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• Analyses of marine biotoxins in mussels by LC-MS/HRMS and LC-MS/MS. • Identification of unknown toxin analogs in shellfish. • Implementation of molecular networking for mapping and identifying toxin analogs. • Production of semi-synthesized fatty acid esters of portimine-A. • Discovery of 13- O -acyl esters of portimine-A (n = 13) and a new pinnatoxin analog. Vulcanodinium rugosum is a benthic dinoflagellate known for producing pinnatoxins, pteriatoxins, portimines and kabirimine. In this study, we aimed to identify unknown analogs of these emerging toxins in mussels collected in the Ingril lagoon, France. First, untargeted data acquisitions were conducted by means of liquid chromatography coupled to hybrid quadrupole-orbitrap mass spectrometry. Data processing involved a molecular networking approach, and a workflow dedicated to the identification of biotransformed metabolites. Additionally, targeted analyses by liquid chromatography coupled to triple quadrupole mass spectrometry were also implemented to further investigate and confirm the identification of new compounds. For the first time, a series of 13- O -acyl esters of portimine-A (n = 13) were identified, with fatty acid chains ranging between C12:0 and C22:6. The profile was dominated by the palmitic acid conjugation. This discovery was supported by fractionation experiments combined with the implementation of a hydrolysis reaction, providing further evidence of the metabolite identities. Furthermore, several analogs were semi-synthesized, definitively confirming the discovery of these metabolization products. A new analog of pinnatoxin, with a molecular formula of C 42 H 65 NO 9 , was also identified across the year 2018, with the highest concentration observed in August (4.5 μg/kg). The MS/MS data collected for this compound exhibited strong structural similarities with PnTX-A and PnTX-G, likely indicating a substituent C 2 H 5 O 2 in the side chain at C33. The discovery of these new analogs will contribute to deeper knowledge of the chemodiversity of toxins produced by V. rugosum or resulting from shellfish metabolism, thereby improving our ability to characterize the risks associated with these emerging toxins. [ABSTRACT FROM AUTHOR]

• V. rugosum presence was associated with seawater temperature. • PnTX G concentration in mussels was associated with seawater temperature and V. rugosum presence. • PnTX G persisted for several months in mussels and in seawater. • Predicted human health risk associated with PnTX G in mussels was maximum in the warmest periods of the year. Consumption of seafood contaminated by phycotoxins produced by harmful algae is a major issue in human public health. Harmful algal blooms are driven by a multitude of environmental variables; therefore predicting human dietary exposure to phycotoxins based on these variables is a promising approach in health risk management. In this study, we attempted to predict the human health risks associated with Vulcanodinium rugosum and its neurotoxins, pinnatoxins (PnTXs), which have been regularly found in Mediterranean lagoons since their identification in 2011. Based on environmental variables collected over 1 year in four Mediterranean lagoons, we developed linear mixed models to predict the presence of V. rugosum and PnTX G contamination of mussels. We found that the occurrence of V. rugosum was significantly associated with seawater temperature. PnTX G contamination of mussels was highest in summer but persisted throughout the year. This contamination was significantly associated with seawater temperature and the presence of V. rugosum with a time lag, but not with dissolved PnTX G in seawater. By using the contamination model predictions and their potential variability/uncertainty, we calculated the human acute dietary exposures throughout the year and predicted that 25% of people who consume mussels could exceed the provisional acute benchmark value during the warmest periods. We suggest specific recommendations to monitor V. rugosum and PnTX G. [ABSTRACT FROM AUTHOR]

Vulcanodinium rugosum , a dinoflagellate developing in Ingril Lagoon (Mediterranean, France) is responsible for shellfish intoxications due to the neurotoxin pinnatoxin G. A one year survey (March 2012–April 2013) was conducted in this oligotrophic shallow lagoon and key environmental parameters were recorded (temperature, salinity and nutrients). The spatio-temporal distribution of V. rugosum in water column and on macrophytes was also determined. Planktonic cells of V. rugosum were observed at all sampling stations, but in relatively low concentrations (maximum of 1000 cell/L). The highest abundances were observed from June to September 2012. There was a positive correlation between cell densities and both temperature and salinity. Non-motile cells were detected on macrophytes, with a maximum concentration of 6300 cells/g wet weight. Nitrite and ammonium were negatively related to V. rugosum abundance whereas total nitrogen, total phosphorus and phosphates showed a positive correlation. Altogether , in situ results suggest that V. rugosum is rather thermophilic and that organic nutrients should be considered when studying the nutrition requirements for this noxious expanding dinoflagellate. [ABSTRACT FROM AUTHOR]

Harmful Algal Bloom species are ubiquitous and their blooms occur in the Arabian Gulf. In this study, two cruises were performed in 2012 and 2013 to collect phytoplankton samples from 4 sites in the Arabian Gulf. Toxin analyses of phytoplankton samples for 32 algal toxins from 5 different toxin groups were conducted on the samples using both enzyme linked immunosorbent assay (ELISA) and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Results demonstrated, for the first time, the presence of paralytic shellfish toxins (PSTs), diarrhetic shellfish toxin (DST), amnesic shellfish toxin (AST), cyclic imines (CIs) and polyether-lactone toxins in freeze-dried phytoplankton samples. Four Vulcanodinium rugosum cultures were established from field samples and these proved to contain between 603 and 981 ng pinnatoxin (PnTx) H per mg dry weight in addition to being positive for portimine. These strains from Qatar clustered with strains from Japan and Florida based on large subunit rRNA and rRNA internal transcribed spacer gene sequences. [ABSTRACT FROM AUTHOR]

The transport of microalgae in the ballast tanks of shipping vessels is of global concern because many algal species can survive in ballast tanks as nonmotile or cyst stages increasing the likelihood of introductions into foreign ports. In 2004 a peridinoid dinoflagellate was collected and isolated from ballast residuals of the merchant vessel Southern Fighter in Port Tampa Bay, Florida, USA. Light and electron microscopy of the motile and nonmotile peridinoid cells indicated the dinoflagellate was Vulcanodinium rugosum . Bayesian and maximum-parsimony (MP) phylogenetic analyses of V. rugosum strain CCFWC516 showed that sequenced regions of its LSU and ITS matched those of V. rugosum strains from Japan but were divergent from those strains from New Zealand and France. LC–MS analyses indicated that strain CCFWC516 did not produce the neurotoxin pinnatoxin, an ability that has been reported for other strains of this species. Analyses did show, however, that strain CCFWC516 did produce portimine, a cyclic imine produced by all other strains of this species. The M/V Southern Fighter visited numerous ports along the coast of Japan and the North Sea before sailing to Florida. The phylogenetic match to Japanese strains, as well as the ship's ballast exchange history, suggests that strain CCFWC516 originated from Japan. In light of the increase in global shipping traffic, increases in vessel size and capacity, and the increased connectivity between the Pacific Ocean and the Caribbean and Gulf of Mexico that will result from the widening of the Panama Canal, the introduction of nonnative, harmful algal species is an area of heightened concern and calls for increased vigilance. [ABSTRACT FROM AUTHOR]