As the number of legal and illegal gold mining sites increases in the Andes–Amazonia region, integrative methods to evaluate the effects of mining pollution on freshwater ecosystems are of paramount importance. Here, we sampled water and sediments in 11 sites potentially affected by gold mining activities in the Napo province (Ecuador). The environmental impacts were evaluated using the following lines of evidence (LOEs): water physicochemical parameters, metal exposure concentrations, macroinvertebrate community response (AAMBI), and toxicity by conducting bioassays with Lactuca sativa and Daphnia magna. Dissolved oxygen and total suspended solids were under (<80%) and above (>130 mg/Ls) quality standards 65% of the sites. Ag, Al, As, Cd, Cu, Fe, Mn, Pb, and Zn in water and V, B, and Cr in sediments were detected above quality standards at sampled sites. Nine out of eleven sites were classified as having bad environmental quality based on the AAMBI. L. sativa seed germination in both water (37% to 70%) and sediment (0% to 65%) indicate significant toxicity. In five sites, neonates of D. magna showed a 25% reduction in survival compared to the control. Our integrated LOEs index ranked sites regarding their environmental degradation. We recommend environmental impact monitoring of the mining expansion at the Andes–Amazonia region using multiple LOEs.

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