters. Cultures of Coolia malayensis and Coolia tropicalis were also established and exhibited acute in vivo toxicity to adults of Artemia salina, while C. palmyrensis and C. santacroce were non-toxic. The presence of 30 yessotoxin analogues, 7 metabolites of Coolia and 44 Gambierdiscus metabolites was screened in 14 strains of Coolia. 44-methyl gambierone (formerly referred to as MTX3) and a new isomer of this compound were detected only in C. tropicalis, using both low- and high-resolution LC-MS/MS. To our knowledge, this is the first report of gambierone analogues in dinoflagellates other than Gambierdiscus; the role of C. tropicalis in ciguatera poisoning thus deserves to be considered in further investigations. [ABSTRACT FROM AUTHOR]

Ciguatera poisoning is a foodborne illness caused by the consumption of seafood contaminated with ciguatoxins (CTXs) produced by dinoflagellates from the genera Gambierdiscus and Fukuyoa. The suitability of Solid Phase Adsorption Toxin Tracking (SPATT) technology for the monitoring of dissolved CTXs in the marine environment has recently been demonstrated. To refine the use of this passive monitoring tool in ciguateric areas, the effects of deployment time and sampler format on the adsorption of CTXs by HP20 resin were assessed in Anaho Bay (Nuku Hiva Island, French Polynesia), a well-known ciguatera hotspot. Toxicity data assessed by means of the mouse neuroblastoma cell-based assay (CBA-N2a) showed that a 24 h deployment of 2.5 g of resin allowed concentrating quantifiable amounts of CTXs on SPATT samplers. The CTX levels varied with increasing deployment time, resin load, and surface area. In addition to CTXs, okadaic acid (OA) and dinophysistoxin-1 (DTX1) were also detected in SPATT extracts using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), consistent with the presence of Gambierdiscus and Prorocentrum species in the environment, as assessed by quantitative polymerase chain reaction (qPCR) and high-throughput sequencing (HTS) metabarcoding analyses conducted on passive window screen (WS) artificial substrate samples. Although these preliminary findings await further confirmation in follow-up studies, they highlight the usefulness of SPATT samplers in the routine surveillance of CP risk on a temporal scale, and the monitoring of other phycotoxin-related risks in ciguatera-prone areas. [ABSTRACT FROM AUTHOR]

Over the last decade, knowledge has significantly increased on the taxonomic identity and distribution of dinoflagellates of the genera Gambierdiscus and Fukuyoa. Additionally, a number of hitherto unknown bioactive metabolites have been described, while the role of these compounds in ciguatera poisoning (CP) remains to be clarified. Ciguatoxins and maitotoxins are very toxic compounds produced by these dinoflagellates and have been described since the 1980s. Ciguatoxins are generally described as the main contributors to this food intoxication. Recent reports of CP in temperate waters of the Canary Islands (Spain) and the Madeira archipelago (Portugal) triggered the need for isolation and cultivation of dinoflagellates from these areas, and their taxonomic and toxicological characterization. Maitotoxins, and specifically maitotoxin-4, has been described as one of the most toxic compounds produced by these dinoflagellates (e.g., G. excentricus) in the Canary Islands. Thus, characterization of toxin profiles of Gambierdiscus species from adjacent regions appears critical. The combination of liquid chromatography coupled to either low- or high-resolution mass spectrometry allowed for characterization of several strains of Gambierdiscus and Fukuyoa from the Mediterranean Sea and the Canary Islands. Maitotoxin-3, two analogues tentatively identified as gambieric acid C and D, a putative gambierone analogue and a putative gambieroxide were detected in all G. australes strains from Menorca and Mallorca (Balearic Islands, Spain) while only maitotoxin-3 was present in an F. paulensis strain of the same region. An unidentified Gambierdiscus species (Gambierdiscus sp.2) from Crete (Greece) showed a different toxin profile, detecting both maitotoxin-3 and gambierone, while the availability of a G. excentricus strain from the Canary Islands (Spain) confirmed the presence of maitotoxin-4 in this species. Overall, this study shows that toxin profiles not only appear to be species-specific but probably also specific to larger geographic regions. [ABSTRACT FROM AUTHOR]

Pinnatoxin G (PnTx-G) is a marine cyclic imine toxin produced by the dinoflagellate Vulcanodinium rugosum, frequently detected in edible shellfish from Ingril Lagoon (France). As other pinnatoxins, to date, no human poisonings ascribed to consumption of PnTx-G contaminated seafood have been reported, despite its potent antagonism at nicotinic acetylcholine receptors and its high and fast-acting toxicity after intraperitoneal or oral administration in mice. The hazard characterization of PnTx-G by oral exposure is limited to a single acute toxicity study recording lethality and clinical signs in non-fasted mice treated by gavage or through voluntary food ingestion, which showed differences in PnTx-G toxic potency. Thus, an acute toxicity study was carried out using 3 h-fasted CD-1 female mice, administered by gavage with PnTx-G (8–450 µg kg−1). At the dose of 220 µg kg−1 and above, the toxin induced a rapid onset of clinical signs (piloerection, prostration, hypothermia, abdominal breathing, paralysis of the hind limbs, and cyanosis), leading to the death of mice within 30 min. Except for moderate mucosal degeneration in the small intestine recorded at doses of 300 µg kg−1, the toxin did not induce significant morphological changes in the other main organs and tissues, or alterations in blood chemistry parameters. This acute oral toxicity study allowed to calculate an oral LD50 for PnTx-G equal to 208 μg kg−1 (95% confidence limits: 155–281 µg kg−1) and to estimate a provisional NOEL of 120 µg kg−1. [ABSTRACT FROM AUTHOR]

Ciguatera poisoning (CP) is a foodborne disease caused by the consumption of seafood contaminated with ciguatoxins (CTXs) produced by dinoflagellates in the genera Gambierdiscus and Fukuyoa. The toxin production and toxin profiles were explored in four clones of G. polynesiensis originating from different islands in French Polynesia with contrasted CP risk: RIK7 (Mangareva, Gambier), NHA4 (Nuku Hiva, Marquesas), RAI-1 (Raivavae, Australes), and RG92 (Rangiroa, Tuamotu). Productions of CTXs, maitotoxins (MTXs), and gambierone group analogs were examined at exponential and stationary growth phases using the neuroblastoma cell-based assay and liquid chromatography–tandem mass spectrometry. While none of the strains was found to produce known MTX compounds, all strains showed high overall P-CTX production ranging from 1.1 ± 0.1 to 4.6 ± 0.7 pg cell−1. In total, nine P-CTX analogs were detected, depending on strain and growth phase. The production of gambierone, as well as 44-methylgamberione, was also confirmed in G. polynesiensis. This study highlighted: (i) intraspecific variations in toxin production and profiles between clones from distinct geographic origins and (ii) the noticeable increase in toxin production of both CTXs, in particular CTX4A/B, and gambierone group analogs from the exponential to the stationary phase. [ABSTRACT FROM AUTHOR]

Ostreopsis cf. ovata is a toxic marine benthic dinoflagellate responsible for harmful blooms affecting ecosystem and human health, mostly in the Mediterranean Sea. In this study we report the occurrence of a summer O. cf. ovata bloom in Currais, a coastal archipelago located on the subtropical Brazilian coast (~25° S). This bloom was very similar to Mediterranean episodes in many aspects: (a) field-sampled and cultivated O. cf. ovata cells aligned phylogenetically (ITS and LSU regions) along with Mediterranean strains; (b) the bloom occurred at increasing temperature and irradiance, and decreasing wind speed; (c) cell densities reached up to 8.0 × 104 cell cm−2 on fiberglass screen and 5.6 × 105 cell g−1 fresh weight on seaweeds; (d) and toxin profiles were composed mostly of ovatoxin-a (58%) and ovatoxin-b (32%), up to 35.5 pg PLTX-eq. cell−1 in total. Mussels were contaminated during the bloom with unsafe toxin levels (up to 131 µg PLTX-eq. kg−1). Ostreopsis cells attached to different plastic litter, indicating an alternate route for toxin transfer to marine fauna via ingestion of biofilm-coated plastic debris. [ABSTRACT FROM AUTHOR]

Ciguatoxins (CTXs), potent neurotoxins produced by dinoflagellates of the genera Gambierdiscus and Fukuyoa , accumulate in commonly consumed fish species, causing human ciguatera poisoning. Field collections of Pacific reef fish reveal that consumed CTXs undergo oxidative biotransformations, resulting in numerous, often toxified analogs. Following our study showing rapid CTX accumulation in flesh of an herbivorous fish, we used the same laboratory model to examine the tissue distribution and metabolization of Pacific CTXs following long-term dietary exposure. Naso brevirostris consumed cells of Gambierdiscus polynesiensis in a gel food matrix over 16 weeks at a constant dose rate of 0.36 ng CTX3C equiv g -1 fish d -1 . CTX toxicity determination of fish tissues showed CTX activity in all tissues of exposed fish (eight tissues plus the carcass), with the highest concentrations in the spleen. Muscle tissue retained the largest proportion of CTXs, with 44% of the total tissue burden. Moreover, relative to our previous study, we found that larger fish with slower growth rates assimilated a higher proportion of ingested toxin in their flesh (13% vs. 2%). Analysis of muscle extracts revealed the presence of CTX3C and CTX3B as well as a biotransformed product showing the m / z transitions of 2,3-dihydroxyCTX3C. This is the first experimental evidence of oxidative transformation of an algal CTX in a model consumer and known vector of CTX into the fish food web. These findings that the flesh intended for human consumption carries the majority of the toxin load, and that growth rates can influence the relationship between exposure and accumulation, have significant implications in risk assessment and the development of regulatory measures aimed at ensuring seafood safety.

Gymnodimine-A (GYM-A) is a fast-acting microalgal toxin and its production of certified materials requires an efficient harvesting technology from the large-scale cultures of toxigenic microalgae. In this study the recoveries of GYM-A were compared between several liquid-liquid extraction (LLE) treatments including solvents, ratios and stirring times to optimize the LLE technique for harvesting GYM-A from Karenia selliformis cultures, of which the dichloromethane was selected as the extractant and added to microalgal cultures at the ratio 55 mL L -1 (5.5%, v / v ). The recovery of GYM-A obtained by the LLE technique was also compared with filtration and centrifugation methods. The stability of GYM-A in culture media were also tested under different pH conditions. Results showed that both the conventional filter filtration and centrifugation methods led to fragmentation of microalgal cells and loss of GYM-A in the harvesting processes. A total of 5.1 µg of GYM-A were obtained from 2 L of K. selliformis cultures with a satisfactory recovery of 88%. Interestingly, GYM-A obviously degraded in the culture media with the initial pH 8.2 and the adjusted pH of 7.0 after 7 days, but there was no obvious degradation in the acidic medium at pH 5.0. Therefore, the LLE method developed here permits the collection of large-volume cultures of K. selliformis and the high-efficiency extraction of GYM-A. This work provides a simple and valuable technique for harvesting toxins from large-scale cultures of GYM-producing microalgae.

Palytoxin (PLTX) and its congeners are emerging toxins held responsible for a number of human poisonings following the inhalation of toxic aerosols, skin contact, or the ingestion of contaminated seafood. Despite the strong structural analogies, the relative toxic potencies of PLTX congeners are quite different, making it necessary to isolate them individually in sufficient amounts for toxicological and analytical purposes. Previous studies showed poor PLTX recoveries with a dramatic decrease in PLTX yield throughout each purification step. In view of a large-scale preparative work aimed at the preparation of PLTX reference material, we have investigated evaporation as a critical-although unavoidable-step that heavily affects overall recoveries. The experiments were carried out in two laboratories using different liquid chromatography-mass spectrometry (LC-MS) instruments, with either unit or high resolution. Palytoxin behaved differently when concentrated to a minimum volume rather than when evaporated to complete dryness. The recoveries strongly depended on the solubility as well as on the material of the used container. The LC-MS analyses of PLTX dissolved in aqueous organic blends proved to give a peak intensity higher then when dissolved in pure water. After drying, the PLTX adsorption appeared stronger on glass surfaces than on plastic materials. However, both the solvents used to dilute PLTX and that used for re-dissolution had an important role. A quantitative recovery (97%) was achieved when completely drying 80% aqueous EtOH solutions of PLTX under N 2 -stream in Teflon. The stability of PLTX in acids was also investigated. Although PLTX was quite stable in 0.2% acetic acid solutions, upon exposure to stronger acids (pH < 2.66), degradation products were observed, among which a PLTX methyl-ester was identified.

Ciguatera poisoning is a foodborne disease caused by the consumption of seafood contaminated with ciguatoxins (CTXs) produced by dinoflagellates in the genera Gambierdiscus and Fukuyoa . Ciguatera outbreaks are expected to increase worldwide with global change, in particular as a function of its main drivers, including changes in sea surface temperature, acidification, and coastal eutrophication. In French Polynesia, G. polynesiensis is regarded as the dominant source of CTXs entering the food web. The effects of pH (8.4, 8.2, and 7.9), Nitrogen:Phosphorus ratios (24N:1P vs. 48N:1P), and nitrogen source (nitrates vs. urea) on growth rate, biomass, CTX levels, and profiles were examined in four clones of G. polynesiensis at different culture age (D10, D21, and D30). Results highlight a decrease in growth rate and cellular biomass at low pH when urea is used as a N source. No significant effect of pH, N:P ratio, and N source on the overall CTX content was observed. Up to ten distinct analogs of Pacific ciguatoxins (P-CTXs) could be detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in clone NHA4 grown in urea, at D21. Amounts of more oxidized P-CTX analogs also increased under the lowest pH condition. These data provide interesting leads for the custom production of CTX standards.

: Recurrent blooms of Ostreopsis cf. ovata have been reported in Brazil and the Mediterranean Sea with associated ecological, and in the latter case, health impacts. Molecular data based on the D1-D3 and D8-D10 regions of the LSU rDNA and ITS loci, and the morphology of O . cf. ovata isolates and field populations from locations along the Brazilian tropical and subtropical coastal regions and three oceanic islands are presented. Additional ITS sequences from three single cells from the tropical coast are provided. Toxin profiles and quantities of PLTX and their analogues; OVTXs; contained in cells from two clonal cultures and two field blooms from Rio de Janeiro were investigated. Morphology was examined using both light and epifluorescence microscopy. Morphometric analysis of different strains and field populations from diverse locations were compared. Molecular analysis showed that six of the seven sequences grouped at the large "Atlantic/Mediterranean/Pacific" sub-clade, while one sequence branched in a sister clade with sequences from Madeira Island and Greece. The toxin profile of strains and bloom field samples from Rio de Janeiro were dominated by OVTX-a and -b, with total cell quotas (31.3 and 39.3 pg cell -1 ) in the range of that previously reported for strains of O . cf. ovata ., Competing Interests: The authors declare no conflict of interest.

Background: Azaspiracids (AZAs) are marine toxins that are produced by Azadinium and Amphidoma dinoflagellates that can contaminate edible shellfish inducing a foodborne poisoning in humans, which is characterized by gastrointestinal symptoms. Among these, AZA1, -2, and -3 are regulated in the European Union, being the most important in terms of occurrence and toxicity. In vivo studies in mice showed that, in addition to gastrointestinal effects, AZA1 induces liver alterations that are visible as a swollen organ, with the presence of hepatocellular fat droplets and vacuoles. Hence, an in vitro study was carried out to investigate the effects of AZA1, -2, and -3 on liver cells, using human non-tumor IHH hepatocytes., Results: The exposure of IHH cells to AZA1, -2, or -3 (5 × 10 -12 -1 × 10 -7 M) for 24 h did not affect the cell viability and proliferation (Sulforhodamine B assay and 3 H-Thymidine incorporation assay), but they induced a significant concentration-dependent increase of mitochondrial dehydrogenases activity (MTT reduction assay). This effect depends on the activity of mitochondrial electron transport chain complex I and II, being counteracted by rotenone and tenoyl trifluoroacetone, respectively. Furthermore, AZAs-increased mitochondrial dehydrogenase activity was almost totally suppressed in the K + -, Cl - -, and Na + -free media and sensitive to the specific inhibitors of K ATP and hERG potassium channels, Na + /K + , ATPase, and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels., Conclusions: These results suggest that AZA mitochondrial effects in hepatocytes derive from an imbalance of intracellular levels of K + and, in particular, Cl - ions, as demonstrated by the selective reduction of toxin effects by CFTR chloride channel inhibition.

Ciguatera is a foodborne disease caused by the consumption of seafood contaminated with ciguatoxins (CTXs). Ciguatera-like poisoning events involving giant clams ( Tridacna maxima ) are reported occasionally from Pacific islands communities. The present study aimed at providing insights into CTXs tissue distribution and detoxification rate in giant clams exposed to toxic cells of Gambierdiscus polynesiensis , in the framework of seafood safety assessment. In a first experiment, three groups of tissue (viscera, flesh and mantle) were dissected from exposed individuals, and analyzed for their toxicity using the neuroblastoma cell-based assay (CBA-N2a) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The viscera, flesh, and mantle were shown to retain 65%, 25%, and 10% of the total toxin burden, respectively. All tissues reached levels above the safety limit recommended for human consumption, suggesting that evisceration alone, a practice widely used among local populations, is not enough to ensure seafood safety. In a second experiment, the toxin content in contaminated giant clams was followed at different time points (0, 2, 4, and 6 days post-exposure). Observations suggest that no toxin elimination is visible in T. maxima throughout 6 days of detoxification.

Photosynthetic species of the genus Dinophysis are obligate mixotrophs with temporary plastids (kleptoplastids) that are acquired from the ciliate Mesodinium rubrum , which feeds on cryptophytes of the Teleaulax-Plagioselmis-Geminigera clade. A metabolomic study of the three-species food chain Dinophysis-Mesodinium-Teleaulax was carried out using mass spectrometric analysis of extracts of batch-cultured cells of each level of that food chain. The main goal was to compare the metabolomic expression of Galician strains of Dinophysis acuminata and D. acuta that were subjected to different feeding regimes (well-fed and prey-limited) and feeding on two Mesodinium (Spanish and Danish) strains. Both Dinophysis species were able to grow while feeding on both Mesodinium strains, although differences in growth rates were observed. Toxin and metabolomic profiles of the two Dinophysis species were significantly different, and also varied between different feeding regimes and different prey organisms. Furthermore, significantly different metabolomes were expressed by a strain of D. acuminata that was feeding on different strains of the ciliate Mesodinium rubrum . Both species-specific metabolites and those common to D. acuminata and D. acuta were tentatively identified by screening of METLIN and Marine Natural Products Dictionary databases. This first metabolomic study applied to Dinophysis acuminata and D.acuta in culture establishes a basis for the chemical inventory of these species.

The sea urchin Tripneustes gratilla ( Toxopneustidae , Echinoids) is a source of protein for many islanders in the Indo-West Pacific. It was previously reported to occasionally cause ciguatera-like poisoning; however, the exact nature of the causative agent was not confirmed. In April and July 2015, ciguatera poisonings were reported following the consumption of T. gratilla in Anaho Bay (Nuku Hiva Island, Marquesas archipelago, French Polynesia). Patient symptomatology was recorded and sea urchin samples were collected from Anaho Bay in July 2015 and November 2016. Toxicity analysis using the neuroblastoma cell-based assay (CBA-N2a) detected the presence of ciguatoxins (CTXs) in T. gratilla samples. Gambierdiscus species were predominant in the benthic assemblages of Anaho Bay, and G. polynesiensis was highly prevalent in in vitro cultures according to qPCR results. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed that P-CTX-3B was the major ciguatoxin congener in toxic sea urchin samples, followed by 51-OH-P-CTX-3C, P-CTX-3C, P-CTX-4A, and P-CTX-4B. Between July 2015 and November 2016, the toxin content in T. gratilla decreased, but was consistently above the safety limit allowed for human consumption. This study provides evidence of CTX bioaccumulation in T. gratilla as a cause of ciguatera-like poisoning associated with a documented symptomatology., Competing Interests: The authors declare no conflict of interest.

Ciguatera fish poisoning (CFP) is a foodborne disease caused by the consumption of seafood (fish and marine invertebrates) contaminated with ciguatoxins (CTXs) produced by dinoflagellates in the genus Gambierdiscus . The report of a CFP-like mass-poisoning outbreak following the consumption of Tectus niloticus (Tegulidae, Gastropod) from Anaho Bay on Nuku Hiva Island (Marquesas archipelago, French Polynesia) prompted field investigations to assess the presence of CTXs in T. niloticus . Samples were collected from Anaho Bay, 1, 6 and 28 months after this poisoning outbreak, as well as in Taiohae and Taipivai bays. Toxicity analysis using the neuroblastoma cell-based assay (CBA-N2a) detected the presence of CTXs only in Anaho Bay T. niloticus samples. This is consistent with qPCR results on window screen samples indicating the presence of Gambierdiscus communities dominated by the species G. polynesiensis in Anaho Bay. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed that P-CTX-3B was the major congener, followed by P-CTX-3C, P-CTX-4A and P-CTX-4B in toxic samples. Between July 2014 and November 2016, toxin content in T. niloticus progressively decreased, but was consistently above the safety limit recommended for human consumption. This study confirms for the first time T. niloticus as a novel vector of CFP in French Polynesia., Competing Interests: The authors declare no conflict of interest.

Azaspiracid (AZA) poisoning has been reported following consumption of contaminated shellfish, and is of human health concern. Hence, it is important to have sustainable amounts of the causative toxins available for toxicological studies and for instrument calibration in monitoring programs, without having to rely on natural toxin events. Continuous pilot scale culturing was carried out to evaluate the feasibility of AZA production using Azadinium spinosum cultures. Algae were harvested using tangential flow filtration or continuous centrifugation. AZAs were extracted using solid phase extraction (SPE) procedures, and subsequently purified. When coupling two stirred photobioreactors in series, cell concentrations reached 190,000 and 210,000 cell · mL(-1) at steady state in bioreactors 1 and 2, respectively. The AZA cell quota decreased as the dilution rate increased from 0.15 to 0.3 day(-1), with optimum toxin production at 0.25 day(-1). After optimization, SPE procedures allowed for the recovery of 79 ± 9% of AZAs. The preparative isolation procedure previously developed for shellfish was optimized for algal extracts, such that only four steps were necessary to obtain purified AZA1 and -2. A purification efficiency of more than 70% was achieved, and isolation from 1200 L of culture yielded 9.3 mg of AZA1 and 2.2 mg of AZA2 of >95% purity. This work demonstrated the feasibility of sustainably producing AZA1 and -2 from A. spinosum cultures.

Azaspiracids (AZA) are polyether marine toxins that accumulate in various shellfish species and have been associated with severe gastrointestinal human intoxications since 1995. This toxin class has since been reported from several countries, including Morocco and much of western Europe. A regulatory limit of 160 microg AZA/kg whole shellfish flesh was established by the EU in order to protect human health; however, in some cases, AZA concentrations far exceed the action level. Herein we discuss recent advances on the chemistry of various AZA analogs, review the ecology of AZAs, including the putative progenitor algal species, collectively interpret the in vitro and in vivo data on the toxicology of AZAs relating to human health issues, and outline the European legislature associated with AZAs.