Effect of Ether Perfluoro Carboxyl Acids (PFECAs) on Innate Immunity in Earthworms (Eisenia fetida)

Per‑ and polyfluoroalkyl substances (PFAS) persist in soils, yet their effects on invertebrate immunity remain poorly resolved. We compared a legacy congener, perfluorooctanoic acid (PFOA), with three short‑chain ether acids GenX (C6), MOBA (C5) and MOPrA (C4) using a 72 h OECD‑207 filter‑paper assay in the earthworm Eisenia fetida. Endpoints spanned cellular and humoral defence: amoebocyte morphometry, oxidative burst (ROS production), phenol‑oxidase (PO) activity, and transcription of the lectin CCF‑1 and the pore‑forming protein lysenin. MOBA and MOPrA enlarged amoebocytes by ~ 70% (p < 0.001), whereas PFOA and GenX had no morphometric impact. Oxidative burst fell significantly for all congeners; GenX caused a 45% drop at 0.6 µM, while MOPrA declined monotonically from − 18% (0.6 µM) to − 62% (229 µM). PO inhibition followed the same potency order (MOPrA > GenX > MOBA ≫ PFOA) with near‑complete loss at 229 µM MOPrA. CCF‑1 showed dose‑dependent or U‑shaped induction, whereas lysenin peaked at 31 µM MOPrA but was unchanged at 0.6 and 229 µM, suggesting an energetically costly yet inefficient compensatory response. Integrating these findings with published superoxide‑dismutase inhibition and catalase hyper‑activation reveals an oxidative–antioxidant–immune cascade that compromises earthworm defence at concentrations ≥ 0.6 µM (≈ 250 µg kg⁻¹ soil), levels already reported in AFFF‑impacted sites. The combined biomarker panel—amoebocyte size, ROS, CAT, PO, CCF‑1 and lysenin—offers a concise framework for assessing terrestrial PFAS risk and guiding remediation monitoring.