HFPO-DA and PFOA exert differential combined toxicity with Cd in Paramecium: growth, behavior and molecular insights

Hexafluoropropylene oxide dimer acid (HFPO-DA) has come into use as an alternative to perfluorooctanoic acid (PFOA), but it remains elusive whether their comparative toxicities in aquatic organisms under co-occurring heavy metals like cadmium (Cd), particularly one ecologically important microorganism, the protists. Here, we compared the combined toxicity of PFOA and HFPO-DA in euryhaline ciliate Paramecium duboscqui under Cd-contaminated conditions. Chronic exposure (14 days) to field-realistic concentrations of Cd (50 μg/L) and PFOA/HFPO-DA (1 μg/L) revealed similarly adverse effects on growth performance. When combined with Cd, HFPO-DA caused greater disruption to membrane permeability thus enhancing Cd availability in ciliates, and amplifying oxidative stress and apoptosis activation. Moreover, combining HFPO-DA and Cd significantly reduced the motility distance, mean and max velocity in ciliates compared to PFOA. Transcriptomic analysis consequently revealed that disruptions in the core energy metabolism pathways were likely responsible for the insufficient supply of ATP energy in P. duboscqui . Additionally, the simultaneous downregulation of ciliary motility-related gene set and pathway, with the most pronounced suppression observed under HFPO-DA and Cd co-exposure mechanistically linked transcriptional changes to behavioral consequences. Our findings suggest that the combined effects of PFOA/HFPO-DA and Cd pose compounded ecological risks, underscoring the need to consider PFOA and its alternative HFPO-DA in regulatory frameworks along with heavy metals. • Chronic co-exposure had synergistic toxicity on growth and morphology of ciliates. • The addition of HFPO-DA enhanced the Cd availability in P. duboscqui . • HFPO-DA posed greater oxidative stress and damage to energy and motility processes. • Growth inhibition and hypoactive behavior were the combined effects of HFPO-DA and Cd.