Developmental and Neurobehavioural Effects of Bisphenol S in Zebrafish (Danio rerio)

Bisphenol S (BPS) has emerged as a widespread environmental contaminant and a widely used structural substitute for bisphenol A. Despite being marketed as a safer alternative, considerable evidence indicates that BPS exhibits endocrine-disrupting and neurotoxic effects in aquatic organisms and in some cases these effects persist across generations. This thesis investigates the developmental and neurobehavioural toxicity of BPS in zebrafish (Danio rerio) across three interconnected studies spanning embryonic, adult, and intergenerational life stages, with the aim of elucidating the molecular and biochemical mechanisms through which environmentally relevant BPS exposures disrupt neural development and function. In the first study (Chapter 2), zebrafish embryos were exposed to an environmentally relevant concentration of BPS (30 μg/L) from 4 to 120 hours post-fertilization (hpf). BPS exposure significantly elevated reactive oxygen species (ROS) production, lipid peroxidation, and apoptotic cell death in larvae at 120 hpf. Behavioural assessment revealed impaired spontaneous tail coiling at 20 hpf, reduced thigmotaxis, and impaired locomotor activity during the light phase of the light-dark preference test. Biochemical analysis demonstrated elevated whole-body serotonin (5-HT) and acetylcholine concentrations. Transcript abundance (mRNA level) analysis identified widespread dysregulation of serotonergic, cholinergic, and dopaminergic pathway genes, alongside upregulation of neuroinflammatory cytokines (il-1β, il-6) and the apoptotic executor caspase-3, indicating that BPS induces oxidative stress, neuroinflammation, and apoptosis that collectively disrupt neurotransmitter signalling and larval behaviour. The second study (Chapter 3) investigated the long-term consequences of the same embryonic BPS exposure in adult zebrafish assessed at six months of age. While anxiety-like behaviour and locomotion were not significantly altered, adult fish displayed a selective impairment in social affiliation, spending significantly less time associating with conspecific shoals. Persistent brain lipid peroxidation and upregulation of the antioxidant gene gpx1a confirmed chronic oxidative stress. Adult brains also exhibited significantly reduced dopamine concentrations, downregulation of neurotrophic factors (bdnf, manf), continued dysregulation of serotonergic and cholinergic gene expression, and upregulation of the neuroinflammatory mediators il-1β and tnf-α, indicating a chronic subclinical neuroinflammatory state that persisted long after exposure had ended. The third study (Chapter 4) examined the intergenerational consequences of embryonic BPS exposure by generating maternal, paternal, and parental F₁ lineages from F₀ fish exposed during embryogenesis. F₁ larvae reared in clean water exhibited significantly elevated ROS, lipid peroxidation, and apoptosis across all three lineages relative to controls. Serotonergic and cholinergic transcript abundance was broadly dysregulated in F₁ offspring, and neurotransmitter concentrations were selectively elevated in the parental and maternal lineages. These molecular and biochemical alterations were passed on through both maternal and paternal lineages, suggesting that epigenetic mechanisms may play a role in their transmission. However, despite these underlying changes, no obvious neurobehavioural abnormalities were observed in the larvae. The findings of this thesis demonstrate that exposure to environmentally relevant concentration of BPS can adversely affect the zebrafish nervous system across multiple life stages. Although the specific outcomes varied with developmental stage, oxidative stress, neuroinflammation, apoptosis, and disruptions in neurotransmitter systems consistently emerged as key underlying mechanisms. Importantly, some effects persisted into adulthood and were even transmitted to unexposed offspring, raising concerns about the long-term and intergenerational consequences of BPS exposure. These findings challenge the perception of BPS as a safe alternative to BPA and emphasize the need for regulatory frameworks that consider long-term and multigenerational neurotoxicity when assessing the environmental risks of bisphenol compounds.