*2.4. Ames Test*

The method was applied according to Ames [45] using *Salmonella typhimurium* (*S. typhimurium*) TA98 and TA100 strains to evaluate possible mutagenicity. The cultures were grown in Oxoid nutrient broth No. 2. The samples of water were diluted in four different concentrations 25%, 50%, 75%, and 100% (*v*/*v*). The mutagenic effect was evaluated from the number of revertant colonies per plate. The plates were prepared in triplicate for every test sample, and the result presented was the mean of triplicate observation. The mutagenic activity was detected after 120 h of exposure at 37 (±2) ◦C. The revertant colonies' readings were counted using an automatic colony counter (Industrial Scientific). For accuracy of the results, mutagenic index (MI) values greater than or equal to two (≥2) were considered mutagenic [46].

## *2.5. Statistic Analysis*

To establish whether there is a relationship between evaluated physicochemical parameters and heavy metals with results of bioassays, a one-way analysis of variance (ANOVA) was performed with a significance level of *p* < 0.05.
