Massive Occurrence of the Harmful Benthic Dinoflagellate Ostreopsis cf. ovata in the Eastern Adriatic Sea.

In September 2015, a massive occurrence of the Ostreopsis species was recorded in central Adriatic Kastela 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).

A Novel Sensitive Cell-Based Immunoenzymatic Assay for Palytoxin Quantitation in Mussels.

The marine algal toxin palytoxin (PLTX) and its analogues are some of the most toxic marine compounds. Their accumulation in edible marine organisms and entrance into the food chain represent their main concerns for human health. Indeed, several fatal human poisonings attributed to these compounds have been recorded in tropical and subtropical areas. Due to the increasing occurrence of PLTX in temperate areas such as the Mediterranean Sea, the European Food Safety Authority (EFSA) has suggested a maximum limit of 30 µg PLTX/kg in shellfish meat, and has recommended the development of rapid, specific, and sensitive methods for detection and quantitation of PLTX in seafood. Thus, a novel, sensitive cell-based ELISA was developed and characterized for PLTX quantitation in mussels. The estimated limits of detection (LOD) and quantitation (LOQ) were 1.2 × 10-11 M (32.2 pg/mL) and 2.8 × 10-11 M (75.0 pg/mL), respectively, with good accuracy (bias = 2.5%) and repeatability (15% and 9% interday and intraday relative standard deviation of repeatability (RSDr), respectively). Minimal interference of 80% aqueous methanol extract allows PLTX quantitation in mussels at concentrations lower than the maximum limit suggested by EFSA, with an LOQ of 9.1 µg PLTX equivalent/kg mussel meat. Given its high sensitivity and specificity, the cell-based ELISA should be considered a suitable method for PLTX quantitation.

A revisited hemolytic assay for palytoxin detection: Limitations for its quantitation in mussels.

Palytoxin (PLTX) and its analogues have been detected as seafood contaminants associated with a series of human foodborne poisonings. Due to a number of fatalities ascribed to the ingestion of PLTX-contaminated marine organisms, the development of methods for its detection in seafood has been recommended by the European Food Safety Authority (EFSA). Due to its feasibility, the spectrophotometric hemolytic assay is widely used to detect PLTX in different matrices, even though a standardized protocol is still lacking. Thus, on the basis of available assay procedures, a new standardized protocol was set up using purified human erythrocytes exposed to PLTX (working range: 3.9 × 10(-10)-2.5 × 10(-8) M) in a K(+)-free phosphate buffered saline solution, employing a 5 h incubation at 41 °C. An intra-laboratory characterization demonstrated its sensitivity (limit of detection, LOD = 1.4 × 10(-10) M and quantitation, LOQ = 3.4 × 10(-10) M), accuracy (bias = -0.8%), repeatability (RSDr = 15% and 6% for intra- and inter-day repeatability, respectively) and specificity. However, the standardized method seems not to be suitable for PLTX quantitation in complex matrices, such as mussel (Mytilus galloprovincialis) extracts, at least below the limit suggested by EFSA (30 µg PLTXs/Kg shellfish meat). Thus, the hemolytic assay for PLTX quantitation in seafood should be used only after a careful evaluation of the specific matrix effects.

Palytoxin-Containing Aquarium Soft Corals as an Emerging Sanitary Problem.

Palytoxin (PLTX), one the most potent marine toxins, and/or its analogs, have been identified in different marine organisms, such as Palythoa soft corals, Ostreopsis dinoflagellates, and Trichodesmium cyanobacteria. Although the main concern for human health is PLTXs entrance in the human food chain, there is growing evidence of adverse effects associated with inhalational, cutaneous, and/or ocular exposure to aquarium soft corals contaminated by PLTXs or aquaria waters. Indeed, the number of case reports describing human poisonings after handling these cnidarians is continuously increasing. In general, the signs and symptoms involve mainly the respiratory (rhinorrhea and coughing), skeletomuscular (myalgia, weakness, spasms), cardiovascular (electrocardiogram alterations), gastrointestinal (nausea), and nervous (paresthesia, ataxia, tremors) systems or apparates. The widespread phenomenon, the entity of the signs and symptoms of poisoning and the lack of control in the trade of corals as aquaria decorative elements led to consider these poisonings an emerging sanitary problem. This review summarizes literature data on human poisonings due to, or ascribed to, PLTX-containing soft corals, focusing on the different PLTX congeners identified in these organisms and their toxic potential.

Ovatoxin-a, A Palytoxin Analogue Isolated from Ostreopsis cf. ovata Fukuyo: Cytotoxic Activity and ELISA Detection.

This study provides the first evaluation of the cytotoxic effects of the recently identified palytoxin (PLTX) analog, ovatoxin-a (OVTX-a), the major toxin produced by Ostreopsis cf. ovata in the Mediterranean Sea. Its increasing detection during Ostreopsis blooms and in seafood highlights the need to characterize its toxic effects and to set up appropriate detection methods. OVTX-a is about 100 fold less potent than PLTX in reducing HaCaT cells viability (EC50 = 1.1 × 10(-9) M vs 1.8 × 10(-11) M, MTT test) in agreement with a reduced binding affinity (Kd = 1.2 × 10(-9) vs 2.7 × 10(-11) M, saturation experiments on intact cells). Similarly, OVTX-a hemolytic effect is lower than that of the reference PLTX compound. Ost-D shows the lowest cytotoxicity toward HaCaT keratinocytes, suggesting the lack of a hydroxyl group at C44 as a critical feature for PLTXs cytotoxic effects. A sandwich ELISA developed for PLTX detects also OVTX-a in a sensitive (LOD = 4.2 and LOQ = 5.6 ng/mL) and accurate manner (Bias = 0.3%), also in O. cf. ovata extracts and contaminated mussels. Although in vitro OVTX-a appears less toxic than PLTX, its cytotoxicity at nanomolar concentrations after short exposure time rises some concern for human health. The sandwich ELISA can be a viable screening method for OVTXs detection in monitoring program.