Development of Water Cluster-Secondary Ion Mass Spectrometry and Particle Induced X-ray Emission to Investigate Spatially Resolved Biological Responses to Nanopolystyrene in Zebrafish Larvae

Nanoplastics (NPs) are increasingly recognised as an emerging class of environmental contaminants due to their persistence, mobility, and evidence of – although in limited capacity - ability to cross biological barriers. Their nanoscale size and high surface reactivity enable interactions with cells and tissues in ways distinct from larger microplastics, raising concerns for both ecosystem and human health. Although their occurrence has been documented across aquatic systems, most experimental studies have relied on unrealistically high exposure concentrations and bulk analytical methods that obscure tissue-specific effects. This has left critical knowledge gaps in our understanding of NP uptake at environmentally realistic levels and their capacity to disrupt organ-level elemental and molecular homeostasis. This thesis addresses these gaps by developing and applying a multimodal analytical framework to investigate NP interactions in zebrafish (Danio rerio) embryos, a well established vertebrate model in developmental toxicology. Key methodological advances included cryosectioning protocols that preserved tissue integrity and analyte localisation, as well as embedding and substrate strategies compatible with both Induced X-ray Emission (PIXE) and Secondary Ion Mass Spectrometry (SIMS) imaging. PIXE enabled quantitative, spatially resolved mapping of biologically relevant elements such as calcium (Ca), potassium (K), phosphorus (P), sulphur (S), zinc (Zn), and iron (Fe), while Gas Cluster Ion Beam SIMS (GCIB-SIMS) provided sub-cellular molecular imaging of lipids and metabolites. These workflows were complemented by radiolabelling with [14C]-NPs, offering independent uptake quantification and validating imaging data. Collectively, these approaches demonstrate that correlative elemental and molecular imaging is feasible, reproducible, and highly informative in small vertebrate models. The results revealed several important findings. PIXE analysis identified localised calcium accumulation in the heart and potassium perturbations in NP-exposed and PFOS co-exposed embryos, suggesting that NPs can translocate into embryos or at least interfere with biological processes significantly. This also suggests that cardiac ion regulation can be a sensitive target of NP exposure. GCIB-SIMS imaging uncovered reproducible lipidomic alterations, including downregulation of sphingomyelins and iv phosphatidylcholines in brain regions. Strikingly, these molecular effects were detected at exposure levels in the low parts-per-trillion range, underscoring the sensitivity of SIMS imaging and revealing the potential for subtle yet biologically meaningful impacts at environmentally relevant doses. Radiolabelling experiments confirmed size-dependent uptake, with 50 nm particles showing significantly greater internalisation than 250 nm particles, and uptake dynamics aligning with the elemental and molecular disruptions observed by imaging. Overall, this work shows that exposure to nanoplastics in developing zebrafish embryos is associated with measurable disruptions in elemental and molecular regulation at environmentally relevant concentrations. These findings demonstrate that such exposures can induce subtle, spatially resolved changes in biological chemistry during early development. However, the downstream consequences of this dysregulation for embryo development, physiology, or long-term survival remain unknown and were not assessed in the present study. Beyond these biological observations, this thesis establishes robust and adaptable workflows for multimodal, spatially resolved imaging that enable the combined investigation of molecular and elemental distributions within the same organism. These methodological advances provide new tools for probing tissue-specific responses in zebrafish and other model systems, and have the potential to support future studies aimed at elucidating toxicological mechanisms, developmental outcomes, and, ultimately, informing environmental risk assessment. Importantly, this work highlights the value of spatially resolved analytical approaches for detecting subtle exposure related changes that may not be evident using bulk analytical methods.