Amputation in Enchytraeus crypticus - a New Approach Methodology (NAM) - assessing the longer-term effects of Cadmium under regeneration stress events

In natural environments, organisms frequently experience injuries caused by physical disturbance, contamination, or extreme climatic conditions, which may alter their sensitivity to toxic stress. Understanding how such conditions, like injuries, affect organism performance is particularly relevant in ecotoxicology, where exposure to pollutants can interfere with tissue repair and long-term fitness. Enchytraeus crypticus , a standard test soil species, can regenerate posterior segments and therefore offers the opportunity to integrate regeneration capacity as an additional endpoint in toxicity assays. In the present study, we established a methodology to integrate regeneration as an ecotoxicological testing endpoint, directly in accordance with already established OECD/ISO guidelines for this organism. Furthermore, the impacts of cadmium (Cd) exposure on survival and reproduction were assessed following induced artificial amputation stress, hence under regeneration response processes. Mature adult organisms were amputated and exposed to 0–320 mg Cd kg −1 DW soil in LUFA 2.2 soil. Regeneration success was evaluated at day 7, and survival and reproduction at days 22 and 43. E. crypticus regenerated the complete body in all treatments, except at 320 mg Cd kg −1 DW soil. Survival was similar between amputated and non-amputated organisms at day 22, but at day 43, mortality reached 100% in amputated versus 70% in non-amputated organisms at 320 mg Cd/kg DW soil, indicating increased vulnerability if under prolonged exposure when regeneration is required. Reproduction showed a clear dose-response pattern at both 22 and 43 days. EC 50 values for amputated vs. non-amputated organisms were 42 vs. 82 mg Cd kg −1 DW soil (day 22) and 37 vs. 29 mg Cd kg −1 DW soil (day 43), indicating a shift toward improved reproductive performance after regeneration was completed This suggests a compensatory mechanism, one that reallocates energy toward reproduction once regeneration is complete. These findings emphasize the importance of considering both shorter and longer-term biological responses when evaluating the impact of contaminants on soil organisms. Further research into the molecular mechanisms underlying this plasticity could provide deeper insights into the adaptive strategies of E. crypticus in polluted environments. • Cd exposure impairs regeneration and survival in a dose-dependent manner. • Amputated E. crypticus show full recovery except at the highest Cd concentration. • Reproduction improved post-regeneration, suggesting a compensatory response. • Longer exposure reveals the effects of stress-repair that are not visible in standard assays.