Plastic products, such as bisphenol A (BPA), are widely released into the environment, causing adverse effects in aquatic organisms. In response to regulatory restrictions on BPA, structurally similar bisphenol analogues have been introduced as alternatives. However, these compounds often lack comprehensive toxicological evaluation and may pose comparable or even greater environmental risks.

This study investigated the toxicity of BPA, two BPA analogues (BPAP and BPAF), and one BPA monomer derivative (BADGE·2HCl) using zebrafish liver (ZFL) cell spheroids. ZFL spheroids were exposed to 5 µM for acute exposure (24 hours) and 1–5 µM for chronic exposure (6 days). Afterwards, metabolic activity, changes in gene expression and alterations in the lipidome were evaluated.

For acute exposure, upregulation of cyp1a expression was detected after exposure to BADGE·2HCl, suggesting activation of xenobiotic metabolism pathways. For chronic exposure, ATP levels decreased at 5 µM for BPAP, BPAF, and BADGE·2HCl. Notably, chronic exposure to BADGE·2HCl (1–5 µM) and BPA (5 µM) caused significant alterations in membrane lipid composition, predominantly affecting phosphatidylcholines, ether-linked phosphatidylcholines, and phosphatidylethanolamines, lipids essential for maintaining membrane integrity. Additionally, exposure to 5 µM BADGE·2HCl led to a substantial upregulation of ceramides and sphingomyelins, which are associated with mitochondrial dysfunction and apoptotic pathways.

In conclusion, BADGE·2HCl elicited the most pronounced alterations in lipid metabolism, followed by BPA. The results highlight the need for more rigorous testing and stricter regulatory measures to mitigate the potential environmental and health risks posed by these compounds. Moreover, this study highlights the utility of ZFL spheroids as a robust in vitro model for evaluating the chronic toxicity of environmental contaminants, offering a physiologically relevant alternative to traditional monolayer cultures.