The dry weight of freeze dried plant tissues was determined and total MCs and
CYN extracted following the methods described in Section 2.2 after a process of
freezing at –80 1C, freeze drying and subsequent sonication of plant tissues. MCs
were extracted using 0.1 M acetic acid (10 ml) followed by 20 ml of a mixture of
methanol-chloroform (1:1, v/v) and CYN extracted in distilled water with TFA
(0.1%, v/v). The total MCs and CYN were subsequently quantified using the
commercially available Elisa tests from Abraxis (Los Angeles, California, USA)
specific for the ADDA amino acid in MCs and nodularins and for the CYN molecule.
The cyanotoxin quantifications were performed according to the manufacturer’s
instructions. In each analysis, control samples were introduced (extracts from
leaves and roots of plants not exposed to cyanobacterial cell extracts) to screen for
false positives. The Elisa assays of controls were negative. Moreover a quality
control positive sample from the Elisa kit and also a sample consisting in an
extract (from each matrix spiked) with a known dose of these toxins were
analyzed in order to avoid false negatives.