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.

Identification of New CTX Analogues in Fish from the Madeira and Selvagens Archipelagos by Neuro-2a CBA and LC-HRMS.

Ciguatera Poisoning (CP) is caused by consumption of fish or invertebrates contaminated with ciguatoxins (CTXs). Presently CP is a public concern in some temperate regions, such as Macaronesia (North-Eastern Atlantic Ocean). Toxicity analysis was performed to characterize the fish species that can accumulate CTXs and improve understanding of the ciguatera risk in this area. For that, seventeen fish specimens comprising nine species were captured from coastal waters inMadeira and Selvagens Archipelagos. Toxicity was analysed by screening CTX-like toxicity with the neuroblastoma cell-based assay (neuro-2a CBA). Afterwards, the four most toxic samples were analysed with liquid chromatography-high resolution mass spectrometry (LC-HRMS). Thirteen fish specimens presented CTX-like toxicity in their liver, but only four of these in their muscle. The liver of one specimen of Muraena augusti presented the highest CTX-like toxicity (0.270 ± 0.121 µg of CTX1B equiv·kg-1). Moreover, CTX analogues were detected with LC-HRMS, for M. augusti and Gymnothorax unicolor. The presence of three CTX analogues was identified: C-CTX1, which had been previously described in the area; dihydro-CTX2, which is reported in the area for the first time; a putative new CTX m/z 1127.6023 ([M+NH4]+) named as putative C-CTX-1109, and gambieric acid A.

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.

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.

First toxin profile of ciguateric fish in Madeira Arquipelago (Europe).

Ciguatera fish poisoning (CFP) is a human foodborne intoxication caused by ingestion of tropical fishes contaminated with the potent polyether toxins known as ciguatoxins (CTXs). These toxins are issued from Gambierdiscus species of dinoflagellates. Herbivorous fish accumulate these toxins in their musculature and viscera after ingesting dinoflagellates. Epidemiological studies showed that CFP has been present in areas between 35 degrees North and 35 degrees South latitude, mainly, Indo-pacific and Caribbean areas, but not in waters closed to European and African continent. In the present paper, a specimen of Seriola dumerili weighing 70 kg and a smaller Seriola fasciata specimen, captured in waters belonging to Selvagens Islands (Madeira Arquipelago), were analyzed. Fishes from this genus were implicated in previous suspected ciguatera poisoning outbreaks in the Portuguese Madeira Arquipelago in the North Atlantic Ocean. Analysis was performed by two approaches, a functional method using cerebellar granule cells and by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) method. The study was carried out in one portion of the tail muscle of Seriola fasciata and five parts of the body of Seriola dumerili (tail muscle, head, ventral muscle, mid muscle, and liver). The functional method consisted in the modification of the inward sodium current in cerebellar granule cells and the chemical method was a high resolution chromatography, which allowed elucidating the toxin profile in the samples. In addition, UPLC-MS technique was optimized and used for detecting and quantifying CTXs for the first time. After fish extraction and clean up, the chromatograms revealed the presence of CTX-1B at 1111.6 m/z, CTX-3C at 1023.5 m/z, a CTX analogue at 1040.6 m/z, and a CTX from the Caribbean or Indic waters at 1141.6 m/z. Therefore, the results obtained in the present paper for both methods confirm, for the first time, the presence of CTX in fish from Madeira Arquipelago.