Outbreaks of lipophilic toxins have been recorded in the north Atlantic coast of Morocco since 1999, but are rare in the Mediterranean coast. Samples of mussels from the Atlantic coast where toxicity was detected by mouse bioassay were stored for further research. Chemical analysis by LC-MS conducted in mussels harvested from this region showed, in addition to the presence of okadaic acid (OA) and dinophysistoxin-2 (DTX2), the presence of azaspiracid-2 (AZA2) as the dominant form of the azaspiracid's (AZAs) family, followed by AZA1 (13% to 26%). AZA3 was rarely detected, and maximal concentrations found were between 3% and 8% of total AZA1/3. The presence of AZA2 and AZA1 was confirmed by mass spectra. Time series corresponding to the summer of 2004 and 2005 showed maximal concentration of AZAs appeared in July in both years. Correlation with occurrence of OA and DTX2, showed both toxin families could appear simultaneously in Moroccan mussels, but maximal concentrations found were always separated in time. This is the first report of azaspiracids in Morocco (NW Africa) and the first report outside of European coastlines.

INTRODUCTION

Azaspiracid poisoning (AZP) is a new human gastrointestinal illness discovered in 1995 after consumption of contaminated Irish mussels in the Netherlands (McMahon & Silke 1996). Azaspiracid-1 (AZA1) was the first molecule of the group to be structurally characterized (Satake et al. 1998). Now, at least more than 10 azaspiracid congeners are known (James et al. 2003b). From these, AZA1, AZA2, and AZA3 were the dominant compounds found in shellfish, followed by AZA4 and AZA5. The remaining ones (AZA6-11) were minor components. It is hypothesized so far that these lipophilic toxins accumulate in bivalve molluscs after feeding on the toxic microalgae Protoperidinium crassipes, previously considered to be toxicologically harmless (James et al. 2003a).

The symptoms of acute AZP intoxication include nausea, vomiting, severe diarrhea, and stomach cramps (Satake et al. 1998), which closely resemble the symptoms associated with diarrheic shellfish poisoning (DSP). Toxicological studies showed that repeated administration of AZAs caused prolonged damage in the intestine and induced lung tumors in mice (Ito et al. 2002).

Although shellfish harvested in Ireland provoked the first incidents of human intoxications by azaspiracids, the research in other European countries has led to the discovery of these toxins in shellfish from the United Kingdom, Norway, France, and Spain (James et al. 2002a, Magdalena et al. 2003). Although AZP is a serious concern in Europe, its presence has never been reported outside European waters.

Outbreaks of lipophilic toxins have been recorded in shellfish from the north Atlantic coast of Morocco since 1999, but these toxins are rare in the Mediterranean coast (Taleb 2005). The Moroccan monitoring program to screen for the presence of lipophilic compounds employs the mouse bioassay (MBA), which does not allow distinguishing the contamination from different phycotoxin families, such as DSP or AZP, because these compounds are detected altogether. Although several methodologies exist to study the contamination with DSP, the only methodology available so far to confirm the specific presence of azaspiracids is liquid chromatography coupled to mass spectrometry (LC-MS) (Ofuji et al. 1999). Samples of blue mussels collected in Morocco during the summers of 2004 and 2005 were screened by LC-MS for lipophilic compounds. Besides the presence of DSP toxins (to be reported elsewhere), the presence of azaspiracids was put into evidence and is now reported here for the first time.

MATERIALS AND METHODS

Study Area

Samples of mussels (Mytilus galloprovincialis) were collected regularly from Oulad Ghanem and Dar Hamra at the Atlantic coast of Morocco (south of Casablanca, Fig. 1). Digestive glands (DG) were dissected from whole shellfish, homogenized, and extracted for MBA testing. The samples used for the study were selected from those collected in the period of summer outbreaks of samples positive to lipophilic toxins. Digestive glands of positive samples were stored frozen for ulterior analysis by LC-MS.

[FIGURE 1 OMITTED]

Sample Preparation and LC-MS Analysis

Digestive glands were homogenized, and a 5-g aliquot was taken and extracted by ultraturrax with 20 mL aqueous 90% methanol in a screw-cap plastic centrifuge tube, followed by a 10 min centrifugation at 2500g. A 2-milliliter aliquot of the methanolic supernatant was transferred to 10-mL glass test tubes, washed with hexane (2 x 2 mL), and extracted with dichloromethane (2 x 2 mL). The combined dichloromethane layers were dried with anhydrous sodium sulphate, centrifuged, transferred to small glass test tubes, and dried at 38[degrees]C under reduced pressure on a RapidVap (Labconco, USA). After resuspension with 500-[micro]L aqueous 90% methanol, contents were transferred to a 2 mL autosampler vial. Vortex mixing was used in all liquid-liquid and resuspension steps.