Advances in the Early Warning of Shellfish Toxification by Dinophysis acuminata.

In Western Europe, the incidence of DST is likely the highest globally, posing a significant threat with prolonged bans on shellfish harvesting, mainly caused by species of the dinoflagellate genus Dinophysis. Using a time series from 2014 to 2020, our study aimed (i) to determine the concentration of D. acuminata in water at which shellfish toxin levels could surpass the regulatory limit (160 µg OA equiv kg-1) and (ii) to assess the predictability of toxic events for timely mitigation actions, especially concerning potential harvesting bans. The analysis considered factors such as (i) overdispersion in the data, (ii) distinct periods of presence and absence, (iii) the persistence of cells, and (iv) the temporal lag between cells in the water and toxins in shellfish. Four generalized additive models were tested, with the Tweedie (TW-GAM) model showing superior performance (>85%) and lower complexity. The results suggest existing thresholds currently employed (200 and 500 cells L-1) are well-suited for the Portuguese coast, supported by empirical evidence (54-79% accuracy). The developed algorithm allows for thresholds to be tailored on a case-by-case basis, offering flexibility for regional variations.

A 15-Year Retrospective Review of Ciguatera in the Madeira Islands (North-East Atlantic, Portugal).

The first ciguatera fish poisoning (CFP) in Portugal dates from 2008 when 11 people reported CFP symptoms after consuming a 30 kg amberjack caught around the Selvagens Islands (Madeira Archipelago). Since then, 49 human poisonings have been reported. The emergence of a new threat challenged scientists and regulators, as methods for toxic microalgae analyses and ciguatoxin (CTX) detection were not implemented. To minimise the risk of ciguatera, the Madeira Archipelago authorities interdicted fisheries in Selvagens Islands and banned the capture of amberjacks weighing more than 10 kg in the entire region of Madeira Archipelago. The accurate identification and quantification of the benthic toxin-producing algae species spreading to new areas require efforts in terms of both microscopy and molecular techniques. Two ciguatera-causing dinoflagellates, Gambierdiscus excentricus and Gambierdiscus australes, were identified in the Madeira Island and Selvagens sub-archipelago, respectively. Regarding the CTX analysis (N2a cell-based assay and LC-MS) in fish, the results indicate that the Selvagens Islands are a ciguatera risk area and that fish vectoring CTX are not limited to top predator species. Nevertheless, advances and improvements in screening methods for the fast detection of toxicity in seafood along with certified reference material and sensitive and selective targeted analytical methods for the determination of CTX content are still pending. This study aims to revise the occurrence of ciguatera cases in the Madeira Archipelago since its first detection in 2008, to discuss the risk management strategy that was implemented, and to provide a summary of the available data on the bioaccumulation of CTX in marine fish throughout the marine food web, taking into consideration their ecological significance, ecosystem dynamics, and fisheries relevance.

Bivalve Shellfish Safety in Portugal: Variability of Faecal Levels, Metal Contaminants and Marine Biotoxins during the Last Decade (2011-2020).

Bivalves are a high-value product whose production has markedly increased, reaching 9863 tonnes in Portugal in 2021. Bivalves' habitats-lagoons, estuaries and coastal waters-are exposed to biological and anthropogenic contaminants, which can bioaccumulate in these organisms and pose a significant public health risk. The need to obtain a safe product for human consumption led to the implementation of standardised hygiene regulations for harvesting and marketing bivalve molluscs, resulting in routine monitoring of bivalve production areas for microbial quality, metal contaminants, and marine biotoxins. While excessive levels of biotoxins and metal contamination lead to temporary harvesting bans, high faecal contamination leads to area reclassification and impose post-harvest treatments. In this study, the seasonal and temporal variability of these parameters were analysed using historical data generated by the monitoring programme during the last decade. Moreover, the impact of the monitoring program on bivalve harvesting from 2011 to 2020 was assessed. This program presented a considerable improvement over time, with an increase in the sampling effort and the overall program representativeness. Finally, contamination risk, revising control measures, and defining recommendations for risk mitigation measures are given in the light of ten years' monitoring.

An Update on Ciguatoxins and CTX-like Toxicity in Fish from Different Trophic Levels of the Selvagens Islands (NE Atlantic, Madeira, Portugal).

The Selvagens Islands, which are a marine protected area located at the southernmost point of the Portuguese maritime zone, have been associated with fish harboring ciguatoxins (CTX) and linked to ciguatera fish poisonings. This study reports the results of a field sampling campaign carried out in September 2018 in these remote and rarely surveyed islands. Fifty-six fish specimens from different trophic levels were caught for CTX-like toxicity determination by cell-based assay (CBA) and toxin content analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Notably, high toxicity levels were found in fish with an intermediate position in the food web, such as zebra seabream (Diplodus cervinus) and barred hogfish (Bodianus scrofa), reaching levels up to 0.75 µg CTX1B equivalent kg-1. The LC-MS/MS analysis confirmed that C-CTX1 was the main toxin, but discrepancies between CBA and LC-MS/MS in D. cervinus and top predator species, such as the yellowmouth barracuda (Sphyraena viridis) and amberjacks (Seriola spp.), suggest the presence of fish metabolic products, which need to be further elucidated. This study confirms that fish from coastal food webs of the Selvagens Islands represent a high risk of ciguatera, raising important issues for fisheries and environmental management of the Selvagens Islands.

High Levels of Tetrodotoxin (TTX) in Trumpet Shell Charonia lampas from the Portuguese Coast.

Tetrodotoxin (TTX) is a potent neurotoxin, considered an emerging toxin in Europe where recently a safety limit of 44 µg TTX kg-1 was recommended by authorities. In this study, three specimens of the large gastropod trumpet shell Charonia lampas bought in a market in south Portugal were analyzed using a neuroblastoma cell (N2a) based assay and by LC-MS/MS. N2a toxicity was observed in the viscera of two individuals analyzed and LC-MS/MS showed very high concentrations of TTX (42.1 mg kg-1) and 4,9-anhydroTTX (56.3 mg kg-1). A third compound with m/z 318 and structurally related with TTX was observed. In the edible portion, i.e., the muscle, toxin levels were below the EFSA recommended limit. This study shows that trumpet shell marine snails are seafood species that may reach the markets containing low TTX levels in the edible portion but containing very high levels of TTX in non-edible portion raising concerns regarding food safety if a proper evisceration is not carried out by consumers. These results highlight the need for better understanding TTX variability in this gastropod species, which is critical to developing a proper legal framework for resources management ensuring seafood safety, and the introduction of these gastropods in the markets.

Tetrodotoxin and saxitoxin in two native species of puffer fish, Sphoeroides marmoratus and Lagocephalus lagocephalus, from NE Atlantic Ocean (Madeira Island, Portugal).

The presence in EU waters of invasive tetrodotoxin (TTX) -harbouring puffer fishes has been receiving increasingly attention due to potential new threats posed by this potent neurotoxin. The present study investigates the occurrence of tetrodotoxin, saxitoxin (STX), and their analogues in two native puffer fish species from the NE Atlantic. High TTX content was detected by LC-MS/MS in several tissues of the Guinean puffer Sphoeroides marmoratus from Madeira Island (Portugal), reaching concentrations as high as 15 mg TTX kg-1 in the digestive tract of a male specimen and 7.4 mg TTX kg-1 in gonads of a female specimen. Several TTX analogues were also detected, including the 4-epi-TTX, 4,9-Anhydro-TTX, 5- 11- deoxyTTX and 6,11-dideoxyTTX. Although at low levels, STX was detected in liver of the Oceanic puffer Lagocephalus lagocephalus. Trace levels of decarbamoylsaxitoxin (dcSTX) were also observed in L. lagocephalus. This study reports the presence of TTX and STX in native fish from EU waters, highlighting the need for a proper understating of the origin, distribution and fate of these toxins in NE Atlantic.

New Insights into the Occurrence and Toxin Profile of Ciguatoxins in Selvagens Islands (Madeira, Portugal).

Ciguatoxins (CTXs), endemic from tropical and subtropical regions of the Pacific and Indian Ocean and the Caribbean Sea, have caused several human poisonings during the last decade in Europe. Ciguatera fish poisonings (CFP) in Madeira and Canary Islands appear to be particularly related with consumption of fish caught close to Selvagens Islands, a Portuguese natural reserve composed of three small islands that harbor high fish biomass. In this study, fish specimens considered as potential vectors of CTXs were caught in Madeira and Selvagens archipelagos for toxins determination via sensitive liquid chromatography with tandem mass spectrometry detection (LC⁻MS/MS). CTXs were found in most of the fish samples from Selvagens and none from Madeira. Caribbean ciguatoxin-1 (C-CTX1) was the only toxin congener determined, reaching the highest value of 0.25 µg C-CTX1 kg-1 in a 4.6 kg island grouper (Mycteroperca fusca). This study indicates that a diversity of fish from different trophic levels contains CTXs, Selvagens appear to be one of the most favorable locations for CTXs food web transfer and finally, this study highlights the need of further research based on intensive environmental and biological sampling on these remote islands.

Cuidar da pessoa com transplante renal hospitalizada / Caring for the renal transplant patient in hospital

O enfermeiro acompanha a pessoa na trajectória da Doença Renal Crónica (DRC), desde a fase de tratamento conservador, até à transplantação renal. A transição para o domicílio após o transplante renal leva a que, por vezes, se confrontem com a necessidade de serem novamente internadas em consequência de eventos, nomeadamente processos infecciosos e/ou ameaça de rejeição. Emergem então, um conjunto de sentimentos que são agravados pela necessidade da hospitalização que a pessoa vê como uma ameaça à sua autonomia e à sua capacidade de decisão. Perante esta perspetiva considerou-se pertinente, analisar as percepções e motivações dos enfermeiros sobre os cuidados prestados à pessoa após o transplante renal quando é rehospitalizada. Realizou-se um estudo qualitativo, descritivo, com características de estudo de caso. Numa primeira fase, foram efetivadas quatro entrevistas não estruturadas, posteriormente o esquema de análise obtido foi apresentado, discutido e ampliado com a equipa de enfermagem em três focus grupo. A análise dos dados seguiu as linhas orientadoras definidas por Gibbs (2009). Da informação obtida resultaram cinco temas principais: Acolhimento, descrevendo-se a sua organização e conteúdo, bem como a importância da atitude do enfermeiro; Cuidado quotidiano, abordando as áreas de intervenção, a promoção do autocuidado, a preparação do regresso a casa e as dificuldades inerentes; Gestão do regime terapêutico, salientando-se a promoção da adesão terapêutica; Características dos doentes, analisando-se as diferenças ao nível do seu conhecimento e comportamento e Desafios no processo de cuidados, incluindo a relação com as motivações e vivências pessoais e os constrangimentos no cuidar. Os enfermeiros revelaram que o processo de cuidados à pessoa transplantada renal é complexo, reconhecendo que existem áreas nas quais a sua ação carece de maior sistematização e aprofundamento. Apesar do desgaste que é trabalhar com pessoas com DRC, manifestam motivação e satisfação para continuar a intervir neste domínio. Esta investigação pode ajudar à tomada de decisão do enfermeiro na prestação de cuidados à pessoa com transplante renal bem como motivar ajustes na organização dos cuidados desde a admissão à alta.

Estimating the contribution of N-sulfocarbamoyl paralytic shellfish toxin analogs GTX6 and C3+4 to the toxicity of mussels (Mytilus galloprovincialis) over a bloom of Gymnodinium catenatum.

Gymnodinium catenatum, a dinoflagellate species with a global distribution, is known to produce paralytic shellfish poisoning (PSP) toxins. The profile of toxins of G. catenatum is commonly dominated by sulfocarbamoyl analogs including the C3+4 and GTX6, which to date has no commercial certified reference materials necessary for their quantification via chemical methods, such as liquid chromatography. The aim of this study was to assess the presence of C3+4 and GTX6 and their contribution to shellfish toxicity. C3+4 and GTX6 were indirectly quantified via pre-column oxidation liquid chromatography with fluorescence detection after hydrolysis conversion into their carbamate analogs. Analyses were carried out in mussel samples collected over a bloom of G. catenatum (>63×103cellsl-1) in Aveiro lagoon, NW Portuguese coast. Concentration levels of sulfocarbamoyl toxin analogs were two orders of magnitude higher than decarbamoyl toxins, which were in turn one order of magnitude higher than carbamoyl toxins. Among the sulfocarbamoyl toxins, C1+2 were clearly the dominant compounds, followed by C3+4 and GTX6. The least abundant sulfocarbamoyl toxin was GTX5. The most important compounds in terms of contribution for sample toxicity were C1+2, which justified 26% of the PSP toxicity. The lesser abundant dcSTX constitutes the second most important compound with similar % of toxicity to C1+2, C3+4 and GTX6 were responsible for approximately 11% and 13%, respectively. The median of the sum of C3+4 and GTX6 was 27%. These levels reached a maximum of 60% as was determined for the sample collected closest to the G. catenatum bloom. This study highlights the importance of these low potency PSP toxin analogs to shellfish toxicity. Hydrolysis conversion of C3+4 and GTX6 is recommended for determination of PSP toxicity when LC detection methods are used for PSP testing in samples exposed to G. catenatum.

Paralytic shellfish poisoning due to ingestion of Gymnodinium catenatum contaminated cockles--application of the AOAC HPLC official method.

The potent paralytic shellfish toxins (PSTs) produced by Gymnodinium catenatum have appeared irregularly since the onset in 1986 of a monitoring program aimed at preventing contaminated bivalves from the Portuguese coast to reaching the consumer. In years where high contamination levels were attained, sporadic episodes of human poisonings were also recorded, as in 1994. The reappearance of high contamination led to the appearance of new cases during 2007. This study reports details of toxin ingestion, symptomatology and toxin presence in the fluids of one of these victims, an adult male who ingested several kilograms of cockles. In cockle samples collected the week before and during the week when the intoxication took place, the major PSTs detected by the HPLC method based on AOAC Official Method 2005.06 belonged to the sulfamate (81-68 molar percent) and decarbamoyl groups (19-32 molar percent), comprising GTX5, GTX6, C1,2, C3,4, dcNeo, and dcSTX. In the patient urine sample sulfamate and decarbamoyl derivatives were also found, comprising by GTX5 (28%), GTX6 (25%), dcSTX (24%) and dcNeo (22%), but no C toxins and no dcGTX2,3 were detected. Compared to the cockle samples, there was an increase in the proportion of dcSTX, dcNeo and GTX5 (molar percentage) in the urine sample, but not of GTX6. Overall, compounds which had the presence of an O-sulfate at C11 were absent in urine while being relatively abundant in the bivalve (36.5-47.0 molar percent). In blood plasma PSTs were not detected.

Profiles of paralytic shellfish poisoning toxins in shellfish from Portugal explained by carbamoylase activity.

The presence of paralytic shellfish poisoning (PSP) toxins has not been recorded in the Portuguese coast since 1995. A bloom of Gymnodinium catenatum occurred in the NW coast of Portugal in the autumn of 2005, and PSP profiles were determined in several inshore and offshore shellfish species by HPLC after pre-column oxidation. Most of the species studied contained a complex toxin profile, typically representative of contamination by G. catenatum. However, clams such as Spisula solida contained mainly decarbamoyl toxins, while less extensive transformation was found in Scrobicularia plana. In vitro incubation of S. solida digestive glands with PSP standards revealed a rapid transformation of carbamate and N-sulfocarbamoyl toxins into their corresponding decarbamate analogues. After 24 h, less than 5% of the carbamate or N-sulfocarbamoyl toxins tested remained. After a 24 h in vitro incubation of S. plana digestive glands, no decarbamate analogues were detected. Artificial toxification of S. plana with cultures of G. catenatum revealed the conversion into decarbamoyl analogues progressed slowly: initially dcGTX2+3 and dcSTX accounted only for 5% of total non N-1 hydroxilated toxins, after 6 days these toxins accounted for 41% of the toxin composition. In vitro incubations of digestive glands from other commercial bivalves did not reveal production of decarbamoyl analogues over a 24 h period.