Acid In Mussels Research Articles

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

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

Eavesdropping on plankton—can zooplankton monitoring improve forecasting of biotoxins from harmful algae blooms?

AbstractHarmful algae bloom (HAB) forecasting has developed rapidly over recent decades, but predicting harmful levels of marine biotoxins in shellfish is still a challenge. New discoveries suggest that predator‐prey interactions may be an important driver in the formation of HABs. Key species of harmful algae respond to copepod infochemicals with increased toxin production. In addition, copepods feed selectively on less defended prey, which may further promote harmful taxa. Here we explore if eavesdropping on predator‐prey dynamics by monitoring zooplankton can improve HAB forecasting. We first examine an 8‐yr time series including copepod biomass, harmful algae cells (Dinophysis spp.), and diarrhetic shellfish toxins in blue mussels (Mytilus edulis) using generalized additive models. Models including copepod biomass more accurately predicted okadaic acid in mussels than phytoplankton alone. We then apply this connection more narrowly by analyzing the specific copepod exudates known to induce toxin production, copepodamides, from the mussels sampled in biotoxin monitoring. Adding copepodamide data from shellfish extracts increased model performance compared to copepod biomass. Results suggest that including grazing effects through copepodamide measurements may provide a cost‐efficient way to improve accuracy and lead time for predicting the accumulation of microalgal toxins in shellfish.

A novel microfluidic system for the sensitive and cost-effective detection of okadaic acid in mussels.

Okadaic acid (OA) is produced by marine dinoflagellates and it can be easily accumulated in shellfish, causing intoxications when consumed by humans. Consequently, there is a need for sensitive, reliable and cost-effective methods to detect OA in real samples. In this work, we developed a novel and affordable microfluidic system to detect OA based on the protein phosphatase 1 inhibition colorimetric assay. This enzyme was immobilized in a microfluidic chamber by physisorption in an alumina sol-gel. The results show good enzyme stability over time when maintained at 4 °C. The developed system was sensitive for OA standard solutions, presenting a limit of detection (LOD) of 11.6 nM over a large linear range (43.4 to 3095.8 nM). Our method revealed an LOD as low as 0.2 μg kg-1 and a linear range between 1.47 and 506 μg kg-1 for extracted mussel matrix, detecting OA concentrations in contaminated mussels much lower than the regulated limit (160 μg kg-1). The enzyme stability and reusability along with the simplicity and low cost associated with microfluidics systems make this method very interesting from a commercial point of view.

SPR immunosensor for the detection of okadaic acid in mussels using magnetic particles as antibody carriers

Protein G-coated magnetic particles (MPs) were used as immobilisation supports for an antibody against okadaic acid (MAbOA) and carriers into a surface plasmon resonance (SPR) device for the development of a direct competitive immunosensor for okadaic acid (OA). SPR analysis of MAbOA–MP conjugates demonstrated that conjugations were successful with complete immobilisation of all the antibody biomolecules onto the MPs. Moreover, MAbOA–MP conjugates provided up to 11-fold higher SPR signals, compared to free MAbOA. The use of conjugates in the direct competition assay provided a 3-fold lower LODμg/L (2.6μg of OA/L, equivalent to 12μg of OA/kg mussel meat). The presence of mussel matrix did not interfere in the OA quantification as seen in the calibration curves. Mussel samples, obtained from Ebro Delta's bays (NW Mediterranean) during a diarrheic shellfish poisoning (DSP) event and in the presence of Dinophysis sacculus, an OA producer, in the shellfish production area, were analysed with the MP-based SPR immunosensor. The OA contents correlated with those obtained by liquid chromatography–tandem mass spectrometry (LC–MS/MS) (y=0.984x−5.273, R2=0.789, p<0.001) and by mouse bioassay (MBA).

Novel Sensor Design and Application for Detection of Dangerous Contaminated Marine Biotoxins

We have developed a novel planar interdigital sensing system: using a microcontroller and local fabricated circuits with dual supply and signal processing for data acquisition. The first prototype is called seafood inspection tool or SIT consist of a SiLab C8051F020 microcontroller, a signal processing circuit, novel planar interdigital sensor, an expansion board and dual power supply circuit. A user friendly software was also developed to make SIT easy to be used by users who has a little technical knowledge. SIT was developed to be tested by the fisherman who can do a pre-screening process of the mussels at their ranch site.Our main objective is to sense the presence of dangerous contaminated acid in mussels and other seafoods.

Evaluating the performance of sampling plans to detect okadaic acid in mussels in production areas

INTRODUCTION: Bivalve molluscan shellfish feed on phytoplankton (micro-algae) which might potentially contain biotoxins like okadaic acid. In order to ensure food safety, the level of okadaic acid equivalents in live bivalve molluscs must not exceed 160 μg per kg (Regulation 853/2004/EC). Therefore, sampling plans must be set by official authorities in order to monitor production areas to check the presence of okadaic acid in shellfish, knowing that mussel may be used as an indicator species (Regulation 854/2004/EC). There is a high interindividual variability between mussels, even for mussels taken at the same sampling point and at the same time (Edebo et al., 1988 ; Duinker et al., 2007). However there has never been a probabilistic evaluation of the impact that the sample size and the number of samples taken, have on the analysis results and the corresponding decision for the shellfish lot. Nathalie Wesolek (1), Francois-Gilles Carpentier (1), Dominique Parent-Massin (1), Alain-Claude Roudot (1) (1) Laboratoire de Toxicologie Alimentaire et Cellulaire, Universite de Bretagne Occidentale, UBO, 29200 BREST (email : nathalie.wesolek@univ-brest.fr)

Seasonal Variation in Nutrient Composition of Mytilus coruscus from China

Seasonal variation in the nutrient composition of Mytilus coruscus (thick shell mussel), cultivated in Shengsi Islands, Zhejiang Province, China, was investigated in this study. Proximate composition, mineral and amino acid concentrations, as well as the compositions of the lipid and fatty acid of thick shell mussels were analyzed. Proximate composition and mineral contents were seasonally varied significantly except for calcium (Ca) and lead (Pb). Glycine was the predominant amino acid in mussels throughout all seasons, while lysine, threonine, phenylalanine, and arginine were the main essential amino acids. The predominant lipids were phospholipids, followed by triacylglycerols and sterol esters. Polyunsaturated fatty acids (PUFA) predominated over saturated and monounsaturated fatty acids throughout the year. Docosahexaenoic acid (12.44-18.34% of total fatty acids) and eicosapentaenoic acid (10.79-14.60%) were the most abundant PUFA. Significant seasonal variations were observed in the compositions of most amino acids, lipid classes, and fatty acids. Cultivated Chinese thick shell mussels represent a source of the health benefiting long chain n-3 PUFA, essential amino acids, and minerals for human consumption.

A low cost novel sensing system for detection of dangerous marine biotoxins in seafood

A novel planar interdigital sensor-based sensing system has been developed for detection of dangerous marine biotoxins in seafood. Our main objective is to sense the presence of dangerous contaminated acid in mussels and other seafood by observing the change of reactive impedance of the planar interdigital sensors. Initial studies were conducted with three peptide derivatives namely sarcosine, proline and hydroxylproline. These three chemicals are structurally closely related to our target molecule. The proline molecule is arguably the most important amino acid in peptide conformation, contains the basic structural similarity to the domoic acid. Three novel interdigital sensors have been designed and fabricated. All sensors have the same effective area but having different sensor configurations. The initial results show that sensors respond very well to the chemicals and it is possible to discriminate the different chemicals from the output of the sensor. The sensors were also being tested with three seafood products. Results from the analysis have shown that one configuration has better sensitivity compared to other configurations. The sensor with the best sensitivity was chosen for experiment using proline and mussels. The changes in sensor sensitivity were analyzed with mussels before and after adding the proline. The presence of proline on the mussel surface and also injected proline to the mussels was very clearly detected by the sensor. Further experiment was conducted with small amount of domoic acid (0.5–5.0 μg) injected to a mussel and it was found that the chosen sensor was able to detect small amount of domoic acid (1.0 μg) injected into the mussel sample. The result shows that the sensor was able to detect approximately 12.6 μg/g of domoic acid in mussel meat. The outcomes from the experiments provide chances of opportunity for further research in developing a low cost miniature type of sensors for reliable sensing system for commercial use.

Determination of domoic acid in mussels by HPLC with post-column derivatization using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and fluorescence detection

A new, sensitive method was developed for the determination of the neurotoxin domoic acid (DA) using a reversed phase separation followed by post-column derivatization (PCD) with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and subsequent fluorescence detection. The PCD conditions which involves a two-step reaction was fully optimized for the lowest detection limit. The first reaction occurs between DA and NBD-Cl while the second makes possible the detection of the derivative causing the destruction of the interfering fluorescent 4-hydroxy-7-nitrobenzo-2-oxa-1,3-diazole (NBD-OH) which is the hydrolysis product of NBD-Cl. Kainic acid a similar base structure compound with DA was used as an internal standard. The developed post-column method provides the ability for a fully automated analysis, low detection limits (LOD 25 ppb in real samples of mussel extracts), it requires less sample preparation, and it gives clean simple chromatograms without chromatographic interferences from coeluting compounds such as tryptophan. The method was successfully applied to for the quantitative determination of DA in mussel tissues at quantities as low as 75 μg/kg tissue.

Fatty acid composition of New Zealand green-lipped mussels, Perna canaliculus: Implications for harvesting for n-3 extracts

Marine bivalves offer a potentially important source of long-chained polyunsaturated fatty acids (PUFA) for human health supplements. Lipid extracts from individual New Zealand green-lipped mussels (NZGLM) were analyzed as fatty acid methyl esters (FAME) by gas chromatography to assess geographical and seasonal differences between large (86 ± 1 mm) male and female and small (44 ± 1 mm) mussels. PUFAs dominated in spring and summer, comprising ∼ 50% of total fatty acids. Moreover, the commercially important n-3 fatty acids, 20:5n-3 (eicosapentaenoic acid, EPA) and 22:6n-3 (docosahexaenoic acid, DHA) together accounted for 70–79% of total PUFAs in spring and summer. During winter there was a marked decrease in condition and total n-3 PUFAs and a concomitant increase in saturated fatty acids in mussels, suggesting they had already spawned, had increased metabolic demands and limited PUFA-rich phytoplankton as food. While total n-3 content was not significantly different, there were geographical differences in individual n-3 fatty acids. Mussels collected from the cooler waters of Stewart Island had greater levels of 20:5n-3 (EPA), while those collected in Marlborough had greater concentrations of 22:6n-3 (DHA), which was attributed mainly to differences in phytoplankton composition. Total n-3 content and the condition index varied seasonally with greater concentrations of n-3 PUFAs, especially EPA, recorded in large mussels in spring, coincident with spring diatom blooms. Total PUFA levels and condition indices remained high in summer. There was no significant difference in condition indices, total n-3 content, DHA or EPA levels between large male and female mussels. Conversely, large mussels had significantly greater amounts of n-3 PUFAs than small mussels at Marlborough Sounds, while small mussels had marginally greater total n-3 concentrations than large mussels at Stewart Island. Taken together, these results suggest that the NZGLM offers a potentially important source of n-3 PUFAs for human health supplements. Our findings suggest that optimal harvesting conditions occur in spring when mussel condition and n-3 content peak for large mussels. Although DHA and EPA levels varied geographically, total n-3 content was not significantly different between sites, which implies that harvesting mussels for n-3 extracts would be driven more by logistical considerations.

Detection of the marine toxin okadaic acid in mussels during a diarrhetic shellfish poisoning (DSP) episode in Thermaikos Gulf, Greece, using biological, chemical and immunological methods

An approach involving chemical and biological techniques was taken for the detection and quantification of the marine toxin okadaic acid (OA) in mussels from Thermaikos Gulf, Greece, during a 4-month DSP episode that occurred in 2002. Samples were analyzed using the mouse bioassay, high performance liquid chromatography (HPLC) with fluorimetric detection and an enzyme-linked immunosorbent assay (ELISA). Okadaic acid was quantifiable at three different sites of Thermaikos Gulf reaching a maximum concentration of 36 μg/g hepatopancreas. High correlation was revealed between results derived from HPLC and ELISA ( R 2 = 0.998), while 91% consistency between HPLC and the mouse bioassay results was observed.

Identification of minor fatty acids in mussels ( Mytilus galloprovincialis) by GC–MS of their 2-alkenyl-4,4-dimethyloxazoline derivatives

The fatty acid composition of mussel lipids was determined by gas chromatography–mass spectrometry (GC–MS) of their corresponding 2-alkenyl-4,4-dimethyloxazoline derivatives. Silver ion thin layer chromatography isolated enriched fractions of minor fatty acids extracted from mussels. Several non-methylene interrupted (NMI) fatty acids were singled out, and the non-methylene interrupted trienoic (NMIT) fatty acids 5,11,14–20:3 and 7,13,16–22:3 have been detected in marine mussels for the first time. This discovery strongly supports a previously suggested biosynthetic route implying the desaturation and subsequent elongation of 20:2 ω6.

Methodological improvement of the protein phosphatase inhibition assay for the detection of okadaic acid in mussels.

A simplified procedure for the enzyme inhibition assay to measure okadaic acid and DTX-1 in mussels, based on the use of a commercially available enzyme preparation, is presented. The detection limit is 10 ng of toxin per g of digestive glands. Using Certified Reference Material (MUS-2), high accuracy and good precision is demonstrated for contamination levels higher than 32 ng g(-1). Twenty samples can be processed in about 9 h by one operator, at the cost of US$ 10 per sample. Some possibilities for further enhancing the sensitivity and reducing the processing time are discussed and a monitoring example is presented.

Semi-automated membrane based chemiluminescent immunosensor for flow injection analysis of okadaic acid in mussels

A chemiluminescent immunosensor integrated in a flow injection analysis system was developed for the detection of the ‘diarrheic shellfish poisoning’ (DSP) toxin okadaic acid (OA). Anti-OA monoclonal antibodies were labelled with horseradish peroxidase for their use in a competitive assay, in which the free antigen of the sample competes with immobilised OA. Based on commercially available polyethersulfone membranes, this bioanalytical system exhibits low non-specific binding of antibodies in the presence of mussel homogenate. The immunosensor was used in a semi-automated analysis procedure in which the free OA containing sample was injected in the flow system concomitantly with the labelled antibodies. With an overall measurement time of 20 min, the immunosensor has a detection limit of 0.2 μg OA/100 g mussel homogenate (0.1 μg OA/L in the assay solution), and measurements could be performed over three decades, from 0.2 to 200 μg OA/100 g of mussel homogenate. The operational stability of the sensor for the detection of critically contaminated mussels (40 μg/100 g of mussel homogenate) has been investigated over 38 OA determination cycles. A stable response was obtained under the 34 first measurements with a CV of 11.7%. Moreover, no significant change in the immunosensor performances was observed during short and long term storage periods of the membranes (1 month). The performances of five immunosensors (five different membranes) showed a good repeatability with a variation of the negative control and the critically contaminated signal equal to 7% and 12.6%, respectively.

Evaluation of the fluorometric protein phosphatase inhibition assay in the determination of okadaic acid in mussels

The protein phosphatase inhibition assay for okadaic acid, the major DSP toxin, modified to use the fluorescence substrates methylumbelliferyl phosphate (MUP) and fluorescein diphosphate (FDP), was compared to the assay using p-nitrophenylphosphate ( p-NPP) and the bioluminescence assay using luciferin phosphate (L–P). Under the standard assay conditions used okadaic acid inhibited the enzyme activity dose-dependently with IC 50 values of 1.5 nM (MUP) and 1.2 nM (FDP). This compares to IC 50 values of 0.9 and 6 nM using L–P and p-NPP respectively. CDP- star, a chemiluminescence substrate, was not hydrolysed by the enzyme. Decreasing the enzyme concentration lowered the IC 50 for the colorimetric method (IC 50=2 nM [ p-NPP], 0.75 nM enzyme) but no shift was observed with fluorimetry. However at enzyme concentrations <1.5 nM (standard assay) the error margin was too great for routine analysis. The method using fluorimetry allowed detection of okadaic acid concentrations to levels ≤1 μg/100 g of mussel tissue which is well below the limit of 20 μg/100 g (mouse bioassay) set by some regulatory agencies. Determination of the toxin content in naturally contaminated mussels in three separate experiments gave coefficients of variance ranging from 16 to 29% (MUP) and from 8 to 78% ( p-NPP). Multicomparison studies showed that concentrations of okadaic acid in naturally contaminated mussel samples determined by fluorescence generally agreed with those obtained using ELISA and LC–MS procedures, and with the mouse bioassay. However using the mouse bioassay as the standard, values determined by the ELISA, PP-2A and LC–MS all scored false negative results compared to those for the mouse bioassay in the range 20–40 μg/100 g mussel, and at the limit of the mouse bioassay the values by the other three methods were substantially less. With few exceptions the methods scored okadaic acid with highest to lowest values in the following order: mouse bioassay>ELISA>PP-2A>LC–MS. The fluorimetric assay was both more sensitive and accurate than the colorimetric assay (the latter showed a propensity towards false positives in the region 20 μg/100 g), and the moderate increase in equipment cost appears to be outweighed by the performance of the method.

Concentration and dispersal of a Pseudo-nitzschia bloom in Penn Cove, Washington, USA.

A bloom of the pennate diatom Pseudo-nitzschia, several species of which are associated with the production of the potent excitotoxin domoic acid, was observed in a Puget Sound, Washington embayment in July and August of 1997. Penn Cove, which receives nutrients from the nearby Skagit River and abundant sunshine during summer months due to its location in the rain shadow of the Olympic Mountains, is the home of a commercial mussel farm which supplies shellfish to many coastal areas of the USA. Levels of domoic acid in mussels increased to 3 ppm on 6 and 10 July, corresponding to the observation of a brown algal bloom in Penn Cove. Four species of Pseudo-nitzschia (P. pungens, P. multiseries, P. australis, and P. pseudodelicatissima) were present in our samples from the cove, corresponding to levels of domoic acid in seawater ranging from 0.1-0.8 mirog l(-1) as measured by a receptor binding assay. The highest Pseudo-nitzschia concentration during the time of our sampling was 13 million cells per liter on 28 July. The bloom of Pseudo-nitzschia occurred after a period of strong discharge from the Skagit River and rain accompanied by elevated south and southeasterly winds. Stratification of the cove, providing optimal bloom conditions, was facilitated by weak winds, sunshine, and a freshwater lens at the mouth of the cove. The position of the Pseudo-nitzschia bloom was influenced by buoyancy fronts caused by exchange of water within the cove with that of Saratoga Passage. The decay of this bloom in Penn Cove was accompanied by decreasing nitrate levels at all measured depths. These and future observations aid in the development of a model for prediction of toxic bloom events in the shallow embayments of Puget Sound.

Automatic HPLC-UV determination of domoic acid in mussels and algae

A rapid and sensitive automatic method is presented for the determination of domoic acid (DA) using HPLC with a column-switching system and UV-detection. Interfering peaks resulting from matrix protein components are excluded by use of an especially designed reversed-phase HPLC column for pre-separation. The method is suitable for extracts both from mussels and from algae. Sample material is extracted with pure water and the crude extract is injected directly. Application of a column-switching system eliminates the need for any further sample clean-up after extraction.

Suitability of the MTT-based cytotoxicity assay to detect okadaic acid contamination of mussels

The suitability of a cytotoxicity assay based on the MTT colorimetric method has been evaluated for the detection of okadaic acid in mussels. On KB cells, okadaic acid exhibited a dose-dependent cytotoxic effect, the IC 50 being inversely related to the exposure time ( IC 50 = 6.3 ng/ml, 4.0 ng/ml and 1.1 ng/ml after 24, 48 and 72 hr of contact, respectively). Using a contact time of 24 hr, the MTT cytotoxicity assay is suitable for revealing okadaic acid concentrations in mussel samples as low as 50 ng/g of digestive glands, with a sensitivity higher than that of the commercially available kits for enzyme-linked immunosorbent assay (ELISA). In the okadaic acid concentration range from 50 to 1500 ng/g of digestive glands the MTT cytotoxicity assay showed satisfactory accuracy and reproducibility. A high degree of correlation was found between the okadaic acid content of 16 naturally contaminated samples measured by the MTT cytotoxicity assay and by an ELISA.

A protein phosphatase 2A inhibition assay for a fast and sensitive assessment of okadaic acid contamination in mussels

The specific inhibitory activity exerted by okadaic acid on protein phosphatase 2A was used to assess the presence of okadaic acid in mussels, using a commercially available protein phosphatase 2A preparation. Under the conditions used, okadaic acid inhibits the enzymatic activity dose-dependently, with an IC 50 = 0.26 ng/ml (0.32 nM). The assay is accurate and reproducible. Okadaic acid was detected in concentrations as low as 0.063 ng/ml in aqueous solutions and 2 ng/g in mussel digestive glands. Thirty naturally contaminated mussel samples were submitted to the protein phosphatase 2A inhibition assay as well as to an ELISA assay and to a MTT cytotoxicity assay, with similar results. The proposed assay is sensitive, rapid and does not require expensive equipment. These characteristics make it a good candidate for employment in the routine assessment of okadaic acid shellfish contamination.

Comparison of mouse bioassay, HPLC and enzyme immunoassay methods for determining diarrhetic shellfish poisoning toxins in mussels

R. Draisci, L. Croci, L. Giannetti, L. Cozzi, L. Lucentini, D. De Medici and A. Stacchini. Comparison of mouse bioassay, HPLC and enzyme immunoassay methods for determining diarrhoetic shellfish poisoning toxins in mussels. Toxicon 32, 1379–1384, 1994.—Mussel specimens ( Mytilus galloprovincialis) collected from two different areas of the Adriatic Sea were analysed for diarrhoetic shellfish poisoning (DSP) toxin by three methods: mouse bioassay, the DSP Check enzyme immunoassay kit, and high-performance liquid chromatography (HPLC). The results obtained confirm that Yasumoto's mouse bioassay, capable of detecting all the components of the DSP group, is still necessary to determine the wholesomeness of the product. The ELISA method has not always given quantitatively reliable results. The HPLC method is advantageous in terms of sensitivity, accuracy, specificity and rapidity. However, its application is limited so far to the determination of okadaic acid in mussels.