Optimization of isolation and concentration of the common freshwater cyanobacterial toxins ATX-a, CYN and MC-LR using standard techniques, optimization of cyanobacteria growth.

Some of the most commonly identified freshwater toxins are anatoxin-a (ATX-a), cylindrospermopsin (CYN), and microcystin-LR (MC-LR). The aim of this paper was to compare different methods of extracting and concentrating these cyanotoxins and check the impact of selected physical factors on the accumulation of biomass of Dolichospermum flos-aquae, Microcystis aeruginosa, and Raphidiopsis raciborskii. The effect of different cyanobacteria cultivation conditions on the amount of cyanotoxins synthesized showed no significant changes over time in the average concentration of all tested toxins in the medium compared to the control. Mixing cultures increases the intracellular content of ATX-a. Aerating also positively affects the concentration of MC-LR intracellularly. In order to optimize the solid phase extraction (SPE) process of toxins, the C18 phase or activated carbon was used. In general, higher toxin recoveries were achieved when using the C18 phase. The best result was achieved for ATX-a, 94% recovery with elution using methanol with 0.1% trifluoroacetic acid (TFA). For MC-LR, the best recovery was 59%, and for CYN 22%. The study evaluated the various methods to release cyanotoxins from cyanobacteria showed that: the highest ATX-a concentration (0.60 μg/mg d.w) was obtained using MilliQ water and microwave treatment for 10-15 s. For MC-LR, the highest extracted amount (6.73 μg/mg d.w) resulted from methanol treatment and boiling at 100 °C for 15 min. CYN extraction was the most effective by using MilliQ water and alternative freezing/thawing (1.54 μg/mg d.w). In conclusion, changing the optimal parameters of cyanobacterial cultivation, only slightly affects the increase in biomass accumulation and synthesis of cyanobacterial toxins. In the case of ATX, the key is the use of the TFA additive in the SPE process. No single method has been identified as the ideal approach for isolating various intracellular cyanotoxins.