Single versus combined lethal and sublethal effects of polyhydroxybutyrate nanoplastics and the antidepressant sertraline to Xenopus laevis: In vivo and in vitro approaches

The increasing presence of micro(nano)plastics and pharmaceuticals in aquatic environments raises concerns about their individual and combined effects on non-target organisms, particularly amphibians. Biodegradable plastics such as polyhydroxybutyrate (PHB) are often proposed as environmentally friendly alternatives, however, their individual effects and interactions with co-occurring contaminants and associated ecological risks remain poorly understood. In this context, sertraline (SER), a widely used antidepressant frequently detected in aquatic systems, was selected to evaluate its interaction with PHB nanoplastics (PHB-NPs). This study assessed the lethal and sublethal effects of PHB-NPs and SER, individually and in mixture, during 96 h exposures using early life stages of Xenopus laevis (embryos and tadpoles). Endpoints included mortality, malformations, growth (body length and mass), and heart rate. In addition, in vitro 72 h cytotoxicity assays using A6 and XTC-2 amphibian cell lines were applied as a preliminary toxicity screening tool. The PHB-NPs (0.01–100 μg L −1 ) did not induce significant effects on embryos or tadpoles. SER showed higher toxicity, with tadpoles demonstrating greater sensitivity than embryos (96 h LC 50 : 0.46 and 2.58 mg L −1 , respectively). Sublethal effects were observed across all surviving treatments, including reduced growth in both body length and mass in tadpoles and in body length in embryos. In vitro assays confirmed SER cytotoxicity, with XTC-2 cells showing sensitivity comparable to that of embryos (72 h EC 50 : 3.78 mg L −1 ). Furthermore, co-exposure assays suggested interactions between PHB-NPs and SER, warranting further investigation into the ecological implications of their combined occurrence in aquatic environments. Overall, this study highlights the importance of assessing both individual and combined effects of emerging contaminants across different life stages. Cell-based assays may serve as complementary tools for preliminary toxicity screening, supporting the 3R principles by helping reduce the need for animal testing in subsequent experimental phases. Background Biodegradable plastics such as polyhydroxybutyrate (PHB) are increasingly promoted as sustainable alternatives to conventional plastics, although their environmental hazard, particularly in nanosized form (PHB-NPs), remains poorly understood. Sertraline (SER), a widely prescribed antidepressant, is frequently detected in aquatic environments due to continuous input and incomplete removal during wastewater treatment. The co-occurrence of these contaminants raises concerns regarding potential combined effects on aquatic organisms. Objectives This study aimed to evaluate the individual and combined effects of SER and PHB-NPs on early life stages of the amphibian Xenopus laevis , and to assess the applicability of amphibian cell-based assays as a preliminary screening tool within an ecotoxicological framework. Methods Embryos (following FETAX with modifications) and tadpoles of X. laevis were exposed for 96 h to SER (0.1–5 mg L −1 ) and, individually and in mixture. Endpoints included mortality, malformations, and growth (body length and mass). In parallel, 72 h cytotoxicity assays were conducted using A6 and XTC-2 amphibian cell lines. Median lethal (LC 50 ) and effective (EC 50 ) concentrations were determined, and potential interactions under co-exposure were explored. Results PHB-NPs induced no significant effects on embryos or tadpoles. SER exerted toxic effects, with tadpoles displaying higher sensitivity than embryos (96 h LC 50 = 0.46 and 2.58 mg L −1 , respectively). Sublethal effects were observed in all surviving treatments, namely in terms of reduced growth in tadpoles (body length and mass) and in embryos (body length). In vitro assays confirmed SER cytotoxicity, with XTC-2 cells showing sensitivity comparable to embryos (72 h EC 50 = 3.78 mg L −1 ) and revealed interactions between SER and PHB-NPs under co-exposure conditions. Conclusions This study provides additional data on the effects of co-occurring nanoplastics and pharmaceuticals across amphibian life stages. While interactions between SER and PHB-NPs were observed, the underlying mechanisms remain unclear. Cell-based assays showed potential as complementary tools for preliminary toxicity screening, contributing to the reduction of animal use in early testing phases.