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Ecotoxicity of Nanomaterials in Relation to the Freshwater Blackworm, Lumbriculus Variegatus Simon Robert Little Submitted Ecotoxicity of nanomaterials in relation to the freshwater blackworm, Lumbriculus variegatus Simon Robert Little Submitted for the degree of Doctor of Philosophy Heriot-Watt University School of Energy, Geoscience, Infrastructure and Society July 2017 The copyright in this thesis is owned by the author. Any quotation from the thesis or use of any of the information contained in it must acknowledge this thesis as the source of the quotation or information. i Abstract Nanotechnologies are increasing being applied within consumer products, science, medicine, engineering and industrial processes. Due to their small size and high reactivity, nanoparticles (NPs) possess a potentially unique risk towards humans and the environment. Although the aquatic environment is predicted to be the final recipient of nano-wastes, little is known regarding the potential toxicity of NPs towards aquatic species. Within this thesis, the toxicity of two reference nanoparticles: silver (NM-300K) and titanium dioxide (NM-104) were investigated in relation to the freshwater oligochaete, Lumbriculus variegatus in artificial water and sediment. Greater toxicity was observed for NM-300K towards L. variegatus in both short-term (96-hour) water and long-term (28-days) sediment exposures. Sub-lethal NM-300K aquatic exposures (0.2 mg/L) led to an inhibition of L. variegatus swimming behaviours designed for the escape of predation as well as increases in the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT). Increases in SOD and CAT indicate the onset of oxidative stress, however, no oxidative damage (as measured by lipid peroxidation) was observed. The inclusion of Suwannee River humic acid (5mg/L) as a natural organic matter (NOM) source generally reduced NM-300K toxicity in aquatic exposures – improving swimming behaviours and limiting SOD and CAT activities to those recorded in control exposures. Although NM-300K displayed greater toxicity than NM-104 in sediment exposures, 100% L. variegatus mortality was only observed at extremely high, potentially environmentally irrelevant concentrations (1333.33 mg/kg). The results of this thesis demonstrate the importance of water and sediment NP toxicity testing in relation to L. variegatus using traditional and newly emerging endpoints and variations to abiotic conditions. ii Acknowledgements I would like to thank my supervisors, Prof. Teresa Fernandes and Dr. Helinor Johnston for providing me with the opportunity to conduct this PhD and for their continued encouragement, advice and support throughout. I would also like to thank Prof. James Ranville for the opportunity to work in his lab at the Colorado Schhol of Mines and for his generosity, hospitality and help during my stay. Finally, I would like to thank Miriam, my parents and sisters who have all made this possible through their patience, love and support. iii ACADEMIC REGISTRY Research Thesis Submission Name: Simon Robert Little School: School of Energy, Geoscience, Infrastructure and Society Version: (i.e. First, Final Degree Sought: PhD Resubmission, Final) Declaration In accordance with the appropriate regulations I hereby submit my thesis and I declare that: 1) the thesis embodies the results of my own work and has been composed by myself 2) where appropriate, I have made acknowledgement of the work of others and have made reference to work carried out in collaboration with other persons 3) the thesis is the correct version of the thesis for submission and is the same version as any electronic versions submitted*. 4) my thesis for the award referred to, deposited in the Heriot-Watt University Library, should be made available for loan or photocopying and be available via the Institutional Repository, subject to such conditions as the Librarian may require 5) I understand that as a student of the University I am required to abide by the Regulations of the University and to conform to its discipline. 6) I confirm that the thesis has been verified against plagiarism via an approved plagiarism detection application e.g. Turnitin. * Please note that it is the responsibility of the candidate to ensure that the correct version of the thesis is submitted. Signature of Date: Candidate: Submission Submitted By (name in capitals): SIMON ROBERT LITTLE Signature of Individual Submitting: Date Submitted: For Completion in the Student Service Centre (SSC) Received in the SSC by (name in capitals): Method of Submission (Handed in to SSC; posted through internal/external mail): E-thesis Submitted (mandatory for final theses) Signature: Date: i Table of contents Chapter 1: Introduction ............................................................................................................. 1 1.1 Nanomaterial and nanoparticle definitions ............................................................. 1 1.2 Nanotechnology ..................................................................................................... 2 1.3 Nanoparticles in the environment ........................................................................... 2 1.4 Silver....................................................................................................................... 4 1.4.1 Occurrence and applications of silver ......................................................... 4 1.4.2 Silver health effects.................................................................................... 5 1.4.3 Silver in the environment ........................................................................... 6 1.4.4 Application of AgNPs .................................................................................. 6 1.4.5 AgNPs in the environment ......................................................................... 8 1.4.6 Toxicity of AgNPs ..................................................................................... 10 1.5 Titanium dioxide ................................................................................................... 12 1.5.1 Titanium dioxide nanoparticles ................................................................ 12 1.5.2 TiO2NPs in the environment ..................................................................... 13 1.5.3 Toxicity of TiO2NPs ................................................................................... 14 1.6. Lumbriculus variegatus ........................................................................................ 16 1.6.1 Biology and ecological role ....................................................................... 16 1.6.2 Reproduction and autotomy .................................................................... 18 1.6.3 Use in ecotoxicological testing ................................................................. 18 1.7 The MARINA project ............................................................................................. 19 1.8 Aims ..................................................................................................................... 20 i Chapter 2: Characterisation of engineered Ag (NM-300K) and TiO2 (NM-104) nanoparticle .... 21 2.1 Introduction .......................................................................................................... 21 2.1.1 Dynamic light scattering ........................................................................... 22 2.1.2 Differential centrifugal sedimentation ..................................................... 24 2.13 Zeta potential ........................................................................................... 25 2.1.4 Transmission electron microscope ........................................................... 26 2.1.5 Energy dispersive x-ray spectroscopy ....................................................... 27 2.1.6 Dissolution ............................................................................................... 27 2.1.7 Aims......................................................................................................... 28 2.2 Methods ............................................................................................................... 29 2.2.1 Nanoparticles ........................................................................................... 29 2.2.2 DLS .......................................................................................................... 29 2.2.3 DCS .......................................................................................................... 31 2.2.4 TEM and EDS ............................................................................................ 31 2.2.5 Dissolution ............................................................................................... 32 2.2.6 Statistical analysis ..................................................................................... 33 2.3 Results .................................................................................................................. 34 2.3.1 DLS ........................................................................................................... 34 2.3.2 DCS ........................................................................................................... 38 2.3.3 TEM and EDS ............................................................................................ 40 2.3.4 NM-300K dissolution................................................................................. 46 2.4 Discussion ............................................................................................................. 47 2.4.1 NM-300K TEM analysis
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