Screening sensitive testing endpoints for Cr(VI) responses in earthworms using cross-species cumulative distributions

Cr(VI) is a priority soil contaminant, yet current earthworm toxicity assessments still rely largely on single-species apical endpoints. In this study, we systematically compared the sensitivity of 12 earthworm species, representing epigeic, endogeic and anecic life forms, to Cr(VI) using four response indicators spanning multiple levels of biological organization: growth inhibition (EC₅₀), acetylcholinesterase (AChE) inhibition, protein secondary-structure alteration (circular dichroism, CD), and coelomic fluid osmolality. Species-specific exposure-response curves were fitted with log-logistic models to derive EC₅₀ values for each endpoint. Species-specific EC₅₀ values were summarized using cumulative distributions. The growth-inhibition distribution was interpreted as an SSD, whereas the biochemical and physiological endpoints were summarized as cumulative frequency distributions. Across the 12 species, EC₅₀ values showed pronounced interspecific variability, ranging from a ∼2.6-fold span for growth inhibition to a ∼27-fold span for AChE inhibition. For AChE inhibition, the most responsive endpoint in the present dataset, Lumbricus rubellus was the most sensitive species (EC₅₀ = 10.21 mg·kg^-1), whereas Lumbricus terrestris was the least sensitive species (EC₅₀ = 280.44 mg·kg^-1). The biochemical and physiological response indicators showed lower 5th-percentile response concentrations than the 28-d growth-inhibition endpoint, with the following lower-tail response ranking: AChE inhibition < protein secondary-structure alteration < coelomic-fluid osmolality deviation < growth inhibition. The AChE-based P₅ was approximately one order of magnitude lower than the growth-inhibitory HC₅, indicating that the AChE biomarker responded at substantially lower nominal Cr(VI) concentrations than the 28-d growth response. Results identified the AChE-based P₅ as the lowest endpoint-specific lower-tail percentile among the assessed biochemical and physiological response indicators, while the 50th-percentile comparison further supported their greater concentration-based responsiveness relative to growth inhibition, providing mechanistic information for endpoint selection in soil Cr(VI) effect assessment.