Characterization Of The Toxicity Of Carbon-Coated Iron Oxide Nanoparticles In Zebrafish: Neurophysiological Effects And Underlying Molecular Mechanisms

Abstract

Iron oxide nanoparticles (IONPs) are widely utilized in biomedical, industrial, and environmental applications; however, their increasing release into aquatic systems raises concerns regarding their potential neurotoxic effects on aquatic organisms. This study investigated the physiological, behavioural, molecular, and electrophysiological impacts of early life IONP exposure in zebrafish (Danio rerio) to elucidate concentration-dependent and developmental outcomes. Zebrafish embryos were exposed to sublethal concentrations of IONPs (0, 1, and 5 mg IONPs/L) from 4 hours post-fertilization to 6 days post-fertilization (dpf), and raised to 30 dpf under normal conditions, to assess potential persistent effects. Physiological analyses revealed no significant mortality or deformity, suggesting the absence of gross morphological toxicity. Nevertheless, trace metal analysis revealed elevated Fe accumulation in tissues, consistent with nanoparticle uptake and bioavailability. Behavioural assessments were consistent with increased locomotor activity and elevated thigmotaxis in 6 dpf larvae exposed to 1 mg/L IONPs, whereas larvae exposed to 5 mg/L exhibited locomotor activity comparable to controls while maintaining elevated thigmotaxis. Overall, behavioural effects appeared more pronounced at the lower exposure concentration. In juveniles (30 dpf), behavioural effects persisted in a non linear manner: 1 mg/L exposure caused hypoactivity and anxiety-like responses, while 5 mg/L exposure induced hyperactivity and reduced social preference. At the molecular level, IONP exposure altered the transcript abundance of oxidative stress-related genes (Superoxide dismutase 1, Catalase, and Glutathione S-transferase) and dopaminergic markers (Tyrosine hydroxylase)). Larvae exhibited elevated cat and gstp1.2 mRNA expression, consistent with increased oxidative stress, while juveniles showed th1 upregulation and gstp1.2 downregulation, consistent with altered dopaminergic and antioxidant defence pathways, respectively. Electrophysiological analyses using multi-electrode arrays revealed significantly increased local field potentials, burst event frequencies and higher number of electrical spikes in 5 mg/L larvae, potentially suggesting enhanced neural excitability. Collectively, these findings demonstrate that early-life exposure to environmentally relevant concentrations of IONPs may induce lasting, life stage- specific neurophysiological and behavioural alterations. This integrated assessment underscores the potential ecological and developmental risks of IONPs and emphasizes the need for long-term, multilevel evaluations in nanoparticle toxicology.