Investigations into the bioavailability of manufactured nanoparticles in fish

Abstract

The field of nanotoxicology has emerged as a discipline in parallel with the rapid expansion of nanotechnology and the use of nanomaterials in modern life. Assessing the potential impacts of manufactured nanoparticles (MNPs) on the environment and human health is critical to the sustainable development of the nano-industry. Current knowledge on the ecological implications of nanotoxicology has major uncertainties surrounding the fate and behaviour of nanomaterials in the exposure environment. Bioavailability, uptake and partitioning of nanomaterials to organisms are key determinates to toxicity, yet these foundations of basic data are only now just starting to emerge in any useful and coherent manner for aquatic animals. This thesis work set out to address this gap in knowledge and further our understanding of these important principles for fish. In an attempt to develop a high through-put screening system for toxicity of MNPs, studies assessing the utility of primary isolated rainbow trout (Oncorhynchus mykiss) hepatocytes found they showed very limited responses to a range of MNPs. There was a lack of any evidence for either lipid peroxidation or xenobiotic detoxification activity. In these studies isolated trout hepatocytes were found to be unresponsive to the induction of these biological responses after exposure to positive controls. The findings demonstrated that the MNPs tested showed low toxicity generally and that fish hepatocytes do not provide a useful system for the screening of potential toxic effects of MNPs. In this cell culture work, coherent anti-Stokes Raman scattering (CARS) microscopy was applied to demonstrate that the particles supplied in the culture medium did cross the cell membrane and enter into the exposed cells. In the second phase of the work in this thesis CARS was investigated as an experimental technique for tracing a wide range of metal and metal oxide MNPs into cells and tissues. CARS was applied to evaluate initial detection of different MNPs and investigate the imaging capability on a range of cells, tissues and organisms. Finally, CARS was applied to assess localisation ability of MNPs within biological matrices. MNPs were shown to be taken into trout hepatocytes using a 3D reconstruction to determine the origin of the MNP signal within the cell. Uptake of MNPs was established into trout gill and kidney tissue, corophium and daphnia species and were shown to have different partitioning in zebrafish embryos. In summary CARS showed great potential for tracing particle uptake and bio-distribution both in vitro and in vivo. Particular benefits include imaging MNPs in living organisms, without the need for labelling or fixing the material. Limitations of the CARS technique are also discussed. In chapter 4, the consequences of the presence of natural organic matter (NOM) were investigated on the uptake of MNPs into fish. Carp (Cyprinus carpio) were exposed to cerium dioxide (CeO2) MNPs in combination with NOM over 28 days. Elevated levels of uptake of cerium were measured in the brain, gill and kidney tissue by induction coupled plasma mass spectroscopy (ICP-MS) for fish exposed to 50 μg/l CeO2 MNPs in combination with 250 μg/l of NOM. There were no such effects of the NOM enhancing uptake for the bulk CeO2 particles. Detailed studies on the behaviour of the CeO2 MNPs in the exposure medium demonstrated the highly complex and dynamic nature of the interactions with NOM. This study discusses some of the difficulties in the techniques, analysis and interpretation of data derived from studies of this nature. The finding that NOM may enhance MNP uptake presents a potential issue for current risk assessment criteria for MNPs that do not consider natural conditions. The final experimental chapter considered maternal transfer as a potentially important route for exposure of embryos and early life stage animals to MNPs in live bearing animals. In this work guppies (Poecilia reticulata) were exposed to 7 nm silver citrate stabilised particles and citrate stabilised bulk sized particles, dosed via the diet for a full gestation cycle. Maternal transfer of Ag to the larvae was significantly higher for the nanoparticulate treatment compared with the bulk and control treatments and larval burden was significantly higher compared with the maternal sires. However, there was no impact of Ag on larval survival, birth weights, or on indices of body condition in the exposed adults. The enhanced uptake of nano Ag compared to bulk Ag particles into the guppy offspring emphasises the potential for exposure to sensitive early life stages of organisms, which to date has not been widely considered and suggests greater research is needed in this area. Collectively, the studies conducted in this thesis contribute to the science base of nanotoxicology, specifically in areas where data are especially lacking and with a focus on bioavailability. These studies have identified that fish hepatocytes do not offer an effective screen for MNPs, and the data produced further suggests that the MNPs tested are not toxic in that form. Working with CARS I have helped advance the understanding on its utility for nanotoxicology studies, with regards to its application and limitation for uptake analyses. The study of MNPs in combination with NOM has identified the fundamental change that real life exposure scenarios may instigate for toxicity assessments of MNPs, with significant impact on risk assessment criteria. Finally, I’ve established that maternal transfer is an exposure route for MNPs that requires further study, with evidence of transfer to sensitive life stages in a non-mammalian system.DEFR