Field-derived PFAS mixtures impair biological functions across organisational levels in a terrestrial invertebrate

: PFAS contamination of terrestrial environments poses ecological risks, yet the biological responses of soil invertebrates to environmentally realistic PFAS mixtures remain poorly characterised. Here we assessed field-collected PFAS-contaminated soils from a firefighting training site in Trelleborg (Sweden) on Eisenia fetida, using a multi-endpoint approach spanning molecular, enzymatic, behavioural and reproductive responses. The soils spanned nearly three orders of magnitude in total PFAS (1.2-955 μg/kg dw), providing field-relevant exposure without laboratory manipulation. Catalase activity was significantly reduced at three of the five contaminated sites (B4, p < 0.001; B5, p < 0.0001; B6, p < 0.05) and phenol oxidase at a further four (B1, B4 and B5, p < 0.01; B6, p < 0.05), indicating disruption of antioxidant and innate-immune pathways, with non-linear changes in the immune transcripts CCF-1 and Lysenin. Escape latency was markedly prolonged at B1 and B4 (median 141 s and 170 s versus 83 s in controls), while acetylcholinesterase activity showed only a marginal, non-monotonic trend (p < 0.1), and biomass loss and reproductive output below the OECD Test 222 validity threshold occurred at every site except the least contaminated (B7). An integrated, non-parametric multivariate analysis unified these endpoints: all contaminated sites except B7 separated from the control, and phenol oxidase activity declined with the internal PFAS burden (ρ = -0.63) rather than with the soil trace-metal load, indicating that the responses tracked internal PFAS rather than co-occurring contaminants. To our knowledge this is the first study to use AFFF-contaminated field soils as the exposure matrix within an OECD Test 222 framework alongside a multi-tier sublethal endpoint battery in E. fetida, previously characterised mainly with spiked or agricultural soils. The results show that AFFF-derived PFAS mixtures impair earthworm function across multiple levels of biological organisation and support including chronic, integrative endpoints in soil ecological risk assessment for PFAS.