13-desmethyl Spirolide Research Articles

Detection of Cyclic Imines (CIs) Toxins in Whole Body of Shellfishes: First Monitoring Report of CIs in South Korea Shellfishes

Cyclic imines (CIs) produced by microalgal species that accumulate in the food chains of marine organisms are novel biotoxins that do not belong to the classic group of marine biotoxins. In the past, CIs were found only in limited areas; however, in recent years, rapid changes in marine ecosystems have led to widespread CIs and increased exposure to toxic risks. In this study, we analyzed seven CI toxins, GYM-A, SPX (13-desmethyl spirolide C, 13, 19-dideMe spirolide C, 20-methyl spirolide G), and PnTX-E, F, and G, using LC/MRM-MS. Shellfish samples were purchased from a domestic Korean fish market (67 samples in 2021 and 216 samples in 2022). The entire body of the shellfish was ground and extracted with 50% methanol, followed by lipophilic-specific SPE. Only GYM-A, PnTX-G, and 13-desmethyl spirolide C were detected in all analyzed samples. The maximum concentrations of GYM-A is maximum 179 ppt (ng/kg) in Crassostrea nippona (March 2022), PnTX-G is maximum 7 ppt in Anadara broughtonii (April 2022), 13-desmethyl SPX C is maximum 58 ppt in Crassostrea nippona (April 2022). The southern coast exhibited the highest frequency of detection of these toxins, which was attributed to elevated sea-surface temperatures, aligned with conducive conditions for toxin-producing phytoplankton. According to the monitoring results, there were no significant CI toxins in the shellfish; however, it is important to monitor CI toxin accumulation in shellfish because of their high risk of toxicity.

Occurrence and distribution of phycotoxins in the Antarctic Ocean

Lipophilic phycotoxins (LPTs) and domoic acid (DA) in Antarctic seawater, as well as parts of the South Pacific and the Southern Indian Oceans were systematically investigated. DA and six LPTs, namely pectenotoxin-2 (PTX2), okadaic acid (OA), yessotoxin (YTX), homo-yessotoxin (h-YTX), 13-desmethyl spirolide C (SPX1), and gymnodimine (GYM), were detected. PTX2, as the dominant LPTs, was widely distributed in seawater surrounding Antarctica, whereas OA, YTX, and h-YTX were irregularly distributed across the region. The total concentration of LPTs in surface seawater ranged from 0.10 to 13.57 ng/L (mean = 2.20 ng/L). ∑LPT levels were relatively higher in the eastern sea areas of Antarctica than in the western sea areas. PTX2 was the main LPT in the vertical profiles, and the PTX2 concentration was significantly higher in the epipelagic zone than water depths below 200 m. The predominant sources of PTX2 and OA in Antarctic sea areas are likely to be Dinophysis.

Lipophilic Shellfish Poisoning Toxins in Marine Invertebrates from the Galician Coast.

For the purpose of assessing human health exposure, it is necessary to characterize the toxins present in a given area and their potential impact on commercial species. The goal of this research study was: (1) to screen the prevalence and concentrations of lipophilic toxins in nine groups of marine invertebrates in the northwest Iberian Peninsula; (2) to evaluate the validity of wild mussels (Mytilus galloprovincialis) as sentinel organisms for the toxicity in non-bivalve invertebrates from the same area. The screening of multiple lipophilic toxins in 1150 samples has allowed reporting for the first time the presence of 13-desmethyl spirolide C, pinnatoxin G, okadaic acid, and dinophysistoxins 2 in a variety of non-traditional vectors. In general, these two emerging toxins showed the highest prevalence (12.5-75%) in most of the groups studied. Maximum levels for 13-desmethyl spirolide C and pinnatoxin G were found in the bivalves Magallana gigas (21 µg kg-1) and Tellina donacina (63 µg kg-1), respectively. However, mean concentrations for the bivalve group were shallow (2-6 µg kg-1). Okadaic acid and dinophysistoxin 2 with lower prevalence (1.6-44.4%) showed, on the contrary, very high concentration values in specific species of crustaceans and polychaetes (334 and 235 µg kg--1, respectively), to which special attention should be paid. Statistical data analyses showed that mussels could be considered good biological indicators for the toxicities of certain groups in a particular area, with correlations between 0.710 (for echinoderms) and 0.838 (for crustaceans). Polychaetes could be an exception, but further extensive surveys would be needed to draw definitive conclusions.

Occurrence and phase distribution of lipophilic marine algal toxins in the bottom boundary layer and sediment-porewater system of two mariculture sites

To date, understanding the fate of lipophilic marine algal toxins (LMATs) in benthic environments on which cultivated shellfish depend is still limited. In this work, the occurrence, concentration levels, and phase distributions of LMATs in the benthic environments of two mariculture sites (Sishili and Rongcheng Bays) in China were investigated for the first time. Five LMATs: okadaic acid (OA), pectenotoxin-2 (PTX2), gymnodimine, 13-desmethyl spirolide C, and azaspiracid-2 (AZA2) and three derivatives: dinophysistoxin-1 isomer (DTX1-iso), pectenotoxin-2 seco acid, and 7-epi- pectenotoxin-2 seco acid were detected in different environmental samples. OA and PTX2 were the dominant LMATs in the bottom boundary layer (BBL) and sediment, whereas AZA2 was present in the sediment only. Notably, DTX1-iso was found for the first time to be widely distributed in the benthic environments of the bays. In BBL, the average proportion of LMATs in the dissolved phase (99.20%) was much higher than in the particulate phase (0.80%). Partition of LMATs was more balanced between sediment porewater (57.80% average proportion) and sediment (42.20%). The concentrations of ∑LMATs in the BBL seawater ranged from 19.09 ng/L to 41.57 ng/L (mean of 32.67 ng/L), and the spatial distribution trend was higher in offshore than nearshore. ∑LMATs concentrations in the sediment and porewater of the two bays ranged from 17.04 ng/kg to 150.13 ng/kg (mean of 53.58 ng/kg) and from 8.29 ng/L to 120.58 ng/L (mean of 46.63 ng/L), respectively. Their spatial distributions differed from those in BBL, showing a trend of high concentrations in areas with heavy land-based inputs. ∑LMATs concentrations in porewater were significantly higher than those in BBL seawaters, suggesting that the potential hazards of LMATs to benthic organisms may be underestimated.

Occurrence and distribution of lipophilic marine algal toxins in the coastal seawater of Southeast China and the South China Sea

The composition, levels, and spatial distribution of dissolved lipophilic marine algal toxins (LMATs) including cyclic imines (CIs), yessotoxins (YTXs), okadaic acid (OA) and its derivatives, pectenotoxins (PTXs), azaspiracids (AZAs), and brevetoxins (BTXs) in the coastal waters of Southeast China (Xiamen) and the South China Sea (Hainan Island and Beibu Gulf) were investigated and compared for the first time. Dissolved AZA3 was firstly detected in the coastal seawater of China. OA and PTX2 were widely distributed in the three areas studied. Gymnodimine (GYM), 13-desmethyl spirolide C (SPX1), YTX, and homo-yessotoxins (h-YTX) were found mainly in the South China Sea. The average ∑LMAT concentrations in the coastal waters of Xiamen, Hainan Island, and Beibu Gulf were 10.02 ng/L, 4.21 ng/L, and 44.27 ng/L, respectively. More groups and much higher concentrations of LMATs occurred in the South China Sea than that in the other sea areas of China.

Stereoselective synthesis of the spirocyclic core of 13-desmethyl spirolide C using an aza-Claisen rearrangement and an exo-selective Diels-Alder cycloaddition.

13-Desmethyl spirolide C is a marine natural product of the cyclic imine class that demonstrates remarkable bioactivity against several biomarkers of Alzheimer's Disease, which renders its [7,6]-spirocyclic imine pharmacophore of significant synthetic interest. This work describes a facile and efficient synthesis of the [7,6]-spirocyclic core of 13-desmethyl spirolide C from inexpensive starting materials, featuring an aza-Claisen rearrangement to simultaneously set both stereocentres of the dimethyl moiety with complete atom economy, and a highly exo-selective Diels-Alder cycloaddition to construct the challenging contiguous tertiary and quaternary stereocentres of the spirocyclic core of 13-desmethyl spirolide C. A comprehensive study of the key Diels-Alder reaction was also performed to evaluate the stereoselectivity and reactivity of various functionalised dienes and protected lactam dienophiles, wherein the first successful exo-selective Diels-Alder cycloaddition to construct spirocyclic structures using a bromodiene and α-exo-methylene dienophiles is reported. This strategy not only establishes a more efficient stereoselective access to the spirocyclic core that can be used for the total synthesis of 13-desmethyl spirolide C, but also serves as a sound platform for convenient preparations of a range of spirocyclic analogues required for a comprehensive biological evaluation of this desirable pharmacophore.

Spirolides in Bivalve Mollusk of the Galician (NW Spain) Coast: Interspecific, Spatial, Temporal Variation and Presence of an Isomer of 13-Desmethyl Spirolide C.

Spirolides are cyclic imines whose risks to human health have not been sufficiently evaluated. To determine the possible impact of these compounds in Galicia (NW Spain), their presence and concentration in bivalve mollusk were studied from 2014 to 2021. Only 13-desmethyl spirolide C (13desmSPXC) and an isomer have been detected, and always at low concentrations. Mussel, Mytilus galloprovincialis, was the species which accumulated more spirolides, but the presence of its isomer was nearly restricted to cockle, Cerastoderma edule, and two clam species, Venerupis corrugata and Polititapes rhomboides. On average, the highest 13desmSPXC levels were found in autumn-winter, while those of its isomer were recorded in spring-summer. Both compounds showed decreasing trends during the study period. Geographically, the concentration tends to decrease from the southern to the north-eastern locations, but temporal variability predominates over spatial variability.

Enzymatic Biotransformation of 13-desmethyl Spirolide C by Two Infaunal Mollusk Species: The Limpet Patella vulgata and the Cockle Cerastoderma edule.

The presence of a 13-desmethyl Spirolide C isomer (Iso-13-desm SPX C) is very common in some infaunal mollusks in Galicia contaminated with this toxin. Its possible origin by biological transformation was investigated by incubating homogenates of the soft tissues of limpets and cockles spiked with 13-desmethyl Spirolide C (13-desm SPX C). The involvement of an enzymatic process was also tested using a raw and boiled cockle matrix. The enzymatic biotransformation of the parent compound into its isomer was observed in the two species studied, but with different velocities. The structural similarity between 13-desm SPX C and its isomer suggests that epimerization is the most likely chemical process involved. Detoxification of marine toxins in mollusks usually implies the enzymatic biotransformation of original compounds, such as hydroxylation, demethylation, or esterification; however, this is the first time that this kind of transformation between spirolides in mollusks has been demonstrated.

Novel Non-paralytic Shellfish Toxin and Non-spirolide Toxicity to Finfish, Brine Shrimp, and Rotifer Observed in a Culture of the Dinoflagellate Alexandrium insuetum Isolated From the Coastal Water of China

The genus Alexandrium is one of the major harmful algal blooms (HABs)-forming dinoflagellate group and at least half of ~40 described species have been reported to produce paralytic shellfish toxins (PSTs). The potentially harmful species Alexandrium insuetum has been reported from many countries of Asia and Europe, and to have paralytic shellfish poisoning toxicity, but no mortality of marine animals was observed during its bloom. Therefore, it is ecologically important to characterize the possible toxicity and toxins of this organism. In this study, based on the establishment of two clonal cultures through cyst germination collected from the Yellow Sea, we identified A. insuetum from China as the first record via light microscopy (LM) and scanning electron microscopy (SEM) observations and phylogenetic analyses. The cultures of A. insuetum were further observed to be toxic to finfish and zooplankton and deleterious to rotifer eggs via laboratory bioassays. The exposure bioassays using rotifer (Brachionus plicatilis), brine shrimp (Artemia salina), and larval finfish (Oryzias melastigma) demonstrated that A. insuetum caused significant lethal effects on finfish and zooplankton species. Rotifer bioassays using cell-free culture medium, heat-treated cultures, and water, methanol, and trichloromethane extracts of algal cells revealed that A. insuetum produced heat-labile, water-soluble toxin(s) that could be excreted from A. insuetum cells and steadily accumulated in the medium during the growth phases. Hatching success of rotifer eggs was also found to be seriously affected by the exposure to A. insuetum. Importantly, ultra-high performance liquid chromatography-tandem mass spectrometry [UPLC (or LC)-MS-MS] analyses suggest the above-described toxicity of A. insuetum was caused by neither PSTs nor spiroimines (13-desmethyl spirolide C and gymnodimine). Collectively, our findings demonstrated the novel toxicity to finfish and zooplankton in A. insuetum, which is ecologically important in not only possibly contributing to population dynamics and even the formation of HABs of the species, but also affecting the on-the-spot survival and the reproduction potency of marine animals. The present work is believed to set a cornerstone for the monitoring and risk assessment of the species along the coastal waters of China and for understanding the general ecology of A. insuetum.

Potent allelopathy and non-PSTs, non-spirolides toxicity of the dinoflagellate Alexandrium leei to phytoplankton, finfish and zooplankton observed from laboratory bioassays.

The dinoflagellate genus Alexandrium has been well known for causing paralytic shellfish poisoning (PSP) worldwide. Several non-PSP toxin-producing species, however, have shown to exhibit fish-killing toxicity. Here, we report the allelopathic activity of Alexandrium leei from Malaysia to other algal species, and its toxicity to finfish and zooplankton, via laboratory bioassays. Thirteen microalgal species that co-cultured with Al. leei revealed large variability in the allelopathic effects of Al. leei on the test algae, with the growth inhibition rates ranging from 0 to 100%. The negative allelopathic effects of Al. leei on microalgae included loss of flagella and thus the motility, damages of chain structure, deformation in cell morphology, and eventually cell lysis. The finfish experienced 100% mortality within 24h exposed to the live culture (2000-6710cells·mL-1), while the rotifer and brine shrimp exhibited 96-100% and 90-100% mortalities within 48h when exposed to 500-6000cells·mL-1 of Al. leei. The mortality of the test animals depended on the Al. leei cell density exposed, leading to a linear relationship between mortality and cell density for the finfish, and a logarithmic relationship for the two zooplankters. When exposed to the treatments using Al. leei whole live culture, cell-free culture medium, extract of algal cells in the f/2-Si medium, extract of methanol, and the re-suspended freeze-and-thaw algal cells, the test organisms (Ak. sanguinea and rotifers) all died at the cell density of 8100cells·mL-1 within 24h. Toxin analyses by HILIC-ESI-TOF/MS and LC-ESI-MS/MS demonstrated that Al. leei did not produce PSP-toxins and 13-desmethyl spirolide C. Overall, our findings demonstrated potent allelopathy and toxicity of Al. leei, which do not only pose threats to the aquaculture industry, fisheries, and marine ecosystems but may also play a part role in the population dynamics and bloom formation of this species.

Gymnodimine A in mollusks from the north Atlantic Coast of Spain: Prevalence, concentration, and relationship with spirolides.

Gymnodimine A has been found in mollusks obtained along the whole northern coast of Spain from April 2017 to December 2019. This is the first time that this toxin is detected in mollusks from the Atlantic coast of Europe. The prevalence of the toxin was, in general, low, being detected on average in approximately 6% of the obtained samples (122 out of 1900). The concentrations recorded were also, in general, low, with a median of 1.3μgkg-1, and a maximum value of 23.93μgkg-1. The maxima of prevalence and concentration were not geographically coincident, taking place the first at the easternmost part of the sampled area and the second at the westernmost part. In most cases (>94%), gymnodimine A and 13-desmethyl spirolide C were concurrently detected, suggesting that Alexandrium ostenfeldii could be the responsible producer species. The existence of cases in which gymnodimine A was detected alone suggests also that a Karenia species could also be involved. The geographical heterogeneity of the distribution suggests that blooms of the producer species are mostly local. Not all bivalves are equally affected, clams being less affected than mussels, oysters, and razor clams. Due to their relatively low toxicity, and their low prevalence and concentration, it seems that these toxins do not pose an important risk for the mollusk consumers in the area.

Gymnodimine A and 13-desMethyl Spirolide C Alter Intracellular Calcium Levels via Acetylcholine Receptors.

Gymnodimines and spirolides are cyclic imine phycotoxins and known antagonists of nicotinic acetylcholine receptors (nAChRs). We investigated the effect of gymnodimine A (GYM A) and 13-desmethyl spirolide C (SPX 1) from Alexandrium ostenfeldii on rat pheochromocytoma (PC12) cells by monitoring intracellular calcium levels ([Ca]i). Using whole cells, the presence of 0.5 µM of GYM A or SPX 1 induced an increase in [Ca]i mediated by acetylcholine receptors (AChRs) and inhibited further activation of AChRs by acetylcholine (ACh). To differentiate the effects of GYM A or SPX 1, the toxins were applied to cells with pharmacologically isolated nAChRs and muscarinic AChRs (mAChRs) as mediated by the addition of atropine and tubocurarine, respectively. GYM A and SPX 1 activated nAChRs and inhibited the further activation of nAChRs by ACh, indicating that both toxins mimicked the activity of ACh. Regarding mAChRs, a differential response was observed between the two toxins. Only GYM A activated mAChRs, resulting in elevated [Ca]i, but both toxins prevented a subsequent activation by ACh. The absence of the triketal ring system in GYM A may provide the basis for a selective activation of mAChRs. GYM A and SPX 1 induced no changes in [Ca]i when nAChRs and mAChRs were inhibited simultaneously, indicating that both toxins target AChRs.

Development of a data dependent acquisition-based approach for the identification of unknown fast-acting toxins and their ester metabolites

Phycotoxins in the marine food-web represent a serious threat to human health. Consumption of contaminated shellfish and/or finfish poses risk to consumer safety: several cases of toxins-related seafood poisoning have been recorded so far worldwide. Cyclic imines are emerging lipophilic toxins, which have been detected in shellfish from different European countries. Currently, they are not regulated due to the lack of toxicological comprehensive data and hence the European Food Safety Authority has required more scientific efforts before establishing a maximum permitted level in seafood. In this work, a novel data dependent liquid chromatography – high resolution mass spectrometry (LC-HRMS) approach has been successfully applied and combined with targeted studies for an in-depth investigation of the metabolic profile of shellfish samples. The proposed analytical methodology has allowed: i) to discover a plethora of unknown fatty acid esters of gymnodimines and ii) to conceive a brand new MS-based strategy, termed as backward analysis, for discovery and identification of new analogues. In particular, the implemented analytical workflow has broadened the structural diversity of cyclic imine family through the inclusion of five new congeners, namely gymnodimine –F, -G, –H, –I and -J. In addition, gymnodimine A (376.5 μg/kg), 13-desmethyl spirolide C (11.0–29.0 μg/kg) and pinnatoxin G (3.1–7.7 μg/kg) have been detected in shellfish from different sites of the Mediterranean basin (Tunisia and Italy) and the Atlantic coast of Spain, with the confirmation of the first finding of pinnatoxin G in mussels harvested in Sardinia (Tyrrhenian Sea, Italy).

Detection of Cyclic Imine Toxins in Dietary Supplements of Green Lipped Mussels (Perna canaliculus) and in Shellfish Mytilus chilensis.

Seafood represents a significant part of the human staple diet. In the recent years, the identification of emerging lipophilic marine toxins has increased, leading to the potential for consumers to be intoxicated by these toxins. In the present work, we investigate the presence of lipophilic marine toxins (both regulated and emerging) in commercial seafood products from non-European locations, including mussels Mytilus chilensis from Chile, clams Tawerea gayi and Metetrix lyrate from the Southeast Pacific and Vietnam, and food supplements based on mussels formulations of Perna canaliculus from New Zealand. All these products were purchased from European Union markets and they were analyzed by UPLC-MS/MS. Results showed the presence of the emerging pinnatoxin-G in mussels Mytilus chilensis at levels up to 5.2 µg/kg and azaspiracid-2 and pectenotoxin-2 in clams Tawera gayi up to 4.33 µg/kg and 10.88 µg/kg, respectively. This study confirms the presence of pinnatoxins in Chile, one of the major mussel producers worldwide. Chromatograms showed the presence of 13-desmethyl spirolide C in dietary supplements in the range of 33.2–97.9 µg/kg after an extraction with water and methanol from 0.39 g of the green lipped mussels powder. As far as we know, this constitutes the first time that an emerging cyclic imine toxin in dietary supplements is reported. Identifying new matrix, locations, and understanding emerging toxin distribution area are important for preventing the risks of spreading and contamination linked to these compounds.

Cyclic Imines (CIs) in Mussels from North-Central Adriatic Sea: First Evidence of Gymnodimine A in Italy.

Cyclic imines (CIs) are emerging marine lipophilic toxins (MLTs) occurring in microalgae and shellfish worldwide. The present research aimed to study CIs in mussels farmed in the Adriatic Sea (Italy) during the period 2014–2015. Twenty-eight different compounds belonging to spirolides (SPXs), gymnodimines (GYMs), pinnatoxins (PnTXs) and pteriatoxins (PtTXs) were analyzed by the official method for MLTs in 139 mussel samples collected along the Marche coast. Compounds including 13-desmethyl spirolide C (13-desMe SPX C) and 13,19-didesmethyl spirolide C (13,19-didesMe SPX C) were detected in 86% of the samples. The highest levels were generally reported in the first half of the year reaching 29.2 µg kg−1 in January/March with a decreasing trend until June. GYM A, for the first time reported in Italian mussels, was found in 84% of the samples, reaching the highest concentration in summer (12.1 µg kg−1). GYM A and SPXs, submitted to tissue distribution studies, showed the tendency to accumulate mostly in mussel digestive glands. Even if SPX levels in mussels were largely below the European Food Safety Authority (EFSA) reference of 400 μg SPXs kg−1, most of the samples contained CIs for the large part of the year. Since chronic toxicity data are still missing, monitoring is surely recommended.

Occurrence and distribution of lipophilic phycotoxins in a subtropical bay of the South China Sea.

Lipophilic phycotoxins (LPs) pose significant threats to the health of marine mammals, birds, and human beings. The distribution and components of lipophilic phycotoxins contamination in subtropical area in the South China Sea are rarely known. This study systematically assessed the composition, concentration, and distribution of typical LPs in a typical subtropical bay, Daya Bay located in the South China Sea. Phytoplankton, seawater, suspended particulate matter, sediments, and shellfish samples were simultaneously collected from Daya Bay, and analyzed using liquid chromatography with tandem mass spectrometry. Okadaic acid, dinophysistoxins-1, pectenotoxins-2, yessotoxin and its derivate homo-yessotoxin, azaspiracid-2, 13-desmethyl spirolide C and gymnodimine were widely spread in multiple media in Daya Bay. Pectenotoxins-2 was the most widely distributed and highly concentrated toxin in the marine environments of Daya Bay. Toxin homo-yessotoxin was only detected in sediments and shellfish samples, and none of yessotoxin group components were found in phytoplankton and seawater, indicating that sediments were the major source of yessotoxin in shellfish. The study strongly demonstrated the lipophilic phycotoxins accumulated in shellfish are multisource, not only derived from toxigenic algae, but also from other marine media containing lipophilic phycotoxins. This study systematically distinguished multi-pathways of bioaccumulation of LPs in the marine shellfish.

Distribution, partitioning, and seasonal variation of lipophilic marine algal toxins in aquatic environments of a typical semi-closed mariculture bay

Lipophilic marine algal toxins (LMATs) pose a potential threat to the health of marine shellfish consumers and marine breeding industries. In this study, LMATs in dissolved phases (DP) and particulate phases (PP) in the seawater of Jiaozhou Bay were accurately determined over four seasons to understand their composition, level, phase partitioning, spatiotemporal variation, and potential sources in aquatic environments of a typical semi-closed mariculture bay. Various LMATs, such as okadaic acid (OA), dinophysistoxin-1 (DTX1), dinophysistoxin-2 (DTX2), gymnodimine (GYM), 13-desmethyl spirolide C (SPX1), pectenotoxin-2 (PTX2), pectenotoxin-2 seco acid (PTX2 SA), and pectenotoxin-11 (PTX11), were detected in DP and PP; of these, OA and PTX2 were the dominant LMATs in DP and PP, respectively. The average proportion of ΣLMATs in DP (97.5%) was significantly higher than that in PP (2.5%), which indicates that LMATs are predominantly partitioned into DP. The total concentrations of LMATs in DP ranged from 4.16 ng/L to 23.19 ng/L (mean, 13.35 ng/L) over four seasons. The highest levels of LMATs in DP and PP were found in summer (mean, 16.71 ng/L) and spring, respectively, while the maximum variety of LMATs was found in autumn. This result suggests that seasonal changes could influence the composition, concentration, and phase partitioning of LMATs in aquatic environments of a coastal semi-closed mariculture bay. ΣLMAT concentrations were higher in the western region than in the eastern region of the bay, where shellfish may have a greater risk of exposure. Dinophysis acuminata, Dinophysis fortii, and Prorocentrum minimum were the potential sources of LMATs in the aquaculture seawater. Overall, various LMATs occurred in the semi-closed mariculture bay, and the persistence and bioavailability of these toxins in aquaculture seawater should be determined in future research.

LC-MS/MS Analysis of the Emerging Toxin Pinnatoxin-G and High Levels of Esterified OA Group Toxins in Galician Commercial Mussels.

The occurrence of marine harmful algae is increasing worldwide and, therefore, the accumulation of lipophilic marine toxins from harmful phytoplankton represents a food safety threat in the shellfish industry. Galicia, which is a commercially important EU producer of edible bivalve mollusk have been subjected to recurring cases of mussel farm closures, in the last decades. This work aimed to study the toxic profile of commercial mussels (Mytilus galloprovincialis) in order to establish a potential risk when ingested. For this, a total of 41 samples of mussels farmed in 3 Rías (Ares-Sada, Arousa, and Pontevedra) and purchased in 5 local markets were analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS). Chromatograms showed the presence of okadaic acid (OA), dinophysistoxin-2 (DTX-2), pectenotoxin-2 (PTX-2), azaspiracid-2 (AZA-2), and the emerging toxins 13-desmethyl spirolide C (SPX-13), and pinnatoxin-G (PnTX-G). Quantification of each toxin was determined using their own standard calibration in the range 0.1%–50 ng/mL (R2 > 0.99) and by considering the toxin recovery (62–110%) and the matrix correction (33–211%). Data showed that OA and DTX-2 (especially in the form of esters) are the main risk in Galician mollusks, which was detected in 38 samples (93%) and 3 of them exceeded the legal limit (160 µg/kg), followed by SPX-13 that was detected in 19 samples (46%) in quantities of up to 28.9 µg/kg. Analysis from PTX-2, AZA-2, and PnTX-G showed smaller amounts. Fifteen samples (37%) were positive for PTX-2 (0.7–2.9 µg/kg), 12 samples (29%) for AZA-2 (0.1–1.8 µg/kg), and PnTX-G was detected in 5 mussel samples (12%) (0.4 µg/kg–0.9 µg/kg). This is the first time Galician mollusk was contaminated with PnTX-G. Despite results indicating that this toxin was not a potential risk through the mussel ingestion, it should be considered in the shellfish safety monitoring programs through the LC–MS/MS methods.

Contamination status of lipophilic marine toxins in shellfish samples from the Bohai Sea, China

Lipophilic marine toxins in shellfish pose significant threats to the health of seafood consumers. To assess the contamination status of shellfish by lipophilic marine toxins in the Bohai Sea, nine species of shellfish periodically collected from five representative aquaculture zones throughout a year were analyzed with a method of liquid chromatography-tandem mass spectrometry (LC–MS/MS). Lipophilic marine toxins, including okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), yessotoxin (YTX), homo-yessotoxin (homo-YTX), azaspiracids (AZA2 and AZA3), gymnodimine (GYM), and 13-desmethyl spirolide C (13-DesMe-C), were detected in more than 95 percent of the shellfish samples. Toxins PTX2, YTX, 13-DesMe-C and GYM were predominant components detected in shellfish samples. Scallops, clams and mussels accumulated much higher level of lipophilic marine toxins compared to oysters. Toxin content in shellfish samples collected from different sampling locations showed site-specific seasonal variation patterns. High level of toxins was found during the stages from December to February and June to July in Hangu, while from March to April and August to September in Laishan. Some toxic algae, including Dinophysis acuminata, D. fortii, Prorocentrum lima, Gonyaulax spinifera and Lingulodinium polyedrum, were identified as potential origins of lipophilic marine toxins in the Bohai Sea. The results will offer a sound basis for monitoring marine toxins and protecting the health of seafood consumers.

Association of the Toxigenic Dinoflagellate Alexandrium ostenfeldii With Spirolide Accumulation in Cultured Mussels (Mytilus galloprovincialis) From Northwest Mexico

Spirolides are polyether cyclic imines considered as “fast acting toxins”. Long term human health consequences of spirolide ingestion are uncertain, and hence regulatory limits for human consumption have not been established. Nevertheless, monitoring these toxins in shellfish is essential because they can interfere with detection by mouse bioassay of lipophilic regulated toxins. Todos Santos Bay (TSB), in the northeast of the Baja California Peninsula, is an important shellfish cultivation and fish-farming area in Mexico. The toxin analog 13-desmethyl spirolide C has been reported in cultivated mussels (Mytilus galloprovincialis) from TSB, but the causative species associated with accumulation of this toxin has not been previously identified. We assessed the occurrence of Alexandrium ostenfeldii, the unique known producer of spirolides, by inverted light microscopy and by PCR with species-specific oligonucleotides designed for the ITS and 18S rDNA. We determined the presence and abundance of this species at the surface and at the thermocline from samples collected over two annual sampling periods (2013 - 2014 and 2016 – 2017). During the 2013-2014 period, A. ostenfeldii was found in 50% of the samples analyzed by light microscopy. The highest cell abundance (about 3.6 x 103 cells L-1) occurred in October 2013. During 2016 – 2017 the dinoflagellate was present in low cell abundances (<5 x 102 cells L-1) and was detected in only 20.9% of the samples. Cells of this species were usually found when sea surface temperature ranged from 17 to 20 °C. We also evaluated spirolide accumulated in cultivated mussels from TSB by tandem mass spectrometry (LC-MS/MS). The only spirolide detected was 13-desmethyl spirolide C, found mainly during the 2013-2014 sampling period, with the highest concentration (1.05 μg kg-1) in June 2014. During winter, toxin concentration was at or below the detection limit. During 2016-2017, spirolides were below the detection limit, coinciding with the absence of the causative species. Cell abundance of A. ostenfeldii and spirolide concentration in mussels did not present a clear correlation. This study represents the first record of A. ostenfeldii in TSB and provides evidence that this species is the primary origin of spirolides accumulated in mussels.