Optimization and Validation of a High Throughput UHPLC-MS/MS Method for Determination of the EU Regulated Lipophilic Marine Toxins and Occurrence in Fresh and Processed Shellfish.

Under the name of lipophilic marine toxins, there are included more than 1000 toxic secondary metabolites, produced by phytoplankton, with the common chemical property of lipophilicity. Due to toxicological effects and geographical distribution, in European legislation relevant compounds are regulated, and their determination is accomplished with the reference liquid chromatography-tandem mass spectrometry method. In this study a modified ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed for the identification and quantification of EU-regulated lipophilic toxins. The method optimization included a refinement of SPE-C18 clean-up, in order to reduce matrix interferences. Improved LC conditions and upgraded chromatographic ammonia-based gradient ensured the best separation of all analytes and, in particular, of the two structural isomers (OA and DTX2). Also, different MS parameters were tested, and confirmation criteria finally established. The validation studies confirmed that all parameters were satisfactory. The requirements for precision (RSD% < 11.8% for each compound), trueness (recoveries from 73 to 101%) and sensitivity (limits of quantification in the range 3−8 µg kg−1) were fulfilled. The matrix effect, ranging from −9 to 19%, allowed the use of a calibration curve in solvent (3−320 µg kg−1 in matrix) for quantification of real samples. Method relative uncertainty ranged from 12 to 20.3%. Additionally, a total of 1000 shellfish samples was analysed, providing a first preliminary surveillance study that may contribute to the knowledge of lipophilic marine toxins contamination. Increase in algae proliferation events and intoxication cases, EFSA suggestions for modification of maximum permitted levels and toxicity equivalency factors, and new studies of important toxic effects underline that implementation of reference methods still represents an important task for health and food safety laboratories.

Quantitation Overcoming Matrix Effects of Lipophilic Toxins in Mytilus galloprovincialis by Liquid Chromatography-Full Scan High Resolution Mass Spectrometry Analysis (LC-HR-MS).

The analysis of marine lipophilic toxins in shellfish products still represents a challenging task due to the complexity and diversity of the sample matrix. Liquid chromatography coupled with mass spectrometry (LC-MS) is the technique of choice for accurate quantitative measurements in complex samples. By combining unambiguous identification with the high selectivity of tandem MS, it provides the required high sensitivity and specificity. However, LC-MS is prone to matrix effects (ME) that need to be evaluated during the development and validation of methods. Furthermore, the large sample-to-sample variability, even between samples of the same species and geographic origin, needs a procedure to evaluate and control ME continuously. Here, we analyzed the toxins okadaic acid (OA), dinophysistoxins (DTX-1 and DTX-2), pectenotoxin (PTX-2), yessotoxin (YTX) and azaspiracid-1 (AZA-1). Samples were mussels (Mytilus galloprovincialis), both fresh and processed, and a toxin-free mussel reference material. We developed an accurate mass-extracted ion chromatogram (AM-XIC) based quantitation method using an Orbitrap instrument, evaluated the ME for different types and extracts of mussel samples, characterized the main compounds co-eluting with the targeted molecules and quantified toxins in samples by following a standard addition method (SAM). An AM-XIC based quantitation of lipophilic toxins in mussel samples using high resolution and accuracy full scan profiles (LC-HR-MS) is a good alternative to multi reaction monitoring (MRM) for instruments with HR capabilities. ME depend on the starting sample matrix and the sample preparation. ME are particularly strong for OA and related toxins, showing values below 50% for fresh mussel samples. Results for other toxins (AZA-1, YTX and PTX-2) are between 75% and 110%. ME in unknown matrices can be evaluated by comparing their full scan LC-HR-MS profiles with those of known samples with known ME. ME can be corrected by following SAM with AM-XIC quantitation if necessary.

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.

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.

Cobalt versus Osmium: Control of Both trans and cis Selectivity in Construction of the EFG Rings of Pectenotoxin†4.

Catalytic oxidative cyclisation reactions have been employed for the synthesis of the E and F rings of the complex natural product target pectenotoxin 4. The choice of metal catalyst (cobalt- or osmium-based) allowed for the formation of THF rings with either trans or cis stereoselectivity. Fragment union using a modified Julia reaction then enabled the synthesis of an advanced synthetic intermediate containing the EF and G rings of the target.

Influence of Temperature on Growth and Production of Pectenotoxin-2 by a Monoclonal Culture of Dinophysis caudata.

The effects of temperature on growth and production of Lipophilic Toxins (LT) by a monoclonal culture of Dinophysis caudata was studied. The cell density of D. caudata increased significantly with increasing temperature, and was the highest under 27, 30, and 32.5 °C. Temperature affected the average specific growth rate (µ) during the exponential growth phase (EG), which increased from 15 °C to 30 °C, and then decreased at 32.5 °C. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that this strain of D. caudata produced only pectenotoxin-2 (PTX-2) whose concentration increased significantly with incubation period, except at 32.5 °C. It was significantly different between temperatures ≤18 °C, ≥21 °C, and 32.5 °C. The cellular toxin production (CTP, pg·cell(-1)·day(-1)) showed variation with growth phase and temperature, except at 32.5 °C. The average net toxin production (Rtox) was not affected by temperature. During EG, the average specific toxin production rate (µtox) increased significantly with increase in temperature, reaching a peak of 0.66 ± 0.01 day(-1) at 30 °C, and then decreased. Over the entire growth span, µtox was significantly correlated to µ, and this correlation was most significant at 27 and 30 °C. During EG, µtox was affected by both temperature and growth. This study shows that temperature affects growth and toxin production of this strain of D. caudata during EG. In addition, a positive correlation was found between toxin production and growth.

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.

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.

Mortality and histopathology in sheepshead minnow (Cyprinodon variegatus) larvae exposed to pectenotoxin-2 and Dinophysis acuminata.

Toxic species of the dinoflagellate genus Dinophysis can produce diarrheic toxins including okadaic acid (OA) and dinophysistoxins (DTXs), and the non-diarrheic pectenotoxins (PTXs). Okadaic acid and DTXs cause diarrheic shellfish poisoning (DSP) in human consumers, and also cause cytotoxic, immunotoxic and genotoxic effects in a variety of mollusks and fishes at different life stages in vitro. The possible effects of co-produced PTXs or live cells of Dinophysis to aquatic organisms, however, are less understood. Effects on an early life stage of sheepshead minnow (Cyprinodon variegatus), a common finfish in eastern USA estuaries, were evaluated using a 96-h toxicity bioassay. Three-week old larvae were exposed to PTX2 concentrations from 50 to 4000 nM, live Dinophysis acuminata culture (strain DAVA01), live cells resuspended in clean medium or culture filtrate. This D. acuminata strain produced mainly intracellular PTX2 (≈ 21 pg cell-1), with much lower levels of OA and dinophysistoxin-1. No mortality or gill damages were observed in larvae exposed to D. acuminata (from 5 to 5500 cells mL-1), resuspended cells and culture filtrate. However, exposure to purified PTX2 at intermediate to high concentrations (from 250 to 4000 nM) resulted in 8 to 100% mortality after 96 h (24-h LC50 of 1231 nM). Histopathology and transmission electron microscopy of fish exposed to intermediate to high PTX2 concentrations revealed important gill damage, including intercellular edema, necrosis and sloughing of gill respiratory epithelia, and damage to the osmoregulatory epithelium, including hypertrophy, proliferation, redistribution and necrosis of chloride cells. Tissue damage in gills is likely caused by the interaction of PTX2 with the actin cytoskeleton of the affected gill epithelia. Overall, the severe gill pathology observed following the PTX2 exposure suggested death was due to loss of respiratory and osmoregulatory functions in C. variegatus larvae.

Co-occurrence of marine and freshwater phycotoxins in oysters, and analysis of possible predictors for management.

Oysters (Crassostrea virginica) were screened for 12 phycotoxins over two years in nearshore waters to collect baseline phycotoxin data and to determine prevalence of phycotoxin co-occurrence in the commercially and ecologically-relevant species. Trace to low concentrations of azaspiracid-1 and -2 (AZA1, AZA2), domoic acid (DA), okadaic acid (OA), and dinophysistoxin-1 (DTX1) were detected, orders of magnitude below seafood safety action levels. Microcystins (MCs), MC-RR and MC-YR, were also found in oysters (maximum: 7.12 µg MC-RR/kg shellfish meat wet weight), warranting consideration of developing action levels for freshwater phycotoxins in marine shellfish. Oysters contained phycotoxins that impair shellfish health: karlotoxin1-1 and 1-3 (KmTx1-1, KmTx1-3), goniodomin A (GDA), and pectenotoxin-2 (PTX2). Co-occurrence of phycotoxins in oysters was common (54%, n = 81). AZAs and DA co-occurred most frequently of the phycotoxins investigated that are a concern for human health (n = 13) and PTX2 and KmTxs co-occurred most frequently amongst the phycotoxins of concern for shellfish health (n = 9). Various harmful algal bloom (HAB) monitoring methods and tools were assessed for their effectiveness at indicating levels of phycotoxins in oysters. These included co-deployed solid phase adsorption toxin tracking (SPATT) devices, toxin levels in particulate organic matter (POM, >1.5 µm) and whole water samples and cell concentrations from water samples as determined by microscopy and quantitative real-time PCR (qPCR). The dominant phycotoxin varied between SPATTs and all other phycotoxin sample types, and out of the 11 phycotoxins detected in oysters, only four and seven were detected in POM and whole water respectively, indicating phycotoxin profile mismatch between ecosystem compartments. Nevertheless, there were correlations between DA in oysters and whole water (simple linear regression [LR]: R2 = 0.6, p < 0.0001, n = 40), and PTX2 in oysters and SPATTs (LR: R2 = 0.3, p = 0.001, n = 36), providing additional monitoring tools for these phycotoxins, but oyster samples remain the best overall indicators of seafood safety.

Growth, Toxin Content and Production of Dinophysis Norvegica in Cultured Strains Isolated from Funka Bay (Japan).

The successful cultivation of Dinophysis norvegica Claparède & Lachmann, 1859, isolated from Japanese coastal waters, is presented in this study, which also includes an examination of its toxin content and production for the first time. Maintaining the strains at a high abundance (>2000 cells per mL-1) for more than 20 months was achieved by feeding them with the ciliate Mesodinium rubrum Lohmann, 1908, along with the addition of the cryptophyte Teleaulax amphioxeia (W.Conrad) D.R.A.Hill, 1992. Toxin production was examined using seven established strains. At the end of the one-month incubation period, the total amounts of pectenotoxin-2 (PTX2) and dinophysistoxin-1 (DTX1) ranged between 132.0 and 375.0 ng per mL-1 (n = 7), and 0.7 and 3.6 ng per mL-1 (n = 3), respectively. Furthermore, only one strain was found to contain a trace level of okadaic acid (OA). Similarly, the cell quota of pectenotoxin-2 (PTX2) and dinophysistoxin-1 (DTX1) ranged from 60.6 to 152.4 pg per cell-1 (n = 7) and 0.5 to 1.2 pg per cell-1 (n = 3), respectively. The results of this study indicate that toxin production in this species is subject to variation depending on the strain. According to the growth experiment, D. norvegica exhibited a long lag phase, as suggested by the slow growth observed during the first 12 days. In the growth experiment, D. norvegica grew very slowly for the first 12 days, suggesting they had a long lag phase. However, after that, they grew exponentially, with a maximum growth rate of 0.56 divisions per day (during Days 24-27), reaching a maximum concentration of 3000 cells per mL-1 at the end of the incubation (Day 36). In the toxin production study, the concentration of DTX1 and PTX2 increased following their vegetative growth, but the toxin production still increased exponentially on Day 36 (1.3 ng per mL-1 and 154.7 ng per mL-1 of DTX1 and PTX2, respectively). The concentration of OA remained below detectable levels (≤0.010 ng per mL-1) during the 36-day incubation period, with the exception of Day 6. This study presents new information on the toxin production and content of D. norvegica, as well as insights into the maintenance and culturing of this species.

Effects of Two Toxin-Producing Harmful Algae, Alexandrium catenella and Dinophysis acuminata (Dinophyceae), on Activity and Mortality of Larval Shellfish.

Harmful algal bloom (HAB) species Alexandrium catenella and Dinophysis acuminata are associated with paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP) in humans, respectively. While PSP and DSP have been studied extensively, less is known about the effects of these HAB species or their associated toxins on shellfish. This study investigated A. catenella and D. acuminata toxicity in a larval oyster (Crassostrea virginica) bioassay. Larval activity and mortality were examined through 96-h laboratory exposures to live HAB cells (10−1000 cells/mL), cell lysates (1000 cells/mL equivalents), and purified toxins (10,000 cells/mL equivalents). Exposure to 1000 cells/mL live or lysed D. acuminata caused larval mortality (21.9 ± 7.0%, 10.2 ± 4.0%, respectively) while exposure to any tested cell concentration of live A. catenella, but not lysate, caused swimming arrest and/or mortality in >50% of larvae. Exposure to high concentrations of saxitoxin (STX) or okadaic acid (OA), toxins traditionally associated with PSP and DSP, respectively, had no effect on larval activity or mortality. In contrast, pectenotoxin-2 (PTX2) caused rapid larval mortality (49.6 ± 5.8% by 48 h) and completely immobilized larval oysters. The results indicate that the toxic effects of A. catenella and D. acuminata on shellfish are not linked to the primary toxins associated with PSP and DSP in humans, and that PTX2 is acutely toxic to larval oysters.

Progress on the investigation and monitoring of marine phycotoxins in China.

Marine phycotoxins associated with paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), ciguatera fish poisoning (CFP), tetrodotoxin (TTX), palytoxin (PLTX) and neurotoxin ß-N-methylamino-L-alanine (BMAA) have been investigated and routinely monitored along the coast of China. The mouse bioassay for monitoring of marine toxins has been progressively replaced by the enzyme-linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which led to the discovery of many new hydrophilic and lipophilic marine toxins. PSP toxins have been detected in the whole of coastal waters of China, where they are the most serious marine toxins. PSP events in the Northern Yellow Sea, the Bohai Sea and the East China Sea are a cause of severe public health concern. Okadaic acid (OA) and dinophysistoxin-1 (DTX1), which are major toxin components associated with DSP, were mainly found in coastal waters of Zhejiang and Fujian provinces, and other lipophilic toxins, such as pectenotoxins, yessotoxins, azaspiracids, cyclic imines, and dinophysistoxin-2(DTX2) were detected in bivalves, seawater, sediment, as well as phytoplankton. CFP events mainly occurred in the South China Sea, while TTX events mainly occurred in Jiangsu, Zhejiang and Fujian provinces. Microalgae that produce PLTX and BMAA were found in the phytoplankton community along the coastal waters of China.

Pectenotoxin-2 from marine sponges: a potential anti-cancer agent-a review.

Pectenotoxin-2 (PTX-2), which was first identified as a cytotoxic entity in marine sponges, has been reported to display significant cytotoxicity to human cancer cells where it inhibits mitotic separation and cytokinesis through the depolymerization of actin filaments. In the late stage of endoreduplication, the effects of PTX-2 on different cancer cells involves: (i) down-regulation of anti-apoptotic Bcl-2 members and IAP family proteins; (ii) up-regulation of pro-apoptotic Bax protein and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (DR4/DR5); and (iii) mitochondrial dysfunction. In addition, PTX-2 induces apoptotic effects through suppression of the nuclear factor κB (NF-κB) signaling pathway in several cancer cells. Analysis of cell cycle regulatory proteins showed that PTX-2 increases phosphorylation of Cdc25c and decreases protein levels of Cdc2 and cyclin B1. Cyclin-dependent kinase (Cdk) inhibitor p21 and Cdk2, which are associated with the induction of endoreduplication, were upregulated. Furthermore, it was found that PTX-2 suppressed telomerase activity through the transcriptional and post-translational suppression of hTERT. The purpose of this review was to provide an update regarding the anti-cancer mechanism of PTX-2, with a special focus on its effects on different cellular signaling cascades.

Spatiotemporal distribution of phycotoxins and their co-occurrence within nearshore waters.

Harmful algal blooms (HABs), varying in intensity and causative species, have historically occurred throughout the Chesapeake Bay, U.S.; however, phycotoxin data are sparse. The spatiotemporal distribution of phycotoxins was investigated using solid-phase adsorption toxin tracking (SPATT) across 12 shallow, nearshore sites within the lower Chesapeake Bay and Virginia's coastal bays over one year (2017-2018). Eight toxins, azaspiracid-1 (AZA1), azaspiracid-2 (AZA2), microcystin-LR (MC-LR), domoic acid (DA), okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), and goniodomin A (GDA) were detected in SPATT extracts. Temporally, phycotoxins were always present in the region, with at least one phycotoxin group (i.e., consisting of OA and DTX1) detected at every time point. Co-occurrence of phycotoxins was also common; two or more toxin groups were observed in 76% of the samples analyzed. Toxin maximums: 0.03 ng AZA2/g resin/day, 0.25 ng DA/g resin/day, 15 ng DTX1/g resin/day, 61 ng OA/g resin/day, 72 ng PTX2/g resin/day, and 102,050 ng GDA/g resin/day were seasonal, with peaks occurring in summer and fall. Spatially, the southern tributary and coastal bay regions harbored the highest amount of total phycotoxins on SPATT over the year, and the former contained the greatest diversity of phycotoxins. The novel detection of AZAs in the region, before a causative species has been identified, supports the use of SPATT as an explorative tool in respect to emerging threats. The lack of karlotoxin in SPATT extracts, but detection of Karlodinium veneficum by microscopy, however, emphasizes that this tool should be considered complementary to, but not a replacement for, more traditional HAB management and monitoring methods.

Spatial distribution and source of biotoxins in phytoplankton from the South China Sea, China.

Marine phycotoxins severely threaten ecosystem health and mariculture. This study investigates the spatial distribution and source of diverse phycotoxins in the South China Sea (SCS), during four 2019/2020 cruises. Saxitoxin (STX) and okadaic acid (OA) -groups, azaspiracids, cyclic imines, pectenotoxins (PTX), yessotoxins, and domoic acid (DA) toxins were analyzed in microalgal samples. PTX2 occurred with the highest (93.5%) detection rate (DR) during all cruises, especially in the Pearl River Estuary (PRE) in June 2019. Homo-yessotoxin (hYTX) and DA were found during three cruises in August 2020, and high DR of hYTX (67.7%, 29.3%) and DA (29.0%, 29.3%) in the PRE and Guangdong coast, respectively, in June 2019 and 2020, peaking at concentrations of 777 pg hYTX L-1 and 38514 pg DA L-1. The phycotoxin distribution demonstrated that DA-producing microalgae gathered close to the PRE and Guangdong coast, while hYTX-producing microalgae distributed relatively far offshore. Microalgae producing PTX2- and STX-group toxins were more widely living in the SCS. High-throughput sequencing results suggested that Alexandrium pacificum and Gonyaulax spinifera were responsible for STX-group toxins and hYTX, respectively, while Pseudo-nitzschia cuspidata was the main source of DA. Widely distributed PTX2, hYTX, and DA were reported for the first time in the SCS.

Role of enteric glial cells in the toxicity of phycotoxins: Investigation with a tri-culture intestinal cell model.

Lipophilic phycotoxins are secondary metabolites produced by phytoplankton. They can accumulate in edible filtering-shellfish and cause human intoxications, particularly gastrointestinal symptoms. Up to now, the in vitro intestinal effects of these toxins have been mainly investigated on simple monolayers of intestinal cells such as the enterocyte-like Caco-2 cell line. Recently, the combination of Caco-2 cells with mucus secreting HT29-MTX cell line has been also used to mimic the complexity of the human intestinal epithelium. Besides, enteric glial cells (EGC) from the enteric nervous system identified in the gut mucosa have been largely shown to be involved in gut functions. Therefore, using a novel model integrating Caco-2 and HT29-MTX cells co-cultured on inserts with EGC seeded in the basolateral compartment, we examined the toxicological effects of two phycotoxins, pectenotoxin-2 (PTX2) and okadaic acid (OA). Cell viability, morphology, barrier integrity, inflammation, barrier crossing, and the response of some specific glial markers were evaluated using a broad set of methodologies. The toxicity of PTX2 was depicted by a slight decrease of viability and integrity as well as a slight increase of inflammation of the Caco-2/HT29-MTX co-cultures. PTX2 induced some modifications of EGC morphology. OA induced IL-8 release and decreased viability and integrity of Caco-2/HT29-MTX cell monolayers. EGC viability was slightly affected by OA. The presence of EGC reinforced barrier integrity and reduced the inflammatory response of the epithelial barrier following OA exposure. The release of GDNF and BDNF gliomediators by EGC could be implicated in the protection observed.

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

ABSTRACT: 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.

Cyclodextrin polymers as passive sampling materials for lipophilic marine toxins in Prorocentrum lima cultures and a Dinophysis sacculus bloom in the NW Mediterranean Sea.

Cyclodextrins, cyclic oligomers that form a conical structure with an internal cavity, are proposed as new and sustainable materials for passive sampling of lipophilic marine toxins. Two applicability scenarios have been tested. First, disks containing ß-cyclodextrin-hexamethylene diisocyanate (ß-CD-HDI) and ß-cyclodextrin-epichlorohydrin (ß-CD-EPI) polymers were immersed in Prorocentrum lima cultures for different days (2, 12 and 40). LC-MS/MS analysis showed capture of free okadaic acid (OA) and dinophysistoxin-1 (DTX1) by cyclodextrins at contents that increased with immersion time. Cyclodextrins resulted more efficient in capturing DTX1 than OA. In a second experiment, disks containing ß-CD-HDI, ß-CD-EPI, γ-CD-HDI and γ-CD-EPI were deployed in harbor waters of El Masnou (NW Mediterranean Sea) during a Dinophysis sacculus bloom in February 2020. Free OA and pectenotoxin-2 (PTX2) were captured by cyclodextrins. Toxin contents were higher at sampling points and sampling weeks with higher D. sacculus cell abundance. In this case, PTX2 capture with cyclodextrins was more efficient than OA capture. Therefore, cyclodextrins have provided information regarding the toxin profile of a P. lima strain and the spatial and temporal dynamics of a D. sacculus bloom, proven efficient as passive sampling materials for environmental monitoring.

Investigation of pectenotoxin profiles in the Yellow Sea (China) using a passive sampling technique.

Pectenotoxins (PTXs) are a group of lipophilic algal toxins. These toxins have been found in algae and shellfish from Japan, New Zealand, Ireland, Norway and Portugal. PTX profiles vary with geographic location of collection site. The aim of the present study was to investigate PTX profiles from the Yellow Sea, China. The sampling location was within an aquatic farm (N36 degrees 12.428', E120 degrees 17.826') near the coast of Qingdao, China, in the Yellow Sea from 28 July to 29 August 2006. PTXs in seawater were determined using a solid phase adsorption toxin tracking (SPATT) method. PTXs were analyzed by HPLC-MSMS. PTX-2, PTX-2 sec acid (PTX-2 SA) and 7-epi-PTX-2 SA were found in seawater samples. The highest levels of PTXs (107 ng/g of resin PTX-2, 50 ng/g of resin PTX-2 SA plus 7-epi-PTX-2 SA) in seawater were found on 1 August, 2006. From 1 August to 29 August, the levels of PTX-2 and PTX-2 SA decreased. In the same area, the marine algae, Dinophysis acuminata was found in the seawater in the summer months of 2006. This indicated that Dinophysis acuumuta might be the original source of PTXs. PTX-11 and PTX-12a/b were not found in seawater.