Environmental fate Aggregation Nanoecotoxicology Aquatic invertebrates Mytilus edulis Haemocytes Lysosomal biomarker

............................................................................................................................................. iii Acknowledgements ......................................................................................................................... v List of Abbreviations ..................................................................................................................... ix 1. Introduction .............................................................................................................................. 1 1.1 Nanotechnology .............................................................................................................................. 1 1.2 Nanotoxicology ................................................................................................................................ 5 1.3 Objectives ........................................................................................................................................ 10 1.4 Outline .............................................................................................................................................. 11 2. Background ............................................................................................................................. 12 2.1 The blue mussel ............................................................................................................................ 12 2.1.1 Feeding and digestion .......................................................................................................................... 13 2.1.2 Circulation ................................................................................................................................................. 14 2.1.3 Defence mechanism .............................................................................................................................. 14 2.2 Titanium dioxide nanoparticles .............................................................................................. 15 2.3 Fate of nanoparticles in the aquatic environment ............................................................ 16 2.3.1 Nanoparticles entering the aquatic system ................................................................................ 16 2.3.2 Persistence ................................................................................................................................................ 17 2.3.3 Behaviour in the water – Aggregation .......................................................................................... 17 2.4 Interactions between nanoparticles and aquatic invertebrates .................................. 23 2.4.1 Uptake across the respiratory epithelium ................................................................................... 23 2.4.2 Ingestion and subsequent cellular uptake .................................................................................. 24 2.4.3 Bioaccumulation ..................................................................................................................................... 27 2.4.4 Transfer along food chain ................................................................................................................... 28 2.5 Biological effects ........................................................................................................................... 29 2.5.1 Lethal responses of nano-­‐TiO2 ......................................................................................................... 30 2.5.2 Biochemical effects of nanoparticles ............................................................................................. 30 2.5.3 Physiologic effects of nanoparticles ............................................................................................... 34 2.5.4 Interactive effects ................................................................................................................................... 35 3. Materials and methods ....................................................................................................... 36 3.1 Behaviour of nanoparticles in seawater .............................................................................. 36 3.2 Transmission electron microscopy ........................................................................................ 36 3.3 Concentration of nano-­‐TiO2 in seawater .............................................................................. 37 3.4 Acute toxicity test ......................................................................................................................... 39 3.5 Chronic toxicity test ..................................................................................................................... 39 3.6 Haemolymph sampling ............................................................................................................... 40 3.7 Neutral red retention time ........................................................................................................ 40 3.8 Collection of histology samples ............................................................................................... 41 3.9 Data management and statistical analysis .......................................................................... 42 3.9.1 Turbidity and concentration ............................................................................................................. 42 3.9.2 Neutral Red Retention Time .............................................................................................................. 42 Nanostructured titanium dioxide: Fate in the aquatic environment and effects on the blue mussel Mytilus edulis Master’s thesis in Biological Chemistry viii 4. Results ...................................................................................................................................... 43 4.1 Solubility of nano-­‐TiO2 in water .............................................................................................. 43 4.2 Transmission electron microscopy ........................................................................................ 45 4.3 Determination of nano-­‐TiO2 concentrations in seawater .............................................. 46 4.4 Short-­‐term exposure ................................................................................................................... 47 4.5 Long-­‐term exposure .................................................................................................................... 48 4.6 Variations in exposure concentrations ................................................................................. 49 5. Discussion ............................................................................................................................... 52 5.1 Future work .................................................................................................................................... 59 6. Conclusion ............................................................................................................................... 61 References ....................................................................................................................................... 62 List of figures .................................................................................................................................. 71 List of tables ................................................................................................................................... 72 Appendices ..................................................................................................................................... 73 Appendix 1. Turbidity measurements of nano-­‐TiO2 samples .................................................. 73 Appendix 2. Data on test organisms ................................................................................................. 74 Nanostructured titanium dioxide: Fate in the aquatic environment and effects on the blue mussel Mytilus edulis Master’s thesis in Biological Chemistry ix List of Abbreviations CNT Carbon nanotube EC50 Effect concentration, 50% ERA Environmental risk assessment GST Glutathione S-transferase ISO International Organization for Standardization Kow Octanol-water partition coefficient LC50 Lethal concentration, 50% LMS Lysosomal membrane stability LOEC Lowest observable effect concentration LPO Lipid peroxidation Nano-SiO2 Silicon dioxide nanoparticles Nano-TiO2 Titanium dioxide nanoparticles NOEC No observable effect concentration NTU Nephelometric turbidity units PAH Polycyclic aromatic hydrocarbons PEC Predicted environmental concentration PNEC Predicted no effect concentration ROS Reactive oxygen species SOP Standard operating procedure TEM Transmission electron microscopy Nanostructured titanium dioxide: Fate in the aquatic environment and effects on the blue mussel Mytilus edulis Master’s thesis in Biological Chemistry x Nanostructured titanium dioxide: Fate in the aquatic environment and effects on the blue mussel Mytilus edulis Master’s thesis in Biological Chemistry 1