Neurobehavioral and Mechanistic Sub-lethal Studies in Aquatic Toxicology on Potential Micro-pollutants. A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Milanga Wasana Walpitagama B.Sc. – Bangalore University, India. School of Science College of Science, Engineering and Health RMIT University September 2019 i Declaration I certify that except where due acknowledgement has been made, the work is that of the author alone; the work has not been submitted previously, in whole or in part, to qualify for any other academic award; the content of the thesis is the result of work which has been carried out since the official commencement date of the approved research program; any editorial work, paid or unpaid, carried out by a third party is acknowledged; and, ethics procedures and guidelines have been followed. I acknowledge the support I have received for my research through the provision of an Australian Government Research Training Program Scholarship for PhD studies. Milanga Wasana Walpitagama September 2019. ii Acknowledgements I have been able to present this thesis and my research work with the help and support of many individuals, all of whom had my best interests at heart. I would like to offer my sincere gratitude to all those who helped me in concluding this research successfully as well as presenting it on time. First of all, I wish to thank my senior supervisor, Associate Professor Donald Wlodkowic, for accepting me as his research student and providing me with this opportunity. Thank you very much for guiding me through the difficulties I faced during my research and for the direct and honest constructive criticism, which made me a better student and researcher. Furthermore, I would like to thank Dr. Jan Kaslin, my associate supervisor from Monash University for all the assistance he provided, especially with allowing me to access Monash University’s fish facility and providing me with guidance and knowledge whenever necessary. I would like to express my gratitude to my wife, Nadeesha Gunarathna, for her continued moral and financial support, which made it possible for me to continue my research work undisturbed. Thank you for being at my side during this whole endeavour and for being my strength when I needed it the most. Moreover, I would like to thank my parents, family, and friends for their ongoing moral support in all my life’s achievements. In addition, I wish to thank all the academic staff at RMIT University Bundoora (Building 223), including Dr. Anna Walduck, Professor Andy Ball, Associate Professor Jeff Shimeta, and Professor Dayanthi Nugegoda. Thank you all for the support and guidance you have offered me over the last 4 years. I am also thankful to the following RMIT academic support staff, Dr. Lisa Dias, Leanne Bickford, Emma Stockham, and Shannon Fernandes for all the help they provided whenever I required it. Huge thanks go to the following technical staff from Monash University FishCore facility, Rodney Glanvill, Neal Ashcroft, and research assistant Alon Douek, for their continuous support throughout my research. Last but not least I would like to express my appreciation for all the support and friendship I received from my colleagues from the Phenomics lab and ecotoxicology research group: Rhys, Feng, Yushi, Yutao, and Jason. Thank you for all the help during the last 4 years. Finally, a special thanks go to Charlene, Ana, and Abhi for all their endless support and friendship. iii Table of Contents DECLARATION ...................................................................................................................... II ACKNOWLEDGEMENTS ..................................................................................................... III TABLE OF CONTENTS ......................................................................................................... IV LIST OF FIGURES .................................................................................................................. X LIST OF TABLES ............................................................................................................. XVIII NOMENCLATURE ............................................................................................................. XIX ABSTRACT ........................................................................................................................ XXII PUBLICATIONS ............................................................................................................... XXIV CHAPTER 1. INTRODUCTION ........................................................................................... 1 1.1. Introduction to global plastic production and consumption ................................................................ 1 1.2. Environmental impact of plastic waste in the aquasphere ................................................................... 4 1.2.1. Mechanical impact of plastics on marine wildlife ............................................................................ 5 1.2.2. Sorption of toxicants to plastics ........................................................................................................ 6 1.2.3. Effects of microplastics as environmental pollutants ........................................................................ 8 1.3. Toxicity of plastic additives .................................................................................................................. 11 1.3.1. Toxicity of plastic additives to aquatic biota .................................................................................. 14 1.3.1.1. Induction of oxidative stress ....................................................................................................... 14 1.3.1.2. Endocrine-disrupting effects ...................................................................................................... 16 1.3.1.3. Developmental toxicity .............................................................................................................. 18 1.3.1.4. Neurotoxicity and behaviour alterations ..................................................................................... 20 1.4. Three-dimensional (3D) printed plastics as emerging environmental hazards ................................ 21 1.4.1. Methods of plastic 3D printing ....................................................................................................... 23 1.4.1.1. Fused deposition modelling (FDM) ........................................................................................... 24 1.4.1.2. Stereolithography (SLA) ............................................................................................................ 25 1.4.1.3. Digital micro-mirror device-based projection printing (DMD–PP) ........................................... 27 1.4.1.4. Multi-jet modelling (MJM) ........................................................................................................ 28 1.4.1.5. Selective laser sintering (SLS) ................................................................................................... 29 1.4.2. Unexplored environmental impacts of 3D-printed polymers .......................................................... 30 1.4.2.1. Toxicity of 3D-printed plastics ................................................................................................... 31 1.5. Mechanisms of environmental toxicant action .................................................................................... 34 1.5.1. Sublethal effects of toxicants .......................................................................................................... 35 1.5.2. DNA and RNA damage .................................................................................................................. 37 1.5.3. Oxidative stress ............................................................................................................................... 39 1.5.4. Mitochondrial toxicity .................................................................................................................... 41 iv 1.5.5. Caspase-dependent apoptosis.......................................................................................................... 43 1.5.5.1. A mechanistic link between oxidative stress and programmed cell death .................................. 46 1.5.6. Alternative modes of cell death ...................................................................................................... 47 1.5.7. Zebrafish as an in vivo model for analysis of programmed cell death ............................................ 48 1.5.7.1. Transgenic zebrafish models ...................................................................................................... 49 1.5.7.2. Small molecule fluorescent and luminance probes..................................................................... 50 1.6. Neurotoxicity and developmental neurotoxicity of industrial chemicals .......................................... 51 1.6.1. Neurotoxicity of 3D printing plastic additives ................................................................................ 54 1.6.2. Zebrafish as a proxy model for neurotoxicity testing ..................................................................... 55 1.6.2.1. Development of zebrafish’ central nervous system (CNS)......................................................... 56 1.6.2.2. Biological markers of neurotoxicity ..........................................................................................