Mixtures of legacy and replacement perfluorosulphonic acids (PFSAs) demonstrate ratio-, concentration- and endpoint-dependent synergistic interactions in vitro.

ABSTRACT

The extensive use of poly- and per-fluoroalkyl substances (PFASs) has les to their widespread presence in the environment, raising concerns about potential toxicity. While certain PFASs of concern have been phased-out or banned, new PFASs continue to be produced. Two such substances are perfluoroethylcyclohexane sulphonate (PFECHS) and perfluorobutane sulphamide (FBSA), replacements of perfluoroctanesulphonic acid (PFOS) that have recently been detected in multiple environmental media around the globe. Despite PFASs generally occurring in the environment as mixtures, few data are available outlining the effects of PFAS mixtures. Therefore, this research investigated the interaction potential of binary and ternary mixtures of emerging and legacy PFASs. The immortalized rainbow trout gill cell line (RTgill-W1) was chosen as the experimental model to investigate two apical endpoints cytotoxicity and phospholipidosis. RTgill-W1 cells were exposed for 24 h to each compound to obtain endpoint-specific effect concentrations (LCx; ECx). These values were then applied to formulate mixture predictions following the Loewes Additivity and Steel and Peckham methods. Based on cytotoxicity, relative potencies of individual compounds were PFOS > PFECHS > FSBA. PFOS and PFECHS had nearly identical effects on phospholipidosis, while FSBA did not have any effects. Most mixtures had a synergistic effect on cytotoxicity, but the effect was both dose- and ratio-dependent. PFOS and PFECHS were additive at lower concentrations (LC10) and synergistic at higher concentrations (LC50; 31, 11, and 13). PFECHS and FSBA mixtures were synergistic at all doses and ratios (31, 11, 13), while FBSA and PFOS were mainly synergistic at higher concentrations and at ratios favouring PFOS (11, 13). Tertiary combinations were mainly synergistic. For phospholipidosis, mixtures were strictly additive. These results are strongly suggestive of synergism between emerging PFAS replacements and highlight that independent apical mechanisms of different PFASs could combine to induce unexpected toxicity. Considering that emerging replacements are continuing to increase in concentration in the environment, such mixture scenarios are also likely to continue to increase in probability.