Search Results (17)

Palytoxins (PLTXs) and ovatoxins (OVTXs) are a group of highly potent marine toxins that pose significant health risks through seafood contamination and environmental exposure. OVTX-producing algae have been linked to dermatitis and respiratory distress in Mediterranean beachgoers, while serious public health concerns are related to PLTX accumulation in seafood. In 2009, the European Food Safety Authority highlighted the need for analytical detection methods of the PLTX group of toxins and for the preparation of reference materials. This study investigates the stability of the palytoxin signal using liquid chromatography tandem mass spectrometry (LC-MRM-MS) and UV-Vis spectrophotometry under different experimental conditions: three concentrations (10, 1, and 0.5 µg/mL), three methanol–water mixtures (10%, 50%, and 90%), and two temperatures (6 °C and 25 °C). The results showed that the PLTX signal response is significantly influenced by the experimental conditions used. LC-MRM-MS analysis revealed the optimal response of PLTX in 50% and 90% MeOH at 25 °C, with minimal signal loss occurring over 16 h (9% and 6%). UV-Vis data indicated reduced absorbance in 10% MeOH, but a stable spectral intensity over 21 h in all the tested solvent mixtures. These results underscore the necessity of carefully controlled experimental conditions to ensure accurate and reproducible PLTX detection in environmental and food safety monitoring. Full article

Palytoxins are a group of highly potent and structurally complex marine toxins that rank among some of the most toxic substances known to science. Palytoxins are naturally synthesized by a variety of marine organisms, including Palythoa zoanthids, Ostreopsis dinoflagellates, and Trichodesmium cyanobacteria, and are widely distributed in tropical and temperate regions where they can bioaccumulate in marine life. The evolution of research on palytoxins has been an intricate exchange between interdisciplinary fields, drawing insights from chemistry, biology, medicine, and environmental science in efforts to better understand and mitigate the health risks associated with this family of toxins. In this review, we begin with a brief history covering the discovery of this group of toxins and the events that led to its isolation. We then focus on the chemical structure of these compounds and their proposed mechanism of action. Finally, we review in vitro, ex vivo, and in vivo studies related to their toxicity, with the aim to provide a broad overview of the current knowledge on palytoxin toxinology. Full article

This study aimed to improve the understanding of the nutrient modulation of Ostreopsis cf. ovata toxin content. During the 2018 natural bloom in the NW Mediterranean, the total toxin content (up to ca. 57.6 ± 7.0 pg toxin cell⁻¹) varied markedly. The highest values often coincided with elevated O. cf. ovata cell abundance and with low inorganic nutrient concentrations. The first culture experiment with a strain isolated from that bloom showed that cell toxin content was higher in the stationary than in the exponential phase of the cultures; phosphate- and nitrate-deficient cells exhibited similar cell toxin variability patterns. The second experiment with different conditions of nitrogen concentration and source (nitrate, urea, ammonium, and fertilizer) presented the highest cellular toxin content in the high-nitrogen cultures; among these, urea induced a significantly lower cellular toxin content than the other nutrient sources. Under both high- and low-nitrogen concentrations, cell toxin content was also higher in the stationary than in the exponential phase. The toxin profile of the field and cultured cells included ovatoxin (OVTX) analogues -a to -g and isobaric PLTX (isoPLTX). OVTX-a and -b were dominant while OVTX-f, -g, and isoPLTX contributed less than 1-2%. Overall, the data suggest that although nutrients determine the intensity of the O. cf. ovata bloom, the relationship of major nutrient concentrations, sources and stoichiometry with cellular toxin production is not straightforward. Full article

The dinoflagellate Ostreopsis cf. ovata produces several families of toxic polyketides. Despite only a few field measurements of these phycotoxins in seawater and aerosols, they are believed to be responsible for dermatitis and the toxic inhalations reported during blooms of this species. Therefore, the stability of these compounds in seawater is essential to understanding the causes of these symptoms, however, this has never been assessed. In the current study, the optimization of a solid phase extraction (SPE) procedure was first performed to ensure the most efficient extraction of all phycotoxins known to be produced by this strain, including the recently described liguriatoxins. The SPE cartridge SDBL^® under non acidified conditions offered the best option. The stability of the ovatoxins and the liguriatoxins under biotic and abiotic stress was assessed by exposing the spent medium of a culture of Ostreopsis cf. ovata to its bacterial consortium and natural sunlight. A rapid biotic transformation was detected for both families of compounds. When exposed to bacteria, the half-lives of the ovatoxins were reached before 10 h and at 36 h, 97% of these toxins had been transformed. The half-lives of the liguriatoxins were 10 h under these conditions. Photolysis (abiotic degradation) of the ovatoxins (T_1/2 < 36 h) was faster than for the liguriatoxins (T_1/2 > 62 h). Although none of the catabolites of these phycotoxins were thoroughly identified, an untargeted metabolomics approach combined with molecular networking highlighted the presence of several compounds exhibiting structural similarities with the ovatoxins. Additional work should confirm the preliminary findings on these potential ovatoxins’ catabolites and their biological properties. The rapid transformation of O. cf. ovata’s phycotoxins introduces questions concerning their presence in seawater and their dispersion in the sea spray aerosols. The compounds involved in the toxic inhalations and dermatitis often experienced by beachgoers may stem from the catabolites of these toxins or even unrelated and as yet unidentified compounds. Full article

Intense blooms of the toxic dinoflagellate Ostreopsis producing palytoxin (PlTX) analogs, mainly ovatoxins (OVTXs), have been a recurrent phenomenon along several Mediterranean coasts. Although there is evidence of seafood contamination by these toxins, the dynamics of their bioaccumulation during Ostreopsis blooms is not yet clear. Toxin concentrations in wild mussels at two sites in the Conero Riviera, along the northern Adriatic Sea (Portonovo and Passetto), were analyzed from August to October 2021, throughout an Ostreopsis cf. ovata bloom, to investigate their relationships with the bloom pattern and abundances. Contaminated mussels showed the typical toxin profile dominated by OVTX-a and -b, with levels lower than the threshold considered unsafe for human consumption (30 µg PlTX-equivalent kg⁻¹ soft tissue). The toxin content reached 36.4 µg PlTX kg⁻¹ soft tissue only in a single sampling date. A clear correlation between the bioaccumulation of OVTXs in mussels and the abundance of Ostreopsis was observed. Our results highlighted, however, that bioaccumulation in the mussels is not affected merely by the abundance of toxic microalgae, since higher toxin levels were recorded at Portonovo, where the cell abundances were lower than at Passetto. The results from this study indicate that the Italian guidelines for the management of Ostreopsis blooms in bathing waters are also useful in managing the risks of human intoxication through ingestion, as mussel contamination was detected only during the alert phase (10,000–30,000 cells L⁻¹). Full article

Blooms of the benthic toxic dinoflagellate genus Ostreopsis have been recorded more frequently during the last two decades, particularly in warm temperate areas such as the Mediterranean Sea. The proliferation of Ostreopsis species may cause deleterious effects on ecosystems and can impact human health through skin contact or aerosol inhalation. In the eastern Atlantic Ocean, the toxic O. cf. ovata has not yet been reported to the north of Portugal, and the only species present further north was O. cf. siamensis, for which the toxic risk is considered low. During summer blooms of unidentified Ostreopsis species on the French Basque coast (Atlantic) in 2020 and 2021, people suffered from irritations and respiratory disorders, and the number of analyzed cases reached 674 in 2021. In order to investigate the causes, sampling was carried out during summer 2021 to (i) taxonomically identify Ostreopsis species present using a molecular approach, (ii) isolate strains from the bloom and culture them, and (iii) characterize the presence of known toxins which may be involved. For the first time, this study reports the presence of both O. cf. siamensis and O. cf. ovata, for which the French Basque coast is a new upper distribution limit. Furthermore, the presence of ovatoxins a, b, c, and d in the environmental sample and in a cultivated strain in culture confirmed the toxic nature of the bloom and allowed identifying O. cf. ovata as the producer. The present data identify a new health risk in the area and highlight the extended distribution of some harmful dinoflagellates, presumably in relation to climate change. Full article

In France, four groups of lipophilic toxins are currently regulated: okadaic acid/dinophysistoxins, pectenotoxins, yessotoxins and azaspiracids. However, many other families of toxins exist, which can be emerging toxins. Emerging toxins include both toxins recently detected in a specific area of France but not regulated yet (e.g., cyclic imines, ovatoxins) or toxins only detected outside of France (e.g., brevetoxins). To anticipate the introduction to France of these emerging toxins, a monitoring program called EMERGTOX was set up along the French coasts in 2018. The single-laboratory validation of this approach was performed according to the NF V03-110 guidelines by building an accuracy profile. Our specific, reliable and sensitive approach allowed us to detect brevetoxins (BTX-2 and/or BTX-3) in addition to the lipophilic toxins already regulated in France. Brevetoxins were detected for the first time in French Mediterranean mussels (Diana Lagoon, Corsica) in autumn 2018, and regularly every year since during the same seasons (autumn, winter). The maximum content found was 345 µg (BTX-2 + BTX-3)/kg in mussel digestive glands in November 2020. None were detected in oysters sampled at the same site. In addition, a retroactive analysis of preserved mussels demonstrated the presence of BTX-3 in mussels from the same site sampled in November 2015. The detection of BTX could be related to the presence in situ at the same period of four Karenia species and two raphidophytes, which all could be potential producers of these toxins. Further investigations are necessary to understand the origin of these toxins. Full article

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. Full article

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. Full article

In September 2015, a massive occurrence of the Ostreopsis species was recorded in central Adriatic Kaštela Bay. In order to taxonomically identify the Ostreopsis species responsible for this event and determine their toxin profile, cells collected in seawater and from benthic macroalgae were analyzed. Conservative taxonomic methods (light microscopy and SEM) and molecular methods (PCR-based assay) allowed the identification of the species Ostreopsis cf. ovata associated with Coolia monotis. The abundance of O. cf. ovata reached 2.9 × 10⁴ cells L⁻¹ in seawater, while on macroalgae, it was estimated to be up to 2.67 × 10⁶ cells g⁻¹ of macroalgae fresh weight and 14.4 × 10⁶ cells g⁻¹ of macroalgae dry weight. An indirect sandwich immunoenzymatic assay (ELISA) and liquid chromatography–high-resolution mass spectrometry (LC-HRMS) were used to determine the toxin profile. The ELISA assay revealed the presence of 5.6 pg palytoxin (PLTX) equivalents per O. cf. ovata cell. LC-HRMS was used for further characterization of the toxin profile, which showed that there were 6.3 pg of the sum of ovatoxins (OVTXs) and isobaric PLTX per O. cf. ovata cell, with a prevalence of OVTXs (6.2 pg cell⁻¹), while the isobaric PLTX concentration was very low (0.1 pg cell⁻¹). Among OVTXs, the highest concentration was recorded for OVTX-a (3.6 pg cell⁻¹), followed by OVTX-b (1.3 pg cell⁻¹), OVTX-d (1.1 pg cell⁻¹), and OVTX-c (0.2 pg cell⁻¹). Full article

Palytoxin (PlTX) and analogues are produced by certain dinoflagellates, sea anemones, corals and cyanobacteria. PlTX can accumulate in the food chain and when consumed it may cause intoxication with symptoms like myalgia, weakness, fever, nausea, and vomiting. The analysis of PlTXs is challenging, and because of the large molecular structure, it is difficult to develop a sensitive and selective liquid chromatography-mass spectrometry (LC-MS/MS) method. In this work, an LC-MS/MS method was developed to analyse PlTXs with use of lithium iodine and formic acid as additives in the mobile phase. For method development, initially, LC-hrMS was used to accurately determine the elemental composition of the precursor and product ions. The main adduct formed was [M + H + 2Li]³⁺. Fragments were identified with LC-hrMS and these were incorporated in the LC-MS/MS method. A method of 10 min was developed and a solid phase extraction clean-up procedure was optimised for shellfish matrix. The determined limits of detection were respectively 8 and 22 µg PlTX kg⁻¹ for mussel and oyster matrix. Oysters gave a low recovery of approximately 50% for PlTX during extraction. The method was successfully in-house validated, repeatability had a relative standard deviation less than 20% (n = 5) at 30 µg PlTX kg⁻¹ in mussel, cockle, and ensis, and at 60 µg PlTX kg⁻¹ in oyster. Full article

Harmful benthic dinoflagellates, usually developing in tropical areas, are expanding to temperate ecosystems facing water warming. Reports on harmful benthic species are particularly scarce in the Southern Mediterranean Sea. For the first time, three thermophilic benthic dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima and Coolia monotis) were isolated from Bizerte Bay (Tunisia, Mediterranean) and monoclonal cultures established. The ribotyping confirmed the morphological identification of the three species. Maximum growth rates were 0.59 ± 0.08 d⁻¹ for O. cf. ovata, 0.35 ± 0.01 d⁻¹ for C. monotis and 0.33 ± 0.04 d⁻¹ for P. lima. Toxin analyses revealed the presence of ovatoxin-a and ovatoxin-b in O. cf. ovata cells. Okadaic acid and dinophysistoxin-1 were detected in P. lima cultures. For C. monotis, a chromatographic peak at 5.6 min with a mass m/z = 1061.768 was observed, but did not correspond to a mono-sulfated analogue of the yessotoxin. A comparison of the toxicity and growth characteristics of these dinoflagellates, distributed worldwide, is proposed. Full article

During the summer of 2010, 31 species including fish, echinoderms, gastropods, crustaceans, cephalopods and sponges were sampled in the Bay of Villefranche on the French Mediterranean coast and screened for the presence of PLTX-group toxins using the haemolytic assay. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for confirmatory purposes and to determine the toxin profile. The mean toxin concentration in the whole flesh of all sampled marine organisms, determined using the lower- (LB) and upper-bound (UB) approach was 4.3 and 5.1 µg·kg⁻¹, respectively, with less than 1% of the results exceeding the European Food Safety Authority (EFSA) threshold of 30 µg·kg⁻¹and the highest values being reported for sea urchins (107.6 and 108.0 µg·kg⁻¹). Toxins accumulated almost exclusively in the digestive tube of the tested species, with the exception of octopus, in which there were detectable toxin amounts in the remaining tissues (RT). The mean toxin concentration in the RT of the sampled organisms (fishes, echinoderms and cephalopods) was 0.7 and 1.7 µg·kg⁻¹ (LB and UB, respectively), with a maximum value of 19.9 µg·kg⁻¹ for octopus RT. The herbivorous and omnivorous organisms were the most contaminated species, indicating that diet influences the contamination process, and the LC-MS/MS revealed that ovatoxin-a was the only toxin detected. Full article

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. Full article

Here we summarize the current knowledge on the transfer and accumulation of harmful algal bloom (HAB)-related toxins in cephalopods (octopods, cuttlefishes and squids). These mollusks have been reported to accumulate several HAB-toxins, namely domoic acid (DA, and its isomers), saxitoxin (and its derivatives) and palytoxin (and palytoxin-like compounds) and, therefore, act as HAB-toxin vectors in marine food webs. Coastal octopods and cuttlefishes store considerably high levels of DA (amnesic shellfish toxin) in several tissues, but mainly in the digestive gland (DG)—the primary site of digestive absorption and intracellular digestion. Studies on the sub-cellular partitioning of DA in the soluble and insoluble fractions showed that nearly all DA (92.6%) is found in the cytosol. This favors the trophic transfer of the toxins since cytosolic substances can be absorbed by predators with greater efficiency. The available information on the accumulation and tissue distribution of DA in squids (e.g., in stranded Humboldt squids, Dosidicus gigas) is scarcer than in other cephalopod groups. Regarding paralytic shellfish toxins (PSTs), these organisms accumulate them at the greatest extent in DG >> kidneys > stomach > branchial hearts > posterior salivary glands > gills. Palytoxins are among the most toxic molecules identified and stranded octopods revealed high contamination levels, with ovatoxin (a palytoxin analogue) reaching 971 μg kg⁻¹ and palytoxin reaching 115 μg kg⁻¹ (the regulatory limit for PlTXs is 30 μg kg⁻¹ in shellfish). Although the impacts of HAB-toxins in cephalopod physiology are not as well understood as in fish species, similar effects are expected since they possess a complex nervous system and highly developed brain comparable to that of the vertebrates. Compared to bivalves, cephalopods represent a lower risk of shellfish poisoning in humans, since they are usually consumed eviscerated, with exception of traditional dishes from the Mediterranean area. Full article