Characterization of Atmospheric Processes of Brevetoxins in Sea Spray Aerosols from Red Tide Events.

Atmospheric processes can affect the longevity of harmful toxins in sea spray aerosols (SSA). This study characterized the degradation of brevetoxin (BTx) in SSA under different environmental conditions. The samples of seawater collected during a Karenia brevis bloom in Manasota, Florida, were nebulized into a large outdoor photochemical chamber to mimic the atmospheric oxidation of aerosolized toxins and then aged in the presence or absence of sunlight and/or O3. Aerosol samples were collected during the aging process using a Particle-Into-Liquid Sampler. Their BTx concentrations were measured using an enzyme-linked immuno-sorbent assay (ELISA) and high-performance liquid chromatography/tandem mass spectroscopy. The BTx ozonolysis rate constant measured by ELISA was 5.74 ± 0.21 × 103 M-1 s-1. The corresponding lifetime for decay of 87.5% BTx in the presence of 20 ppb of O3 was 7.08 ± 0.26 h, suggesting that aerosolized BTx can still travel long distances at night before SSA deposition. BTx concentrations in SSA decreased more rapidly in the presence of sunlight than in its absence due to oxidation with photochemically produced OH radicals.

Structural Characterization of Maitotoxins Produced by Toxic Gambierdiscus Species.

Identifying compounds responsible for the observed toxicity of the Gambierdiscus species is a critical step to ascertaining whether they contribute to ciguatera poisoning. Macroalgae samples were collected during research expeditions to Rarotonga (Cook Islands) and North Meyer Island (Kermadec Islands), from which two new Gambierdiscus species were characterized, G. cheloniae CAWD232 and G. honu CAWD242. Previous chemical and toxicological investigations of these species demonstrated that they did not produce the routinely monitored Pacific ciguatoxins nor maitotoxin-1 (MTX-1), yet were highly toxic to mice via intraperitoneal (i.p.) injection. Bioassay-guided fractionation of methanolic extracts, incorporating wet chemistry and chromatographic techniques, was used to isolate two new MTX analogs; MTX-6 from G. cheloniae CAWD232 and MTX-7 from G. honu CAWD242. Structural characterization of the new MTX analogs used a combination of analytical chemistry techniques, including LC-MS, LC-MS/MS, HR-MS, oxidative cleavage and reduction, and NMR spectroscopy. A substantial portion of the MTX-7 structure was elucidated, and (to a lesser extent) that of MTX-6. Key differences from MTX-1 included monosulfation, additional hydroxyl groups, an extra double bond, and in the case of MTX-7, an additional methyl group. To date, this is the most extensive structural characterization performed on an MTX analog since the complete structure of MTX-1 was published in 1993. MTX-7 was extremely toxic to mice via i.p. injection (LD50 of 0.235 µg/kg), although no toxicity was observed at the highest dose rate via oral administration (155.8 µg/kg). Future research is required to investigate the bioaccumulation and likely biotransformation of the MTX analogs in the marine food web.

A mussel tissue certified reference material for multiple phycotoxins. Part 5: profiling by liquid chromatography-high-resolution mass spectrometry.

A freeze-dried mussel tissue-certified reference material (CRM-FDMT1) was prepared containing the marine algal toxin classes azaspiracids, okadaic acid and dinophysistoxins, yessotoxins, pectenotoxins, cyclic imines, and domoic acid. Thus far, only a limited number of analogues in CRM-FDMT1 have been assigned certified values; however, the complete toxin profile is significantly more complex. Liquid chromatography-high-resolution mass spectrometry was used to profile CRM-FDMT1. Full-scan data was searched against a list of previously reported toxin analogues, and characteristic product ions extracted from all-ion-fragmentation data were used to guide the extent of toxin profiling. A series of targeted and untargeted acquisition MS/MS experiments were then used to collect spectra for analogues. A number of toxins previously reported in the literature but not readily available as standards were tentatively identified including dihydroxy and carboxyhydroxyyessotoxin, azaspiracids-33 and -39, sulfonated pectenotoxin analogues, spirolide variants, and fatty acid acyl esters of okadaic acid and pectenotoxins. Previously unreported toxins were also observed including compounds from the pectenotoxin, azaspiracid, yessotoxin, and spirolide classes. More than one hundred toxin analogues present in CRM-FDMT1 are summarized along with a demonstration of the major acyl ester conjugates of several toxins. Retention index values were assigned for all confirmed or tentatively identified analogues to help with qualitative identification of the broad range of lipophilic toxins present in the material.

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.

Lipophilic Toxins in Wild Bivalves from the Southern Gulf of California, Mexico.

Most of the shellfish fisheries of Mexico occur in the Gulf of California. In this region, known for its high primary productivity, blooms of diatoms and dinoflagellates are common, occurring mainly during upwelling events. Dinoflagellates that produce lipophilic toxins are present, where some outbreaks related to okadaic acid and dinophisystoxins have been recorded. From January 2015 to November 2017 samples of three species of wild bivalve mollusks were collected monthly in five sites in the southern region of Bahía de La Paz. Pooled tissue extracts were analyzed using LC-MS/MS to detect lipophilic toxins. Eighteen analogs of seven toxin groups, including cyclic imines were identified, fortunately individual toxins did not exceed regulatory levels and also the total toxin concentration for each bivalve species was lower than the maximum permitted level for human consumption. Interspecific differences in toxin number and concentration were observed in three species of bivalves even when the samples were collected at the same site. Okadaic acid was detected in low concentrations, while yessotoxins and gymnodimines had the highest concentrations in bivalve tissues. Although in low quantities, the presence of cyclic imines and other lipophilic toxins in bivalves from the southern Gulf of California was constant.

Characterization of maitotoxin-4 (MTX4) using electrospray positive mode ionization high-resolution mass spectrometry and UV spectroscopy.

RATIONALE: The dinoflagellate genera Gambierdiscus and Fukuyoa are producers of toxins responsible for Ciguatera Poisoning (CP). Although having very low oral potency, maitotoxins (MTXs) are very toxic following intraperitoneal injection and feeding studies have shown they may accumulate in fish muscle. To date, six MTX congeners have been described but two congeners (MTX2 and MTX4) have not yet been structurally elucidated. The aim of the present study was to further characterize MTX4. METHODS: Chemical analysis was performed using liquid chromatography coupled to a diode-array detector (DAD) and positive ion mode high-resolution mass spectrometry (LC/HRMS) on partially purified extracts of G. excentricus (strain VGO792). HRMS/MS studies were also carried out to tentatively explain the fragmentation pathways of MTX and MTX4. RESULTS: The comparison of UV and HRMS (ESI+ ) spectra between MTX and MTX4 led us to propose the elemental formula of MTX4 (C157 H241 NO68 S2 , as the unsalted molecule). The comparison of the theoretical and measured m/z values of the doubly charged ions of the isotopic profile in ESI+ were coherent with the proposed elemental formula of MTX4. The study of HRMS/MS spectra on the tri-ammoniated adduct ([M - H + 3NH4 ]2+ ) of both molecules gave additional information about structural features. The cleavage observed, probably located at C99 -C100 in both MTX and MTX4, highlighted the same A-side product ion shared by the two molecules. CONCLUSIONS: All these investigations on the characterization of MTX4 contribute to highlighting that MTX4 belongs to the same structural family of MTXs. However, to accomplish a complete structural elucidation of MTX4, an NMR-based study and LC/HRMSn investigation will have to be carried out.

A Strategy to Replace the Mouse Bioassay for Detecting and Identifying Lipophilic Marine Biotoxins by Combining the Neuro-2a Bioassay and LC-MS/MS Analysis.

Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place for testing shellfish on a regular basis. In some countries testing is performed using the so-called mouse bioassay, an assay that faces ethical concerns not only because of animal distress, but also because it lacks specificity and results in high amounts of false positives. In Europe, for lipophilic marine biotoxins (LMBs), a chemical analytical method using LC-MS/MS was developed as an alternative and is now the reference method. However, safety is often questioned when relying solely on such a method, and as a result, the mouse bioassay might still be used. In this study the use of a cell-based assay for screening, i.e., the neuro-2a assay, in combination with the official LC-MS/MS method was investigated as a new alternative strategy for the detection and quantification of LMBs. To this end, samples that had been tested previously with the mouse bioassay were analyzed in the neuro-2a bioassay and the LC-MS/MS method. The neuro-2a bioassay was able to detect all LMBs at the regulatory levels and all samples that tested positive in the mouse bioassay were also suspect in the neuro-2a bioassay. In most cases, these samples contained toxin levels (yessotoxins) that explain the outcome of the bioassay but did not exceed the established maximum permitted levels.

Relative molar response of lipophilic marine algal toxins in liquid chromatography/electrospray ionization mass spectrometry.

RATIONALE: Accurate quantitative analysis of lipophilic toxins by liquid chromatography/mass spectrometry (LC/MS) requires calibration solution reference materials (RMs) for individual toxin analogs. Untargeted analysis is aimed at identifying a vast number of compounds and thus validation of fully quantitative untargeted methods is not feasible. However, a semi-quantitative approach allowing for profiling is still required and will be strengthened by knowledge of the relative molar response (RMR) of analogs in LC/MS with electrospray ionization (ESI). METHODS: RMR factors were evaluated for toxins from the okadaic acid (OA/DTXs), yessotoxin (YTX), pectenotoxin (PTX), azaspiracid (AZA) and cyclic imine (CI) toxin groups, in both solvent standards and environmental sample extracts. Since compound ionization and fragmentation influences the MS response of toxins, RMRs were assessed under different chromatographic conditions (gradient, isocratic) and MS acquisition modes (SIM, SRM, All-ion, target MS/MS) on low- and high-resolution mass spectrometers. RESULTS: In general, RMRs were not significantly impacted by chromatographic conditions (isocratic vs gradient), with the exception of DTX1. MS acquisition modes had a more significant impact, with PnTX-G and SPX differing notably. For a given toxin group, response factors were generally in the range of 0.5 to 2. The cyclic imines were an exception. CONCLUSIONS: Differences in RMRs between toxins of a same chemical base structure were not significant enough to indicate major issues for non-targeted semi-quantitative analysis, where there is limited or no availability of standards for many compounds, and where high degrees of accuracy are not required. Differences in RMRs should be considered when developing methods that use a standard of a single analogue to quantitate other toxins from the same group.

A mussel tissue certified reference material for multiple phycotoxins. Part 4: certification.

A freeze-dried mussel tissue (Mytilus edulis) reference material (CRM-FDMT1) was produced containing multiple groups of shellfish toxins. Homogeneity and stability testing showed the material to be fit for purpose. The next phase of work was to assign certified values and uncertainties to 10 analytes from six different toxin groups. Efforts involved optimizing extraction procedures for the various toxin groups and performing measurements using liquid chromatography-based analytical methods. A key aspect of the work was compensating for matrix effects associated with liquid chromatography-mass spectrometry through standard addition, dilution, or matrix-matched calibration. Certified mass fraction values are reported as mg/kg of CRM-FDMT1 powder as bottled for azaspiracid-1, -2, and -3 (4.10 ± 0.40; 1.13± 0.10; 0.96 ± 0.10, respectively), okadaic acid, dinophysistoxin-1 and -2 (1.59 ± 0.18; 0.68 ± 0.07; 3.57± 0.33, respectively), yessotoxin (2.49 ± 0.28), pectenotoxin-2 (0.66 ± 0.06), 13-desmethylspirolide-C (2.70 ± 0.26), and domoic acid (126 ± 10). Combined uncertainties for the certified values include contributions from homogeneity, stability, and characterization experiments. The commutability of CRM-FDMT1 was assessed by examining the extractability and matrix effects for the freeze-dried material in comparison with its equivalent wet tissue homogenate. CRM-FDMT1 is the first shellfish matrix CRM with certified values for yessotoxins, pectenotoxins or spirolides, and is the first CRM certified for multiple toxin groups. CRM-FDMT1 is a valuable tool for quality assurance of phycotoxin monitoring programs and for analytical method development and validation. Graphical Abstract CRM-FDMT1 is a multi-toxin mussel tissue certified reference material (CRM) to aid in development and validation of analytical methods for measuring the levels of algal toxins in seafood.

A mussel (Mytilus edulis) tissue certified reference material for the marine biotoxins azaspiracids.

Azaspiracids (AZAs) are lipophilic biotoxins produced by marine algae that can contaminate shellfish and cause human illness. The European Union (EU) regulates the level of AZAs in shellfish destined for the commercial market, with liquid chromatography-mass spectrometry (LC-MS) being used as the official reference method for regulatory analysis. Certified reference materials (CRMs) are essential tools for the development, validation, and quality control of LC-MS methods. This paper describes the work that went into the planning, preparation, characterization, and certification of CRM-AZA-Mus, a tissue matrix CRM, which was prepared as a wet homogenate from mussels (Mytilus edulis) naturally contaminated with AZAs. The homogeneity and stability of CRM-AZA-Mus were evaluated, and the CRM was found to be fit for purpose. Extraction and LC-MS/MS methods were developed to accurately certify the concentrations of AZA1 (1.16 mg/kg), AZA2 (0.27 mg/kg), and AZA3 (0.21 mg/kg) in the CRM. Quantitation methods based on standard addition and matrix-matched calibration were used to compensate for the matrix effects in LC-MS/MS. Other toxins present in this CRM at lower levels were also measured with information values reported for okadaic acid, dinophysistoxin-2, yessotoxin, and several spirolides.

Potential threats posed by new or emerging marine biotoxins in UK waters and examination of detection methodology used in their control: brevetoxins.

Regular occurrence of brevetoxin-producing toxic phytoplankton in commercial shellfishery areas poses a significant risk to shellfish consumer health. Brevetoxins and their causative toxic phytoplankton are more limited in their global distribution than most marine toxins impacting commercial shellfisheries. On the other hand, trends in climate change could conceivably lead to increased risk posed by these toxins in UK waters. A request was made by UK food safety authorities to examine these toxins more closely to aid possible management strategies, should they pose a threat in the future. At the time of writing, brevetoxins have been detected in the Gulf of Mexico, the Southeast US coast and in New Zealand waters, where regulatory levels for brevetoxins in shellfish have existed for some time. This paper reviews evidence concerning the prevalence of brevetoxins and brevetoxin-producing phytoplankton in the UK, together with testing methodologies. Chemical, biological and biomolecular methods are reviewed, including recommendations for further work to enable effective testing. Although the focus here is on the UK, from a strategic standpoint many of the topics discussed will also be of interest in other parts of the world since new and emerging marine biotoxins are of global concern.

Occurrence of lipophilic marine toxins in shellfish from Galicia (NW of Spain) and synergies among them.

Lipophilic marine toxins pose a serious threat for consumers and an enormous economic problem for shellfish producers. Synergistic interaction among toxins may play an important role in the toxicity of shellfish and consequently in human intoxications. In order to study the toxic profile of molluscs, sampled during toxic episodes occurring in different locations in Galicia in 2014, shellfish were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), the official method for the detection of lipophilic toxins. The performance of this procedure was demonstrated to be fit for purpose and was validated in house following European guidelines. The vast majority of toxins present in shellfish belonged to the okadaic acid (OA) group and some samples from a particular area contained yessotoxin (YTX). Since these toxins occur very often with other lipophilic toxins, we evaluated the potential interactions among them. A human neuroblastoma cell line was used to study the possible synergies of OA with other lipophilic toxins. Results show that combination of OA with dinophysistoxin 2 (DTX2) or YTX enhances the toxicity triggered by OA, decreasing cell viability and cell proliferation, depending on the toxin concentration and incubation time. The effects of other lipophilic toxins as 13-desmethyl Spirolide C were also evaluated in vitro.

Responses of sympatric Karenia brevis, Prorocentrum minimum, and Heterosigma akashiwo to the exposure of crude oil.

Impacts of the Deepwater Horizon oil spill on phytoplankton, particularly, the tolerability and changes to the toxin profiles of harmful toxic algal species remain unknown. The degree to which oil-affected sympatric Karenia brevis, Prorocentrum minimum, and Heterosigma akashiwo, all of which are ecologically important species in the Gulf of Mexico, was investigated. Comparison of their tolerability to that of non-toxic species showed that the toxin-production potential of harmful species does not provide a selective advantage. Investigated toxin profiles for K. brevis and P. minimum demonstrated an increase in toxin productivity at the lowest crude oil concentration (0.66 mg L(-1)) tested in this study. Higher crude oil concentrations led to significant growth inhibition and a decrease in toxin production. Findings from this study could assist in the assessment of shellfish bed closures due to high risk of increased toxin potential of these phytoplankton species, especially during times of stressed conditions.

Synchronic distribution of the dinoflagellate Protoceratium reticulatum and yessotoxins in a high stratified fjord system: Tidal or light modulation?

Protoceratium reticulatum is the main yessotoxin-producer along the Chilean coast. Thus far, the yessotoxin levels recorded in this region have not posed a serious threat to human health. However, a bloom of P. reticulatum during the austral summer of 2022 caused the first ban of shellfish collection, due to the high toxin levels. A bloom of P. reticulatum during the austral summer of 2020 allowed an evaluation of the fine-scale distribution of the dinoflagellate during a tidal cycle. High-resolution measurements of biophysical properties were carried out in mid-summer (February 18-19) at a fixed sampling station in Puyuhuapi Fjord, Chilean Patagonia, as part of an intensive 24-h biophysical experiment to monitor the circadian distributions of P. reticulatum vegetative cells and yessotoxins. High P. reticulatum cell densities (>20 × 103 cells L-1) were found in association with a warmer (14.5-15 °C) and estuarine (23.5-24.5 g kg-1) sub-surface water layer (6-8 m). P. reticulatum cell numbers and yessotoxins followed a synchronic distribution pattern consistent with the excursions of the pycnocline. Nevertheless, the surface aggregation of the cells was modulated by the light cycle, suggesting daily vertical migration. The yessotoxin content per P. reticulatum cell ranged from 9.4 to 52.2 pg. This study demonstrates both the value of fine-scale resolution measurements of biophysical properties in a highly stratified system and the potential ecosystem impact of P. reticulatum strains producing high levels of yessotoxins.

Novel electrochemical immunoassay for quantitative monitoring of biotoxin using target-responsive cargo release from mesoporous silica nanocontainers.

A novel homogeneous immunoassay protocol was designed for quantitative monitoring of small molecular biotoxin (brevetoxin B, PbTx-2, as a model) by using target-responsive cargo release from polystyrene microsphere-gated mesoporous silica nanocontainer (MSN). Initially, monoclonal mouse anti-PbTx-2 capture antibody was covalently conjugated onto the surface of MSN (mAb-MSN), and the electroactive cargo (methylene blue, MB) was then trapped in the pores of mAb-MSN by using aminated polystyrene microspheres (APSM) based on the electrostatic interaction. Upon addition of target PbTx-2, the positively charged APSM was displaced from the negatively charged mAb-MSN because of the specific antigen-antibody reaction. Thereafter, the molecular gate was opened, and the trapped methylene blue was released from the pores. The released methylene blue could be monitored by using a square wave voltammetry (SWV) in a homemade microelectrochemical detection cell. Under optimal conditions, the SWV peak current increased with the increasing of PbTx-2 concentration in the range from 0.01 to 3.5 ng mL(-1) with a detection limit (LOD) of 6 pg mL(-1) PbTx-2 at the 3Sblank criterion. Intra- and interassay coefficients of variation with identical batches were ≤6% and 9.5%, respectively. The specificity and sample matrix interfering effects were acceptable. The analysis in 12 spiked seafood samples showed good accordance between results obtained by the developed immunoassay and a commercialized enzyme-linked immunosorbent assay (ELISA) method. Importantly, the target-responsive controlled release system-based electrochemical immunoassay (CRECIA) offers a promising scheme for the development of advanced homogeneous immunoassay without the sample separation and washing procedure.

Displacement-type quartz crystal microbalance immunosensing platform for ultrasensitive monitoring of small molecular toxins.

A novel displacement-type quartz crystal microbalance (QCM) immunosensing strategy, based on glucose and its analogue dextran for concanavalin A (ConA) binding sites, was designed for ultrasensitive monitoring of small molecular biotoxins (brevetoxin B, PbTx-2, used as a model) with signal amplification on a graphene-functionalized sensing interface. To construct such a QCM immunosensing platform, phenoxy-functionalized dextran (DexP) was initially assembled onto the surface of graphene-coated QCM probe via the π-stacking interaction, and ConA-labeled monoclonal mouse anti-PbTx-2 capture antibody was then immobilized on the DexP-modified probe by dextran-ConA binding. Gold nanoparticle heavily functionalized with glucoamylase and bovine serum albumin-PbTx-2 (PbTx-2-BSA) conjugate was employed as the trace tag. A competitive-type immunoassay format was adopted for the online monitoring of PbTx-2 between anti-PbTx-2 antibody immobilized on the QCM probe and PbTx-2-BSA labeled on the gold nanoparticle. Accompanying the gold nanoparticle, the carried glucoamylase could hydrolyze amylopectin in glucose. The produced glucose competed with dextran for ConA and displaced the ConA-streptavidin-anti-PbTx-2 complex from the QCM probe, resulting in the frequency change. Under optimal conditions, the frequency of the QCM immunosensor was indirectly proportional to the concentration of target PbTx-2 in the sample and exhibited a dynamic range from 1.0 pg·mL(-1) to 10 ng·mL(-1) with a detection limit (LOD) of 0.6 pg·mL(-1) at the 3Sblank level. Intra- and interassay coefficients of variation were below 7.5% and 9.5%, respectively. In addition, the methodology was evaluated for analysis of PbTx-2 in 15 spiked seafood samples and showed good accordance between results obtained by the displacement-type QCM immunosensor and a commercialized enzyme-linked immunosorbent assay (ELISA) method.

[Application of a fluorescent dye method based on changes in membrane potential in detecting shellfish toxins].

OBJECTIVE: To develop a rapid and sensitive assay for detecting the common sea food toxins including paralytic shellfish poisoning toxin (PST), tetrodotoxin (TTX) and neurotoxic shellfish poisoning toxin (NST) based on their toxicological character. METHODS: Neuroblastoma cells were incubated with the fluorescent dye bis-oxonol, whose distribution across the membrane was potential-dependent. Changes in membrane potential of the cells induced by gonyautoxins (GTX2,3), brevetoxin (BTX) and TTX were observed respectively, using bis-oxonol. RESULTS: Within 2 - 200 nmol/L of GTX2,3 or 20 - 600 nmol/L of TTX, veratridine-induced depolarization was shown to be inhibited by GTX2,3 or TTX in dose-dependent manner. Within 15 - 400 ng/ml, there was a dose-dependent relationship between the NSP-induced depolarization and toxin concentration. CONCLUSION: It was likely to find a rapid, specific, and reliable method with bis-oxonol for detecting GTX2,3, TTX and BTX in sea food.

Toxin levels and profiles in microalgae from the north-Western Adriatic Sea--15 years of studies on cultured species.

The Northern Adriatic Sea is the area of the Mediterranean Sea where eutrophication and episodes related to harmful algae have occurred most frequently since the 1970s. In this area, which is highly exploited for mollusk farming, the first occurrence of human intoxication due to shellfish consumption occurred in 1989, nearly 10 years later than other countries in Europe and worldwide that had faced similar problems. Until 1997, Adriatic mollusks had been found to be contaminated mostly by diarrhetic shellfish poisoning toxins (i.e., okadaic acid and dinophysistoxins) that, along with paralytic shellfish poisoning toxins (i.e., saxitoxins), constitute the most common marine biotoxins. Only once, in 1994, a toxic outbreak was related to the occurrence of paralytic shellfish poisoning toxins in the Adriatic coastal waters. Moreover, in the past 15 years, the Adriatic Sea has been characterized by the presence of toxic or potentially toxic algae, not highly widespread outside Europe, such as species producing yessotoxins (i.e., Protoceratium reticulatum, Gonyaulax spinifera and Lingulodinium polyedrum), recurrent blooms of the potentially ichthyotoxic species Fibrocapsa japonica and, recently, by blooms of palytoxin-like producing species of the Ostreopsis genus. This review is aimed at integrating monitoring data on toxin spectra and levels in mussels farmed along the coast of the Emilia-Romagna region with laboratory studies performed on the species involved in the production of those toxins; toxicity studies on toxic or potentially toxic species that have recently appeared in this area are also reviewed. Overall, reviewed data are related to: (i) the yessotoxins producing species P. reticulatum, G. spinifera and L. polyedrum, highlighting genetic and toxic characteristics; (ii) Adriatic strains of Alexandrium minutum, Alexandrium ostenfeldii and Prorocentrum lima whose toxic profiles are compared with those of strains of different geographic origins; (iii) F. japonica and Ostreopsis cf. ovata toxicity. Moreover, new data concerning domoic acid production by a Pseudo-nitzschia multistriata strain, toxicity investigations on a Prorocentrum cf. levis, and on presumably ichthyotoxic species, Heterosigma akashiwo and Chattonella cf. subsalsa, are also reported.

Determination of brevetoxins in shellfish by LC/MS/MS: single-laboratory validation.

A single-laboratory validation is reported for an LC/MS/MS quantification of six brevetoxins in four matrixes (Greenshell mussel, eastern oyster, hard clam, and Pacific oyster). Recovery and precision data were collected from seven analytical batches using shellfish flesh at 0.05 mg/kg. Method recoveries and within-laboratory reproducibility ranged from 73 to 112%, with an RSD between 14 and 18% for brevetoxin-3, brevetoxin B5, brevetoxin B2, and S-desoxy brevetoxin B2. The recovery and within-laboratory reproducibility for brevetoxin-2 was 61%, with an RSD of 27%. Brevetoxin B1 gave an RSD of 12%, but no reference material was available and this toxin was only recorded in a hard clam sample naturally contaminated with brevetoxins. One naturally contaminated sample of each shellfish matrix, with brevetoxin levels ranging from 0.012 to 9.9 mg/kg, was tested in multiple batches, and the RSDs were similar to those for fortified samples at 0.05 mg/kg. Comparisons with limited data for the neurotoxic shellfish poisoning mouse bioassay for four naturally contaminated shellfish samples showed that the regulatory action limit of 0.8 mg/kg is conservative with respect to the bioassay regulatory limit of 20 mouse units/100 g.

Brevetoxicosis: red tides and marine mammal mortalities.

Potent marine neurotoxins known as brevetoxins are produced by the 'red tide' dinoflagellate Karenia brevis. They kill large numbers of fish and cause illness in humans who ingest toxic filter-feeding shellfish or inhale toxic aerosols. The toxins are also suspected of having been involved in events in which many manatees and dolphins died, but this has usually not been verified owing to limited confirmation of toxin exposure, unexplained intoxication mechanisms and complicating pathologies. Here we show that fish and seagrass can accumulate high concentrations of brevetoxins and that these have acted as toxin vectors during recent deaths of dolphins and manatees, respectively. Our results challenge claims that the deleterious effects of a brevetoxin on fish (ichthyotoxicity) preclude its accumulation in live fish, and they reveal a new vector mechanism for brevetoxin spread through food webs that poses a threat to upper trophic levels.