Improved Isolation Procedures for Okadaic Acid Group Toxins from Shellfish (Mytilus edulis) and Microalgae (Prorocentrum lima).

Okadaic acid (OA) group toxins may accumulate in shellfish and can result in diarrhetic shellfish poisoning when consumed by humans, and are therefore regulated. Purified toxins are required for the production of certified reference materials used to accurately quantitate toxin levels in shellfish and water samples, and for other research purposes. An improved procedure was developed for the isolation of dinophysistoxin-2 (DTX2) from shellfish (M. edulis), reducing the number of purification steps from eight to five, thereby increasing recoveries to ~68%, compared to ~40% in a previously reported method, and a purity of >95%. Cell densities and toxin production were monitored in cultures of Prorocentrum lima, that produced OA, DTX1, and their esters, over ~1.5 years with maximum cell densities of ~70,000 cells mL-1 observed. Toxin accumulation progressively increased over the study period, to ~0.7 and 2.1 mg L-1 of OA and DTX1 (including their esters), respectively, providing information on appropriate harvesting times. A procedure for the purification of OA and DTX1 from the harvested biomass was developed employing four purification steps, with recoveries of ~76% and purities of >95% being achieved. Purities were confirmed by LC-HRMS, LC-UV, and NMR spectroscopy. Additional stability observations led to a better understanding of the chemistry of these toxins.

Risk Assessment of Pectenotoxins in New Zealand Bivalve Molluscan Shellfish, 2009-2019.

Pectenotoxins (PTXs) are produced by Dinophysis spp., along with okadaic acid, dinophysistoxin 1, and dinophysistoxin 2. The okadaic acid group toxins cause diarrhetic shellfish poisoning (DSP), so are therefore regulated. New Zealand currently includes pectenotoxins within the DSP regulations. To determine the impact of this decision, shellfish biotoxin data collected between 2009 and 2019 were examined. They showed that 85 samples exceeded the DSP regulatory limit (0.45%) and that excluding pectenotoxins would have reduced this by 10% to 76 samples. The incidence (1.3%) and maximum concentrations of pectenotoxins (0.079 mg/kg) were also found to be low, well below the current European Food Safety Authority (EFSA) safe limit of 0.12 mg/kg. Inclusion within the DSP regulations is scientifically flawed, as pectenotoxins and okadaic acid have a different mechanism of action, meaning that their toxicities are not additive, which is the fundamental principle of grouping toxins. Furthermore, evaluation of the available toxicity data suggests that pectenotoxins have very low oral toxicity, with recent studies showing no oral toxicity in mice dosed with the PTX analogue PTX2 at 5000 µg/kg. No known human illnesses have been reported due to exposure to pectenotoxins in shellfish, a fact which combined with the toxicity data indicates that they pose negligible risk to humans. Regulatory policies should be commensurate with the level of risk, thus deregulation of PTXs ought to be considered, a stance already adopted by some countries.

A fluorescence microplate assay based on molecularly imprinted silica coated quantum dot optosensing materials for the separation and detection of okadaic acid in shellfish.

Molecularly imprinted polymers (MIPs) are attracting substantial interest as artificial plastic antibodies because of their biometric capability for targeting small molecules. In this study, molecularly imprinted silica material-coated quantum dots (MIS-QDs) with selective recognition capability to okadaic acid (OA) were developed and characterized. The synthesized MIS-QDs with specific imprinting cavities exhibited excellent recognition capability similar to those of biological antibodies and high fluorescence (FL) quenching selectivity for OA. Furthermore, the MIS-QDs with unsaturated bonds were immobilized onto the surface of 96-well microplates by cold plasma-induced grafting. A novel direct competitive microplate assay strategy was then proposed. The FL quenching properties of the developed microplate assay showed an excellent linear relationship with OA in the range of 10.0-100.0 µg/kg with a correlation coefficient of 0.9961. The limit of detection for OA was 0.25 µg/kg in the shellfish samples. The mean quantitative recoveries were 92.5%-101.0% and 92.9%-101.3%, with relative standard deviations of <7.7% and 7.6% for pure solvents and purified shellfish samples, respectively. The established microplate assay strategy can be used as a rapid and high-throughput method for analyzing OA marine toxins in biological samples.

Ultrastable nitrogen-doped carbon nanotube encapsulated cobalt nanoparticles for magnetic solid-phase extraction of okadaic acid from aquatic samples.

An easy-prepared adsorbent with high stable and good dispersibility is especially valuable for the development of magnetic solid-phase extraction (MSPE) techniques. In this study, magnetic nitrogen-doped carbon nanotube cages (N-CNTCs) were synthesized via direct carbonization of cobalt (II)-containing metal-organic frameworks. During carbonization, cobalt ions inside the MOFs were converted into magnetic functional nanoparticles of N-CNTCs. Simultaneously, large amounts of nitrogen, originating from the organic ligands, were doped into the carbon framework. This unique structure gave the N-CNTCs excellent chemical stability, high affinity, and good dispersibility. The synthesized magnetic N-CNTCs were then used for MSPE of okadaic acid (OA) from aquatic samples. A simple, efficient, and sensitive method for detecting and quantitating OA was developed by combining the above sample pretreatment technique with high-performance liquid chromatography -tandem mass spectrometry (HPLC-MS/MS). The resulting method boasts a linear dynamic range of 3.0-1000.0 pg mL-1 with good linearity (R2 ≥ 0.9994). The limit of detection (LOD) and limit of quantification (LOQ) were 1.3 pg mL-1 and 3.0 pg mL-1, respectively. Several shellfish and seafood samples were analyzed using the developed method, showing satisfactory recoveries (82.0-107.0%) and relative standard deviations (<4.5%). The developed method was also used to investigate the OA distribution in crab tissues. Our results demonstrate that magnetic N-CNTCs are promising adsorbents for providing reliable support for the early warning and tracing to the source of algae toxins.

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.

Recyclable magnetic covalent organic framework for the extraction of marine biotoxins.

A novel procedure for the preparation of magnetic covalent organic frameworks (COFs) is reported. In situ functionalization of Fe3O4 with dopamine rapidly afforded amino-functionalized magnetic nanoparticles, which after decoration with a COF building block and subsequent COF growth gave access to magnetic composite mTpBD-Me2. The optimized synthesis conditions yielded crystalline and superparamagnetic material with no loss in surface area as compared to bulk COF. The composite material was employed for the first time in magnetic solid-phase extraction of marine biotoxins from seawater with high efficiency, where calculated maximum adsorption capacities of 812 mg g-1 and 830 mg g-1 were found for okadaic acid (OA) and dinophysistoxin-1 (DTX-1), respectively, corresponding to an increase of ∼500-fold for OA and ∼300-fold for DTX-1 as compared to the commonly used non-magnetic macroporous resins. Nearly quantitative desorption efficiency of both biotoxins was obtained using 2-propanol as solvent, rendering the composite materials recyclable with merely minor losses in adsorption capacity after five consecutive cycles of adsorption/desorption. In addition, retention of crystallinity after the adsorption cycles highlights the stability of the composite in seawater. These results illustrate the great efficiency of the novel material in biotoxin adsorption and show great promise for its application in environmental monitoring programs.

Combined effect of temperature and nutritional regime on the elimination of the lipophilic toxin okadaic acid in the naturally contaminated wedge shell Donax trunculus.

The influence of nutritional regime and water temperature on depuration rates of OA-group toxins in the wedge shell Donax trunculus was examined by exposing naturally contaminated specimens to three nutritional regimes (microalgae, commercial paste of microalgae, and starvation) for 14 days at 16 °C and 20 °C. Total OA was quantified in the whole soft tissues of the individuals collected in days 2, 4, 6, 8, 10, 12 and 14. Mortality, dry weight, condition index, gross biochemical composition and gametogenic stages were surveyed. Low variation of glycogen and carbohydrates during the experiments suggest that wedge shells were under non-dramatic stress conditions. Wedge shells fed with non-toxic diets showed similar depuration rates being 15 and 38% higher than in starvation, at 16 and 20 °C, respectively. Depuration rates under non-toxic diets at 20 °C were 71% higher than at 16 °C. These results highlight the influence of water temperature on the depuration rate of total OA accumulated by D. trunculus, even when the increase is of only 4 °C, as commonly observed in week time scales in the southern Portuguese coastal waters. These results open the possibility of a faster release of OA in harvested wedge shells translocated to depuration systems when under a slight increase of water temperature.

Separation and purification of two minor typical diarrhetic shellfish poisoning toxins from harmful marine microalgae via combined liquid chromatography with mass spectrometric detection.

A novel method was developed for the purification of two typical diarrhetic shellfish poisoning toxins from toxin-producing marine microalgae using macroporous resin, high-speed countercurrent chromatography-mass spectrometry, and semipreparative high-performance liquid chromatography-mass spectrometry. Analytical high-performance liquid chromatography-mass spectrometry was used for identification and purity analysis of okadaic acid and dinophysistoxin-1 because they exhibit no visible or ultraviolet absorption. First, four kinds of macroporous resins were investigated, and HP-20 macroporous resin was selected for the preenrichment and cleanup of the two target toxins. Second, the resin-purified sample was further purified using high-speed countercurrent chromatography coupled with a mass spectrometer. The purities of the obtained okadaic acid and dinophysistoxin-1 were 89.0 and 83.0%, respectively, as determined through analytical high-performance liquid chromatography-mass spectrometry. Finally, further purification was carried out using semipreparative high-performance liquid chromatography with mass spectrometry, and the purities of the final okadaic acid and dinophysistoxin-1 products were both over 98.0% based on the analytical high-performance liquid chromatography-mass spectrometry chromatograms and fraction spectra. This work demonstrates that the proposed purification process is a powerful method for the preparation of high-purity okadaic acid and dinophysistoxin-1 from toxin-producing marine microalgae. Moreover, it is particularly important for the purification and preparation of minor toxins that exhibit no visible or ultraviolet absorption from harmful marine algae.

Limaol: A Polyketide from the Benthic Marine Dinoflagellate Prorocentrum lima.

Limaol (1), along with a dinophysistoxin 1 derivative and an okadaic acid (OA) derivative, was isolated from the large-scale cultivation of the benthic marine dinoflagellate Prorocentrum lima. The structure of 1 was determined by a combination of NMR spectroscopy and mass spectrometry and contained tetrahydropyran, 1,3,5,7-tetra(methylene)heptane, and octahydrospiro[pyran-2,2'-pyrano[3,2-b]pyran] moieties. The absolute configuration of 1 was completely elucidated on the basis of ROESY correlations, J-based configuration analysis, and modified Mosher's ester analysis. Limaol showed moderate cytotoxicity when compared to OA against three cancer cell lines.

Two-year study of lipophilic marine toxin profile in mussels of the North-central Adriatic Sea: First report of azaspiracids in Mediterranean seafood.

Since the late 1980s, the North-central Adriatic Sea has frequently experienced blooms of harmful algal species, producing marine lipophilic toxins (MLTs) which accumulate in mussels and pose a serious threat to consumer health. Here, we present a 2-year LC-MS/MS study (2012-2014) of the MLT profile in mussels from the North-central Adriatic Sea in the context of the presence of toxic phytoplankton concentrations in seawater. Okadaic acid increased in mussels from all areas during the summer and autumn-winter periods with a rising trend between 2012 and 2014. In the same periods, Dinophysis sp. increased in abundance in seawater, but the highest densities of algae did not always coincide with the highest levels of toxins in mussels. Yessotoxins (YTXs) content in mussel increased sharply in the autumn-winter periods even exceeding the legal limit; although this accumulation did not always correlated with the YTX-producers in water (such as Lingulodinium polyedrum and Protoceratium reticulatum) a massive bloom of Gonyaulax spinifera was reported in November 2013, suggesting the role of this species in YTXs shellfish contamination. Traces of Azaspiracid 2 (AZA-2) were observed often in mussels during the study period, confirming for the first time the presence of this biotoxin in Mediterranean seafood.

New method for the analysis of lipophilic marine biotoxins in fresh and canned bivalves by liquid chromatography coupled to high resolution mass spectrometry: a quick, easy, cheap, efficient, rugged, safe approach.

A new method for the analysis of lipophilic marine biotoxins (okadaic acid, dinophysistoxins, azaspiracids, pectenotoxins, yessotoxins, spirolids) in fresh and canned bivalves has been developed. A QuEChERS methodology is applied; i.e. the analytes are extracted with acetonitrile and clean-up of the extracts is performed by dispersive solid phase extraction with C18. The extracts are analyzed by ultra-high performance liquid chromatography coupled to a hybrid quadrupole-Orbitrap mass spectrometer, operating in tandem mass spectrometry mode, with resolution set at 70,000 (m/z 200, FWHM). Separation of the analytes, which takes about 10min, is carried out in gradient elution mode with a BEH C18 column and mobile phases based on 6.7mM ammonia aqueous solution and acetonitrile mixtures. For each analyte the molecular ion and 1 or 2 product ions are acquired, with a mass accuracy better than 5ppm. The quantification is performed using surrogate matrix matched standards, with eprinomectin as internal standard. The high-throughput method, which has been successfully validated, fulfills the requirements of European Union legislation, and has been implemented as a routine method in a public health laboratory.

Acuminolide A: structure and bioactivity of a new polyether macrolide from dinoflagellate Dinophysis acuminata.

Acuminolide A (1), along with pectenotoxin II (PTX-2), dinophysistoxin I (DTX-1), okadaic acid (OA), and 7-epi-PTX-2 seco acid, was isolated from a large-scale cultivation of the dinoflagellate Dinophysis acuminata. The new 33-membered macrolide 1 was characterized by detailed analysis of 2D NMR and MS data. Its relative stereochemistry was elucidated on the basis of ROESY correlations and J-based analysis. In contrast to the other well-known toxins that were isolated, 1 showed no cytotoxicity against four cancer cell lines but caused potent stimulation of actomyosin ATPase activity.

Graphene based pipette tip solid phase extraction of marine toxins in shellfish muscle followed by UPLC-MS/MS analysis.

Graphene is a novel carbonic material with great potentials for the use as sorbent due to its ultrahigh surface area. Herein, we report the use of graphene as sorbent in solid-phase extraction (SPE) using pipette tip as cartridge namely GPT-SPE, together with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), for the analysis of lipophilic marine toxins (LMTs), including yessotoxins (YTX), okadaic acid (OA), dinophysistoxin-1 (DTX1), gymnodimine (GYM), spirolides-1 (SPX1), pectenotoxin-2 (PTX2) and azaspiracid-1 (AZA1) in shellfish. The GPT-SPE procedure was optimized and the performance of graphene was fully validated. Results with high-sensitivity and good reproducibility was obtained and compared with that of other sorbents like C18 silica, multi-walled carbon nanotubes (MWCNTs), commercial Oasis HLB, and Strata-X for the extraction of LMTs, which showed superiority and advantages of graphene, such as good recoveries, stability and compatibility with various solvents. In order to exhibit the potentials of graphene as an excellent sorbent material, 67 mussel samples from six coastal cities of China were analyzed. OA was found to be the dominant contaminant, while YTX was also detected with low level.

First report of the toxin profile of Dinophysis sacculus Stein from LC-MS analysis of laboratory cultures.

Dinophysis sacculus is associated with DSP outbreaks especially in the Mediterranean Sea and is supposed to be mildly toxic based on few toxin results from field samples. First report of LC-MS analysis of D. sacculus cultures from Galicia (NW Spain) showed moderate amounts of OA (7.8 pg cell(-1)) comparable to those found in Dinophysis acuminata from the same region, PTX2 (13.2 pg cell(-1)) and trace amounts of DTX1 (0.8 pg OA equiv. cell(-1)). The contribution of D. sacculus to DSP outbreaks in the Galician Northern Rías should not be underestimated.

Okadaic acid meet and greet: an insight into detection methods, response strategies and genotoxic effects in marine invertebrates.

Harmful Algal Blooms (HABs) constitute one of the most important sources of contamination in the oceans, producing high concentrations of potentially harmful biotoxins that are accumulated across the food chains. One such biotoxin, Okadaic Acid (OA), is produced by marine dinoflagellates and subsequently accumulated within the tissues of filtering marine organisms feeding on HABs, rapidly spreading to their predators in the food chain and eventually reaching human consumers causing Diarrhetic Shellfish Poisoning (DSP) syndrome. While numerous studies have thoroughly evaluated the effects of OA in mammals, the attention drawn to marine organisms in this regard has been scarce, even though they constitute primary targets for this biotoxin. With this in mind, the present work aimed to provide a timely and comprehensive insight into the current literature on the effect of OA in marine invertebrates, along with the strategies developed by these organisms to respond to its toxic effect together with the most important methods and techniques used for OA detection and evaluation.

Diarrhetic shellfish toxins and other lipophilic toxins of human health concern in Washington State.

The illness of three people in 2011 after their ingestion of mussels collected from Sequim Bay State Park, Washington State, USA, demonstrated the need to monitor diarrhetic shellfish toxins (DSTs) in Washington State for the protection of human health. Following these cases of diarrhetic shellfish poisoning, monitoring for DSTs in Washington State became formalized in 2012, guided by routine monitoring of Dinophysis species by the SoundToxins program in Puget Sound and the Olympic Region Harmful Algal Bloom (ORHAB) partnership on the outer Washington State coast. Here we show that the DSTs at concentrations above the guidance level of 16 µg okadaic acid (OA) + dinophysistoxins (DTXs)/100 g shellfish tissue were widespread in sentinel mussels throughout Puget Sound in summer 2012 and included harvest closures of California mussel, varnish clam, manila clam and Pacific oyster. Concentrations of toxins in Pacific oyster and manila clam were often at least half those measured in blue mussels at the same site. The primary toxin isomer in shellfish and plankton samples was dinophysistoxin-1 (DTX-1) with D. acuminata as the primary Dinophysis species. Other lipophilic toxins in shellfish were pectenotoxin-2 (PTX-2) and yessotoxin (YTX) with azaspiracid-2 (AZA-2) also measured in phytoplankton samples. Okadaic acid, azaspiracid-1 (AZA-1) and azaspiracid-3 (AZA-3) were all below the levels of detection by liquid chromatography tandem mass spectrometry (LC-MS/MS). A shellfish closure at Ruby Beach, Washington, was the first ever noted on the Washington State Pacific coast due to DSTs. The greater than average Fraser River flow during the summers of 2011 and 2012 may have provided an environment conducive to dinoflagellates and played a role in the prevalence of toxigenic Dinophysis in Puget Sound.

New invertebrate vectors for PST, spirolides and okadaic acid in the North Atlantic.

The prevalence of poisoning events due to harmful algal blooms (HABs) has declined during the last two decades through monitoring programs and legislation, implemented mainly for bivalves. However, new toxin vectors and emergent toxins pose a challenge to public health. Several locations on the Portuguese coast were surveyed between 2009 and 2010 for three distinct biotoxin groups [saxitoxin (PST), spirolide (SPX) and okadaic acid (OA)], in 14 benthic species of mollusks and echinoderms. Our main goals were to detect new vectors and unravel the seasonal and geographical patterns of these toxins. PSTs were analyzed by the Lawrence method, SPXs by LC-MS/MS, and OA by LC-MS/MS and UPLC-MS/MS. We report 16 new vectors for these toxins in the North Atlantic. There were differences in toxin contents among species, but no significant geographical or seasonal patterns were found. Our results suggest that legislation should be adjusted to extend the monitoring of marine toxins to a wider range of species besides edible bivalves.

[Isolation and identification of shellfish toxins from contaminated blue mussel (Mytilus edulis) from the East China Sea].

OBJECTIVE: To investigate the contamination of shellfish poisoning of mussels, the poisonous constituents in that were isolated and identified. METHODS: The mussel tissue homogenate was extracted by acetone, and then the acetone extract was partitioned between diethyl ether and water. The ether extract was fractionated by column chromatography over silica gel and further isolated by semi-preparative RP-HPLC, monitored by ultra performance liquid chromatography coupled with triple quadrupole mass spectrometry. RESULTS: Four poisonous constituents were isolated. Two of them were elucidated as pectenotoxin-2 seco acid and 7-epi-pectenotoxin-2 seco acid, respectively, on the basis of mass spectral data and compared with the production of enzymatic hydrolysis of PTX-2, and others were identified as okadaic acid and dinophysistoxin-1 by UPLC-MS/MS analysis compared with standard substances. CONCLUSION: OA, DTX-1, 7-epi-PTX-2sa and PTX-2sa had been isolated from the mussel, respectively. The concentrations of free OA, DTX-1 and total OA in which were surpassed the maximum permitted levels in EU. OA and DTX-1 were confirmed to be the main toxins responsible for this DSP outbreak.

First detection and seasonal variation of lipophilic toxins okadaic acid, dinophysistoxin-1, and yessotoxin in Korean gastropods.

Okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2, and yessotoxin (YTX) are classes of lipophilic toxins found in marine animals. OA and DTX1 accumulation causes diarrhetic shellfish poisoning, a worldwide public health problem. Diarrhetic shellfish poisoning has not previously been reported in gastropods, which are widely consumed in Korea. Seasonal variation in marine lipophilic toxins in gastropods was investigated using liquid chromatography-tandem mass spectrometry. Eighty specimens of Neptunea cumingii, 65 specimens of Rapana venosa, and 95 specimens of Batillus cornutus were collected at the Tongyeong fish market on the southern coast of Korea between May 2009 and December 2010. OA, DTX1, and YTX were detected in meat and digestive glands in all gastropod species studied. Pectenotoxin-2 was not found in any sample tested. Lipophilic toxins were detected in the digestive glands of gastropods; no lipophilic toxin was detected in the salivary glands of the carnivorous gastropods, N. cumingii and R. venosa. The highest concentrations of OA (21.5 ng/g) and DTX1 (8.4 ng/g) were detected in the digestive glands of R. venosa, and the maximum concentration of YTX (13.7 ng/g) was found in the digestive glands of N. cumingii. The maximum toxicities in gastropod tissues were lower than the European standard for acceptable levels. The concentrations of lipophilic toxins in carnivorous gastropods showed a high degree of seasonal variation; lipophilic toxins in carnivorous gastropods were found predominantly in spring and summer. This is the first report of the occurrence of lipophilic toxins in Korean gastropods.

A simple colorimetric enzymatic-assay for okadaic acid detection based on the immobilization of protein phosphatase 2A in sol-gel.

Okadaic acid (OA), a lipophilic toxin, is produced by Dinophysis and Prorocentrum, and causes diarrheic shellfish poisoning to humans. The mechanism of OA action is based on the reversible inhibition of protein phosphatase type 2A (PP2A) by the toxin. Therefore, this inhibition could be used to develop assay for OA detection. In this work, a colorimetric test based on the PP2A inhibition was developed for OA detection. PP2A from GTP and Millipore was immobilized on silica sol-gel, and the detection was performed. A limit of detection of 0.29 and 1.14 µg/L was respectively observed for enzyme from GTP and Millipore. The immobilization technique provided a tool to preserve the enzymatic activity, which is very unstable in solution. The PP2A immobilized sol-gel exhibited a storage stability of near 5 months, when microtiter plate with enzyme-immobilized polymer was kept at -18C°. The combination of the simplicity of the colorimetric method, along with long storage stability achieved by sol-gel immobilization, demonstrated the potentiality of this technique to be used for commercial purpose.