Per- and polyfluoroalkyl substances (PFAS) occur in terrestrial ecosystems as complex mixtures, and their ecological effects are more accurately characterized through the integration of multiple complementary biomarkers rather than single-endpoint assessments. In earthworms, innate immune and stress-response pathways provide sensitive indicators of contaminant-induced physiological alterations across different levels of biological organization. The present study aimed to investigate the potential synergistic or antagonistic effects of PFAS mixtures in earthworms using a multi-biomarker approach. To this end, a suite of molecular and cellular biomarkers was evaluated, including haemocyte morphometric alterations, reactive oxygen species (ROS) production, catalase (CAT) activity, phenoloxidase (PO) activity, acetylcholinesterase (AChE) activity, and γ-aminobutyric acid (GABA) levels. Biomarker responses were assessed in earthworms exposed either to five individual PFAS compounds or to an equimolar five-component mixture under a one-fifth dosing design (individual PFAS: 0.12, 0.84, and 45.8 µM; PFAS mixture: 0.6, 4.2, and 229 µM total concentration).

Across all endpoints allowing direct comparison, the observed effects of the PFAS mixture were consistently lower than the sum of the corresponding single-compound effects, indicating predominantly sub-additive interactions. Observed-to-additive ratios ranged from approximately 0.18 to 0.66 for haemocyte morphometry, AChE activity, and ROS production, while PO activity exhibited an even stronger sub-additive response. No evidence of synergistic interactions was detected for any biomarker. Multivariate ordination analysis integrating the five fully crossed endpoints revealed a two-dimensional response pattern, with PFOA and the PFAS mixture clustering separately from ether-based and short-chain PFAS congeners. Together with the observed haemocyte spreading phenotype, these findings indicate a coherent, multi-level disruption of immune and oxidative stress pathways in earthworms. Overall, the results suggest that PFAS mixtures act predominantly through sub-additive mechanisms, with the combined biological response largely driven by the most toxic constituent present in the mixture.