Behavior-Based Toxicity Assessment of Heavy Metals in Earthworm Reveals Copper as High Potent Metallic Elements
Earthworms are important soil organisms that contribute to nutrient cycling, organic matter decomposition, soil aeration, and water infiltration. Because of their permeable skin and continuous contact with soil, they are particularly sensitive to metal contamination and are widely used as bioindicators in terrestrial ecotoxicology. In this study, the acute toxicity of 23 selected metallic elements, applied in their chloride forms, was evaluated in the earthworm Eisenia andrei using a 72-h filter-paper contact assay. In addition to mortality, video-based locomotor tracking was used to quantify eight behavioral endpoints: average speed, average angular velocity, entropy, fractal dimension, freezing-time ratio, meandering, thigmotaxis, and time spent in the central area. No mortality was observed at 1 ppm, although several metals induced sublethal alterations in locomotor activity and movement patterns. At 10 ppm, antimony, copper, and vanadium caused mortality, while other metals produced concentration-dependent behavioral disturbances, including changes in activity, movement complexity, freezing, and wall-preference behavior. Exploratory RNA-seq profiling of the Cu-, Sb-, and V-exposed groups further identified both shared and metal-specific candidate stress-response signatures. Vanadium produced the broadest transcriptomic perturbation, whereas copper and antimony shared enrichment of protein-quality-control functions. The three metals also differed in their oxidative-stress, ion-regulatory, and xenobiotic-response profiles, suggesting that similar organism-level toxicity may arise through distinct cellular disturbances. Overall, the integration of automated behavioral phenotyping with exploratory transcriptomic profiling provides a sensitive framework for identifying both sublethal and severe effects of metal exposure in earthworms.