Marine Biotoxins Research Articles

Inhalable particle-bound marine biotoxins in a coastal atmosphere: Concentration levels, influencing factors and health risks

Characterizing marine biotoxins (MBs) composition in coastal aerosol particles has become essential to tracking sources of atmospheric contaminants and assessing human inhalable exposure risks to air particles. Here, coastal aerosol particles were collected over an almost 3-year period for the analysis of eight representative MBs, including brevetoxin (BTX), okadaic acid (OA), pectenotoxin-2 (PTX-2), domoic acid (DA), tetrodotoxin (TTX), saxitoxin (STX), ciguatoxin (CTX) and ω-Conotoxin. Our data showed that the levels of inhalable airborne marine biotoxins (AMBs) varied greatly among the subcategories and over time. Both in daytime and nighttime, a predominance of coarse-mode AMB particles was found for all the target AMBs. Based on the experimental data, we speculate that an ambient AMB might have multiple sources/production pathways, which include air-sea aerosol production and direct generation and release from toxigenic microalgae/bacteria suspended in surface seawater or air, and different sources may make different contribution. Regardless of the subcategory, the highest deposition efficiency of an individual AMB was found in the head airway region, followed by the alveolar and tracheobronchial regions. This study provides new information about inhalable MBs in the coastal atmosphere. The coexistence of various particle-bound MBs raises concerns about potential health risks from exposure to coastal air particles.

Novel Methodologies for Providing In Situ Data to HAB Early Warning Systems in the European Atlantic Area: The PRIMROSE Experience

Harmful algal blooms (HABs) cause harm to human health or hinder sustainable use of the marine environment in Blue Economy sectors. HABs are temporally and spatially variable and hence their mitigation is closely linked to effective early warning. The European Union (EU) Interreg Atlantic Area project “PRIMROSE”, Predicting Risk and Impact of Harmful Events on the Aquaculture Sector, was focused on the joint development of HAB early warning systems in different regions along the European Atlantic Area. Advancement of the existing HAB forecasting systems requires development of forecasting tools, improvements in data flow and processing, but also additional data inputs to assess the distribution of HAB species, especially in areas away from national monitoring stations, usually located near aquaculture sites. In this contribution, we review different novel technologies for acquiring HAB data and report on the experience gained in several novel local data collection exercises performed during the project. Demonstrations include the deployment of autonomous imaging flow cytometry (IFC) sensors near two aquaculture areas: a mooring in the Daoulas estuary in the Bay of Brest and pumping from a bay in the Shetland Islands to an inland IFC; and several drone deployments, both of Unmanned Aerial Vehicles (UAV) and of Autonomous Surface vehicles (ASVs). Additionally, we have reviewed sampling approaches potentially relevant for HAB early warning including protocols for opportunistic water sampling by coastguard agencies. Experiences in the determination of marine biotoxins in non-traditional vectors and how they could complement standard routine HAB monitoring are also considered.

A Novel SELEX Based on Immobilizing Libraries Enables Screening of Saxitoxin Aptamers for BLI Aptasensor Applications.

Saxitoxin (STX) is one of the potent marine biotoxins that has high rate of lethality. However, there are no effective treatments at present, and the existing detection methods need to be further explored because of ethical problems or technical limitations. In this work, oligonucleotide aptamers toward STX were screened based on immobilizing libraries on Immobilized Metal-Chelate (IMC), such as Ni-NTA Sepharose, and the IMC-SELEX was conducted by the G-quadruplex library and the random library, respectively. Aptamer 45e (from the G-quadruplex library) and aptamer 75a were obtained after optimization, and aptamer 45e turned out to have a higher affinity toward STX. Furthermore, it was found that the hydrogen bonding and the van der Waals forces (VDW) played major roles in the high efficiency and specificity between STX and 45e by means of molecular docking and dynamics simulation. Based on this, aptamer 45e-1 with the Kd value of 19 nM was obtained by further optimization, which was then used to construct a simple, label-free and real-time optical BLI aptasensor for the detection of STX. This aptasensor showed good reproducibility and stability. In summary, with the advantages of screening aptamers of high efficiency and specificity toward the targets, the proposed IMC-SELEX provides a promising screening strategy for discovering aptamers, which could be used as the potential molecular recognition elements in the fields of biomedicine, food safety and environmental monitoring.

Food Safety Risk in Germany From Mislabeled Imported Fish: Ciguatera Outbreak Trace-Back, Toxin Elucidation, and Public Health Implications

Ciguatera poisoning (CP) is a prevalent food related health risk, caused by the consumption of seafood contaminated with ciguatoxins (CTXs). Seafood is the most traded food commodity worldwide, and since 2012, imported snapper fish (Lutjanidae) were the leading cause of CP in Germany. Following a Germany wide CP outbreak in 2017, a product trace-back investigation was conducted for imported fish labeled as “Red Snapper” (Lutjanus malabaricus). Forty-five fish muscle-tissue portions from the implicated batch and two meal remnants were analyzed for CTXs. All samples were positive for “CTX-like toxicity” containing a range of 0.23–11.4 ng CTX3C equivalents per gram of wet tissue, determined by an in vitro cell assay [N2a-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) chromatograms revealed the (potential) presence of several marine biotoxins of the class CTX in all batch samples. All samples exceeded current multi-national product legal requirements and recommended guidelines for CTXs. DNA barcoding confirmed the fish sold was mislabeled and was identified as L. bohar, a species frequently involved in CP. Consequently, the mislabeled food and contaminants risk focuses attention on the importance of correct food labeling. Processes for food authentication and CTX contaminant analysis exist and can be used to potentially prevent, stop, and remove foods from commerce for further evaluation to ensure consumer safety. This study further demonstrates their necessity.

Emerging Marine Biotoxins in European Waters: Potential Risks and Analytical Challenges.

Harmful algal blooms pose a challenge regarding food safety due to their erratic nature and forming circumstances which are yet to be disclosed. The best strategy to protect human consumers is through legislation and monitoring strategies. Global warming and anthropological intervention aided the migration and establishment of emerging toxin producers into Europe’s temperate waters, creating a new threat to human public health. The lack of information, standards, and reference materials delay effective solutions, being a matter of urgent resolution. In this work, the recent findings of the presence of emerging azaspiracids, spirolildes, pinnatoxins, gymnodimines, palitoxins, ciguatoxins, brevetoxins, and tetrodotoxins on European Coasts are addressed. The information concerning emerging toxins such as new matrices, locations, and toxicity assays is paramount to set the risk assessment guidelines, regulatory levels, and analytical methodology that would protect the consumers.

Selection, Characterization, and Optimization of DNA Aptamers against Challenging Marine Biotoxin Gymnodimine-A for Biosensing Application.

Gymnodimines (GYMs), belonging to cyclic imines (CIs), are characterized as fast-acting toxins, and may pose potential risks to human health and the aquaculture industry through the contamination of sea food. The existing detection methods of GYMs have certain defects in practice, such as ethical problems or the requirement of complicated equipment. As novel molecular recognition elements, aptamers have been applied in many areas, including the detection of marine biotoxins. However, GYMs are liposoluble molecules with low molecular weight and limited numbers of chemical groups, which are considered as “challenging” targets for aptamers selection. In this study, Capture-SELEX was used as the main strategy in screening aptamers targeting gymnodimine-A (GYM-A), and an aptamer named G48nop, with the highest KD value of 95.30 nM, was successfully obtained by screening and optimization. G48nop showed high specificity towards GYM-A. Based on this, a novel aptasensor based on biolayer interferometry (BLI) technology was established in detecting GYM-A. This aptasensor showed a detection range from 55 to 1400 nM (linear range from 55 to 875 nM) and a limit of detection (LOD) of 6.21 nM. Spiking experiments in real samples indicated the recovery rate of this aptasensor, ranging from 96.65% to 109.67%. This is the first study to report an aptamer with high affinity and specificity for the challenging marine biotoxin GYM-A, and the new established aptasensor may be used as a reliable and efficient tool for the detection and monitoring of GYMs in the future.

Reducing environmental impacts of marine biotoxin monitoring: A laboratory report

Laboratories globally contribute significantly to consumption of resources, greenhouse gas emissions, and generation of waste. Shellfish destined for human consumption are required to be tested for the presence of regulated marine biotoxins, that can be harmful to human health. Whilst running the national monitoring program for the detection of biotoxins in shellfish, efforts were made to increase resource efficiencies by reducing waste and energy consumption leading to reduced environmental and financial costs. Methods were verified to allow transitions to more sustainable and environmentally-friendly consumables, replacing plastics with paperboard and glass alternatives, leading to a reduction in the consumption of single-use plastics by 69%. A shift to polystyrene recycling and composting non-toxic shellfish waste led to an overall reduction in non-chemical waste of >95%. Adoption of green analytical chemistry principles to procurement and preparation of chemical solutions led to a reduction in hazardous chemical waste by ~23%. A further reduction in printing (~81%) was achieved by transitioning to digital document control. Strategies to reduce energy consumption through ‘switch off’ campaigns and improved fume hood and cold storage equipment management were also implemented. Fume hood and cold storage equipment energy consumption was reduced by 30%. The strategies implemented could be adopted by other laboratories e.g., monitoring and research laboratories dealing with pharmaceutical, biological, and environmental samples.

Concentrations of Tetrodotoxin (TTX) and Its Analogue 4,9-Anhydro TTX in Different Tissues of the Silver-Cheeked Pufferfish (Lagocephalus sceleratus, Gmelin, 1789) Caught in the South-Eastern Mediterranean Sea, Lebanon.

Pufferfishes are among the best-known marine organisms that accumulate marine biotoxins such as Tetrodotoxin (TTX). In the Mediterranean Sea, the silver-cheeked toadfish Lagocephalus sceleratus is the most reported TTX-bearer, causing many fatal and non-fatal cases. In Lebanon, no previous studies have measured TTX levels although the possibility of TTX-poisoning is high since L. sceleratus is caught in different sizes and can be mistaken with other small fishes. Hence, this study reports TTX and its analogue 4,9-anhydro TTX in L. sceleratus collected from Lebanese waters in the Eastern Mediterranean Sea. The results show that TTX concentrations in fish tissues varied between 0.10 and 252.97 µg/g, while those of 4,9-anhydro TTX oscillated between 0.01 and 43.01 µg/g. Internal organs of L. sceleratus were the most toxic parts of its body, with the highest TTX levels found in gonads (mainly ovaries) and liver, followed by the muscles and skin with concentrations always exceeding the safety level. Toxicity fluctuations of L. sceleratus, its expansion, ecological and economic effects were also elucidated. Based on the present findings, it has been confirmed that L. sceleratus constitutes a health, ecological and economic risks, and therefore its trade in seafood markets should be banned to avoid any potential intoxication.

Bayesian Networks modeling of diarrhetic shellfish poisoning in Mytilus edulis harvested in Bantry Bay, Ireland

Diarrhetic Shellfish Poisoning (DSP) results from the human consumption of contaminated shellfish with marine biotoxins, which are produced by some species of marine dinoflagellates, mainly belonging to the genus Dinophysis. Shellfish contamination with marine biotoxins not only pose a threat to human health, but also lead to financial loss to aquaculture operations from the temporary closure of production areas when toxin concentrations exceed regulatory levels. In this study, we developed a Bayesian Network (BN) model for forecasting the short-term variations of DSP toxins in blue mussels (Mytilus edulis) from Bantry Bay, Southwest Ireland. Data inputs to a BN model from 10 production sites in Bantry Bay included plankton cell densities in sea water, DSP toxin concentration in mussels and sea surface temperature. The model was trained with data from 2014 to 2018, and validated with data of 2019. Validation consisted of predicting the DSP toxin concentration at one production site using the model parameters from the other locations as input values. Model validation showed that the prediction accuracy was higher than 86%. Sensitivity analysis indicated that in general, DSP toxin concentration was more relevant than plankton abundance. This initial work has demonstrated the usefulness of BN modeling as an approach to short term forecasting. Further work is ongoing to use the model for scenario testing and to increase the number of environmental parameters used as inputs to the model.

Synergistic Effect of Environmental Food Pollutants: Pesticides and Marine Biotoxins

Synergistic Effect of Environmental Food Pollutants: Pesticides and Marine Biotoxins

Safety of oil from Schizochytrium sp. (strain ATCC 20889) for use in infant and follow-on formula as a novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the safety of Schizochytrium sp. oil as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF which is the subject of the application is an oil rich in docosahexaenoic acid (DHA) that is produced by the microalgae Schizochytrium sp. (strain ATCC‐20889). The applicant proposed to add the NF in infant formulae (IF) and follow‐on formulae (FOF) at use levels in accordance with Regulation (EU) No 609/2013. The evidence provided by the applicant does not demonstrate to which species the strain Schizochytrium sp. ATCC 20889 belongs. As the source organism of the NF is not characterised at species level, no assessment for inclusion in the Qualified Presumption of Safety (QPS) list can be performed by EFSA. Marine biotoxins (including cyanotoxins) in the NF were below their limits of quantification. However, since it is unknown to which species the strain Schizochytrium sp. ATCC 20889 belongs, the concern that this strain has the potential to produce other toxins remains. No toxicological studies with the NF were provided by the applicant. Toxicological studies are available with DHA‐rich algal oils produced from other strains of Schizochytrium sp. However, the Panel considers that those toxicological studies cannot be used to establish the safety of the oil produced by the strain which is under assessment in this application (Schizochytrium sp. ATCC 20889). Therefore, based on the information provided by the applicant, the Panel concludes that the safety of the NF has not been established.

Tetrodotoxin in live bivalve mollusks from Europe: Is it to be considered an emerging concern for food safety?

Tetrodotoxins (TTXs) are a group of potent neurotoxins named after the Tetraodontidae fish family (pufferfish). TTXs have been reported in several animal taxa, both terrestrial and marine. The ingestion of TTX-contaminated flesh can cause serious neurotoxic symptomatology and can eventually lead to death. Traditionally, TTXs have been associated with Asian countries, in particular with pufferfish consumption. However, they have also been reported in bivalve mollusks farmed in the Pacific area and, recently, in European seas. In Europe, different countries have reported TTXs, especially those bordering the Mediterranean Sea. As a consequence, in 2017 the European Food Safety Authority (EFSA) released an opinion with reference to TTX present in marine gastropods and bivalves, proposing a safety limit of 44 µg/kg TTXs in shellfish meat, below which no adverse effects should be observed in humans. Nevertheless, this limit has been exceeded on many occasions in European shellfish and, while for bivalves there have been no registered human intoxications, that is not the case for marine gastropods. However, TTXs have not yet been included in the list of marine biotoxins officially monitored in live bivalve mollusks within the European Union (EU). Thus, the aims of this manuscript are to discuss the increasing occurrence of TTXs in live bivalve mollusks from European sea waters, to acknowledge the still ongoing knowledge gaps that should be covered and to stimulate constructive debate on the eventuality of adopting a shared regulatory context, at least in the EU, for monitoring and managing this potential threat to food safety.

Determination of lipophilic marine biotoxins in shellfish foods by ultra performance liquid chromatography-hybrid triple quadrupole linear ion trap-mass spectrometry

An ultra performance liquid chromatography-hybrid triple quadrupole linear ion trap-mass spectrometry(UPLC-QqLIT-MS) was established for determination of lipophilic marine biotoxins in shellfish. And the 12 lipophilic marine biotoxins in shellfish were surveyed. The lipophilic marine biotoxins in homogenized shellfish were ultrasonically extracted by methanol in super-sonic instrument, and cleaned up by solid phase extraction of Strata-X column, and eluted with methanol(containing 0.3% ammonia water). The elution was diluted with water, and cleaned by 0.22 μm millipore filter. The filtrate was separated on a Waters ACQUITY UPLC BEH C_(18) column(150 mm×2.1 mm, 1.7 μm)by gradient elution in 12 minutes with acetronitrile-water(containing 0.01%(V/V) ammonia and 2 mmol/L ammonium formate) as mobile phase, and detected by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS), identified by electrospray ionization(ESI) in simultaneous scanning mode of positive and negative ions using multiple reaction monitoring, and quantified with external standards. Information dependent acquisition scan function(IDA) combined with enhanced production scan(EPI) was used to confirm the 12 lipophilic marine biotoxins. The calibration curves of 12 lipophilic marine biotoxins showed good linearity in the range of 0.5-50 μg/L with correlation coefficients were 0.9984-0.9999.The detection limits of the method were 0.15-0.29 μg/kg. The recoveries of three spiking levels ranged from 80.0% to 116.0%, and the relative standard deviation(RSD) were 0.6%-6.4%(n=7). The method for determination of 12 lipophilic marine biotoxins in shellfish by UPLC-QqLIT-MS was of operation convenience, less interference from impurities and good accuracy, which could meet the requirements for the determination of 12 lipophilic marine biotoxins residues in sea foods.

Natural Products as Potential Antiviral Drugs: The Specific Case of Marine Biotoxins.

To fight against various viral infections researchers turned to new chemical structures resulting from natural medicinal plants and more recently from “marine origin” as sources of active molecules against viral infections. The present manuscript describes complex marine origin drugs, their chemical complex structure, their therapeutic use, and their antiviral properties. Emphasis is placed more particularly on the properties of ionic channels (Na+, K+, Ca2+) blockers compounds from marine origin, named Dinotoxins, derived from “dinoflagellates microalgae”. These compounds are of particular pharmaceutical interest since ionic channels blockers could be used to fight against a wide diversity of viruses, including SARS-CoV2 virus.

Climate change and health impact: is food missing in the equation?

Abstract It has been generally recognized by researchers and regulators that climate change constitutes an important driver affecting the food sector, representing a public health issue that deserves particular attention. Climate change is associated with increasing temperatures and more extreme rainfall; it alters relationships among crops, pests, pathogens, and weeds; and it exacerbates several trends including declines in pollinating insects, increasing water scarcity, increasing ground-level ozone concentrations, and fishery declines. In one hand, climate change threatens our ability to ensure global food security, eradicate poverty and achieve sustainable development. Agricultural productivity is affected by changing rainfall patterns, drought, flooding and the geographical redistribution of pests and diseases, with consequent implications in food availability, a key requirement for food security. On the other hand, despite less debated, climate change could also affect food safety, namely through the occurrence of food safety hazards at various stages of the food chain, from “farm to fork”. The tendency to increase the use of agrochemicals to balance the effects of more frequent extreme weather events and water scarcity in some regions could become more frequent. In addition to pesticide residues, both chemical and microbiological risks are expected to impair food and feed safety as a consequence of climate change: in particular mycotoxins, marine biotoxins (phycotoxins), trace metals, among others. Here, all these concepts will be discussed and integrated, to highlight the potential impact of climate change in food systems, focusing particularly on the contribution to human health especially in what concerns food safety.

Digital Technologies and Open Data Sources in Marine Biotoxins' Risk Analysis: The Case of Ciguatera Fish Poisoning.

Currently, digital technologies influence information dissemination in all business sectors, with great emphasis put on exploitation strategies. Public administrations often use information systems and establish open data repositories, primarily supporting their operation but also serving as data providers, facilitating decision-making. As such, risk analysis in the public health sector, including food safety authorities, often relies on digital technologies and open data sources. Global food safety challenges include marine biotoxins (MBs), being contaminants whose mitigation largely depends on risk analysis. Ciguatera Fish Poisoning (CFP), in particular, is a MB-related seafood intoxication attributed to the consumption of fish species that are prone to accumulate ciguatoxins. Historically, CFP occurred endemically in tropical/subtropical areas, but has gradually emerged in temperate regions, including European waters, necessitating official policy adoption to manage the potential risks. Researchers and policy-makers highlight scientific data inadequacy, under-reporting of outbreaks and information source fragmentation as major obstacles in developing CFP mitigation strategies. Although digital technologies and open data sources provide exploitable scientific information for MB risk analysis, their utilization in counteracting CFP-related hazards has not been addressed to date. This work thus attempts to answer the question, “What is the current extent of digital technologies’ and open data sources’ utilization within risk analysis tasks in the MBs field, particularly on CFP?”, by conducting a systematic literature review of the available scientific and grey literature. Results indicate that the use of digital technologies and open data sources in CFP is not negligible. However, certain gaps are identified regarding discrepancies in terminology, source fragmentation and a redundancy and downplay of social media utilization, in turn constituting a future research agenda for this under-researched topic.

Guidance Level for Brevetoxins in French Shellfish

Brevetoxins (BTXs) are marine biotoxins responsible for neurotoxic shellfish poisoning (NSP) after ingestion of contaminated shellfish. NSP is characterized by neurological, gastrointestinal and/or cardiovascular symptoms. The main known producer of BTXs is the dinoflagellate Karenia brevis, but other microalgae are also suspected to synthesize BTX-like compounds. BTXs are currently not regulated in France and in Europe. In November 2018, they have been detected for the first time in France in mussels from a lagoon in the Corsica Island (Mediterranean Sea), as part of the network for monitoring the emergence of marine biotoxins in shellfish. To prevent health risks associated with the consumption of shellfish contaminated with BTXs in France, a working group was set up by the French Agency for Food, Environmental and Occupational Health & Safety (Anses). One of the aims of this working group was to propose a guidance level for the presence of BTXs in shellfish. Toxicological data were too limited to derive an acute oral reference dose (ARfD). Based on human case reports, we identified two lowest-observed-adverse-effect levels (LOAELs). A guidance level of 180 µg BTX-3 eq./kg shellfish meat is proposed, considering a protective default portion size of 400 g shellfish meat.

Rapid electrochemical detection of domoic acid based on polydopamine/reduced graphene oxide coupled with in-situ imprinted polyacrylamide

Domoic acid, namely amnesic shellfish toxin, is a highly neurotoxic substance to marine animals and humankind. To reduce the incidence of poisoning accidents, the exploitation of specific and rapid detection method for domoic acid monitoring is highly required. Herein, an electrochemical molecularly imprinted polymer (MIP) sensor based on polydopamine-reduced graphene oxide/polyacrylamide composite (PDA-rGO/PAM) was constructed successfully to detect domoic acid. The domoic acid molecule could be recognized in imprinted cavities of PAM reversibly through hydrogen bonding. PDA-rGO promoted the loading capacity of PAM and improved the charge transfer rate, which amplified the electrical signal response of the MIP sensor. The screen-printed electrode (SPE) modified with PDA-rGO/PAM displayed satisfactory response toward toxin contaminated sample at a linear range from 1 to 600 nM and a low detection limit of 0.31 nM, demonstrating the prospective application of the transducer as a portable sensing platform for the on-site detection of hazardous marine biotoxin. Moreover, benefiting from the superior specificity and stability of MIP, the fabricated sensor could be utilized to detect the domoic acid content in mussel extracts directly without complex pretreatment operation.

New Trends in the Occurrence of Yessotoxins in the Northwestern Adriatic Sea.

Yessotoxins (YTXs) are polycyclic toxic ether compounds produced by phytoplanktonic dinoflagellates which accumulate in filter-feeding organisms. We know that the water temperature in our areas Northwestern Adriatic Sea is optimal for the growth of potentially toxic algae (around 20 °C). In recent years, these temperatures have remained at these levels for longer and longer periods, probably due to global warming, which has led to an excessive increase in toxin levels. The interruption of mussel harvesting caused by algae negatively affects farmers’ revenues and the availability of local fish, causing a major economic loss in Italy’s main shellfish sector. Methods: In the nine years considered, 3359 samples were examined: 1715 marine waters, 73 common clams; 732 mussels; 66 oysters; and 773 veracious clams. Bivalve molluscs were examined for the presence of marine biotoxins, including YTXs, while potentially toxic algae, including those producing YTXs, were searched for and counted in marine waters. The method adopted for the quantification of lipophilic toxins involves the use of an LC-MS/MS system. The enumeration of phytoplankton cells was performed according to the Utermhöl method. Results: Between 2012 and 2020, 706 molluscs were tested for YTXs. In total, 246 samples tested positive, i.e., 34.84%. Of the positive samples, 30 exceeded the legal limit. Conclusion: In this regard, it is essential to develop and activate, as soon as possible, an “early warning” system that allows a better control of the production areas of live bivalve molluscs, thus allowing an optimal management of the plants in these critical situations.

Extraction and LC-MS/MS Analysis of Ciguatoxins: A Semi-Targeted Approach Designed for Fish of Unknown Origin.

Ciguatoxins (CTXs) are polyether marine biotoxins that can cause ciguatera poisoning (CP) after the consumption of fish or invertebrates containing sub ppb levels; concentrations that present a challenge for current extraction and analysis methods. Here, a newly developed and (partly) validated single-day extraction protocol is presented. First, the fish sample is broken-down by enzymatic digestion, followed by extraction and extract clean-up by defatting and two solid-phase extractions. Final extracts were investigated using two different CTX-analysis methods; an in vitro cytotoxicity assay (N2a-assay) and by LC-MS/MS. Validation was performed for both fillet and freeze-dried samples of snapper, parrotfish, and grouper spiked with CTX1B, 52-epi-54-deoxyCTX1B, 54-deoxyCTX1B, and CTX3C. Based on recovery rates (35–88%) and matrix effects (66–116%) determined by LC-MS/MS, the enzyme protocol is applicable to various matrices. The protocol was applied to naturally contaminated fish tissue (Lutjanus bohar) obtained during a CP incident in Germany. Several potential CTX congeners were identified by a two-tier LC-MS/MS approach (screening of sodium adducts, high-resolution or low-resolution confirmation via ammonium adducts). Inclusion of >30 known CTX congeners into the LC-MS/MS methods and single-day sample preparation make the method suitable for analysis of ciguatera suspect samples at sub ppb levels also with undisclosed CTX profiles.