Toxic and genotoxic effects of potassium dichromate in Pseudokirchneriella subcapitata detected by microscopy and AFLP marker analysis

The effect of chromium on Pseudokirchneriella subcapitata has been assessed using different approaches: growth rate, metabolic activity microscopy analyses, and amplified fragment length polymorphism (AFLP) assessment of DNA damage. Starting from 24 h of treatment all the tested concentrations resulted in consistent algal growth inhibition. The average daily growth rate after 72 h calculated for the control (0.53 ± 0.01) was statistically higher than those estimated for cell treated with 1, 2.5, 5 and 7.5 μg g⁻¹ potassium dichromate (0.46 ± 0.02; 0.32 ± 0.01; 0.25 ± 0.01 and −0.02 ± 0.04, respectively). A reduction of viable cell numbers, estimated with FDA approach, was also observed after 24 h treatment for all the tested chromium concentrations apart from the lowest (1 μg g⁻¹ potassium dichromate). A recovery of esterase activity was detected after 48 and 72 h for all treatments with the except of the 7.5 μg g⁻¹ potassium dichromate treated samples showing a very modest recovery. This data suggests that potassium dichromate is, even from the lowest tested concentration, highly toxic to P. subcapitata, and that this algal strain is a sensitive organism suitable for monitoring chromium in water. Our data also suggest that although algal counting by microscope and the FDA test gave similar indications concerning chromium effect, the FDA test was not completely reliable. In fact, we observed a decrease in the FDA stained cell numbers in control samples after 48 and 72 h of treatment, when most cells were actively proliferating. This lack of fluorescence could be explained by an uncontrolled fluorescein efflux due to the interaction of many experimental factors. The genotoxic effects of the different chromium concentrations were investigated by analyses of the DNA from control and treated algal samples, 72 h after inoculation. AFLP analysis revealed a total of 258 bands, 109 of which were polymorphics. Analysis of the AFLP matrix suggests that potassium dichromate is a powerful genotoxic agent, inducing genetic mutations also at the lowest tested concentration (0.35 μg g⁻¹). Furthermore, we observed a correlation between the polymorphic bands with increasing chromium concentration. Finally, the absence of preferential mutation sites suggests that the chromium induced DNA changes are randomly distributed in the genome.