The Fundão mine tailings dam rupture of 2015, in the Rio Doce basin, Brazil, resulted in the deposition of tailings downstream of the dam. Dispute arose as to whether metals associated with the tailings may have potentially contributed to toxicity to organisms, consisting in a time bomb that could be ticking. Published data on toxicity to benthic and aquatic organisms did not enable an informed assessment using a weight-of-evidence based approach. This study was conducted to ascertain if sediments at ‘hot spots’ that received Fundão tailings reflected elevated concentrations of metals sufficient to result in toxicity to freshwater organisms. The lines of evidence considered included assessing metals concentrations in relation to sediment quality criteria, biogeochemical characterizations, an evaluation of potential metal release upon resuspension to provide information on bioavailability, and acute and chronic toxicity effects using sensitive native species for waters (water flea, Daphnia similis) and sediments (burrowing midge larvae, Chironomus sancticaroli). Only porewater concentrations of iron and manganese exceeded Brazilian surface water criteria, while most trace elements showed no enrichment or elevated environmental indices. The concentrations of bioavailable metals were assessed to be low, and metal concentrations did not increase in the overlying water upon resuspension; rather, they decreased through time. Toxicity testing in resuspended waters and bulk sediments resulted in no acute or chronic toxicity to either benthic or aquatic species. The low metal bioavailability and absence of toxicity of the tailings-enriched sediments was attributed to the strong binding and rapid removal of potentially toxic metal ions due to by oxyhydroxides and particles in the presence of iron rich particulates. The findings of these sediment “hot spot” studies indicate the Fundão dam release of tailings over six years ago is not resulting in current release of toxic concentrations of metals into the freshwaters of the Rio Doce.