Search

Abstract Analysis of marine biotoxins in high-lipid bivalves has several limitations due to the coexistence of lipid matrices, low biotoxin concentrations, and the different physiochemical properties of the various biotoxins. In this study, a sensitive and reliable method to simultaneously determine six diarrhetic shellfish poisoning (DSP) toxins (five acidic and one neutral biotoxins) in oysters and mussels was developed based on UHPLC-MS/MS-multiple ion reaction monitoring (MRM) using time segment polarity switching. For effective sample pretreatment, three protocols including syringe filtration, Strata™-X solid phase extraction (SPE), and freezing lipid filtration combined with SPE (FLF + SPE) were evaluated, and FLF + SPE method was shown to be the most effective with high lipid removal efficiency. Aging effects of the UHPLC mobile phase on MS detection sensitivity and retention time shift of DSP toxins were carefully examined. The developed method provided good recoveries ranging from 81.0 to 119.6%, showing no significant matrix effect and assay was linear with coefficients of determination above R2 > 0.99 for all analytes. Validation tests using a certified reference material (CRM) and spiking DSP toxins (OA and DTX-1 at 1.07 μg/g, and DTX-2 at 0.86 μg/g) showed this method to be acceptable, with a relative standard deviation of <0.17% and recovery ranging from 87.29 to 109.97%. Finally, the established method was successfully applied to quantify DSP toxins in 40 oyster and mussel samples collected from Korean fishery markets, ensuring sea food safety. Highlights • Three extraction protocols were examined for determining lipophilic DSP toxins in high-lipid bivalves • SPE combined with FLF was shown the best analytical performance in terms of cleanup efficiency, matrix effect, and sensitivity of DSP toxins. • Simultaneously determined different polarity DSP toxins in a single run using time segment polarity switching mode • Validated quantification of trace DSP toxins through CRM analysis and spiking experiments • Successful monitoring the levels of DSP toxins in high-lipid bivalves [ABSTRACT FROM AUTHOR]

Polar marine toxins are more challenging to analyze by mass spectrometry-based methods than lipophilic marine toxins, which are now routinely measured in shellfish by multiclass reversed-phase liquid chromatography-tandem mass spectrometry (MS/MS) methods. Capillary electrophoresis (CE)-MS/MS is a technique that is well suited for the analysis of polar marine toxins, and has the potential of providing very high resolution separation. Here, we present a CE-MS/MS method developed, with use of a custom-built interface, for the sensitive multiclass analysis of paralytic shellfish toxins, tetrodotoxins, and domoic acid in seafood. A novel, highly acidic background electrolyte (5 M formic acid) was designed to maximize protonation of analytes and to allow a high degree of sample stacking to improve the limits of detection. The method was applied to a wide range of regulated and less common toxin analogues, and exhibited a high degree of selectivity between toxin isomers and matrix interference. The limits of detection in mussel tissue were 0.0052 mg/kg for tetrodotoxins, 0.160 mg/kg for domoic acid, and between 0.0018 and 0.120 mg/kg for paralytic shellfish toxins, all of which showed good linearity. Minimal ionization suppression was observed when the response from neat and mussel-matrix-matched standards was corrected with multiple internal standards. Analysis of shellfish matrix reference materials and spiked samples demonstrated good accuracy and precision. Finally, the method was transferred to a commercial CE-MS/MS system to demonstrate its widespread applicability for use in both R & D and routine regulatory settings. The approach of using a highly acidic background electrolyte is of broad interest, and can be considered generally applicable to simultaneous analysis of other classes of small, polar molecules with differing pKa values.ᅟ [ABSTRACT FROM AUTHOR]

Evaluation of shellfish aquaculture for potential contaminants is essential for consumer confidence and safety. Every three years, between 1999 and 2014, bivalve shellfish from aquaculture zones in up to 31 estuaries across 2,000 km of Australia's east coast were tested for cadmium, copper, lead, mercury, selenium and zinc. Inorganic arsenic was included in the analyses in 2002, and total arsenic was used as a screen for the inorganic form in subsequent years. Concentrations of inorganic arsenic, cadmium, lead and mercury were low and did not exceed maximum limits mandated in the Australia New Zealand Food Standards Code. As maximum limits have not been assigned to copper, selenium and zinc, accepted international dietary guidelines were used as a benchmark. Dietary exposure assessments for these elements demonstrated that shellfish from the aquaculture areas investigated do not present a food safety risk. Continued surveillance is essential, given increasing pressure on Australia's coastal resources. [ABSTRACT FROM AUTHOR]

This work reports the first communication relating to the presence of yessotoxins in Mytilus galloprovincialis from coastal mussel farms (Sardinia, western Mediterranean) detected during 2008 and 2013 through a monitoring programme. The paper emphasizes how the changes both in yessotoxin permitted limits and used methods, established by legislation, have influenced the interpretation of the obtained results. Consequently, the samples that resulted negative during 2008 would have been positive until August 2013 and negative from September 2013 up to now, and the samples that were positive in 2013 would have been positive in 2008 and negative nowadays, according to Regulation currently in force. Regular monitoring of biotoxins demonstrated that, although yessotoxins have been rarely present in the past in Sardinia, they may cause toxicity in shellfish. So, it's important to keep up on legislation's changing and laboratory methods. [ABSTRACT FROM AUTHOR]

The identification of a new suite of toxins, called azaspiracids (AZA), as the cause of human illnesses after the consumption of shellfish from the Irish west coast in 1995, resulted in interest in understanding the global distribution of these toxins and of species of the small dinoflagellate genus Azadinium , known to produce them. Clonal isolates of four species of Azadinium, A. poporum, A. cuneatum, A. obesum and A. dalianense were obtained from incubated sediment samples collected from Puget Sound, Washington State in 2016. These Azadinium species were identified using morphological characteristics confirmed by molecular phylogeny. Whereas AZA could not be detected in any strains of A. obesum, A. cuneatum and A. dalianense , all four strains of A. poporum produced a new azaspiracid toxin, based on LC–MS analysis, named AZA-59. The presence of AZA-59 was confirmed at low levels in situ using a solid phase resin deployed at several stations along the coastlines of Puget Sound. Using a combination of molecular methods for species detection and solid phase resin deployment to target shellfish monitoring of toxin at high-risk sites, the risk of azaspiracid shellfish poisoning can be minimized. [ABSTRACT FROM AUTHOR]

Paralytic shellfish toxins (PSTs) are neurotoxins produced by dinoflagellates and cyanobacteria that cause paralytic shellfish poisoning in humans. PST quantitation by LC-MS is challenging because of their high polarity, lability as gas-phase ions, and large number of potentially interfering analogues. Differential mobility spectrometry (DMS) has the potential to improve the performance of LC-MS methods for PSTs in terms of selectivity and limits of detection. This work describes a comprehensive investigation of the separation of 16 regulated PSTs by DMS and the development of highly selective LC-DMS-MS methods for PST quantitation. The effects of all DMS parameters on the separation of PSTs from one another were first investigated in detail. The labile nature of 11α-gonyautoxin epimers gave unique insight into fragmentation of labile analytes before, during, and after the DMS analyzer. Two sets of DMS parameters were identified that either optimized the resolution of PSTs from one another or transmitted them at a limited number of compensation voltage (CV) values corresponding to structural subclasses. These were used to develop multidimensional LC-DMS-MS/MS methods using existing HILIC-MS/MS parameters. In both cases, improved selectivity was observed when using DMS, and the quantitative capabilities of a rapid UPLC-DMS-MS/MS method were evaluated. Limits of detection of the developed method were similar to those without DMS, and differences were highly analyte-dependant. Analysis of shellfish matrix reference materials showed good agreement with established methods. The developed methods will be useful in cases where specific matrix interferences are encountered in the LC-MS/MS analysis of PSTs in complex biological samples. [ABSTRACT FROM AUTHOR]

Background Harmful algal blooms produce paralytic shellfish toxins that accumulate in the tissues of filter feeding shellfish. Ingestion of these toxic shellfish can cause a serious and potentially fatal condition known as paralytic shellfish poisoning (PSP). The coast of British Columbia is routinely monitored for shellfish toxicity, and this study uses data from the monitoring program to identify spatiotemporal patterns in shellfish toxicity events and their relationships with environmental variables. Methods The dinoflagellate genus Alexandrium produces the most potent paralytic shellfish toxin, saxitoxin (STX). Data on all STX measurements were obtained from 49 different shellfish monitoring sites along the coast of British Columbia for 2002–2012, and monthly toxicity events were identified. We performed hierarchical cluster analysis to group sites that had events in similar areas with similar timing. Machine learning techniques were used to model the complex relationships between toxicity events and environmental variables in each group. Results The Strait of Georgia and the west coast of Vancouver Island had unique toxicity regimes. Out of the seven environmental variables used, toxicity in each cluster could be described by multivariable models including monthly sea surface temperature, air temperature, sea surface salinity, freshwater discharge, upwelling, and photosynthetically active radiation. The sea surface salinity and freshwater discharge variables produced the strongest univariate models for both geographic areas. Conclusions Applying these methods in coastal regions could allow for the prediction of shellfish toxicity events by environmental conditions. This has the potential to optimize biotoxin monitoring, improve public health surveillance, and engage the shellfish industry in helping to reduce the risk of PSP. [ABSTRACT FROM AUTHOR]

The edible bivalves Perna viridis (green mussel), (n = 100) were analysed for their total Hg, Pb, Cu, Ni, Zn, Co, Fe, Cr, Cd, and Mn concentrations to indicate heavy metal contamination in Paradise Point of Karachi coast using atomic absorption spectrophotometer. There are large seasonal variations in the metal concentrations of Mn (0.025-0.67 μg/g), Fe (0.055-7.740 μg/g), Ni (0.004-0.52 μg/g), Hg (0.0001-0.004 μg/g), Zn (0.04-3.32 μg/g), Cu (0.008-1.66 μg/g), Pb (0.022-2.43 μg/g), Co (0.01-0.044 μg/g), Cd (0.04-0.88 μg/g) and Cr (0.13-1.20 μg/g) recorded in bodies/soft tissues of P. viridis obtained in the samples of the year 1993 and 2012 at the Paradise Point of Karachi coast. The results of heavy metals are in the following descending order of concentration in the samples collected in the year 1993: Fe>Cr>Zn>Mn> Pb>Cd>Cu>Ni>Co>Hg, while Fe>Zn>Pb>Cu>Cr>Cd>Mn>Ni>Co>Hg order was recorded in samples collected in the year 2012. The high accumulation of metals was found mostly in the samples collected in the year 2012 when compared with the samples of the year 1993. This is an indication that the area under study showed signs of being exposed to significant levels of heavy metal pollution due to direct discharge of industrial and domestic wastes along the coast. The concentrations of these heavy metals were lower than the permissible limits for human consumption. However, if this pollution persists, it can prove to be very detrimental in future. [ABSTRACT FROM AUTHOR]

The cyclic imine toxin 20-methyl spirolide G (20-meSPX-G), produced by the toxigenic dinoflagellate Alexandrium ostenfeldii/Alexandrium peruvianum, has been previously reported to contaminate shellfish in various European coastal locations, as revealed by mouse toxicity bioassay. The aim of the present study was to determine its toxicological profile and its molecular target selectivity. 20-meSPX-G blocked nerve-evoked isometric contractions in isolated mouse neuromuscular preparations, while it had no action on contractions elicited by direct electrical stimulation, and reduced reversibly nerve-evoked compound muscle action potential amplitudes in anesthetized mice. Voltage-clamp recordings in Xenopus oocytes revealed that 20-meSPX-G potently inhibited currents evoked by ACh on Torpedo muscle-type and human α7 nicotinic acetylcholine receptors (nAChR), whereas lower potency was observed in human α4β2 nAChR. Competition-binding assays showed that 20-meSPX-G fully displaced [³H]epibatidine binding to HEK-293 cells expressing the human α3β2 (Ki = 0.040 nM), whereas a 90-fold lower affinity was detected in human α4β2 nAChR. The spirolide displaced [125I]α-bungarotoxin binding to Torpedo membranes (Ki = 0.028 nM) and in HEK-293 cells expressing chick chimeric α7-5HT3 nAChR (Ki = 0.11 nM). In conclusion, this is the first study to demonstrate that 20-meSPX-G is a potent antagonist of nAChRs, and its subtype selectivity is discussed on the basis of molecular docking models. [ABSTRACT FROM AUTHOR]

Okadaic acid (OA) and its analogs, dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic biotoxins produced by marine algae that can accumulate in shellfish and cause the human illness known as diarrhetic shellfish poisoning (DSP). Regulatory testing of shellfish is required to protect consumers and the seafood industry. Certified reference materials (CRMs) are essential for the development, validation, and quality control of analytical methods, and thus play an important role in toxin monitoring. This paper summarizes work on research and development of shellfish tissue reference materials for OA and DTXs. Preliminary work established the appropriate conditions for production of shellfish tissue CRMs for OA and DTXs. Source materials, including naturally incurred shellfish tissue and cultured algae, were screened for their DSP toxins. This preliminary work informed planning and production of a wet mussel (Mytilus edulis) tissue homogenate matrix CRM. The homogeneity and stability of the CRM were evaluated and found to be fit-for-purpose. Extraction and LC-tandem MS methods were developed to accurately certify the concentrations of OA, DTX1, and DTX2 using a combination of standard addition and matrix-matched calibration to compensate for matrix effects in electrospray ionization. The concentration of domoic acid was also certified. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization. The presence of other toxins in the CRM was also assessed and information values are reported for OA and DTX acyl esters. [ABSTRACT FROM AUTHOR]

Okadaic acid (OA) and its analogs dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic polyethers produced by marine dinoflagellates. These toxins accumulate in shellfish and cause diarrhetic shellfish poisoning (DSP) in humans. Regulatory testing of shellfish is essential to safeguard public health and for international trade. Certified reference materials (CRMs) play a key role in analytical monitoring programs. This paper presents an overview of the interdisciplinary work that went into the planning, production, and certification of calibration-solution CRMs for OA, DTX1, and DTX2. OA and DTX1 were isolated from large-scale algal cultures and DTX2 from naturally contaminated mussels. Toxins were isolated by a combination of extraction and chromatographic steps with processes adapted to suit the source and concentration of each toxin. New 19-epi-DSP toxin analogs were identified as minor impurities. Once OA, DTX1, and DTX2 were established to be of suitable purity, solutions were prepared and dispensed into flame-sealed glass ampoules. Certification measurements were carried out using quantitative NMR spectroscopy and LC-tandem MS. Traceability of measurements was established through certified external standards of established purity. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization, with components from the measurement, stability, and homogeneity studies being propagated into final combined uncertainties. [ABSTRACT FROM AUTHOR]

The worldwide ongoing increase in the frequency, severity and geographic distribution of harmful algal blooms (HABs), together with the huge economic losses they cause, represent a significant problem in many coastal regions. The consumption of shellfish contaminated with marine toxins can trigger serious and potentially fatal intoxications in human populations; most of them can be prevented through the forced prolonged closure of large harvesting areas to allow shellfish decontamination. To reduce the impact produced on the aquaculture sector, new methods for detecting toxic blooms are constantly being reported, and their effects on their principal vectors, bivalve mollusks, are examined. These organisms seem to be insensitive somehow to harmful effects of these toxins since their viability rates after toxic blooms are in general maintained. Nevertheless, some recent biomonitoring studies proved that they can indeed show a cellular and molecular response against toxin exposure. In the last decade, there has been an increase in the use of biomarkers of effect to estimate the negative impact of marine toxins on bivalve mollusks. Certain cellular and molecular parameters are considered as suitable biomarkers, since they reflect the biological response of these organisms to marine toxins. Thus, cellular biomarkers such as DNA breaks, chromosome alterations or cell viability rates are used to evaluate genotoxic and cytotoxic effects; whereas, molecular biomarkers such as gene expression levels, protein levels and enzyme activities of heat-shock proteins, multi-xenobioticresistance proteins or antioxidant enzymes have been studied to assess the response to these toxins. The aim of this chapter is to provide an overview of the current status of biomarker use in the assessment of the effects of marine toxins on bivalve mollusks. In a general way, this work reviews the studies dealing with the application of cellular and molecular biomarkers for monitoring and assessing the different effects induced by exposure of bivalve mollusks to marine toxins. [ABSTRACT FROM AUTHOR]

The present study used similarity analysis of paralytic shellfish poisoning toxins (PSP-toxins) profile patterns to analyze epidemiological linkage of a group of toxins contaminated bivalves collected between years 2004 and 2015. A batch of suspected PSP-toxins contaminated geoduck clams was obtained from import freight and different markets in Hong Kong, the samples of which were identified originating from an overseas shellfish harvest region. Laboratory PSP tests including PSP rapid immunoassay, mouse and chemical assays on that batch of samples confirmed the presence of PSP-toxins with violated levels exceeding 80 μg STXeq/100g of shellfish tissues. Related shellfish products imported from the affected region and other nonimplicated areas were continuously investigated for PSP-toxins contamination for a period of time after this incident. PSP-toxins in the tainted samples were separated by ion-pair chromatography followed by post-column oxidation to fluorescent derivatives for toxins identification and quantification. STX, NEO and GTX2 were the most abundant toxins found in the samples. These three toxins were also the commonly occurring toxins identified in geoduck clams over the past ten years. Molar distributions (mol%) of PSP-toxins in the samples were analyzed by Unweighted Pair Group Method with Arithmetic averages (UPGMA). The toxin profile patterns of the implicated samples were also compared retrospectively with PSP-contaminated geoduck clam samples found in the past 12 years. A cluster with over 96% of similarity level was identified from the implicated samples collected during the incident, suggesting that the group of samples was probably originated from a closely related source/batch. With the application of PSPtoxins profile as specific biochemical fingerprint, the patterns of toxins profile can provide potentially useful information/clue for tracing the possible origin(s) of toxic shellfish in the market [ABSTRACT FROM AUTHOR]

In areas where toxic algal blooms occur, the organisms primarily affected are shellfish, which, because of their high levels of filtration (clearance rate), accumulate high concentrations of nutrients and toxic phytoplankton in their visceral tissue. This accumulation of toxins in shellfish leads to distribution of these toxins to their different non-visceral tissues. This leads to a variation of assimilated toxic profiles (dinoflagellates) through different biotransformation routes (enzymatic and nonenzymatic) present in the different species of shellfish that may be affected by Harmful algal blooms (HABs), which transforms them into powerful vectors of different groups of toxins. The generated toxic variability can be promoted through the transfer of toxins through the food chain, thusallowing the accumulation of toxins in various living organisms that inhabit in the ocean (zooplankton, fish, gastropods, sea lions and whales). This has been important in determining the potential biotransformation forms and degrees of purification occurring in different marine species affected by toxic blooms. The lipophilic toxins identified in shellfish correspond to a broad spectrum of toxins consisting of the okadaic acid-group (OA-group), Pectenotoxins group (PTX-group), Yessotoxin group (YTX-group) and Azaspiracid group (AZA-group). In the present study, a high performance liquid chromatographic MS/MS method was optimized for the simultaneous determination of OA-group, PTX-group, YTX-group and AZA-group in multiple endemichydrobiological organisms (bivalves and gastropods) of the southern sea of Chile. The toxins were extracted from hydrobiological organisms using standard operating procedures. The result reveals that liquid chromatography coupled to mass spectrometry (LCMS/ MS) allows for the rapid and sensitive determination of lipophilic toxins with minimal sample clean-up, allowing for the detection of biotransformation levels in different hydrobiological organisms with high commercial level. [ABSTRACT FROM AUTHOR]

Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv–pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC–CLEIA (chemiluminescent enzyme immunoassay). The IC 15 was 0.012 ± 0.02 μg/L, and the IC 50 was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv–antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection. [ABSTRACT FROM AUTHOR]

The effect of canning in pickled sauce and autoclaving on weight, toxin content, toxin concentration and toxicity of steamed mussels was studied. Weight decreased by 25.5%. Okadaic acid (OA) and DTX2 content of mussel meat decreased by 24.1 and 42.5%, respectively. The estimated toxicity of the mussel remained nearly unchanged (increased by 2.9%). A part of the toxins lost by the mussels was leached to the sauce but the remaining part should have been thermally degraded. DTX2 underwent more degradation than OA and, in both toxins, free forms more than conjugated ones. This process, therefore, cannot be responsible for the large increments of toxicity of processed mussels –relative to the raw ones-sometimes detected by food processing companies. The final product could be monitored in several ways, but analysing the whole can content or the mussel meat once rehydrated seems to be the most equivalents to the raw mussel controls. [ABSTRACT FROM AUTHOR]

This study investigated the application of a three-dimensional physical hydrodynamic model in a harmful algal bloom forecast system for Bantry Bay, southwest Ireland. Modelled oceanographic conditions were studied and used to help understand observed changes in the chemical and biological patterns from the national biotoxins and phytoplankton monitoring program. The study focused on two toxic events in 2013. An upwelling event was predicted by the model prior to the appearance and population increase of potentially toxic diatoms, Pseudo-nitzschia , and associated domoic acid in shellfish. A downwelling episode was provided as a forecast in the model prior to the arrival of a Dinophysis bloom and detection of its associated biotoxins in Bay shellfish. The modelled forecast products developed included expected surface, mid-depth and bottom current pathways at the mouth of the Bay and on the adjacent shelf. The rate and direction of water volume flow at the mouth and mid-bay sections were produced by the model to examine predicted upwelling and downwelling pulses. The model also calculated the evolution of water properties (temperature, salinity and density) with depth along the Bay axis and on the adjacent continental shelf. Direct measurements of water properties at a fixed point, mid-bay, were comparable to model calculations. The operational model for southwest Ireland produces a reliable 3-day physical hydrodynamic forecast of the dominant regional physical processes that result in water exchange events between Bantry Bay and its adjacent shelf. While simulated physical hydrodynamics were provided as a 3-day forecast, the upwelling and downwelling signals from the model, closely linked to toxic HAB episodes, were evident up to 10 days prior to the contamination of shellfish in the Bay. [ABSTRACT FROM AUTHOR]

Okadaic acid (OA) and their derivatives are marine toxins responsible for the human diarrhetic shellfish poisoning (DSP). To date the amount of toxins ingested in food has been considered equal to the amount of toxins available for uptake by the human body. In this study, the OA fraction released from the food matrix into the digestive fluids (bioaccessibility) was assessed using a static in vitro digestion model. Naturally contaminated mussels ( Mytilus galloprovincialis ) and donax clams ( Donax sp.), collected from the Portuguese coast, containing OA and dinophysistoxin-3 (DTX3) were used in this study. Bioaccessibility of OA total content was 88% and 75% in mussels and donax clams, respectively. Conversion of DTX3 into its parent compound was verified during the simulated digestive process and no degradation of these toxins was found during the process. This is the first study assessing the bioaccessibility of OA-group toxins in naturally contaminated seafood. This study provides relevant new data that can improve and lead to more accurate food safety risk assessment studies concerning these toxins. [ABSTRACT FROM AUTHOR]

Four commercial rapid screening methods for Diarrhetic Shellfish Poisoning were applied to the analysis of naturally contaminated shellfish samples from GB. The performance of each kit was assessed through comparison with the reference LC-MS/MS method on a range of both positive and negative bivalve mollusc samples. A quantitative PP2A protein phosphatase assay was the only assay to show the complete absence of false negative results. It showed a fair correlation with LC-MS/MS but with an overall overestimation of sample toxicity together with some indications of interference from sample matrix, most notably within oyster species. A quantitative competitive ELISA also gave a fair correlation with LC-MS/MS, with no evidence of toxicity overestimation and with a good response to samples containing little or no DST's, although one false negative was recorded. The two qualitative lateral flow assays both provided a high percentage agreement with the LC-MS/MS results and there were no indications of false positive results, although both kits also returned one false negative result. The false negative results returned by the three assays were all associated with samples containing high proportions of DTX2, a toxin which occurs commonly in UK shellfish. The scanners provided with both lateral flow assays were easy to use and the provision of numerical results enables a semi-quantitative assessment of toxicities which would significantly benefit the end user. Whilst key differences exist between the proposed assays they are all rapid, do not require expensive equipment and the work here has provided some evidence for suitability for indicative testing for some species of bivalve shellfish from GB. Further work is required however using a larger number of test kit batches on a greater number of samples, particularly for those containing high proportions of DTX2. [ABSTRACT FROM AUTHOR]

Analysis of bivalve molluscs for Diarrhetic Shellfish Poisoning is challenging for producers of shellfish who are unlikely to have access to LC-MS/MS technology for toxin testing. Four commercial rapid testing kits were applied to the analysis of Argentinean shellfish for DSP toxins, comparing results against the regulatory LC-MS/MS method. On the whole the specificity of the methods was good, with no false positive results produced in relation to the maximum permitted limits of DSP toxins. Two lateral flow assays provided an excellent qualitative assessment of DSP-positive samples whilst the quantitative PP2A and ELISA methods showed good evidence for consumer protection from the quantitative results generated. Results from lateral flow devices were further assessed through use of automated scanning devices, significantly improving the repeatability of data analysis and enabling semi-quantitation of test results. Whilst key differences exist between assays, from these results all four would be suitable for end product testing of shellfish in Argentina for DSP. The choice of method is therefore most likely to relate to the practicalities and applicability associated with each assay. [ABSTRACT FROM AUTHOR]

The multifactorial etiology of massive Crassostrea gigas summer mortalities results from complex interactions between oysters, opportunistic pathogens and environmental factors. In a field survey conducted in 2014 in the Mediterranean Thau Lagoon (France), we evidenced that the development of the toxic dinoflagellate Alexandrium catenella, which produces paralytic shellfish toxins (PSTs), was concomitant with the accumulation of PSTs in oyster fresh and the occurrence of C. gigas mortalities. In order to investigate the possible role of toxic algae in this complex disease, we experimentally infected C. gigas oyster juveniles with Vibrio tasmaniensis strain LGP32, a strain associated with oyster summer mortalities, after oysters were exposed to Alexandrium catenella. Exposure of oysters to A. catenella significantly increased the susceptibility of oysters to V. tasmaniensis LGP32. On the contrary, exposure to the non-toxic dinoflagellate Alexandrium tamarense or to the haptophyte Tisochrysis lutea used as a foraging alga did not increase susceptibility to V. tasmaniensis LGP32. This study shows for the first time that A. catenella increases the susceptibility of Crassostrea gigas to pathogenic vibrios. Therefore, in addition to complex environmental factors explaining the mass mortalities of bivalve mollusks, feeding on neurotoxic dinoflagellates should now be considered as an environmental factor that potentially increases the severity of oyster mortality events. [ABSTRACT FROM AUTHOR]

Paralytic Shellfish Toxin blooms are common worldwide, which makes their monitoring crucial in the prevention of poisoning incidents. These toxins can be monitored by a variety of techniques, including mouse bioassay, receptor binding assay, and liquid chromatography with either mass spectrometric or pre- or post-column fluorescence detection. The post-column oxidation liquid chromatography with fluorescence detection method, used routinely in our laboratory, has been shown to be a reliable method for monitoring paralytic shellfish toxins in mussel, scallop, oyster and clam species. However, due to its high sensitivity to naturally fluorescent matrix interferences, when working with unconventional matrices, there may be problems in identifying toxins because of naturally fluorescent interferences that co-elute with the toxin peaks. This can lead to erroneous identification. In this study, in order to overcome this challenge in echinoderm and gastropod matrices, we optimized the conversion of Gonyautoxins 1 and 4 to Neosaxitoxin with 2-mercaptoethanol. We present a new and less time-consuming method with a good recovery (82.2%, RSD 1.1%, n = 3), requiring only a single reaction step. [ABSTRACT FROM AUTHOR]

Recent studies have examined lipophilic marine toxins (LMTs) in shellfish and toxic algae worldwide, but the occurrence and seasonal variations of LMTs in commercial clams (including Mactra veneriformis, Ruditapes philippinarum, Meretrix meretrix, and Cyclina sinensis) at their major culturing area in Jiangsu, China, remain largely unexplored. In this study, a new solid phase extraction (SPE) in combination with an ultra-fast liquid chromatography and triple-quadrupole linear ion trap mass spectrometry (UFLC-TQ-MS) method was developed to determine the presence of 10 typical LMTs (okadaic acid (OA), yessotoxins (YTXs), azaspiracids (AZA1-3), pectenotoxins (PTX2), gymnodimine (GYM), dinophysistoxins (DTX1&2), and spirolides (SPX1)) in the aforementioned four clam matrices. After confirmation of its sensitivity and precision, this method was used to evaluate the amounts of LMTs in clam samples harvested in five aquaculture zones of the Jiangsu coastal area. Monthly variations of GYM, PTX2, OA, and DTX1&2 in 400 clam samples from the sample areas were determined from January 2014 through August 2015. Peak values were observed during May and August. This is the first systematic report of LMTs detected in clam samples harvested in Jiangsu. Follow-up research and the implementation of protective measures are needed to ensure the safety of clams harvested in this area. [ABSTRACT FROM AUTHOR]

The presence of phytoplankton responsible for the production of lipophilic marine biotoxins is well recognised throughout parts of South America. To date, the quantitation of lipophilic toxins in Argentinean shellfish has been limited to select and highly focussed geographical studies. This work reports the analysis for lipophilic marine biotoxins in shellfish harvested across five regions of Argentina between 1992 and 2012. LC-MS/MS analysis was used for the quantitation of all regulated lipophilic toxins. High concentrations of okadaic acid group toxins were quantified, with a clear dominance of the parent okadaic acid and more than 90% of the toxin present as esters. Results showed DSP toxins in shellfish from the Buenos Aires Province during 2006 and 2007, earlier than previously described. There was also strong evidence linking the presence of okadaic acid to human intoxications. Other lipophilic toxins detected were yessotoxin, pectenotoxin-2 and 13-desMeC spirolide. With evidence published recently for the presence of azaspiracid producers, this work reports the detection of low concentrations of azaspiracid-2 in shellfish. As such the data provides the first published evidence for yessotoxins and azaspiracids in Argentinean shellfish and further evidence for the continuing presence of lipophilic marine toxins in Argentinean waters. [ABSTRACT FROM AUTHOR]

The Scotia Rapid Test for PSP is designed for qualitative identification of saxitoxins at levels in shellfish equivalent to the limit of detection of the biological reference method. However, issues with the method have been reported, including the low assay cross reactivity for some toxins, high numbers of false positive results and the subjective test interpretation. This study focussed on approaches to improve each of these issues. A refined test was found to improve GTX1&4 test sensitivity in samples containing high proportions of GTX1&4. The subjectivity of the test was successfully eliminated through use of an automated scanner, which enabled both the reliable identification of test results as well as the provision of a numerical result which could be utilised for more refined results interpretation. Finally the high proportion of false positive results in comparison with the LC-FLD was investigated, with a modified approach incorporating an additional extract dilution applied to a range of shellfish samples with different toxicities. Results showed highly variable limits of detection of the method and no significant reduction in false positive results when applying the additional dilution, which may be of concern to laboratories in receipt of high numbers of samples containing low concentrations of toxins. [ABSTRACT FROM AUTHOR]

Protoceratium reticulatum is a dinoflagellate with a life cycle that includes a motile planktonic stage and a resting cyst stage in benthic habitat, both with a wide geographical distribution, including southern South America. P. reticulatum produces yessotoxins (YTX) – these can be accumulated in shellfish and show potent cytotoxicity, posing a risk to human health if contaminated shellfish is consumed. YTX have been reported from coastal shellfish of many localities, but until now it was unknown if they were present in the austral Southwestern Atlantic and also if local populations of P. reticulatum have the ability to produce these toxins. In this study we report the presence of YTX in plankton samples and its production in culture by two P. reticulatum strains isolated from the San Jorge Gulf (SJG). In addition, we describe the geographical distribution and seasonal abundance of this species based on data collected over the past two decades. The YTX cell quotas calculated from net hauls (∼10 pg cell −1 ) are in the same range as the toxin cell quotas observed in these two isolates. The phylogenetic analysis of sequences of the hypervariable region of the large subunit (LSU) 28S rDNA showed that the two clonal strains from the SJG were part of a monophyletic clade that subdivides P. reticulatum into two well-supported, divergent sub-clades. The sequences of the two strains of P. reticulatum from the SJG fell in the same clade as the majority of sequences of P. reticulatum , which belong to a geographically widely distributed evolutionary clade. P. reticulatum was occasionally observed from about 35° S in Uruguayan shelf waters up to 53° S on the Patagonian shelf and north of Tierra del Fuego, and it was present from coastal areas up to the shelf break zone. We recorded P. reticulatum in plankton samples during spring, summer and autumn but invariably in low abundance (maximum: 560 cells L −1 ). Viable cysts of the species in surface sediments also showed a wide geographical distribution. Together, the high total abundances and high relative numerical contribution to planktonic dinoflagellate assemblages near frontal areas, emphasize the necessity to pay attention to the dynamics of this species in areas of potential risk of harmful algal bloom development. [ABSTRACT FROM AUTHOR]

A single-laboratory validation study was conducted for the hydrophilic interaction-LC-MS/MS analysis of paralytic shellfish toxins (PSTs) in bivalve shellfish. The method was developed as an alternative to the precolumn oxidation AOAC 2005.06 and postcolumn oxidation AOAC 2011.02 LC with fluorescence detection methods, receptor binding assay AOAC 2011.27, as well as the mouse bioassay AOAC 959.08. PSTs assessed were saxitoxin, neosaxitoxin, deoxydecarbamoylsaxitoxin, decarbamoylsaxitoxin, decarbamoylneosaxitoxin, gonyautoxins 1-6, decarbamoylgonyautoxins 2-3, and N-sulfocarbamoyl gonyautoxins 2&3. The method also included the determination of decarbamoylgonyautoxins 1&4, N-sulfocarbamoyl gonyautoxins 1&4, and M toxins. Twelve commercially produced bivalve species from both New Zealand and the United Kingdom were assessed, including mussels, oysters, scallops, and clams. Validation studies demonstrated acceptable method performance characteristics for specificity, linearity, recovery, repeatability, and within-laboratory reproducibility. LOD and LOQ were significantly improved in comparison to current fluorescence-based detection methods, and the method was shown to be rugged. The method performed well in comparison to AOAC 2005.06, with evidence obtained from both comparative analysis of 1141 PST-contaminated samples and successful participation in proficiency testing schemes. The method is suitable for use in regulatory testing and will be submitted for an AOAC collaborative study. [ABSTRACT FROM AUTHOR]

The presence of highly pathogenic Shiga-toxin-producing Escherichia coli ( STEC) in shellfish, upstream waters and sediment from coastal shellfish sites was evaluated using the ISO/ TS-13136 method. Shellfish (oysters, mussels and cockles), water and sediment samples were collected monthly over a period of 1 year. The method used real-time PCR detection of stx1, stx2 and eae genes and genetic markers corresponding to the five major serogroups (O157, O26, O103, O111 and O145) on enrichment broths and the identification of STEC when these genes and markers were detected. stx genes were detected in the broth of 33% of shellfish batches ( n = 126), 91% of water samples ( n = 117) and 28% of sediment ( n = 39). One stx1+, eae+ O26:H11 strain was isolated from a shellfish batch, and O26:H11, O145:H28 and O103:H2 strains without the stx gene ( n = 9) were isolated from shellfish and waters. In conclusion, this study shows the suitability of the ISO/ TS-13136 method to assess the presence of highly pathogenic E. coli strains in shellfish farming areas. It also highlights a low prevalence of STEC and consequently suggests a reduced corresponding human health risk. Significance and Impact of the Study ( STEC) infections have been reported following ingestion of contaminated food or water or after bathing in contaminated waters. However, to date, few studies concerning their detection in coastal environment and shellfish have been reported. The aim of this work was to assess the presence of STEC in three shellfish-harvesting areas by the ISO/ TS-13136 method, which has recently been used for STEC detection in food. [ABSTRACT FROM AUTHOR]

Neurotoxic shellfish poisoning (NSP) is caused by the consumption of molluscan shellfish meat contaminated with brevetoxins produced by the dinoflagellate, Karenia brevis ( K. brevis ). During a prolonged and intermittent K. brevis bloom starting in 2005 lasting through early 2007 in the Gulf of Mexico off southwest Florida coast, there were 24 confirmed cases of NSP linked to the consumption of clams recreationally harvested in, or in close proximity to, regulated shellfish harvesting areas; these shellfish beds had already been officially closed to harvesting due to the presence of the K. brevis bloom. The majority of NSP cases (78%) were in “visitors,” either non-Florida residents or Florida residents living outside the county of harvest. The number of confirmed NSP cases was likely an underestimate of the actual number of cases. Current management strategy appears to be effective in limiting the number of NSP cases associated with shellfish harvested commercially during red tide events. In contrast, public notification that shellfish beds are closed to harvest, due to red tides or pathogens, is not reaching all recreational shellfish harvesters and consumers, particularly visitors from outside the county or state. The constantly changing closure status of shellfish harvesting areas in combination with overlooked notifications may lead to an apparent disregard of harvesting restrictions. It is important, therefore, to provide the general public, including visitors and those with language barriers, with improved access to up-to-date information concerning the daily openings and closings of shellfish harvesting areas. Furthermore, the risks of consuming potentially toxic shellfish should be disseminated more broadly. [ABSTRACT FROM AUTHOR]

In this study, the cultivation conditions of Prorocentrum lima , including temperature, nutrient concentration, photoperiod, and salinity were observed, and then an effective method for the large-volume cultivation of P. lima using a vertical flat photobioreactor was developed for the first time. The maximum cell concentrations and toxin contents of P. lima cultured in the photobioreactor were reached after a 35 days cultivation. Moreover, a step-wise double-sedimentation centrifugation method was used to harvest the microalgae cells, with the harvest rate of 89%. Toxin analysis of dry microalgal powder indicated that OA and DTX1 contents were 15.2 and 21.6 mg g −1 , respectively. These results verify that the culture method using the proposed photobioreactor is effective to massively produce DSP toxin-containing P. lima . This study may serve as a guide for the large-scale production of toxin-producing red-tide benthic microalgae. [ABSTRACT FROM AUTHOR]

We reported previously that okadaic acid (OA) and dinophysistoxin-1 (DTX1) were responsible for diarrhetic shellfish poisoning (DSP) incidents due to consuming cultivated mussels ( Mytilus galloprovincialis) in coastal cities near the East China Sea in May 2011. Pectenotoxin-2 (PTX2) and its seco acids were also present in these mussels. Causative species of microalgae were not identified because detailed information on the location of the contaminated shellfish was not recorded. In order to explore potential causes for these poisoning events, the lipophilic toxin profiles in picked cells of Dinophysis and in shellfish samples collected from two mariculture zones in the East China Sea were analyzed in the present study. Single-cell isolates (100 cells total for each location) of Dinophysis were collected from the aquaculture zones of Gouqi Island (Ningbo City, Zhejiang Province) and Qingchuan Bay (Ningde City, Fujian Province) in July and September 2013, respectively, for lipophilic toxin profiling. Shellfish samples collected over the course of a year from the Gouqi Island aquaculture zone and mussels ( M. galloprovincialis) collected four times from the Qingchuan Bay aquaculture zone were tested for lipophilic toxins by LC-MS/MS. The Dinophysis cells isolated from both sampling sites were identified under the light microscope as Dinophysis caudata. Average quota of PTX2, the predominant toxin in D. caudata isolated from the coastal waters of Gouqi Island and Qingchuan Bay, was 0.58 and 2.8 pg/cell, respectively. Only trace amounts of OA and DTX1 were detected in D. caudata. PTX2, PTX2sa, 7-epi-PTX2sa, OA, and/or DTX1 were found in samples of mussels ( M. galloprovincialis and Mytilus coruscus) collected in the Gouqi Island aquaculture zone from the end of May to the beginning of July 2013. PTX2, PTX2sa, and 7-epi-PTX2sa were also detected in oyster ( Crassostrea gigas) during that period, but almost no OA and DTX1 were present. Gymnodimine (GYM) was detected in almost all mussel ( M. coruscus) samples, with the highest levels occurring in winter. Trace amounts of pectenotoxins (PTXs) and OAs were also found in mussels ( M. galloprovincialis) collected from Qingchuan Bay. D. caudata is suggested as an important source of PTXs in shellfish cultivated in the East China Sea. This is the first report of toxin profiles for single-cell isolates of Dinophysis in the East China Sea. [ABSTRACT FROM AUTHOR]

Food allergic patients have to deal with an avoidance diet. Confusing labelling terms or precautionary labels can result in misinterpretation and risk-taking behaviour. Even those patients that strictly adhere to their diet experience (sometimes severe) unexpected allergic reactions to food. The frequency, severity and causes of such reactions are unknown. The objective of this review was to describe the frequency, severity and causes of unexpected allergic reactions to food in food allergic patients aged > 12 years, in order to develop improved strategies to deal with their allergy. A systematic review was carried out by two researchers, in six electronic databases ( CINAHL, Cochrane, EMBASE, Medline, Psychinfo and Scopus). The search was performed with keywords relating to the frequency, severity and causes of unexpected allergic reactions to food. This resulted in 24 studies which met the inclusion criteria; 18 observational and six qualitative studies. This review shows that knowledge about the frequency of unexpected reactions is limited. Peanut, nuts, egg, fruit/vegetables and milk are the main causal foods. Severe reactions and even fatalities occur. Most reactions take place at home, but a significant number also take place when eating at friends' houses or in restaurants. Labelling issues, but also attitude and risky behaviour of patients can attribute to unexpected reactions. We conclude that prospective studies are needed to get more insight in the frequency, severity, quantity of unintended allergen ingested and causes of unexpected allergic reactions to food, to be able to optimize strategies to support patients in dealing with their food allergy. Although the exact frequency is not known, unexpected reactions to food occur in a significant number of patients and can be severe. For clinical practice, this means that patient education and dietary instructions are necessary. [ABSTRACT FROM AUTHOR]

Triazophos (TP) is a widespread pollutant in aquatic environments. A sex-specific metabolic response in green-lipped mussel Perna viridis to TP exposure was observed in our previous study, and this led us to investigate the mechanisms associated with its toxicity. P. viridis were subjected to chronic exposure (15 days) to TP at 35 μg/L to compare the sex-biased transcriptomic profiles in the gonads of male and female mussels. We identified 632 differentially expressed genes (DEGs) (348 up-regulated and 284 down-regulated) in TP-exposed males, and only 61 DEGs (9 up-regulated and 52 down-regulated) in TP-exposed females. Many DEGs were found to be involved in the nervous, reproductive endocrine, oxidative stress, and immune systems of P. viridis. Additionally, enzymatic activity analysis indicated TP induced neurotoxic effects and oxidative damage to the mussels. Our results demonstrate that the stress response and molecular mechanisms of TP toxicology are different between female and male mussels. [Display omitted] • Sex-specific response to triazophos was found in Perna viridis. • Males presented more differently expressed genes compared with females. • Triazophos caused neurotoxicity and reproductive endocrine disturbance. • Triazophos induced oxidative damage and immune alterations. • Sex needs to be considered in shellfish toxicology. [ABSTRACT FROM AUTHOR]

Aim: To study the prevalence of Rotavirus in shellfish from Southern Kerala. Materials and Methods: The shellfish samples after processing was concentrated using proteinase K. RNA was isolated from the concentrated samples using phenol chloroform method. Rota viral RNA was detected using reverse transcriptionpolymerase chain reaction. Results: A low prevalence of 2.5% (5/200) was obtained during the study. Rotavirus was detected in 2 out of 60 brown mussels (3.33%), 2 out of 70 yellow clams (2.86%) and 1 out of 70 black clams (1.43%). Conclusion: Low prevalence of Rotavirus was obtained in our study. A more extensive study need to be conducted to estimate the prevalence of enteric virus in shellfish. [ABSTRACT FROM AUTHOR]

Lipophilic marine toxins are produced by harmful microalgae and can accumulate in edible filter feeders such as shellfish, leading to an introduction of toxins into the human food chain, causing different poisoning effects. During the last years, analytical methods, based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), have been consolidated by interlaboratory validations. However, the main drawback of LC-MS/MS methods remains the limited number of compounds that can be analyzed in a single run. Due to the targeted nature of these methods, only known toxins, previously considered during method optimization, will be detected. Therefore in this study, a method based on ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC-HR-Orbitrap MS) was developed. Its quantitative performance was evaluated for confirmatory analysis of regulated lipophilic marine toxins in shellfish flesh according to Commission Decision 2002/657/EC. Okadaic acid (OA), dinophysistoxin-1 (DTX-1), pectenotoxin-2 (PTX-2), azaspiracid-1 (AZA-1), yessotoxin (YTX), and 13-desmethyl spirolide C (SPX-1) were quantified using matrix-matched calibration curves (MMS). For all compounds, the reproducibility ranged from 2.9 to 4.9 %, repeatability from 2.9 to 4.9 %, and recoveries from 82.9 to 113 % at the three different spiked levels. In addition, confirmatory identification of the compounds was effectively performed by the presence of a second diagnostic ion (C). In conclusion, UHPLC-HR-Orbitrap MS permitted more accurate and faster detection of the target toxins than previously described LC-MS/MS methods. Furthermore, HRMS allows to retrospectively screen for many analogues and metabolites using its full-scan capabilities but also untargeted screening through the use of metabolomics software. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Shellfish have been implicated in norovirus (NoV) infection outbreaks worldwide. This study presents data obtained from various batches of shellfish and fishery products from a Belgian seafood company over a 6-month period. For the intact shellfish (oysters, mussels, and clams), 21 of 65 samples from 12 of 34 batches were positive for NoVs; 9 samples contained quantitative NoV levels at 3,300 to 14,300 genomic copies per g. For the semiprocessed fishery products (scallops and common sole rolls with scallop fragments), 29 of 36 samples from all eight batches were positive for NoVs; 17 samples contained quantitative NoV levels at 200 to 1,800 copies per g. This convenience study demonstrated the performance and robustness of the reverse transcription quantitative PCR detection and interpretation method and the added value of NoV testing in the framework of periodic control of seafood products bought internationally and distributed by a Belgian seafood processing company to Belgian food markets. [ABSTRACT FROM AUTHOR]

Background: Crassostrea gigas accumulates diarrheic shellfish toxins (DSP) associated to Prorocentrum lima of which Okadaic acid (OA) causes specific inhibitions of serine and threonine phosphatases 1 and 2A. Its toxic effects have been extensively reported in bivalve mollusks at cellular and physiological levels, but genomic approaches have been scarcely studied. Methodology/Principal Findings: Acute and sub-chronic exposure effects of P. lima were investigated on farmed juvenile C. gigas (3–5 mm). The Pacific oysters were fed with three dinoflagellate concentrations: 0.3, 3, and 30×103 cells mL−1 along with a nontoxic control diet of Isochrysis galbana. The effects of P. lima on C. gigas were followed by analyzing expression levels of a total of four genes, three involved in cell cycle regulation and one in immune response by polymerase chain reaction and real time quantitative PCR, where changes in time and cell concentration were found. The highest expression levels were found in oysters fed 3×103 cells mL−1 at 168 h for the cycle regulator p21 protein (9 fold), chromatin assembly factor 1 p55 subunit (8 fold), elongation factor 2 (2 fold), and lipopolysaccharide/β-1, 3 glucan binding protein (13 fold above base line). Additionally, the transcript level of all the genes decreased in oysters fed wich the mixed diet 30×103 cells mL−1 of dinoflagellate after 72 h and was lowest in the chromatin assembly factor 1 p55 subunit (0.9 fold below baseline). Conclusions: On C. gigas the whole cell ingestion of P lima caused a clear mRNA modulation expression of the genes involved in cell cycle regulation and immune system. Over-expression could be related to DNA damage, disturbances in cell cycle continuity, probably a genotoxic effect, as well as an activation of its innate immune system as first line of defense. [ABSTRACT FROM AUTHOR]

Background: Crassostrea gigas accumulates diarrheic shellfish toxins (DSP) associated to Prorocentrum lima of which Okadaic acid (OA) causes specific inhibitions of serine and threonine phosphatases 1 and 2A. Its toxic effects have been extensively reported in bivalve mollusks at cellular and physiological levels, but genomic approaches have been scarcely studied. Methodology/Principal Findings: Acute and sub-chronic exposure effects of P. lima were investigated on farmed juvenile C. gigas (3–5 mm). The Pacific oysters were fed with three dinoflagellate concentrations: 0.3, 3, and 30×103 cells mL−1 along with a nontoxic control diet of Isochrysis galbana. The effects of P. lima on C. gigas were followed by analyzing expression levels of a total of four genes, three involved in cell cycle regulation and one in immune response by polymerase chain reaction and real time quantitative PCR, where changes in time and cell concentration were found. The highest expression levels were found in oysters fed 3×103 cells mL−1 at 168 h for the cycle regulator p21 protein (9 fold), chromatin assembly factor 1 p55 subunit (8 fold), elongation factor 2 (2 fold), and lipopolysaccharide/β-1, 3 glucan binding protein (13 fold above base line). Additionally, the transcript level of all the genes decreased in oysters fed wich the mixed diet 30×103 cells mL−1 of dinoflagellate after 72 h and was lowest in the chromatin assembly factor 1 p55 subunit (0.9 fold below baseline). Conclusions: On C. gigas the whole cell ingestion of P lima caused a clear mRNA modulation expression of the genes involved in cell cycle regulation and immune system. Over-expression could be related to DNA damage, disturbances in cell cycle continuity, probably a genotoxic effect, as well as an activation of its innate immune system as first line of defense. [ABSTRACT FROM AUTHOR]

Highlights: [•] An improvement for sampling plan assessment is proposed. [•] Scale and location parameters of the lognormal distribution are used. [•] Accumulation/depuration processes of OA in mussel are studied. [•] Bacteria might be related to variability. [•] The best fit sampling plan: two samples of 50 mussels each. [Copyright &y& Elsevier]

The fine scale vertical distribution of Dinophysis spp. and Protoceratium reticulatum (potential producers of lipophilic shellfish toxins, LSTs) and its relation with LSTs in shellfish was studied in Reloncaví fjord, a strongly stratified system in Southern Chile. Samples were taken over two years from late spring to early autumn (2007–2008 period) and from early spring to late summer (2008–2009 period). Dinophysis spp., in particular Dinophysis acuminata, were always detected, often forming thin layers in the region of the salinity driven pycnocline, with cell maxima for D. acuminata of 28.5×103 cellsL−1 in March 2008 and 17.1×103 cellsL−1 in November 2008. During the 2008–2009 sampling period, blooms of D. acuminata co-occurred with high densities of cryptophyceans and the ciliate Mesodinium spp. The highest levels of pectenotoxin-2 (PTX-2; 2.2ng L−1) were found in the plankton in February 2009, associated with moderate densities of D. acuminata, Dinophysis tripos and Dinophysis subcircularis (0.1–0.6×103 cellsL−1). However, only trace levels of PTX-2 were observed in bivalves at that time. Dinophysistoxin (DTX-1 and DTX-3) levels in bivalves and densities of Dinophysis spp. were not well correlated. Low DTX levels in bivalves observed during a major bloom of D. acuminata in March 2008 suggested that there is a large seasonal intraspecific variability in toxin content of Dinophysis spp. driven by changes in population structure associated with distinct LST toxin profiles in Reloncaví fjord during the study period. A heterogeneous vertical distribution was also observed for P. reticulatum, whose presence was restricted to summer months. A bloom of this species of 2.2×103 cellsL−1 at 14m depth in February 2009 was positively correlated with high concentrations of yessotoxins in bivalves (51–496ngg−1) and plankton samples (3.2ngL−1). Our results suggest that a review of monitoring strategies for Dinophysis spp. in strongly stratified fjord systems should be carried out. They also indicate that early warning of LST events based on Dinophysis cell numbers are not reliable for seafood control. [ABSTRACT FROM AUTHOR]

Highlights: [•] Conditioning of a clonal culture of Alexandrium catenella to a factorial combination of environmental change variables; CO2, temperature, phosphorus. [•] Measurements of growth and cellular toxins after eight months of conditioning. [•] Increased CO2 led to enhanced cellular toxicity. [•] Combined high CO2, low temperature and phosphate-limitation were the ‘most toxic’. [Copyright &y& Elsevier]

Highlights: [•] The V genes of an antibody for a shellfish toxin gonyautoxin were cloned using phage display. [•] An open sandwich ELISA was designed with the cloned antibody fragment. [•] The assay had more than three orders of magnitude working range while keeping subnanomolar sensitivity. [ABSTRACT FROM AUTHOR]

Monitoring of amnesic shellfish poisoning (ASP) toxins in shellfish from the Catalan coast started in 2001. No ASP toxins were detected in any of the analyses performed before 2008. On 22 January 2008, domoic acid (DA) was detected in Donax trunculus (0.5 mg kg) and confirmed by rapid resolution liquid chromatography-tandem mass spectrometry (0.6 mg kg). A total of 974 shellfish samples were analyzed from January 2008 to December 2011, covering all the Catalan production areas and the most important marketed species. DA was detected in 23.8 % of the samples and was recorded every month in all areas and all species, except Ostrea edulis, although the percentage of samples with DA and DA content varied widely among samples. DA exceeded the regulatory level of 20 mg kg twice: in Callista chione sampled on February 2008 and in D. trunculus sampled on April 2010. DA content in Bolinus brandaris appeared constant and close to 4.5 mg kg for months in 2009. Mytilus galloprovincialis, Crassostrea gigas, and Ruditapes sp. presented very low concentrations of DA in the Ebro Delta bays, despite 113 alert situations according to Pseudo-nitzschia spp. abundances and the high number of shellfish samples analyzed. The origin of DA in Catalan shellfish remains unknown. [ABSTRACT FROM AUTHOR]

Highlights: [•] Azadinium spinosum was exposed to different environmental and nutritional factors. [•] Growth, biomass and AZA cell quota were observed in continuous and or batch culture. [•] A. spinosum grew in a wide range of conditions. [•] Temperature had the main effect on AZA cell quota. [Copyright &y& Elsevier]

Okadaic acid (OKA) and analogues are frequent contaminants of coastal waters and seafood. Structure analysis of the isolated OKA analogue 19-epi-OKA showed important conformation differences expected to result in lower protein phosphatase (PP) inhibitory potencies than OKA. However, 19-epi-OKA and OKA inhibitory activities versus PP2A were unexpectedly found to be virtually equipotent. To investigate the toxicological relevance of these findings, we tested the effects of 19-epi-OKA on cultured cerebellar cells and compared them with those of OKA and its isomer dinophysistoxin-2. 19-epi-OKA caused degeneration of neurites and neuronal death with much lower potency than its congeners. The concentration of 19-epi-OKA that reduced after 24h the maximum neuronal survival (EC5024) by 50% was ~300nM compared with ~2nM and ~8nM for OKA and dinophysistoxin-2, respectively. Exposure to 19-epi-OKA resulted also in less toxicity for cultured glial cells (EC5024,19-epi-OKA ~ 600nM; EC5024,OKA ~ 20nM). 19-epi-OKA induced apoptotic condensation and fragmentation of chromatin, activation of caspases, and activation of ERK1/2 MAP kinases, features previously reported for OKA and dinophysistoxin-2. Also, differential sensitivity to 19-epi-OKA was observed between neuronal and glial cells, a specific characteristic shared by OKA and dinophysistoxin-2 but not by other toxins. Our results are consistent with 19-epi-OKA being included among the group of toxins of OKA and derivatives and support the suitability of cellular bioassays for the detection of these compounds. [ABSTRACT FROM PUBLISHER]

Abstract: Routine monitoring along the coast of the Gulf of Maine (GoM) reveals shellfish toxicity nearly every summer, but at varying times, locations, and magnitudes. The responsible toxin is known to be produced by the dinoflagellate Alexandrium fundyense, yet there is little apparent association between Alexandrium abundance and shellfish toxicity. One possibility is that toxic cells are persistent in offshore areas and variability in shellfish toxicity is caused not by changes in overall abundance, but rather by variability in transport processes. Measurements of offshore Alexandrium biomass are scarce, so we bypass cell abundance as an explanatory variable and focus instead on the relations between shellfish toxicity and concurrent metrics of GoM meteorology, hydrology, and oceanography. While this yields over two decades (1985–2005) of data representing a variety of interannual conditions, the toxicity data are gappy in spatial and temporal coverage. We address this through a combination of parametric curve fitting and hierarchical cluster analysis to reveal eight archetypical modes of seasonal toxicity timing. Groups of locations are then formed that have similar interannual patterns in these archetypes. Finally, the interannual patterns within each group are related to available environmental metrics using classification trees. Results indicate that a weak cross-shore sea surface temperature (SST) gradient in the summer is the strongest correlate of shellfish toxicity, likely by signifying a hydrological connection between offshore Alexandrium populations and near-shore shellfish beds. High cumulative downwelling wind strength early in the season is revealed as a precursor consistent with this mechanism. Although previous studies suggest that alongshore transport is important in moving Alexandrium from the eastern to western GoM, alongshore SST gradient is not an important correlate of toxicity in our study. We conclude by discussing the implications of our results for designing efficient and effective shellfish monitoring programs along the GoM coast. [Copyright &y& Elsevier]