The eco-risk of roxarsone (ROX) was evaluated using multiple responses of earthworm biomarkers under different ROX concentrations for 28 d. With the increasing total arsenic accumulation (TAs-E), biological responses in earthworm generally showed a two-stage changes of homeostasis dysregulation and dose-dependent alterations. At the early periods, ROX stress increased the reactive oxygen species (ROS) and lipid peroxidation (LPO) in a similar manner, and apparently disrupted mitochondrial calcium ([Ca]). But earthworms regulated their mitochondrial and redox homeostasis through stable mitochondrial membrane potential (MMP) and increase of ATP level, superoxide dismutase (SOD) and catalase (CAT). After 14 d, the positively correlated mitochondrial effects of ROS, [Ca], MMP and ATP were related to the behavioral inhibition of burrow length, depth and reuse rate as well as antioxidant up-regulation of Nrf2, HO-1, sod1 and cat. These results contributed possible biomarkers from the dose-dependent relationship between mitochondrial, antioxidant and behavioral responses. Multiple biological detection in earthworms can better reflect the sub-chronic ecotoxicity of phenylarsenic pollutants in soil.
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