University of the Basque Country UPV/EHU Department of Zoology and Animal Cell Biology Research Center for Experimental Marine Biology and Biotechnology - Plentzia Marine Station (PiE) Marine Environment and Resources Erasmus Mundus PhD Program Towards Intelligent Aquaculture - Development of an early Biological Warning System to monitor exposure to contaminants and fish welfare: from artificial vision to systems modelling International PhD Thesis submitted by HARKAITZ EGUIRAUN MARTINEZ For the degree of Philosophiae Doctor Under the supervision of Prof Iciar Martinez and Dr Karmele Lopez de Ipiña. Plentzia, December 2015 (cc)2016 HARKAITZ EGUIRAUN MARTINEZ (cc by-nc 4.0) For Edur, for Berta, and for Aitite Cesar (in memoriam). CONTENTS 4 CONTENTS Image from http://www.ehu.eus/PIE 5 TTOOWWAARRDDSS IINNTTEELLLLIIGGEENNTT AAQQUUAACCUULLTTUURREE 6 CONTENTS CONTENTS List of figures .............................................................................................................v List of tables ............................................................................................................vii Preface ......................................................................................................................1 Acknowledgements ...................................................................................................3 Financial support .......................................................................................................5 Abstract .....................................................................................................................7 Chapter 1. Introduction ............................................................................................11 1.1. General Context ........................................................................................13 1.2. Fish welfare and fish welfare monitoring ...................................................14 1.3. Challenges in the monitoring of chemical contaminants ...........................19 1.4. Malicious contaminants ............................................................................21 1.5. Emergent chemical contaminants .............................................................22 1.6. The need for a paradigm shift in seafood safety assurance .......................25 1.6.1. Introduction to Biological Warning Systems (BWSs) .........................26 1.7. Understanding, defining and dealing with a system ..................................27 1.8. Biological systems and fish farming ..........................................................31 1.9. Hypothesis ................................................................................................31 1.10. Objectives and contributions ....................................................................31 1.11. Ethical statements ....................................................................................32 1.12. Scientific output ........................................................................................33 1.12.1. JCR peer reviewed scientific journals .................................................33 1.12.2. Refereed published conference proceeding ......................................33 1.12.3. International conferences ..................................................................34 1.12.4. Grants................................................................................................36 Chapter 2. Research methodology ..........................................................................37 2.1. Introduction ..............................................................................................39 2.2. Research questions ...................................................................................39 2.3. Research plan ...........................................................................................40 2.3.1. Informational phase ..........................................................................40 2.3.2. Propositional phase ..........................................................................40 2.3.3. Analytical phase ................................................................................40 2.3.4. Evaluative phase ...............................................................................41 i CONTENTS 2.4. Benefits of the research plan ....................................................................41 2.5. Specific research methods used in this Thesis ..........................................42 2.5.1. Image processing ..............................................................................42 2.5.2. Cluster estimation .............................................................................43 2.5.3. Trajectory magnitude normalization by z-score .................................44 2.5.4. Fractal dimension ..............................................................................44 2.5.5. Entropy ..............................................................................................47 2.5.5.1. Shannon entropy ......................................................................48 2.5.5.2. Permutation entropy .................................................................49 2.5.6. Descriptive statistics. Mean and standard deviation .........................50 2.5.7. Boxplots ............................................................................................50 2.5.8. Curve fitting and goodness of the fit .................................................51 2.5.9. Modelling ..........................................................................................52 2.5.10. Programming ....................................................................................54 2.6. Limitations of the study ............................................................................54 2.6.1. Limitations of the research methods used .........................................54 2.6.2. Limitations of the case studies ..........................................................55 2.6.3. Limitations of the model....................................................................55 Chapter 3. State of the art .......................................................................................57 Chapter 4. Application of entropy and fractal dimension analyses to the pattern recognition of contaminated fish responses in aquaculture ............................63 4.1. Introduction ..............................................................................................65 4.2. Materials and Methods .............................................................................66 4.2.1. Experimental cases ............................................................................66 4.2.2. Image acquisition and processing .....................................................66 4.2.3. Object detection and motion estimation ...........................................68 4.2.4. Clustering and trajectory generation .................................................70 4.3. Results and Discussion .............................................................................73 4.4. Conclusions ..............................................................................................78 Chapter 5. Variation in the Shannon entropy of the trajectory of a European seabass (Dicentrarchus labrax) system in relation to the number of fish .....................79 5.1. Introduction ..............................................................................................81 5.2. Materials and methods .............................................................................83 5.2.1. Animals and acclimation conditions ..................................................83 ii CONTENTS 5.2.2. Experimental conditions ....................................................................84 5.2.3. Experimental setup ...........................................................................85 5.2.3.1. Experiment A - Decreasing the density ......................................86 5.2.3.2. Experiment B - Increasing density .............................................86 5.2.4. Data acquisition ................................................................................87 5.2.5. Image processing ..............................................................................87 5.2.6. Trajectory Estimation ........................................................................88 5.2.7. Non Linear Trajectory Analysis ..........................................................89 5.3. Results ......................................................................................................89 5.3.1. Experiment A .....................................................................................89 5.3.2. Experiment B .....................................................................................89 5.4. Discussion ................................................................................................94 Chapter 6. Application of the tool to an experimental case: variation of the Shannon entropy in a European seabass (Dicentrarchus labrax) system exposed to sodium selenite (Na2SeO3) and/or methylmercury chloride (CH3ClHg) ..........97 6.1. Introduction ..............................................................................................99 6.2. Phase A - Na2SO3 exposure ....................................................................100 6.2.1. Biological material and water/environmental conditions ................100 6.2.2. Experimental setup and exposure to Na2SeO3 ......................................101 6.2.3. Image acquisition, processing, trajectory estimation and non linear trajectory analysis ..........................................................................................101 6.2.4. Results and Discussion ....................................................................101 6.3. Phase B - Exposure to MeHg ...................................................................104 6.3.1. Biological material, water/environmental conditions, experimental setup and exposure to MeHg .........................................................................104 6.3.2. Results and discussion ....................................................................104 6.4. Phase C - Recovery period ......................................................................105 6.4.1. Biological material, water/environmental conditions, image acquisition, processing, trajectory estimation and non linear trajectory analysis ..............105 6.4.2. Results and discussion ....................................................................105 6.5. Overall conclusion...................................................................................109 Chapter 7. Modelling .............................................................................................111 7.1. Introduction ............................................................................................113 7.2. Parameters of the model .........................................................................113 7.3. Construction of the model .......................................................................114 iii CONTENTS 7.3.1. Basal-state reference sub-models ...................................................116 7.3.2. Response to the stochastic event reference sub-models .................118 7.3.3. Basal/Event response reference sub-models ..................................120 7.4. Integrated model ....................................................................................122 7.5. Discussion ..............................................................................................125 Chapter 8. General discussion, Conclusions, Thesis and Future prospects ..........127 8.1. General discussion ..................................................................................129 8.2. Conclusions ............................................................................................132 8.3. Thesis .....................................................................................................133 8.4. Future prospects .....................................................................................133 Laburpena..............................................................................................................135 Bibliography ..........................................................................................................143 iv
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