fish vs toxins

Phytoplankton analyzes
The concentration of chlorophyll a (?g L-1) was measured immediately after sampling in a 1-L integrated water sample using a bbe Algae Online Analyzer (AOA, Version 1.5 E1, bbe- Moldaenke company Kiel, Germany). This analyzer takes measurements based on the determination of the fluorescence spectrum and kinetics of the algae. By analyzing the interaction of chlorophyll a (Chloa) with other pigments, the AOA discriminates among four main groups of algae (green algae, cyanobacteria, cryptophytes and diatoms).
Samples for the determination of phytoplankton biovolume were preserved in Lugol?s solution and sedimented in the laboratory. Phytoplankton were counted in concentrated samples, using a Fusch-Rosenthal counting chamber and a Nikon Optiphot-2, 102 (400x magnification). At least 400 cells or filaments were counted to reduce the error to less than 10% (p=0.05). Phytoplankton biovolume (mm3 L-1) was determined based on a volumetric analysis of cells using geometric approximation.
Genetic analyzes of cyanobacteria
The samples of water for DNA assays (always 100 mL of water was collected) were prepared according to Mankiewicz-Boczek et al. (2006). Filters with cyanobacterial material (pore diameter 0.45 ?m, Millipore, USA) were put into the lysis buffer containing 40 mM EDTA, 400 mM NaCl, 0.75 M sucrose, and 50 mM TRIS-HCl (pH 8.3) and frozen as soon as possible. Genetic material was extracted from the filters according to Giovannoni et al. (1990), with some modifications, which improved the extracted DNA quality and quantity, as described by Mankiewicz-Boczek et al. (2006).
Extracted DNA was used as the template for quantitative (qRT-PCR, quantitative real-time PCR) determination of: the 16S rRNA (250 bp) gene fragment specific for the genus Microcystis

and the mcyA (395 bp) gene fragment specific for toxic Microcystis genotypes.
Chromatographic analyzes of microcystins
For high-performance liquid chromatography (HPLC) analyzes, 1-L water samples
containing cyanobacterial material were filtered through GF/C filters (Whatman, Kent, UK GF/C, 1.20 ?m) immediately after sampling. The preparation of cyanobacterial material and the determination of MCs concentration via HPLC-DAD were performed according to Jurczak et al. (2005).
Protein phosphatase inhibition assay
The protein phosphatase inhibition assay was performed with a MicroCystest screening immuno-bioassay (ZEU-Inmunotec, Zaragoza, Spain) to confirm the biological activity of MCs, i.e. their toxicity.