Single and joint exposure to nanoplastics and bisphenols: a comparative assessment of in vitro hazards

Bisphenol A (BPA) has been a widely used additive in the plastic industry and is now restricted by the European Union due to its proved toxicity, being replaced by analogues such as bisphenol E (BPE) and bisphenol Z (BPZ). These analogues are considered less environmentally harmful surrogates while keeping a similar functionality. Many of them are already being marketed, and its release to the environment has been reported, which raises ecological concerns. To date, the available toxicity data of BPA analogues is scarce, thus, being needed to better understand their potential hazards to the biota to validate if they constitute environmentally safer alternatives. At the same time, another environmental problem with plastic pollution raises concerns: this is related with the presence of nanoplastics in the different environmental compartments and its potential hazardousness to the biota. Poly(lactic) acid (PLA) is a bioplastic widely used to replace fossil-based plastics and is already being commercialised. Its biobased origin contributed to its empirical characterization as being safer for the environment, although, a limited scientific knowledge exists regarding its toxicity to the biota, namely as it regards its nano particles. In addition to its intrinsic toxicity to biota, nanoplastics may serve as vectors for chemicals, acting as scavengers of those chemicals or promoting its incorporation by biota. In this context, the present study aimed to make a comparative assessment of the cytotoxicity of BPA, BPE and BPZ alone and in combination with PLA nanoplastics (PLA-NPs) to two amphibian cell lines (A6 and XTC-2 - cell lines of Xenopus laevis). This biological model was selected because: (i) Amphibia is the class of vertebrates with the highest proportion of species threatened of extinction, with one of the main causes for such a scenario being chemical contamination, and (ii) in vitro assays may constitute adequate non-animal methodologies for preliminary hazard assessments of xenobiotics. To achieve the main goal of the present work, the two amphibian cell lines were exposed for 24 h, 48 h, and 72 h, to eight concentrations of each bisphenol (for single exposure assays) or to a mixture of the LC₅₀‚₇₂ₕ‚ ½ x LC₅₀‚₇₂ₕ‚ 2 x LC₅₀‚₇₂ₕ‚ of each bisphenol plus one of two concentrations of PLA-NPs (10 or 100 μg/L; in the mixture exposure assays). Cell viability was assessed through each time point, by performing the MTT (XTC-2 and A6) and resazurin (XTC-2) assays. Overall, the median lethal concentrations (LC₅₀) revealed that A6 cells are more sensitive to the three tested bisphenols than XTC-2. The obtained data support the premise that BPE and BPZ are less toxic to amphibian cell lines than BPA. Thus, based on the 72h-LC₅₀, the cytotoxicity can be ranked as BPA>BPZ>BPE (45.48 mg/L; 57.1 mg/L and 64.7 mg/L, respectively) for XTC-2 cells. A similar trend was observed for A6 cells (24.6 mg/L; 32.1 mg/L; 41.6 mg/L for A6, for BPA, BPZ and BPE, respectively). The combined exposure of the cell lines to each of the three bisphenols and PLA-NPs revealed no interaction between the xenobiotics, as the toxicity caused by the bisphenols alone or in the presence of PLA-NPs showed no significant differences. In conclusion, the obtained results support that the two studied BPA analogues appear to be less toxic than BPA and PLA-NPs exert no interactions on the toxicity of the tested bisphenols. However, more studies must be performed to confirm these results, namely by performing in vivo assays, to confirm the possibility of extrapolation of these data to the individual level, and by assessing other endpoints in order to fully understand the mod of action of these xenobiotics.