Toxicity of individual and binary mixtures of perfluoroalkyl compounds in freshwater algae Raphidocelis subcapitata

Introduction: Per- and polyfluoroalkyl substances (PFAS) are environmental pollutants of increasing scientific interest. Widely used for several commercial and industrial applications, they are potentially harmful to the environment and biota. Following the ban of legacy PFAS such as perfluorooctanoic sulfonic acid (PFOS), several next-generation substitutes have been developed and introduced in the industry (1). Our study investigated the effects of one PFAS monitored by EFSA and two next-generation substitute compounds, either individually or in binary mixtures, in the unicellular algae model of ecotoxicology Raphidocelis subcapitata. Materials and Methods: Algal toxicity tests for perfluorononanoic acid (PFNA), PFOS, perfluorobutanesulfonic acid (PFBS) and perfluorobutanoic acid (PFBA) were executed using the US-EPA 96h Acute Toxicity Test (2), with minor modifications. Each compound was dissolved in Bold’s Basal Medium (1.0 g/L) and serial dilutions were made using a dilution factor of 2.0. Binary mixtures were set up according to the individually obtained concentration inhibiting the algal growth by 50% (EC 50 ), with the highest concentration represented by the sum of each EC 50 and using a dilution factor of 2.0. Results: Data obtained (four replicates) allowed us to determine the EC 50 for each tested PFAS and the type of interaction (additive effect, synergism or antagonism) in PFAS binary mixtures. PFBA was the most toxic PFAS (EC50 30.3 mg/L), followed by PFOS (47.3 mg/L), PFNA (90.5 mg/L) and PFBS (105.8 mg/L). The EC 50 of binary mixtures were 57.0 mg/L (PFOS-PFBS), 44.5 mg/L (PFOS-PFBA), 116.6 mg/L (PFNA-PFBA), 221.5 mg/L (PFNA-PFBS) and 121.4 mg/L (PFBA-PFBS). Discussion: Overall, PFAS toxicity (e.g., trend to bioaccumulation) is directly proportional to the carboxyl chain length; moreover, it increases with the presence of the sulfonate functional group at the end of the carboxyl chain (3). Our study highlights the highest toxicity of PFBA (a short-chain perfluoroalkyl substitute) on the freshwater algae R. subcapitata. Among the binary mixtures, PFOS-PFBS and PFOS-PFBA combinations were proved additive. However, antagonism was observed in the PFNA-PFBA, the PFNA-PFBS, and the PFBA-BFBS mixtures. We hypothesize this might be due to competition for the same molecular targets. Further studies are needed to shed a light on molecular mechanisms involved in PFAS uptake and toxicity in this ecotoxicology model species.