Paulo Vale

Paralytic shellfish toxins can be quantified in bivalves by the AOAC-2005 method. This method is sensitive for several toxins, but other toxins suffer from strong matrix effects or fluorescence partitioning by multiple oxidation products, reducing their sensitivity. Metals were assessed as candidate catalysts to improve the oxidation reactions, instead of the oyster matrix modifier prescribed in this method. The N1-H sub-group of toxins exhibited increased fluorescence upon doping with iron sulphate. The increase was inversely proportional to the fluorescence reduction caused by matrix suppression. Iron acted as a catalyst, lowering the activation energy of the reaction, which otherwise required heating to achieve a similar boost in the fluorescence yield. The fluorescence due to the N1-hydroxyl sub-group of toxins GTX1 + 4 and GTX6 increased with zinc but decreased with iron. When doping with a metal solution fraction-2 of the carboxylic acid partitioning, both metals reduced the fluorescence, while the eluent (NaCL 0.05 M) enhanced it. As metals are retained by COOH cartridges, sulphonic acid leachables might contribute to this reduction. The reduction observed for GTX1 + 4 was primarily due to the decrease in the proportion of the secondary peak over the primary oxidation peak, which can be reversed by doping with an oyster matrix. Nickel chloride was able to replace the oyster modifier effectively in all five bivalve matrices tested, while zinc chloride was not similarly effective. For dcNEO spiked in several bivalve matrices, adding an oyster modifier derived from Magallana gigas caused a 16-19% reduction in fluorescence. Doping with several metals could not achieve the same fluorescence as doping with 0.1 mM acetic acid. For NEO, with one particular oyster extract, the yield could be higher than using NiCl₂. Depending on the specific oyster extract, the use of the matrix modifier required in the method might not be adequate.

The neurotoxins of the saxitoxin family can be the origin of the human neurological syndrome of paralytic shellfish poisoning via contaminated marine bivalve vectors. A pre-chromatographic oxidation method is the official testing method in the EU, also known as the 'Lawrence method'. It involves several steps, including toxin extraction, solid-phase clean-up, and oxidation to produce fluorescent derivatives. Despite the manual peroxide oxidation involving four pipetting steps, high coefficients of determination were commonly obtained for the calibration curves of the respective toxin oxidation products. However, the 11-hydroxysulphate toxins dcGTX2 + 3, C1 + 2 and GTX2 + 3, often had slightly lower coefficients of determination (i.e. R² < 0.998) than their non-11-hydroxysulphate counterparts dcSTX, GTX5 and STX (i.e. R² > 0.998). Deviations of ±10 s from the respective standard reaction times, caused significant alterations in fluorescent yield for toxins quantified with peroxide but not for those quantified with periodate. The oxidation is an endothermal reaction, and its reaction rate is toxin-specific, with the N11-hydroxysulphate toxins having a slower reaction rate. This was confirmed by incubating GTX2 + 3 and STX at different reaction times. The incubation of toxins in a bivalve matrix also slows down the reaction in comparison with oxidation in dilute acetic acid, giving lower recoveries, as studied here in detail for dcGTX2 + 3. Circumventing the matrix effect by dilution is not possible, as 10% of matrix is enough to cause a reduction to half of the fluorescence yield of dcGTX2 + 3. When heating the N1-H toxins with peroxide, the increase in fluorescence yield is inversely proportional to the recovery values commonly found for each toxin.

Saxitoxins are potent neurotoxins originating the acute human neurological syndrome of paralytic shellfish poisoning (PSP) via bivalve vectors. The official testing method in the European Union, commonly known as the ‘Lawrence method’, involves pre-column oxidation steps. The Portuguese monitoring adopted the hydrogen peroxide oxidation screening approach for bivalves contaminated with Gymnodinium catenatum toxins, which can quantify chromatographically at once 6 out of 10 analogues commonly found in bivalves. Seasonal fluctuation in the fluorescence yield of calibration curves was observed across years in a consistent manner. It correlated with fluctuations in average monthly air temperature in Lisbon, highlighting the importance of recording the room temperature during the oxidation steps as a matter of routine practice. Incubation experiments also showed an increase in fluorescence yield with temperature, more pronounced for the 11-hydroxysulphate analogues (dcGTX2 + 3, C1 + 2, GTX2 + 3) than for the 11-H toxins (dcSTX, GTX5[B1], STX). Temperature can be exploited to increase fluorescence yield, assisting in spectral confirmation, but must not exceed 40–50 °C to avoid toxin decomposition or production of extra oxidation products.

In Portugal, high levels of diarrhetic shellfish toxins (DSTs) originating from Dinophysis spp. are accumulated in bivalves, mainly on the northwest coast (NWC). The occurrence of DSTs in spring and early summer is positively related to precipitation. A decrease in average precipitation after 2003 led to a reduction in DSTs after 2008. However, the accumulation of DSTs in the NWC increased in the period 2020–2022. The hydrological year of 2022 was marked by extreme drought. In cockles from the endemic area of Ria de Aveiro, 37.3% of the weeks tested presented samples above the regulatory limit (RL). The previous record was 31.9% in 2005, also an extremely dry year. The average percentage of weeks above the RL in severe drought years surpassed both the low and high precipitation years. In severe dry years, toxicity was dominated mainly by D. acuta toxins. While D. acuminata grows after the abundant river discharges in spring, D. acuta is capable of growing in summer during upwelling favourable conditions. In the last two decades, extensive forest wildfires were recurrent, but the area burnt reduced sharply after 2017 following tighter fire control measures. Low levels of DSTs or low percentage of weeks with cockles above the RL were related to high burnt areas in the previous year. The recent increase in 2020–2022 of DSTs, a planktonic biomarker in bivalves, was also coincident with the similar temporal increase in either the recruitment or the biomass of Sardina pilchardus, a planktivorous fish in decline after 2005/2006. Both low sardine recruitment and biomass were coincident with low precipitation or high burnt areas. Wildfires degrade coastal water quality, as seems reflected both in the cockle’s DST and the sardine stock time series.

Bivalve molluscs can acquire marine biotoxins by filter-feeding upon certain toxin-producing microalgae. The two most common syndromes observed in temperate coastal waters have been diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP). While DSP is a non-fatal gastrointestinal syndrome, PSP is a neurological syndrome which can lead to death by respiratory paralysis in high intoxication scenarios. In Portugal, the presence of both DSP and PSP toxins leads to recurrent seasonal bans of bivalve harvesting. On a few occasions, the bans were not placed in time, not properly disseminated to the public or were disregarded by recreational harvesters. Several cases of poisonings have been studied in collaboration between health authorities and the laboratory in charge of the biotoxin monitoring programme. Some of the outbreaks have even called the attention of the local media. In several of these recorded cases, a common trait has emerged throughout the years: bivalve harvest had often been done during very low tides attributed to either new or full moons. These tides expose intertidal bivalves more widely, increase harvesting time, and allow picking of larger-sized specimens. In some occasions, the consumers were extremely unfortunate because a noxious coincidence had occurred: larger-sized specimens were available but had attained the highest toxin content of the toxic season. This review alerts that despite costly monitoring programmes have been perfected through the years, human poisonings still take place due to the rapid increase in toxin levels and/or disrespect of harvest bans.

A intoxicação paralisante por marisco é uma síndroma raraem Portugal, coincidente com o consumo de bivalves que sealimentaram de microalgas tóxicas da espécie Gymnodiniumcatenatum. É caracterizada por sintomas marcadamente neurológicos, como parestesias, fala incoerente, ataxia, dispneia,apneia, e que podem culminar na morte por paralisia respiratória. Para evitar a ocorrência desta síndrome, a monitorizaçãoperiódica das biotoxinas intoxicação paralisante por mariscoé obrigatória antes de os bivalves serem colhidos para comercialização. No entanto, o desconhecimento ou desrespeito dasproibições de colheita têm originado intoxicações esporádicas que requereram internamento hospitalar. Desde há váriasdécadas que a ocorrência de contaminação elevada destasbiotoxinas em bivalves da Costa Atlântica Ibérica tem ocorridoem intervalos aproximadamente decadais. Estes anos de toxicidade muito elevada têm sido coincidentes com anos em quea actividade solar (derivada do ciclo de manchas solares de 11anos) está no seu mínimo. A sazonalidade desta contaminaçãoé mais grave no outono, e os internamentos hospitalares conhecidos também têm ocorrido no outono.