Paralytic shellfish toxins in the freshwater cyanobacterium Aphanizomenon flos-aquae, isolated from Montargil reservoir, Portugal.

Montargil reservoir, located in a dry flat area in the centre of Portugal, was filled in 1958 to fulfil agricultural, electric and industrial requirements. In May 1996, an intensive bloom of phytoplankton was detected. The algal community was strongly dominated by cyanobacteria with predominance of Aphanizomenon flos-aquae from May to June and Microcystis aeruginosa from July to August. Extracts of samples collected during the bloom period showed high toxicity by mouse bioassay. During the M. aeruginosa predominance period, the toxicity was ascribed to the presence of hepatotoxins, but clear symptoms of paralytic shellfish poison were observed when A. flos-aquae was the dominant species. In order to confirm the production of neurotoxins a strain of A. flos-aquae was isolated and established in culture. In this manuscript, we show the morphological characteristics and confirm paralytic shellfish toxins production by the strain isolated and maintained in culture. Identification of the saxitoxin analogs was achieved using high performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD) and liquid chromatographic mass spectrometry technique (LC-MS). The toxins found in the culture extract were GTX5 (64.5 mol%), neoSTX (23.0 mol%), dcSTX (6.1 mol%), STX (5.4 mol%) and GTX6 (1.1 mol%). This is, to our knowledge, the first report of unambiguous evidence of paralytic shellfish toxins produced by freshwater cyanobacteria in Portugal. The toxin profile is rather different from the previously reported PSP producing A. flos-aquae and demonstrates its diversity in terms of toxin production.