Lipophilic Marine Biotoxins Research Articles

Determination of lipophilic marine biotoxins (azaspiracids, brevetoxins, and okadaic acid group) and domoic acid in mussels by solid-phase extraction and reversed-phase liquid chromatography with tandem mass spectrometry

An accurate and efficient method was developed for the determination of azaspiracid shellfish toxins (azaspiracids-1, -2, and -3), neurotoxic shellfish toxins (brevetoxins-2 and -3), diarrhetic shellfish toxins (okadaic acid and dinophysistoxins-1 and -2), and the amnesic shellfish toxin (domoic acid) in mussels (Mytilus galloprovincialis). Lipophilic marine biotoxins (azaspiracids, brevetoxins, and okadaic acid group) were extracted with 0.5 % acetic acid in methanol under heating at 60°C to improve the extraction efficiency of okadaic acid group toxins and then cleaned up with a C18 solid-phase extraction cartridge. Domoic acid was extracted with 50 % aqueous methanol and then cleaned up with a graphitized carbon solid-phase extraction cartridge. Lipophilic marine biotoxins and domoic acid were quantified by reversed-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The developed method had insignificant matrix effects for the nine analytes and good recoveries in the range of 79.0 % to 97.6 % at three spiking levels for all analytes except brevetoxin-2 (43.8–49.8 %). The developed method was further validated by analyzing mussel tissue certified reference materials, and good agreement was observed between certified and determined values.

Monthly distribution of lipophilic marine biotoxins and associated microalgae in the South Sea Coast of Korea throughout 2021

Aquaculture farms have been established along the South Sea Coast of Korea, supplying most of the seafood consumed domestically. However, annual harmful algal blooms pose a potential threat to seafood safety. This study aimed to determine the spatial and seasonal distributions of 12 lipophilic marine biotoxins (LMTs) in phytoplankton and mussels in the region in 2021. Solid-phase adsorption toxin tracking (SPATT) was used to monitor the cumulative compositions of LMTs in seawater. LMT concentrations were also determined in twelve commercially available species of domestic shellfish to evaluate the potential risks to human health. Gonyaulux spinifera and Dinophysis acuminata, causative microalgae of yessotoxins (YTXs) and pectenotoxins (PTXs), respectively, showed high densities in the region from May to July. This period corresponded to high LMT concentrations in phytoplankton and mussels. Phytoplankton mainly contained PTX-2 and homo-YTX, with a maximum concentration of 2300 ng g−1 wet weight (ww) in May. In contrast, mussels mainly contained homo-YTX and YTX, with a maximum concentration of 1300 ng g−1 ww in July. LMTs-producing microalgae showed low densities and concentrations after July, whereas mussels accumulated toxins until September. In the SPATT sampler, more diverse LMTs were detected than in seawater, phytoplankton, and mussels. For example, dinophysistoxin-1 and azaspiracid-2 were detected only in SPATT. YTXs were detected in domestic seafood samples, including mussels, red scallops, and pen shells, but the concentrations were below the European Food Safety Agency recommended standard of 3.75 mg YTX-eq. kg−1. Moreover, the hazard quotient was less than 100 in all scenarios, indicating that the human health risk was not significant. This study provides valuable data on monthly distribution patterns of LMTs in the South Sea Coast of Korea and can serve as baseline data for future management policies of marine biotoxins.

Official controls for the determination of lipophilic marine biotoxins in mussels farmed along the Adriatic coast of Central Italy.

In the present study, 334 samples of mussels (Mytilus galloprovincialis) harvested along the coasts of the Central Adriatic Sea during the years 2020-2021 were analyzed for the presence of lipophilic marine biotoxins according to the European Harmonized Standard Operating Procedure. The results showed that 74 (22%) and 84 (25%) samples were positive to okadaic acid and yessotoxin groups, respectively. Among them, only 11 (3.3%) samples resulted as non-compliant, as they exceeded the maximum limits (160 μg okadaic acid equivalent/kg) established by the Regulation (EC) 853/2004. The method applied in this study was able to detect and quantify lipophilic marine biotoxins concentrations, in order to monitor their presence in molluscs and avoid the risk of consumer exposure.

Determination of lipophilic marine biotoxins in aquatic products by liquid chromatography coupled with triple quadrupole mass spectrometry

Lipophilic marine biotoxins include Azaspiracid-1 (AZA-1), Azaspiracid-2 (AZA-2), Azaspiracid-3 (AZA-3), Pectenotoxin-2 (PTX 2), Okadaic acid (OA), Dinophysistoxin-2 (DTX-2), Dinophysistoxin-1 (DTX-1), Yessotoxin (YTX), and 1-Homoyessotoxin (Homo-YTX) were extracted with methanol, followed by cleaning up with solid phase extraction technique (SPE). Lipophilic toxins were confirmed and quantified by liquid chromatography coupled with triple quadrupole mass spectrometry (LC/MS/MS) using calibration curves on the solvent. The quantification limits of this method satisfied the requirements of the European Maximum Residue Limit (MRLs) with 25 µg/kg for the YTX group and 10 µg/kg for AZA, OA, and PTX groups. To validate the effectiveness of this method, matrices of clams, fish, and mixed seafood were collected and analyzed (recovery ranged from 92.4 - 101.5%, and relative standard deviation was less than 20%). The method was used successfully to participate in a proficiency testing program organized by Quasimeme (z-score in the range of ±2)

Spatiotemporal distribution characteristics of yessotoxins and pectenotoxins in phytoplankton and shellfish collected from the southern coast of South Korea

The distribution characteristics of lipophilic marine biotoxins (LMTs), such as yessotoxins (YTXs) and pectenotoxins (PTXs) in phytoplankton, mussels, and commercial seafood were determined for the southern coast of South Korea. Gonyaulax spinifera and Dinophysis acuminata, which are the causative microalgae of YTXs and PTXs, were recorded during summer. Homo-YTX and PTX-2 were predominantly detected in phytoplankton (max: 5.7 μg g−1 ww), whereas only YTXs were detected in mussels (max: 1.1 μg g−1 ww). LMT concentrations in mussels were positively correlated with those in phytoplankton. However, there was a 1-month time gap in maximum LMT concentrations between mussels and phytoplankton. Homo-YTX was detected in commercial seafood, including red scallop and comb pen shell. However, homo-YTX concentrations in shellfish were below the recommended value of the European Food Safety Authority (3.75 mg YTX equivalents kg−1); thus, the consumption of this seafood was not considered to be a significant risk for human health.

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.

Surveillance Plan of Lipophilic Marine Biotoxins in Molluscs from the Croatian Coast

In the present study, a 4-year surveillance plan for the detection of lipophilic marine biotoxins in Mytilus galloprovincialis and Callista chione from different aquaculture farms and natural beds along the Central Adriatic coasts of Croatia was reported. The samples were analyzed by a validated LC–MS/MS method in accordance with the Regulation (EU) No 2019/627. Lipophilic marine biotoxins belonging to the okadaic acid group, except for azaspiracids, as well as yessotoxins, were found at different concentrations, always below the maximum limits established by the Regulation (EC) No 853/2004. The seasonal distribution showed the highest values in summer months, and an interannual variability was also observed, probably due to environmental conditions aging on the density of harmful algal blooms. The comprehensive data reported in this study should help the future research in making advancing prediction models along the Eastern Adriatic coast.

Complex profiles of azaspiracid analogues in two culture strains of Azadinium poporum (Amphidomataceae, Dinophyceae) isolated from Japanese coastal waters determined by LC-MS/MS

Lipophilic marine biotoxins azaspiracids (AZAs) are produced by dinoflagellates Azadinium and Amphidoma. Recently, several strains of Azadinium poporum were isolated from Japanese coastal waters. In our present study, AZA analogues in two strains (mdd421 and HM536) of A. poporum were analyzed by several detection techniques on the liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOFMS). The dominant AZA analogue in the Japanese A. poporum strains was AZA2. Other known AZA analogues were AZA11, AZA35, AZA2 methyl ester and AZA2 phosphate ester. Besides these AZAs, thirteen new AZA analogues were discovered in the two strains. A putative AZA analogue (Compound 1) with the smallest molecular weight ever found in nature was also discovered in the two strains. This is the first report describing detailed AZA profiles in Japanese isolates of A. poporum.

New evidence of pectenotoxins in farmed bivalve molluscs from Sardinia (Italy).

Several planktonic dinoflagellates can produce lipophilic phycotoxins that represent a significant threat to public health as well as to shellfish and fish farming. Poisoning related to some of these toxins is categorised as diarrhetic shellfish poisoning. We analysed 975 shellfish samples from Tortoli in the central-eastern region of Sardinia (Italy) from January 2016 to March 2020, to investigate the prevalence of different lipophilic marine biotoxins in mollusc bivalves. The results highlighted the predominant presence of toxins belonging to the okadaic acid group in all samples with toxin concentrations exceeding legal limits, and revealed the new occurrence of pectenotoxins in oysters and clams with a winter seasonality in recent years. The origin of shellfish toxicity was associated with the same Dinophysis species, mainly D. acuminata. Based on both these results and other precedents, monitoring and recording systems are strongly recommended.

Assessment of the Presence of Lipophilic Phycotoxins in Scallops (Argopecten purpuratus) Farmed along Peruvian Coastal Waters

Some harmful algal blooms produce lipophilic marine biotoxins (LMTs) such as okadaic acid (OA; and its analogs dinophysistoxins [DTXs]), yessotoxins (YTXs), pectenotoxins (PTXs), and azaspiracids (AZAs), all of which may accumulate in filter-feeding bivalve mollusks. European health regulations stipulate a limit of 160 μg/kg for OA or DTXs, PTXs, and AZAs and 3.75 mg/kg for YTXs. Argopecten purpuratus is a valuable commercial marine bivalve exploited in Peru. Despite its importance and the periodic reports of the presence of harmful algal blooms in Peruvian coastal waters, information regarding potential contamination of these scallops by LMTs is lacking. We evaluated LMTs in 115 samples of A. purpuratus collected between November 2013 and March 2015 from 18 production areas distributed along the Peruvian coast. The hepatopancreas, which accumulates most of the toxins in the scallop, was analyzed with liquid chromatography-tandem mass spectrometry to quantify OA in its free form, YTX, AZA-1, and PTX-2. Baseline separation was achieved in 19 min. Linearity (R2 > 0.997), precision (coefficient of variation < 15%), and limits of quantification (0.155 to 0.479 ng/mL) were satisfactory. YTX was found in 72 samples, and PTX-2 was found in 17 samples, but concentrations of both biotoxins were below the regulatory limits. Free OA and AZA-1 were not detected in the scallop samples. This atypical profile (i.e., presence of PTX-2 and absence of OA) may be linked to the presence of the dinoflagellate Dinophysis acuminata. The production of YTX could be associated with the phytoplankton Gonyaulax spinifera and Protoceratium reticulatum. This is the first systematic assessment of the four types of LMTs in shellfish from Peruvian coastal waters. The results suggest low prevalence of LMTs in Peruvian bay scallops but support continued surveillance and analysis of LMTs in Peru.

Determination of lipophilic marine biotoxins by liquid chromatography-tandem mass spectrometry in five shellfish species from Washington State, USA

Low extraction efficiency (60-81%) of okadaic acid (OA) and dinophysistoxin 1 (DTX1) was obtained for 4 out of 5 shellfish species from Washington State (WA), USA, during application of a standard extraction method for determination of lipophilic marine biotoxins by LC-MS/MS as recommended by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). OA and total OA including esters, DTX1, DTX2, and total DTX including esters, azaspiracid 1, 2, and 3 (AZA1, AZA2, and AZA3), pectenotoxin 2 (PTX2), and yessotoxin (YTX) were the toxins examined. Matrix-matched standards prepared from the same control samples used for spike-and-recovery tests were employed to evaluate toxin extraction efficiency and sample clean-up procedures. We adjusted the EURLMB extraction method by either using an acidified methanol extraction or pre-cooking shellfish homogenates at 70 °C for 20 min before EURLMB extraction. Extraction efficiency was improved markedly for OA and DTX1 with both modified methods and for YTX with the pre-cooking step included. However, recoveries were lower for YTX using the acidified methanol extraction and for PTX2 in non-mussel samples with the pre-cooking step. A hexane wash was applied to clean water-diluted non-hydrolyzed samples and a hexane wash was combined with solid-phase extraction for cleaning hydrolyzed samples. Improved sample clean-up, combined with LC-MS/MS adjustments, enabled quantification of U.S. Food and Drug Administration-regulated toxins in five shellfish species from WA with acceptable accuracy using non-matrix matched calibration standards.

Morphological and molecular characterization of multiple new Azadinium strains revealed a high diversity of non‐toxigenic species of Amphidomataceae (Dinophyceae) including two new Azadinium species in Irish waters, North East Atlantic

SUMMARYShellfish contamination with azaspiracids (AZA), which are lipophilic marine biotoxins produced by marine dinoflagellates, is a major and recurrent problem for the Irish shellfish industry. AZA are produced by certain species of Amphidomataceae, but the species diversity of this group of microalgae in Irish waters is poorly known. Here we present a morphological and molecular characterization of multiple new strains of non‐toxigenic Azadinium isolated on an oceanographic survey in 2018. A lack of AZA production for all strains presented here was demonstrated by LC‐MS/MS analysis. One strain of Azadinium caudatum var. margalefii (first strain for the area) confirmed non‐toxigenicity of Atlantic populations of this species. One strain designated as Azadinium cf. zhuanum was similar to Az. zhuanum described from China but differed from the type strain in nucleus position, by the dominant number of apical plates, and by significant differences in rRNA gene sequences. Finally, two new non‐toxigenic Azadinium species are described from the North East Atlantic: Azadinium galwayense sp. nov. and Azadinium perfusorium sp. nov. Azadinium galwayense differed from other Azadinium by a characteristic combination regarding presence and location of the ventral pore (vp; on the right side of the pore plate), of a pyrenoid (located in the episome), and by a pentagonal shape of the median anterior intercalary plate 2a, and lack of contact between plates 1″ and 1a. Azadinium perfusorium shared the same vp position as Az. galwayense and differed by a characteristic combination of a pyrenoid located in the hyposome, a tetragonal shape of plate 2a, and a relatively large size of the two lateral anterior intercalary plates. Molecular phylogeny confirmed the distinctiveness of these two new species and their placement in Azadinium. The present findings significantly increased knowledge on the diversity of Azadinium species in the North East Atlantic.

Lipophilic Marine Biotoxins in Mussels from Bulgarian coast and Dietary Intake of Different Population Groups

AbstractIntroductionLipophilic phycotoxins, produced by toxic phytoplankton species, accumulate in digestive glands of mussels. If contaminated mussels containing marine biotoxins over regulatory levels are consumed, it is assumed that this could result in e.g. gastrointestinal disorders.ObjectivesThe aim of the study was to report contamination level of lipophilic marine biotoxins and to assess the potential health risk based on dietary intake for regular consumers and recreational harvesters from Bulgaria.Method/DesignWild and cultivated mussels were sampled in spring seasons of 2017 and 2018 from the North Black Sea coast. Positive phycotoxins concentrations determined via LC-MS/MS in extracts from digestive glands of the mussels were converted in phycotoxin levels for whole mussel meat (mm) by applying a factor of 5 proposed by EFSA. Dietary intakes were calculated by multiplying the mean positive phycotoxin levels (mg /kg mm) by the food consumption data for an average body weight (bw) of the investigated population group.Results and discussionIn total 20 farmed and 15 wild mussel samples were investigated. Yessotoxins and pectenotoxin-2 were detected. The mean YTX levels in cultivated mussels were measured about two times higher in spring 2018 (1.64 mg/kg) than in 2017 (0.70 mg/kg). The mean YTX level in wild mussels was found much lower - 0.46 mg/kg (spring 2017). In this regard, estimation of mean dietary YTX intake show that exposure of both population groups by consumption of cultivated mussels from spring 2018 is highest. Nevertheless, the regulatory threshold – acute reference dose of 25 μg/kg bw, is much above the highest calculated dietary intake (0.006 μg/kg female consumers). The mean PTX2 level in wild mussel samples from spring 2017 (0.71 mg/kg) was found about six times higher than in farmed mussels.The calculation of mean dietary PTX2 intake indicate that most exposed are females if wild mussels from spring 2017 were consumed. Still, the calculated value – 0.003 μg/kg bw were much lower than the ARfD - 0.8 μg/kg bw. All wild mussel samples from spring 2018 were negative. Lowest exposure to detected toxins were established for recreational harvesters.ConclusionThis study showed in general low contamination level with YTX and PTX2 of investigated mussel samples. Thereon, the estimated dietary intake of selected population groups is also much lower than the regulatory thresholds.

Analysis of Cyclic Imines in Mussels (Mytilus galloprovincialis) from Galicia (NW Spain) by LC-MS/MS.

Cyclic imines (CIs) are being considered as emerging toxins in the European Union, and a scientific opinion has been published by the European Food Safety Authority (EFSA) in which an assessment of the risks to human health related to their consumption has been carried out. Recommendations on the EFSA opinion include the search for data occurrence of CIs in shellfish and using confirmatory methods by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which need to be developed and optimized. The aim of this work is the application of LC-MS/MS to the analysis of gymnodimines (GYMs), spirolides (SPXs), pinnatoxins (PnTXs), and pteriatoxins (PtTXs) in mussels from Galician Rias, northwest Spain, the main production area in Europe, and therefore a representative emplacement for their evaluation. Conditions were adjusted using commercially available certified reference standards of GYM-A, SPX-1, and PnTX-G and evaluated through quality control studies. The EU-Harmonised Standard Operating Procedure for determination of lipophilic marine biotoxins in molluscs by LC-MS/MS was followed, and the results obtained from the analysis of eighteen samples from three different locations that showed the presence of PnTXs and SPXs are presented and discussed. Concentrations of PnTX-G and SPX-1 ranged from 1.8 to 3.1 µg/kg and 1.2 to 6.9 µg/kg, respectively, and PnTX-A was detected in the group of samples with higher levels of PnTX-G after a solid phase extraction (SPE) step used for the concentration of extracts.

Amphidomataceae (Dinophyceae) in the western Greenland area, including description of Azadinium perforatum sp. nov.

ABSTRACTAzaspiracids (AZA) are lipophilic marine biotoxins associated with shellfish poisoning which are produced by some species of Amphidomataceae. Diversity and global biogeography of this family are still poorly known. In summer 2017 plankton samples were collected from the central Labrador Sea and western Greenland coast from 64° N (Gothaab Fjord) to 75° N for the presence of Amphidomataceae and AZA. In the central Labrador Sea, light microscopy revealed small Azadinium-like cells (9200 cells l−1). Clonal strains established from plankton samples and scanning electron microscopy of fixed plankton samples revealed at least eight species of Amphidomataceae: Azadinium obesum, Az. trinitatum, Az. dexteroporum, Az. spinosum, Az. polongum, Amphidoma languida, Azadinium spec., and a new species described here as Azadinium perforatum sp. nov. The new species differed from other Azadinium species by the presence of thecal pores on the pore plate. All samples, including cultured strains, filtered seawater samples, and solid phase adsorption toxin tracking (SPATT) samplers deployed during the expedition in a continuous water-sampling system (FerryBox), were negative for AZA. DNA samples and PCR assays were positive for Amphidomataceae from most stations, whereas species-specific assays for three toxigenic species were rarely positive (two stations for Az. poporum, one station for Am. languida). The results highlight the presence of Amphidomataceae in the area but the lack of toxins and low abundance of toxigenic species currently indicate a low risk of toxic Amphidomataceae blooms in Arctic coastal waters.

Reversed-phase/weak anion exchange magnetic mesoporous microspheres for removal of matrix effects in lipophilic marine biotoxins analysis by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry

Magnetic solid-phase extraction (MSPE), using a new reversed-phase/weak anion exchange mix-mode mesoporous magnetic SiO2 adsorbent, was assessed as an approach for reducing matrix effects in the analysis of six lipophilic marine biotoxins in shellfish using ultrahigh-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The adsorbent showed greater adsorption capacity and selectivity for the analytes and, thus, the MSPE microspheres reduced the matrix effects significantly in the subsequent analysis. In the UPLC-MS/MS analysis, precursor and product ions of the analytes were monitored quantitatively and qualitatively using multiple reaction monitoring and product ion confirmation modes. The proposed method exhibited a linear correlation of 0.9980–0.9991 in the working range for azaspiracids (2.0–200.0 ng/mL) and okadaic acid and its derivatives dinophysistoxins (4.0–200.0 ng/mL) with satisfactory recoveries (82.8–118.6%, RSD < 12%), lower LODs (0.4–1.0 μg/kg) and LOQs (1.0–4.0 μg/kg) than existing methods. In addition, consumption of the adsorbent was reduced, and the MSPE operation is simple and rapid relative to alternatives. These results suggest the proposed method has potential for use in the analysis of lipophilic marine biotoxins in shellfish samples.

High abundance of Amphidomataceae (Dinophyceae) during the 2015 spring bloom of the Argentinean Shelf and a new, non-toxigenic ribotype of Azadinium spinosum

Azaspiracids (AZA) are the most recently discovered group of lipophilic marine biotoxins of microalgal origin, and associated with human incidents of shellfish poisoning. They are produced by a few species of Amphidomataceae, but diversity and occurrence of the small-sized dinophytes remain poorly explored for many regions of the world. In order to analyze the presence and importance of Amphidomataceae in a highly productive area of Argentinean coastal waters (El Rincón area, SW Atlantic), a scientific cruise was performed in 2015 to sample the early spring bloom. In a multi-method approach, light microscopy was combined with real-time PCR molecular detection of Amphidomataceae, with chemical analysis of AZA, and with the establishment and characterization of amphidomatacean strains. Both light microscopy and PCR revealed that Amphidomataceae were widely present in spring plankton communities along the El Rincón area. They were particularly abundant offshore at the shelf front, reaching peak densities of 2.8 × 105 cells L−1, but no AZA were detected in field samples. In total, 31 new strains were determined as Az. dalianense and Az. spinosum, respectively. All Az. dalianense were non-toxigenic and shared the same rRNA sequences. The large majority of the new Az. spinosum strains revealed for the first time the presence of a non-toxigenic ribotype of this species, which is otherwise the most important AZA producer in European waters. One of the new Az. spinosum strains, with a particular slender shape and some other morphological peculiarities, clustered with toxigenic strains of Az. spinosum from Norway and, exceptionally for the species, produced only AZA-2 but not AZA-1. Results indicate a wide diversity within Az. spinosum, both in terms of sequence data and toxin profiles, which also will affect the qualitative and quantitative performance of the specific qPCR assay for this species. Overall, the new data provide a more differentiated perspective of diversity, toxin productivity and occurrence of Amphidomataceae in a poorly explored region of the global ocean.

Determination of Lipophilic Marine Biotoxins in Shellfish by Online Turbulent Flow Chromatography Coupled to Liquid Chromatography–Tandem Mass Spectrometry

A novel method based on online turbulent flow chromatography (TFC) combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been established for simultaneous quantitative determination of 11 lipophilic marine toxins (OA, DTX1, DTX2, YTX, hYTX, AZA1, AZA2, AZA3, PTX2, SPX1, and GYM) in shellfish. Sample preparation, including extraction with 75% methanol aqueous solution, centrifugation, and filtration, was followed by online cleanup using TFC. The total run time was within 15.5 min, including online purification, chromatographic separation, and re-equilibration of the TFC system. Various experimental parameters including extraction, purification, separation, and detection were optimized separately in this study. The developed method was validated, and good performance characteristics were obtained. The linear regression coefficients (R2) of matrix-match calibration standard curves established for quantification were higher than 0.9945. The limits of detection and quantification of this method ranged between 0.5 and 4.0, and 1.5 and 10.0 µg/kg, respectively. The average recoveries of spiked target compounds with different concentrations ranged from 80.1 to 113.8%, the intra- and inter-day precision were lower than 12.7% and 14.9%, respectively. The matrix effects were in the range of 76.1–115.9%. The method accuracy was also confirmed with certified reference materials. The results indicate that the developed method has great potential for the routine laboratory analysis or emergency detection of lipophilic marine toxins in shellfish.

Lipophilic marine biotoxins SERS sensing in solutions and in mussel tissue

To detect and recognise three structurally related marine biotoxins responsible for the diarrheic shellfish poisoning (DSP) symptom, namely okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) respectively, as well as the structurally different yessotoxin (YTX), we developed a novel surface-enhanced micro-Raman scattering (micro-SERS) approach to investigate for the first time their micro-SERS signalling in solution and jointly analysed them in conjunction with the normal and toxic mussel tissue. YTX provided the main SERS feature surprisingly similar to DTX-1 and DTX-2, suggesting similar molecular adsorption mechanism with respect to the AgNPs. A fingerprint SERS band at 1017 cm−1 characteristic for the C-CH3 stretching in DTX-1 and DTX-2 and absent in OA SERS signal, allowed direct SERS discrimination of DTX-1,2 from OA. In acid form or as dissolved potassium salt, OA showed reproducible SERS feature for 0.81 μM to 84.6 nM concentrations respectively, while its ammonium salt slightly changed the overall SERS signature. The inherently strong fluorescence of the shellfish tissue, which hampers Raman spectroscopy analysis, further increases when toxins are present in tissue. Through SERS, tissue fluorescence is partially quenched. Artificially intoxicated mussel tissue with DSP toxins and incubated with AgNPs allowed direct SERS evidence of the toxin presence, opening a novel avenue for the in situ shellfish tracking and warning via micro-SERS. Natural toxic tissue containing 57.91 μg kg−1 YTX (LC-MS confirmed) was micro-SERS assessed to validate the new algorithm for toxins detection. We showed that a portable Raman system was able to reproduce the lab-based SERS results, being suitable for in situ raw seafood screening. The new approach provides an attractive, faster, effective and low-cost alternative for seafood screening, with economic, touristic and sustainable impact in aquaculture, fisheries, seafood industry and consumer trust.

Determination of Lipophilic Marine Biotoxins in Mussels Harvested from the Adriatic Sea by LC-MS/MS.

Lipophilic marine biotoxins include okadaic acid, pectenotoxin, yessotoxin and azaspiracid groups. The consumption of contaminated molluscs can lead to acute food poisoning syndromes depending on the exposure level. Regulatory limits have been set by Regulation (European Community, 2004a) No 853/2004 and LC-MS/MS is used as the official analytical method according to Regulation (European Community, 2011) No 15/2011. In this study specimens of mussels (Mytilus galloprovincialis) were collected along the coasts of the central Adriatic Sea during the years 2015–2017 and analyzed by the European harmonized Standard Operating Procedure. The method was validated for linearity, specificity, repeatability and reproducibility and it revealed able to be used for the detection of the lipophilic marine biotoxins. Levels of okadaic acid, pectenotoxin, yessotoxin and its analogs were detected at different concentrations in 148 (37%) out of a total of 400 samples, always below the maximum limits, except for 11 (4.3%) of them that were non-compliant because they exceeded the regulatory limit. Moreover, some samples were exposed to a multi-toxin mixture with regards to okadaic acid, yessotoxin and 1-Homo yessotoxin. Following these results, the aquaculture farms from which the non-compliant samples derived were closed until the analytical data of two consecutive samplings returned favorable. Besides the potential risk of consumption of mussels contaminated by lipophilic marine biotoxins, these marine organisms can be considered as bio-indicators of the contamination status of the marine ecosystem.