Toxicity evaluation of single and combined exposures to polyhydroxybutyrate nanoparticles and caffeine using Xenopus laevis in vivo and in vitro models

Pollution is regarded as a relevant driver of both Human and Environmental Health. Plastic contaminants have been a target of extensive risk assessment research, mainly because of their persistence and fragmentation to nano sizes in the environment, potentiating its uptake to biota, including humans. Polyhydroxybutyrate (PHB) is a bio-based and biodegradable polyester, praised in the medical field due to its biocompatibility and non-toxicity to humans. Notwithstanding, little is known regarding its toxicity when used as nanoparticles (PHB-NPs). In addition to its individual exposure threat, NPs may serve as vectors for chemicals, promoting its incorporation by Humans (e.g. inhalation of NPs loaded with other chemicals) and other biota. Caffeine is the world's most widely consumed psychoactive drug and a relevant representative of pharmaceutically active pollutants. On mammals, caffeine can induce teratogenic and embryotoxic effects. This work aimed to assess the lethal and sublethal toxicity of these two xenobiotics, in single and mixed exposures, by using in vivo (embryos and tadpoles of Xenopus laevis) and in vitro (two cell lines of X. laevis) biological models. Caffeine was toxic to both life stages of X. laevis. Embryos were more sensitive than tadpoles with LC₅₀ of 196 and 226 mg/L, and EC_{50, malformations} of 124 and 241 mg/L, respectively. PHB-NPs showed no effect when exposed alone and in mixture. Cytotoxicity assays revealed LC_50s of 864 (XTC-2), 587 (A6) and 131 (XTC-2) mg/L after caffeine and PHB-NPs exposure, respectively. In co-exposure, the concentration of PHB-NPs was positively correlated with the toxic effect of the mixture.