Dermal absorption drives phenanthrene lethality in Eisenia fetida: A multi-scale mechanistic framework linking mitochondrial uncoupling to organismal toxicity.

Phenanthrene (PHE) is a priority polycyclic aromatic hydrocarbon posing substantial ecological risks to soil invertebrates, yet the mechanistic basis of its toxicity remains incompletely characterized. This study aimed to evaluate route-dependent PHE toxicity in earthworms by comparing sealed and unsealed soil exposures, and to link organismal responses with tissue-specific oxidative stress and mitochondrial bioenergetic disruption. Earthworms were exposed to PHE-contaminated soil (0-160 mg kg-1) under sealed (oral ingestion blocked) and unsealed conditions for 14 days, integrating lethality, ultrastructural pathology, tissue-specific oxidative stress (malondialdehyde, MDA; glutathione, GSH), and mitochondrial bioenergetics (Ca2+-ATPase, Complexes I and III, ATP production-consumption coupling). Sealed and unsealed survival curves converged at ≥80 mg kg-1, confirming dermal absorption as the exclusive lethal driver, Intestinal MDA consistently exceeded epidermal MDA, reflecting preferential gut accumulation and metabolic activation. GSH peaked at 20 mg kg-1 before declining at 40 mg kg-1, marking the transition from antioxidant induction to exhaustion. At 40 mg kg-1, mitochondrial complex activities were suppressed and the ATP production-consumption gap widened to 14.9 percentage points by day 3, providing evidence for electron leakage and respiratory uncoupling. These findings establish a concentration-gated toxicity continuum wherein mitochondrial uncoupling serves as the central mechanistic node linking oxidative stress to lethality, and propose the ATP production-consumption coupling ratio as a sensitive biomarker for PAH ecological risk assessment.