Species- and time-dependent responses of earthworm and microoganisms during nano-hydroxyapatite remediation of cadmium-contaminated soil

Nano-hydroxyapatite (N-HAP) is widely considered an effective amendment for cadmium immobilization in contaminated soils, but its ecological safety to soil fauna and microbial communities remains insufficiently understood, particularly across different earthworm ecological categories and exposure stages. This study evaluated whether N-HAP-induced cadmium stabilization was accompanied by species- and time-dependent biological responses. Two earthworm species, Eisenia fetida representing an epigeic ecological category and Amynthas robustus representing an endogeic ecological category, were exposed to cadmium-contaminated soil amended with 0%, 1.5%, and 3% N-HAP for 14, 28, and 42 days. Basic soil properties, cadmium fractionation, earthworm physiological responses, and phospholipid fatty acids-based microbial biomass and community structure were quantified. N-HAP at 3% increased soil pH by 0.50-0.55 units and decreased exchangeable and weak acid-extractable cadmium by 21.3-27.6%, indicating effective cadmium stabilization. Biological responses were strongly species- and time-dependent: 3% N-HAP caused a transient reduction in Eisenia fetida survival and biomass at 14 days, whereas Amynthas robustus showed no comparable impairment. Lipid peroxidation exhibited asynchronous trajectories, increasing in Eisenia fetida at 42 days under 3% N-HAP, but in Amynthas robustus at 14-28 days under 1.5% N-HAP. At the microbial level, high N-HAP briefly suppressed total microbial biomass and major groups during early exposure, while the broad phospholipid fatty acids-defined community structure remained relatively conserved. Integrated analyses indicated that exposure duration, rather than N-HAP concentration alone, was the dominant factor structuring earthworm and microbial responses, suggesting that the biological effects of N-HAP were governed by progressive soil-amendment stabilization and time-dependent biotic adjustment. Overall, N-HAP effectively immobilized cadmium but imposed short-lived ecological disturbance at high application rates. These findings highlight a trade-off between remediation efficiency and ecological safety, suggesting that moderate, well-timed, and site-specific N-HAP application are needed to maximize cadmium stabilization while minimizing early-stage stress to soil biota.