Aquatic worms are considered a suitable group to evaluate the effects of contaminants in the environment, although one of the main challenges is using native species. Recently, Pristina longiseta was suggested to be used in acute bioassays for tropical regions. In this context, the aim of this study was to establish a chronic exposure for ecotoxicological bioassays utilizing the tropical native species P. longiseta. Firstly, we tested three exposure times (96h, 7d, and 10d) in the presence and absence of aeration. After determining the best con guration, we evaluated the effects of the chronic exposures using the standardized reference substance potassium chloride, the antibiotic sulfamethoxazole, the ame retardant tetrabromobisphenol-A, and sugarcane vinasse. Our results showed the suitability for applying the chronic exposure using P. longiseta and indicated the sensitivity of the offspring to KCl (EC50 = 0.51 g/L). Sulfamethoxazole and TBBPA caused a signi cant reduction in the offspring of P. longiseta (EC50 = 59.9 µg/L and 166.1 µg/L, respectively). Sugarcane vinasse showed high toxicity for the species, and a fraction of 4.26% of vinasse was calculated as EC50. Therefore, the described protocol was successfully applied as an ecotoxicological assessment to evaluate the effects of contaminants on the reproduction rate of the freshwater worm P. longiseta.