Globally, the livelihoods of over a billion people are affected by changes to marine ecosystems, both structurally and systematically. Resources and ecosystem services, provided by the marine environment, contribute nutrition, income, and health benefits for communities. One threat to these securities is ciguatera poisoning; worldwide, the most commonly reported non-bacterial seafood-related illness. Ciguatera is caused by the consumption of (primarily) finfish contaminated with ciguatoxins, potent neurotoxins produced by benthic single-cell microalgae. When consumed, ciguatoxins are biotransformed and can bioaccumulate throughout the food-web via complex pathways. Ciguatera-derived food insecurity is particularly extreme for small island-nations, where fear of intoxication can lead to fishing restrictions by region, species, or size. Exacerbating these complexities are anthropogenic or natural changes occurring in global marine habitats, e.g., climate change, greenhouse-gas induced physical oceanic changes, overfishing, invasive species, and even the international seafood trade. Here we provide an overview of the challenges and opportunities of the 21st century regarding the many facets of ciguatera, including the complex nature of this illness, the biological/environmental factors affecting the causative organisms, their toxins, vectors, detection methods, human-health oriented responses, and ultimately an outlook towards the future. Ciguatera research efforts face many social and environmental challenges this century. However, several future-oriented goals are within reach, including digital solutions for seafood supply chains, identifying novel compounds and methods with the potential for advanced diagnostics, treatments, and prediction capabilities. The advances described herein provide confidence that the tools are now available to answer many of the remaining questions surrounding ciguatera and therefore protection measures can become more accurate and routine.

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.

Ciguatoxins (CTXs) are a group of marine biotoxins, consisting of over 30 different congeners that can contaminate marine food webs. Generally, the molecular structures among the known congeners are variable by geographical region (i.e., ocean basin) and vector species. Limited information is available regarding the CTX profiles among or within CTX vector species and their capture regions. Within this study, an in-depth investigation based on a semi-targeted LC-MS/MS approach was conducted to investigate 52 tissue samples from a single species Lutjanus bohar (Lutjanidae), a common CTX vector, sourced from two distinct regions (Indian Ocean and Pacific Ocean). All samples revealed the presence of a complex CTX contaminant profile, with samples containing several congeners of the CTX3C-group (2,3,51-trihydroxyCTX3C, 2,3,-dihydroxyCTX3C, 2-hydroxyCTX3C, M-seco-CTX3C, 51-hydroxyCTX3C, CTX3C, and respective 49-epimers in most cases). All samples were previously found to possess a CTX-like toxicity within an in vitro cytotoxicity assay (N2a-bioassay), demonstrating the relevance of CTX3C-group congeners with regard to ciguatera poisoning. Individual samples contained an indistinguishable toxin profile within the species and among the distinct oceanic capture regions. These findings imply either a species-specific CTX metabolism or the emergence of an interoceanic CTX toxin profile. The inter-regional CTX profile demonstrated here provides further evidence that classifying CTX congeners based on ocean basins may be imprecise.

Ciguatoxins (CTXs) are neurotoxic marine biotoxins capable of contaminating marine organisms. Approximately 30 CTX analogues have been described; however, only a few have been documented in ciguatera poisoning (CP) outbreaks. Detecting CTXs from complex matrices at CP-relevant concentrations (<1 µg per kg seafood tissue) is analytically challenging. Analytical standards for CTXs are rare. Even after multi-step sample preparation (including liquid–liquid partition, defatting, and solid-phase extraction (SPE)), extracts can contain undesirable co-eluting matrix components. These limitations can exacerbate discrepancies between results obtained by LC-MS/MS and the N2a-bioassay, which are two common CTX detection methods. Herein, a rapid and simple fractionation method, based on normal phase SPE (silica), is demonstrated. Target CTXs were eluted using solvent mixtures of ascending polarity, passed through the column, and separated into eight fractions. To challenge the method, extracts with eleven naturally incurred CTX analogues among different structural CTX groups (e.g., CTX3C, CTX4A, and C-CTX group) were used. The most complex tissue matrix tested (viscera) was improved the most for extract purity and CTX detection, enhancing the correlation between LC-MS/MS and N2a-bioassay results. This workflow represents an advancement for characterizing CTXs in seafood products and CP outbreaks, irrespective of the responsible CTX analogue and where standards are lacking.

Ciguatoxins (CTXs) are marine biotoxins that can contaminate seafood and if consumed can result in ciguatera poisoning (CP). The analysis of CTXs is challenging, as they occur in complex tissue matrices, cause CP symptoms at trace amounts (<1 μg kg⁻¹), and certified reference materials are not available. Currently, no standard operating protocols exist for sample preparation or instrumental analysis. Five laboratories worldwide participated in this first-time method comparison study, which used sample extracts containing different CTX groups (CTX4A group, CTX3C group, C-CTX-1) to identify factors impacting CTX quantitation using LC-MS/MS and LC-HRMS. Matrix effects were found to significantly influence CTX quantitation, along with factors such as instrument, eluents, or selected precursor ion. CTXs were quantified using commercially available, non-certified CTX1B and CTX3C standards. Analogues of the CTX groups behaved differently with regard to matrix effects and suitable calibrants with differences between laboratories exceeding a factor of 10 in some cases.