Diarrhoetic Shellfish Poisoning Research Articles

Influence of seasonality on the presence of okadaic acid associated with Dinophysis species: A four-year study in Sardinia (Italy).

In Sardinia (Italy), bivalve molluscs production plays an important role in the trade balance. Diarrhoetic shellfish poisoning (DSP), an intoxication caused by the ingestion of bivalve molluscs that have accumulated high levels of Okadaic acid (OA), may represent a serious risk for the public health and a remarkable economic loss for the producers. Aim of this work was to improve knowledge about the repeatability of OA accumulation phenomena in various seasons trying to understand whether or not there was a trend. Also, the interaction between toxic algae and OA accumulation was examined. In this study, data of lipophilic toxins, water temperature and abundance of DSP-producing microalgal species were collected in a four-year period (2015–2018) in coastal production areas of Sardinia. Several episodes of OA positive values (>160 eq μgAO/Kg pe, Reg 853/04) were recorded during the study period in different production areas of Sardinia and in different seasons. A seasonal repeatability of OA accumulation in molluscs was observed in some production areas; moreover, different temporal gaps between the presence of toxic algae and OA accumulation were reported. Toxicity was observed almost exclusively in Mytilus galloprovincialis Lamark (99%), being this matrix the most abundant species bred in Sardinia.

Sampling design for biotoxins monitoring in bivalve molluscs crops of the Santa Catarina coast, Brazil

Sampling design for biotoxins monitoring in bivalve molluscs crops of the Santa Catarina coast, Brazil

Simultaneous determination of biotoxins DSP and AZAs in bivalve molluscs and fish by liquid chromatography/tandem mass spectrometry.

A method has been developed for simultaneous determination of the toxins OA, DTX-1, AZA-1, AZA-2 and AZA-3 in various aquatic products as these can cause diarrhoetic shellfish poisoning (DSP) in humans, an intoxication characterized by vomiting and diarrhea. Separation of the toxins was achieved on a C18 column (150 mm × 2.1 mm, 3.5 µm) using an acetonitrile/water gradient with formic acid as an eluent modifier. Electrospray ionisation (ESI) in negative mode was used to generate the molecule related ion [M-H](-), for OA and DTX-1, while ESI in positive mode was used to generate the molecule related ion [M+H](+) for AZAs. Samples were extracted with 80% methanol, followed by partitioning with ethyl acetate, purified on a Poly-Sery MAX cartridge and finally analyzed by LC/ESI-MS/MS in the multiple reaction monitoring (MRM) mode. The limit of detection (LOD) and limit of qualification (LOQ) of the method were in the range of 0.02-0.79 µg/kg and 0.07-2.64 µg/kg in Scomberomorus niphonius, blood clam and oyster, respectively, recoveries of the toxins at three fortification levels ranged from 71.3% to 104.8% with relative standard deviation from 1.0% to 12.5%. The calibration curves were well linear between the LC peak area of the selected ion pair and the concentration of the toxins, with the correlation coefficient over 0.99. The method was sufficiently sensitive to permit the determination of the toxins DSP and AZA in sea food.

Growth, behaviour and cell toxin quota of Dinophysis acuta during a daily cycle

In 2005, a bloom of the Diarrhoetic Shellfish Poisoning (DSP) causative agent Dino- physis acuta Ehrenberg in the Galician Rias Baixas (NW Spain) started in early August and reached maximum densities (up to 2 × 10 4 cell l -1 ) in mid November. A cell cycle study was carried out over a 22 h period on 9 and 10 November to describe the physiological status and the short-term variability in cell toxin quota of D. acuta at the time of the annual maximum of lipophilic toxins in shellfish. At that time, the population of D. acuta showed an extremely low division rate (μ = 0.03 d -1 ), a high frequency of dead cells (up to 15%) and cells with starch granules (up to 93%), and no evidence of recent mixotrophic behaviour. Still, the cells, which did not perform vertical migration, aggregated around salinity-driven density discontinuities in the top 5 m and had a high cell toxin quota (deter- mined by liquid chromatography-mass spectrometry) for this species. A 3.5-fold difference was found between maximum (during the night) and minimum values of cell toxin quota. Okadaic acid (OA) was the predominant toxin and changes in cell quota of different toxins showed different daily pat- terns in the content of okadaiates and pectenotoxins; thus, the toxin profile is influenced by the time of sampling. Dissolved toxins (adsorbed by resins in passive samplers from 8 to 14 November) also reached annual maxima and reflected the vertical distribution of D. acuta cells. These results empha- size the need to parameterize physiological conditions and behaviour of Dinophysis populations dur- ing different phases of the population growth and hydrodynamic scenarios.

Diarrhoetic shellfish poisoning toxins in Cancer pagurus Linnaeus, 1758 (Brachyura, Cancridae) in Norwegian waters

During the summer of 2002 there were several episodes of human intoxication after consumption of brown crabs ( Cancer pagurus ) caught along the Norwegian south coast. The toxic agent was one of the well-known phycotoxins, the diarrhoetic shellfish poisoning (DSP) complex, routinely found in blue mussels ( Mytilus edulis ). This toxin has not previously been documented in brown crabs. The route and rate of toxin accumulation as well as the rate of toxin depuration in crabs were determined in laboratory experiments. The DSP toxins only accumulate in digestive organs (hepatopancreas, HP) in the crabs. When fed with blue mussels containing more than 1000 μg of okadaic acid equivalents kg −1 , the crabs accumulated 3-30% of the toxin. After 2 weeks, the average toxin level in the crabs exceeded the preliminary limit of DSP toxins at 400 μg okadaic acid equivalents kg −1 HP established by the Norwegian Food Safety Authority. A 50% reduction in toxin level was observed after 14-18 days when the crabs were fed f...

Morphological variability of the potentially toxic dinoflagellate Dinophysis sacculus (Dinophyceae) and its taxonomic relationships with D. pavillardii and D. acuminata

The identity of Dinophysis sacculus Stein has proved to be problematic in recent years, during which this species has been associated with diarrhoetic shellfish poisoning (DSP) events along European coasts. To complement the scarce and at times contradictory existing information, we provide details of morphological variation in D. sacculus throughout its geographical range, which includes the Mediterranean Sea and European Atlantic coasts. Our data clarify its relationship with D. pavillardii Schröder. This species differs from D. sacculus only by the presence of a concavity along the dorsal margin, but transitional morphotypes are frequent in almost all the samples examined. We therefore propose merging the two species and classifying morphotypes with a dorsal concavity as D. sacculus f. reniformis (Pavillard) comb. nov. Despite the high variability of Dinophysis sacculus, it was generally possible to separate it from D. acuminata Claparède & Lachmann. The only character distinguishing the two species is the shape of the large hypothecal plates, which in D. sacculus are elongate, almost rectangular or sac-like, whereas in D. acuminata they are shorter, more convex dorsally and often more slender towards the antapex. Thecal areolation and sulcal list ornamentation are more pronounced in D. acuminata, although these characters are somewhat variable. Other thecal details, such as the shape of sulcal platelets and the structure of the apical pore area, were not useful for circumscription of the two taxa. In a few samples from the Galician Rias and from the Spanish Mediterranean coast, we observed morphotypes intermediate between D. sacculus and D. acuminata, which indicates that the two species can have slightly overlapping ranges of variability.

Morphological variability of the potentially toxic dinoflagellate Dinophysis sacculus (Dinophyceae) and its taxonomic relationships with D. pavillardii and D. acuminata

The identity of Dinophysis sacculus Stein has proved to be problematic in recent years, during which this species has been associated with diarrhoetic shellfish poisoning (DSP) events along European coasts. To complement the scarce and at times contradictory existing information, we provide details of morphological variation in D. sacculus throughout its geographical range, which includes the Mediterranean Sea and European Atlantic coasts. Our data clarify its relationship with D. pavillardii Schröder. This species differs from D. sacculus only by the presence of a concavity along the dorsal margin, but transitional morphotypes are frequent in almost all the samples examined. We therefore propose merging the two species and classifying morphotypes with a dorsal concavity as D. sacculus f. reniformis (Pavillard) comb. nov. Despite the high variability of Dinophysis sacculus, it was generally possible to separate it from D. acuminata Claparède & Lachmann. The only character distinguishing the two species is the shape of the large hypothecal plates, which in D. sacculus are elongate, almost rectangular or sac-like, whereas in D. acuminata they are shorter, more convex dorsally and often more slender towards the antapex. Thecal areolation and sulcal list ornamentation are more pronounced in D. acuminata, although these characters are somewhat variable. Other thecal details, such as the shape of sulcal platelets and the structure of the apical pore area, were not useful for circumscription of the two taxa. In a few samples from the Galician Rias and from the Spanish Mediterranean coast, we observed morphotypes intermediate between D. sacculus and D. acuminata, which indicates that the two species can have slightly overlapping ranges of variability.

Seafood-associated diseases and control in Canada.

Paralytic shellfish poisoning (PSP) has been documented for two centuries and control programmes have been operated for fifty years. Although some illnesses are reported almost every year, the last known death from PSP occurred in 1981. In more recent years, amnesic shellfish poisoning, ciguatera poisoning, diarrhoetic shellfish poisoning, scombroid (histamine) poisoning and tetramine poisoning have been documented. The most frequently observed of these diseases is scombroid poisoning from improperly stored fish, but PSP and ciguatera poisoning have the most serious consequences. Vibrio infections arising from naturally-contaminated shellfish are virtually unknown, and viral illnesses from polluted harvested waters are rare. Control is achieved through monitoring of waters for indicators of human pathogens. Inspection systems based on the hazard analysis and critical control point principles are being introduced into all areas of fish and shellfish harvesting. The Inspection Directorate of the Department of Fisheries and Oceans joined the new Canadian Food Inspection Agency in 1997, which co-ordinates all Federal control measures for food in Canada.

Determination of okadaic acid by micellar electrokinetic chromatography with ultraviolet detection

Micellar electrokinetic chromatography (MEKC) with ultraviolet (UV) detection was applied for the determination of non-derivatized phycotoxins associated with diarrhoetic shellfish poisoning. A detection limit for 40 pg of okadaic acid (OA) was achieved. The UV intensities of this toxin measured at 200 nm showed good linearity in the range 40–640 pg. OA was detected in mussels spiked with 10 ng/g whole tissue. The presence of OA and dinophysistoxin-2 was observed in the crude extract of the dinoflagellate Prorocentrum lima.

A rapid tissue culture assay for the detection of okadaic acid and related compounds in mussels

Okadaic acid, the main toxin of the diarrhoetic shellfish poisoning (DSP) group, shows high cytotoxicity to Buffalo green monkey (BGM) kidney cell cultures after different times according to concentration. A rapid method of detecting the presence of okadaic acid and related compounds in contaminated mussels by means of a cytotoxicity test is presented. The method is based on direct microscopic observation of toxin-induced morphological changes in BGM cell cultures. A high correlation ( r = 0.950; P < 0.001) was found between the official mouse bioassay and the cytotoxicity test conducted on naturally contaminated samples of Mytilus galloprovincialis.

First report of pectenotoxin-2 (PTX-2) in algae ( Dinophysis fortii) related to seafood poisoning in Europe

Pectenotoxin-2 (PTX-2), a polyether-lactone included in the neutral class of diarrhoetic shellfish poisoning (DSP) toxins, has been unambiguously detected in Dinophysis fortii collected in the northern Adriatic Sea (Emilia Romagna coasts). This is the first report of such a toxin in Europe. This lipid soluble toxin was identified both in crude methanolic phytoplankton extract and in the neutral fraction obtained by extract chromatography on a basic alumina column. The techniques used were reversed phase high-performance liquid chromatography followed either by UV diode-array detection (LC-UV-DAD) or by mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS-MS) using an atmospheric-pressure ionization source and an ionspray interface. Okadaic acid (OA) was also found in the D. fortii specimens and quantified as 15 pg/cell. Although quantitation of PTX-2 was not possible due to the lack of pure toxin, the high PTX-2:OA ratio suggested PTX-2 was significant in the D. fortii specimens. The presence of PTX-2 in a region with no previous report of DSP neutral toxic compounds may indicate a risk of human poisoning. Serious efforts should therefore be made to develop suitable routine methods capable of detecting the presence of PTXs in biological materials of marine origin, in order to assure the wholesomeness of seafood products.

Dinophysistoxin-2: The predominant diarrhoetic shellfish toxin in Ireland

Diarrhoetic shellfish poisoning (DSP) in Europe is due mainly to the presence of the dinoflagellate toxin, okadaic acid (OA). However, analysis of cultivated mussels ( Mytilus edulis) from southwest Ireland revealed that an isomer of OA, dinophysistoxin-2, was the major toxin present during DSP episodes. Using fluorimetric HPLC, following derivatisation with 9-anthryldiazomethane, both OA and DTX-2 were found in shellfish during a prolonged toxic episode in 1991. However, examination of similar mussel cultivation locations in 1994 showed that DTX-2 was even more predominant. During this DSP period, OA levels were less than 0.7 μg/g, whereas maximum DTX-2 levels of 6.3 μg/g hepatopancreas were recorded. This toxicity in shellfish occurred soon after high cell counts of Dinophysis acuta were observed. As well as large seasonal variability in toxin levels in rope cultured mussels, substantial variations were also observed, both horizontally and vertically, within the water column.

Detection of diarrhoetic shellfish toxins in mussels from Italy by ionspray liquid chromatography-mass spectrometry

Direct detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1) and some of their related compounds in toxic mussels ( Mytilus galloprovincialis) is reported using ionspray liquid chromatography-mass spectrometry (LC-ISP-MS). This was employed to analyse diarrhoetic shellfish poisoning (DSP) toxins in mussels collected from coastal areas of the northern and southern Adriatic Sea. DTX-1 was found in some samples from both the northern and southern Adriatic and this is the first report of the unambiguous identification of this toxin in Italian mussels. The low levels found indicate that this toxin did not play a significant role in toxicity in these samples. Okadaic acid was found in all the mussels examined, although its concentration was not always sufficient to account for DSP toxicity. Furthermore, two related compounds of OA were detected in all the samples and one related DTX-1 compound was observed in some samples from the northern Adriatic. All three compounds are still to be identified, but it is possible that these substances are involved in mussel DSP toxicity in the Adriatic Sea.

Induction of morphological transformation in BALB/3T3 mouse embryo cells by okadaic acid

Okadaic acid is produced by several types of dinoflagellates (marine plankton) and has been implicated as a causative agent of diarrhoetic shellfish poisoning. Okadaic acid, a known tumour promoter in vivo, has been shown to promote morphological transformation of carcinogen-initiated BALB/3T3 cells. This study shows that okadaic acid is capable of inducing morphological transformation of BALB/3T3 cells in the absence of an initiator.

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.

High-performance liquid chromatography of okadaic acid and free fatty acids in mussels

A high-performance liquid chromatographic method developed for the determination of both okadaic acid (OA) and free fatty acids (FFA) was used for analysing mussel samples collected in the Gulf of Trieste. OA and FFA extracted from mussel hepatopancreas were derivatized prior to their chromatographic separation and spectrofluorimetric detection. 9-Chloromethylanthracene (CA) was used as a fluorescent labelling agent. The presence of toxic fatty acids ( e.g., linolenic acid), which may interfere with the bioassay of diarrhoetic shellfish poisoning (DSP) toxins, in the lipidic fraction of the extract was observed, whereas no OA was detected in the analysed samples.