Impact of Short-Chain Perfluoropropylene Oxide Acids on Biochemical and Behavioural Parameters in Eisenia fetida (Savigny, 1826)

Abstract

Per- and polyfluoroalkyl substances (PFAS) are a class of persistent organic pollutants that pose a growing threat to environmental and human health. Soil acts as a long-term reservoir for PFAS, potentially impacting soil biodiversity and ecosystem function. Earthworms, as keystone species in soil ecosystems, are particularly vulnerable to PFAS exposure. In this study, we investigated the sublethal effects of three short-chain (C4-C6) next-generation perfluoropropylene oxide acids (PFPOAs) on the earthworm Eisenia fetida, using a legacy perfluoroalkyl carboxylic acid (PFCA), perfluorooctanoic acid (PFOA), as a reference. We assessed a suite of biochemical endpoints, including markers for oxidative stress (catalase and superoxide dismutase activity), immunity (phenol oxidase activity), neurotoxicity (acetylcholinesterase activity), and behavioural endpoints (escape test). Results indicate that all tested PFAS, even at sub-micromolar concentrations, elicited significant effects across multiple physiological domains. Interestingly, HFPO-DA demonstrated the most substantial impact across all endpoints tested, indicating broad and significant biochemical and neurotoxic effects. Our findings underscore the potential risks of both legacy and emerging PFAS to soil ecosystems, emphasising the need for further research to understand the long-term consequences of PFAS contamination.

Keywords: acetylcholinesterase; catalase; immune response; phenol oxidase; poly and perfluoroalkyl substances; superoxide dismutase.

Figure 1

CAT (catalase) activity. Data are presented as the mean activity per mg of protein ± standard error of the mean (SEM). Statistical significance was determined by the Kruskal–Wallis test followed by a post hoc Dunn’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not statistically significant. The x-axis reports PFAS concentrations in μM, and Ctr represents vehicle-exposed earthworms. Lines above the histogram bars indicate statistical significance between PFAS-treated groups and the control. Each panel represents results for a different PFAS compound: (a) HFPO-DA, (b) PFMOBA, (c) PFMOPrA, and (d) PFOA.

Figure 2

SOD (superoxide dismutase) activity. Statistical significance was determined by ANOVA followed by a post hoc Holm–Sidak’s multiple comparison test. * p < 0.05; ** p < 0.01; and *** p < 0.001; ns, not statistically significant. The line above the histogram bars indicates statistical significance between PFAS-treated groups and the control. Each panel represents results for a different PFAS compound: (a) HFPO-DA, (b) PFMOBA, (c) PFMOPrA, and (d) PFOA. See caption to Figure 1 for more details.

Figure 3

Phenol oxidase. A semi-quantitative evaluation was performed following the absorption (abs) at 590 nm due to L-DOPA oxidation. Statistical significance was determined by the Kruskal–Wallis test followed by a post hoc Dunn’s multiple comparison test. ** p < 0.01; *** p < 0.001; and **** p < 0.0001; ns, not statistically significant. The line above the histogram bars indicates statistical significance between PFAS-treated groups and the control. Each panel represents results for a different PFAS compound: (a) HFPO-DA, (b) PFMOBA, (c) PFMOPrA, and (d) PFOA. See caption to Figure 1 for more details.

Figure 4

Acetylcholinesterase activity. Statistical significance was determined by the Brown–Forsythe ANOVA test, followed by a post hoc Dunnett’s T3 test. * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001; ns, not statistically significant. The line above the histogram bars indicates statistical significance between PFAS-treated groups and the control. Each panel represents results for a different PFAS compound: (a) HFPO-DA, (b) PFMOBA, (c) PFMOPrA, and (d) PFOA. See caption to Figure 1 for more details.

Figure 5

Escape test. Statistical significance was determined by the Kruskal–Wallis test, followed by a post hoc uncorrected Dunn’s test. * p < 0.05; ns, not statistically significant. The line above the histogram bars indicates statistical significance between PFAS-treated groups and the control. Each panel represents results for a different PFAS compound: (a) HFPO-DA, (b) PFMOBA, (c) PFMOPrA, and (d) PFOA. See caption to Figure 1 for more details.

Figure 6

Histogram of latency times obtained from the escape test. The binned counts show the deviations of PFAS specimens from the symmetrical distribution of the control group. A biphasic response is observed, with a general increase in latency times for PFAS treatment. However, PFOA showed quicker median performance as judged by the U-statistics results (see body text).