Elucidation of the Lipid Degradation Process in Soft Tissue and Fluid During Decomposition, in the Presence and Absence of Insects by Jenna Comstock A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in The Faculty of Science Applied Bioscience Program University of Ontario Institute of Technology August 2014 © Jenna Comstock, 2014 i Certificate of Approval ii Copyright Agreement iii Abstract Decomposition has been extensively studied in the presence of insects, however the process in the absence of insects has been considerably less documented. In this study, the decomposition process was compared between domestic pig carcasses in the presence and the partial and complete absence of insects during three consecutive summers. Fluid was collected from carcasses, from which pH and conductivity were measured. The fluid was analyzed via attenuated total reflectance- infrared spectroscopy (ATR-IR) and gas chromatography-mass spectrometry (GC-MS) to examine the fatty acid degradation trends, in an attempt to identify potential biomarkers for estimating the post mortem interval (PMI). Tissue was also collected, from which pH was measured and fatty acids extracted and analyzed by GC-MS. All results were compared between the carcass groups to investigate the effect of insects on the decomposition process. Observations of the physical changes exhibited by the exclusion groups required new decomposition stages to be established to better characterize the processes. The fluid analyses indicated that insects influenced the trends observed from the pH and conductivity measurements. The effect of insects on the bands detected by spectroscopic analysis was unclear, however GC-MS results demonstrated that insects accelerated the rate of hydrogenation when they were present in large numbers; evidence of hydrogenation was absent in both exclusion groups. Tissue pH was also influenced by insect activity and significant differences were observed between carcass groups in the amount of individual fatty acids detected during the insect activity stages and following the advanced decay and dry stages. iv Characteristic fatty acids were not identified that would allow them to be used to estimate PMI. Further research investigating the complete lipid degradation process is required to establish fatty acids of interest which would aid in establishing accurate and objective methods of estimating PMI, to be used in criminal cases. Keywords: Forensic chemistry, Decomposition, Decomposition stages, Insect exclusion, Decomposition fluid, Soft tissue, Fatty acids, pH, Conductivity, Attenuated total reflectance- infrared (ATR-IR) spectroscopy, Gas chromatography- mass spectrometry (GC-MS) v Acknowledgements “As the builders say, the larger rocks do not lie well without the lesser” – Plato Throughout my studies, I have been quick to learn that the interdisciplinary nature of my project required the knowledge and help of many individuals, and to all of you, I am grateful. I would first like to thank my supervisor, Dr. Shari Forbes, for providing me the opportunity to work in her lab as a summer student, which inspired me to pursue graduate studies. She has provided steady guidance and mentorship over the years, even when she was halfway across the world. I would like to thank Dr. Hélène LeBlanc for adopting me as a student and for the opportunities and insight provided. Thank you also to Dr. Andrea Kirkwood for her willingness to supervise me this past year and for all of her patience and help. Further, I would like to thank my committee member, Dr. Jean-Paul Desaulniers, and I thank Dr. Liliana Trevani for her enthusiasm as an examiner over the course of my studies. Thank you to Mike Allison, Clayton Jakins, and Genevieve Barnes for granting me access to their labs, chemistry supplies, and instruments. Many thanks go to Ed Courville for his creative solutions to my fieldwork dilemmas. I would like to extend my gratitude to Laura Benninger and Lori VanBelle for their assistance both inside and outside of the lab, which continued even after we no longer worked together. I am grateful for the help and friendships of all of my lab mates over the years: Sonja, Heloise, Kevin, Kate, Steph, and Alycia. Lastly, but certainly not least, I would like to thank my friends, family, and my wonderful husband, Ricky. Without their continued love and support, I would have never vi made it through these past five years. Special thanks to Rick and Joanne Scrannage for not only designing my anti-scavenging cages, but for actually building them. Thank you to my parents for their constant encouragement and motivation over the years, especially during the difficult times. Finally, I am immensely and forever grateful to my husband, for putting up with all of the trials and tribulations we have endured throughout my studies; you have been, and always will be, my most important rock. vii Table of Contents Certificate of Approval ........................................................................................................ ii Copyright Agreement ......................................................................................................... iii Abstract............................................................................................................................... iv Acknowledgements ............................................................................................................ vi List of Tables .................................................................................................................... xiii List of Figures................................................................................................................... xiv List of Appendices .......................................................................................................... xviii List of Abbreviations ........................................................................................................ xix Chapter 1 : Introduction ................................................................................................. 1 1.1 Decomposition ........................................................................................................... 3 1.1.1 Characterizing Decomposition Stages ................................................................ 4 1.1.2 Commonly Used Decomposition Stages ............................................................. 7 1.2 Factors That Influence the Rate of Decay ............................................................... 10 1.3 Estimating the Post-Mortem Interval ....................................................................... 12 1.3.1 Early Post-Mortem Period ................................................................................. 13 1.3.2 Entomological Data ........................................................................................... 14 1.3.3 Chemical Biomarkers ........................................................................................ 16 1.4 Lipids in the Body.................................................................................................... 20 1.4.1 Lipids in Porcine Adipose Tissue ..................................................................... 23 1.4.2 Effect of Diet on Tissue Lipids ......................................................................... 24 1.4.3 Effect of Diet on Porcine Tissue Lipids ............................................................ 26 1.5 Lipid Degradation .................................................................................................... 27 1.5.1 Anaerobic Degradation ..................................................................................... 28 1.5.2 Aerobic Degradation ......................................................................................... 29 1.6 Fatty Acids in Insects ............................................................................................... 30 1.7 Body Fluids .............................................................................................................. 32 1.7.1 Decomposition Fluid ......................................................................................... 33 1.8 Study Objectives ...................................................................................................... 34 Chapter 2 : Experimental Design ................................................................................. 35 2.1 Research Trials ........................................................................................................ 36 viii 2.1.1 Experimental Site .............................................................................................. 36 2.1.2 Weather Data ..................................................................................................... 36 2.1.3 Experimental Subjects ....................................................................................... 37 2.2 Method Testing (Trial 1).......................................................................................... 38 2.2.1 Tissue Collection ............................................................................................... 38 2.3 Replicate Trials (2011, 2012, and 2013).................................................................. 39 2.3.1 Experimental Setup ........................................................................................... 39 2.3.2 Observations .....................................................................................................