51 research outputs found
Faster ciguatoxin extraction methods for toxicity screening
No full textAbstract Ciguatera poisoning (CP) is a severe global public health problem caused by the consumption of seafood products contaminated with ciguatoxins (CTXs). The growing demand for seafood products requires high-throughput testing for CTX-susceptible seafood, however complex extraction and slow cleanup methods inhibit this goal. Herein, several methods for extracting CTXs from fish tissue were established and compared; these methods are sensitive, specific, and valid while achieving higher sample extraction throughput than currently established protocols. The trial fish material was generated from multiple species, with different physical conditions (wet and freeze-dried tissue), and naturally contaminated with various CTXs (i.e., CTX-1B, CTX-3C, and C-CTX-1), thus ensuring these methods are robust and broadly applicable. The extraction methods used were based on mechanical maceration with acetone or methanol or enzymatic digestion followed by acetone and ethyl acetate extraction. Crude extracts were investigated for CTX-like toxicity using an in vitro mouse neuroblastoma (N2a) cell-based assay (CBA). Among the three methods, there was no significant difference in toxin estimates (p = 0.219, two-way ANOVA), indicating their interchangeability. For speed (> 16 samples/day), accuracy (100%), and CTX analog retention confirmation by liquid chromatography-tandem mass spectrometry (LC‒MS/MS), the preferred extraction methods were both methanol and enzyme-based. All extraction methods post hoc confirmation of CTX analogs successfully met international seafood market-based CTX contaminant guidance. These methods can drastically increase global CTX screening capabilities and subsequently relieve sample processing bottlenecks, inhibiting environmental and human health-based CTX analysis
Fish Extract Fractionation by Solid Phase Extraction: Investigating Co-Occurring Ciguatoxins by LC-MS/MS and N2a-Bioassay
No full textCiguatoxins (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
Identical Ciguatoxin-3C group profiles in Lutjanus bohar from the Pacific and Indian Oceans - indicating the need to re-evaluate geographical CTX classifications
No full textCiguatoxins (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.</jats:p
Extraction and LC-MS/MS Analysis of Ciguatoxins: A Semi-Targeted Approach Designed for Fish of Unknown Origin
No full textCiguatoxins (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
Ciguatera poisoning in Europe: A traceback to Indian Ocean sourced snapper fish (Lutjanus bohar)
No full textThe consumption of seafood containing marine biotoxins called ciguatoxins (CTXs) can result in ciguatera poisoning (CP), a globally prevalent seafood-born human illness. The southwestern coast of India is a regional source of seafood attributed to isolated and mass outbreaks of CP since 2015, both locally and exported globally. Samples of frozen snapper product (Lutjanus bohar) described herein, were part of a 7000 kg international shipment into the European Union from southwest India and implicated in a CP outbreak in the Netherlands. DNA barcoding confirmed the species as Lutjanus bohar with a base pair identity of 99%. LC-MS/MS and HRMS analyses describe CTX-3C-group compounds with an in vitro Neuro-2a (cell-based) cytotoxicity MTT-assay based toxicity range of 0.79–5.39 ng CTX-3C equivalent (eq.) per g wet tissue eq. This CP traceback includes an investigation and description of the production chain distribution, catch region, outbreak, toxin-group, and follow-up actions for the seafood products associated with the outbreak. Together this in-depth traceback investigation provides an account of the CP outbreak from harvest to consumption for a region of coastal India with a sizable seafood production industry but with limited CP data
Food Safety Risk in Germany From Mislabeled Imported Fish: Ciguatera Outbreak Trace-Back, Toxin Elucidation, and Public Health Implications
No full textCiguatera 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
Ciguatera Mini Review: 21st Century Environmental Challenges and the Interdisciplinary Research Efforts Rising to Meet Them
No full textGlobally, 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
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