Purpose <p>Assessment of aquatic systems using biomarkers can provide early detection of genotoxins and support conservation strategies worldwide. We evaluate the presence of mutagenicity in surface and sediment interstitial waters of Negra Lake, located in Itapuã State Park, southern Brazil, to understand ecological threats to freshwater environments in protected areas.</p> Methods <p>Surface water and sediment samples were collected and tested through the Salmonella microsuspension assay using four strains both with/without metabolic activation.</p> Results <p>We identified basal mutagenic activity in surface and interstitial water. Surface water showed minimal mutagenicity associated with redox imbalance. Interstitial water exhibited cytotoxic and mutagenic properties of both frameshift and base-pair substitution mutations.</p> Conclusion <p>Our findings corroborate sediments as a major source of genotoxins and Salmonella/microsome as a sensitive biomarker for conservation efforts in protected freshwater ecosystems worldwide. We fill a gap guiding future research and insights into background data within the Brazilian Pampa biome.</p> Graphical Abstract <p></p>