Congener-Specific Modulation of Humoral Effector Activity in Eisenia fetida Following PFAS Exposure

Per- and polyfluoroalkyl substances (PFASs) are persistent environmental contaminants of growing concern for soil ecosystems, yet their effects on the humoral arm of innate immunity in soil invertebrates remain poorly characterized. Here, we used the earthworm Eisenia fetida to screen 31 legacy and emerging PFAS congeners for their ability to modulate the hemolytic activity of cell-free coelomic fluid, a functional readout of soluble immune effectors including the pore-forming toxin lysenin. Earthworms were exposed under OECD 207 contact-filter conditions at two concentrations (0.6 and 229 µM) for 72 h, after which decellularized coelomic fluid was tested against sheep erythrocytes. To dissect direct biochemical interference from organism-mediated regulation, the same panel was also applied ex vivo (2.5 µM) to coelomic fluid from unexposed earthworms. In vivo, PFASs produced markedly heterogeneous, congener-specific responses: PFBS, PFBA and PFMOPrA suppressed hemolytic activity, whereas PMDA, PFHxA and HFPO-DA enhanced it. In contrast, ex vivo exposure produced a consistent, broad inhibition of hemolysis, indicating a direct interaction of PFASs with soluble immune proteins. Proteomic profiling of the lysenin family under PFOA and HFPO-DA suggested isoform-level reweighting rather than uniform abundance shifts, although effects did not survive multiple-testing correction. Together, these data show that PFASs act as congener-specific immunomodulators of extracellular humoral defense in E. fetida and identify candidate congeners for confirmatory mechanistic studies.