Investigating the etiology of Haff disease: Optimization and validation of a sensitive LC-MS/MS method for palytoxins analysis in directly associated freshwater and marine food samples from Brazil.

Haff disease typically develops after eating contaminated marine or freshwater species, especially fish. Despite still having an unknown etiology, recent reports have suggested its possible correlation with palytoxins. Therefore, the present work aimed to optimize and perform a validation of a sensitive method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the analysis of palytoxin and some of its analogs, with the main purpose of investigating their presence in marine and freshwater food samples associated with Haff disease in Brazil. The method optimization was performed using a central composite rotatable design and fish samples fortified with the palytoxin standard. Then, the optimized method was validated for different food matrices, including freshwater and marine fish, mollusks, and crustaceans. The sample preparation involved a solid-liquid extraction using methanol and water, solid-phase extraction using Strata-X cartridges, and on-column palytoxin oxidation. The detection of the main oxidized fragments (amino and amide aldehydes) was achieved by LC-MS/MS with electrospray ionization in positive mode, using a C18 column, as well as acetonitrile and water as mobile phases, both acidified with 0.1 % of formic acid. After optimization and validation, the etiological investigation involved the analysis of 16 Brazilian Haff disease-related food samples (in natura and leftover meals) from 2022. The method was demonstrated to be appropriate for quantitative analysis of freshwater and marine species. So far, it has proven to be one of the most sensitive methods related to palytoxin detection (LOD 10 µg/kg), being able to work in a range that includes the provisional ingestion limit (30 µg/kg). Regarding the Haff disease-related samples analysis, there is a strong indication of palytoxin contamination since the amino aldehyde (common fragment for all palytoxins) was detected in 15 of the 16 samples. Selected results were confirmed using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS).

Occurrence of potentially toxic microalgae and diarrhetic shellfish toxins in the digestive tracts of green sea turtles (Chelonia mydas) from southern Brazil.

Algal toxins are involved in the mortality and/or illness of marine organisms via consumption of contaminated prey, or upon direct exposure to toxic cells. In this study, the presence of potentially toxic microalgal cells was investigated within the digestive tract contents of a threatened species of green turtle (Chelonia mydas). Additionally, lipophilic toxins were determined by LC-MS/MS in tissue samples (liver, stomach and/or intestine) of selected animals (n = 39 individuals) found dead-stranded in southern Brazil, from winter/2015 to autumn/2016. Thirteen potentially toxic species of microalgae (both benthic and planktonic), including seven dinoflagellates, six cyanobacteria and one diatom, were found in the digestive tract contents of green turtles. Among them, dinoflagellates belonging to the Dinophysis acuminata species complex were the most frequent (36%) and abundant (maximum average abundance of 566 cells g-1 in spring/2015). Moreover, 23% of the examined sea turtles exhibited detectable levels of the diarrhetic shellfish toxin okadaic acid (OA) in washed digestive tissues. Seven individuals accumulated OA in their intestines (max. 24.1 ng g-1) and two in the stomachs (max. 7.4 ng g-1). Toxin levels in the tissues were directly and significantly (r = 0.70, p < 0.025) associated with the cell abundance of OA-producing D. acuminata and Prorocentrum lima species complexes within the digestive contents of green turtles. Although OA concentrations were relatively low, possible chronic exposure might deteriorate general health conditions of exposed sea turtles, increasing the risk for diseases. Okadaic acid has been regarded as a tumor-promoting compound and an environmental co-factor in the incidence of fibropapillomatosis, a frequent disease in juvenile green turtles inhabiting this geographic region. Even though, only one green turtle containing OA in the digestive tissues (out of six examined) also presented fibropapillomatosis in this study. Notwithstanding, sea turtles are sentinels of ocean health. Monitoring the accumulation of algal toxins and their negative effects on these organisms contributes to conserving biodiversity and marine habitats.

Determination of the toxic variability of lipophilic biotoxins in marine bivalve and gastropod tissues treated with an industrial canning process.

Contamination of shellfish with lipophilic marine biotoxins (LMB), pectenotoxins (PTXs), yessotoxins (YTXs) and okadaic acid (OA) toxin groups in southern Chile is a constant challenge for the development of miticulture considering the high incidence of toxic episodes that tend to occur. This research is focused on using methodologies for assessing the decrease in toxins of natural resources in Chile with high value, without altering the organoleptic properties of the shellfish. The species were processed through steaming (1 min at 121°C) and subsequent canning (5 min at 121°C). Changes in the profiles of toxins and total toxicity levels of LMB in endemic bivalves and gastropods were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total reduction of toxicity (≈ 15%) was not related to the destruction of the toxin, but rather to the loss of LMB on removing the shells and packing media of canned products (***p < 0.001). Industrial processing of shellfish reduces LMB contents by up to 15% of the total initial contents, concomitant only with the interconversion of PTX-group toxins into PTX-2sa. In soft bottom-dwelling species with toxicities beyond the standard for safe human consumption (≥ 160 µg OA-eq kg-1), toxicity can be reduced to safe levels through industrial preparation procedures.