Multilevel investigation of the ecotoxicological effects of sewage sludge biochar on the earthworm Eisenia fetida

The ecological risks of sewage sludge biochar (SSB) after land use is still not truly reflected. Herein, the ecological risks of SSB prepared at different temperature were investigated using the earthworm E. fetida as a model organism from the perspectives of organismal, tissue, cellular, and molecular level. The findings revealed that the ecological risk associated with low-temperature SSB (SSB₃₀₀) was more pronounced compared to medium- and high-temperature SSB (SSB₅₀₀ and SSB₇₀₀), and the ecological risk intensified with increasing SSB addition rates, as revealed by an increase in the integrated biomarker response v2 (IBRv2) value by 2.59–25.41 compared to those of SSB₅₀₀ and SSB₇₀₀. Among them, 10% SSB₃₀₀ application caused significant oxidative stress and neurotoxicity in earthworms compared to CK (p < 0.001). The weight growth rate and cocoon production rate of earthworms were observed to decrease by 25.06% and 69.29%, respectively, while the mortality rate exhibited a significant increase of 33.34% following a 10% SSB₃₀₀ application, as compared to the CK. Moreover, 10% SSB₃₀₀ application also resulted in extensive stratum corneum injury and significant longitudinal muscle damage in earthworms, while also inducing severe collapse of intestinal epithelial cells and disruption of intestinal integrity. In addition, 10% SSB₃₀₀ caused abnormal expression of earthworm detoxification and cocoon production genes (p < 0.001). These results may improve our understanding of the ecotoxicity of biochar, especially in the long term application, and contribute to providing the guidelines for applying biochar as a soil amendment.