RESEARCH REPOSITORY This is the author’s final version of the work, as accepted for publication following peer review but without the publisher’s layout or pagination. The definitive version is available at: Magni PA, Lawn J, Guareschi EE., A practical review of adipocere: Key findings, case studies and operational considerations from crime scene to autopsy, Journal of Forensic and Legal Medicine (2021) doi: https://doi.org/10.1016/j.jflm.2020.102109. http://researchrepository.murdoch.edu.au/59347/ Copyright: © 2020 Published by Elsevier Ltd This article is posted here for your personal use. No further distribution is permitted. Journal Pre-proof A practical review of adipocere: Key findings, case studies and operational considerations from crime scene to autopsy Paola A. Magni, Jessica Lawn, Edda E. Guareschi PII: S1752-928X(20)30216-X DOI: https://doi.org/10.1016/j.jflm.2020.102109 Reference: YJFLM 102109 To appear in: Journal of Forensic and Legal Medicine Received Date: 1 July 2020 Revised Date: 15 December 2020 Accepted Date: 17 December 2020 Please cite this article as: Magni PA, Lawn J, Guareschi EE, A practical review of adipocere: Key findings, case studies and operational considerations from crime scene to autopsy, Journal of Forensic and Legal Medicine (2021), doi: https://doi.org/10.1016/j.jflm.2020.102109. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd. AUTHORSHIP STATEMENT Manuscript title: A practical review of adipocere: key findings, case studies and operational considerations from crime scene to autopsy All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication before its appearance in Forensic Science International. Authorship contributions Category 1 of • Conception and design of study: PA Magni, J Lawn • Acquisition of data: J Lawn, EE Guareschi ro • Analysis and/or interpretation of data: J Lawn, PA Magni, EE Guareschi Category 2 • Drafting the manuscript: PA Magni -p re • Revising the manuscript critically for important intellectual content: J Lawn, EE Guareschi lP Category 3 • Approval of the version of the manuscript to be published (the names of all authors na must be listed): PA Magni, J Lawn, EE Guareschi ur Acknowledgements Jo All persons who have made substantial contributions to the work reported in the manuscript (e.g., technical help, writing and editing assistance, general support), but who do not meet the criteria for authorship, are named in the Acknowledgements and have given us their written permission to be named. If we have not included an Acknowledgements, then that indicates that we have not received substantial contributions from non-authors. This statement is signed by all the authors Author’s name (typed) Author’s signature Date PA Magni 16/12/2020 J Lawn 16/12/2020 EE Guareschi 16/12/2020 1 A practical review of adipocere: key findings, case studies and operational considerations from 2 crime scene to autopsy 3 4 Paola A. Magni1,2, Jessica Lawn1, Edda E. Guareschi1 5 6 1. Discipline of Medical, Molecular & Forensic Sciences, Murdoch University, 90 South Street, 7 Murdoch, Western Australia 6150, Australia 8 2. Murdoch University Singapore, King’s Centre, 390 Havelock Road, Singapore 169662 of 9 ro 10 p.magni@murdoch.edu.au 11 jessjames14@gmail.com 12 edda.guareschi@murdoch.edu.au -p re 13 lP 14 Corresponding author na 15 Paola A. Magni 16 Murdoch University Singapore ur 17 King’s Centre, 390 Havelock Road, Singapore 169662 Jo 18 Tel: (+65) 68380765 19 p.magni@murdoch.edu.au 20 1 Abstract 2 After death, the body begins decomposition, a process that starts with the breakdown of organic 3 matter and typically leads to the complete degradation of a body. Such a process is highly affected by 4 (micro and macro) environmental factors of intrinsic and extrinsic nature. 5 Adipocere is a substance formed from the decomposition of adipose tissue and represents a disruption 6 to the typical decomposition process. 7 Such disruption causes decomposition to slow or arrest completely, placing a body into a state of 8 preservation, and causes complications in the estimation of the time since death (Post-Mortem of 9 Interval, PMI). While several studies have been performed on the nature, the formation and the ro 10 degradation of adipocere, there is still no reliable model to assess the PMI of a body exhibiting it. 11 Case studies are an important source to aid pathologists and investigators during a case. 12 -p This review presents a summary and an update on the knowledge surrounding the chemistry and the re 13 factors affecting adipocere formation and degradation, the timing and the distribution of adipocere lP 14 throughout a body, and the techniques used to investigate it. Furthermore, a table of the most na 15 important case studies involving adipocere since 1950, several images and descriptions of recent cases 16 and operational considerations for the best practice at the crime scene and autopsy are presented to be ur 17 used as a reference to facilitate forensic professionals in adipocere cases. Jo 18 19 Keywords: Adipose tissue; decomposition; preservation; formation; degradation; case-work; best- 20 practice 21 22 1. Introduction 23 Following death, a body will start the natural process of decomposition. Generally, the decomposition 24 process is expected to constitute several subsequent stages that will modify the body from fresh to 25 skeletonised remains1. The rate of decomposition is affected by both the external environment – 26 extrinsic factors e.g. temperature, humidity, type of soil or water where the body is disposed – and the 27 body itself – intrinsic factors, e.g. body size, amount of fat tissue, cause of death2. The stage of 28 decomposition can be used along with other indicators, such as the presence of certain insects, to 29 estimate the time since death (or minPMI, minimum Post-Mortem Interval PMI)3. Estimation of the 30 minPMI is important in a forensic investigation as it may be used in the reconstruction of the events 31 prior to death, and it may corroborate or contradict the alibi of a suspect4, 5. The estimation of the 32 minPMI is more difficult when the typical decomposition process is disrupted, such as in the case of 33 the formation of adipocere6. Adipocere is a postmortem transformative phenomenon which occurs as 34 an alternative to putrefaction. This does not allow a clearly defined allocation for adipocere in most 35 decomposition scoring systems, which are typically based on the characterization of putrefaction- 36 only, in different environments6-8. of 37 The term ‘adipocere’ was first introduced by Fourcroy in 17899, although the “spontaneous ro 38 conversion of animal matter into a substance considerably resembling spermaceti” was apparently 39 already mentioned in the works of Lord Bacon (1561-1626), who may have been the first to have 40 -p made a reference to it9. Adipocere is a substance that forms on decomposing remains through the re 41 decomposition of adipose tissue found within a body. It represents an alteration of the decomposition lP 42 process, slowing it down or halting it up to hundreds or even thousands of years under stable conditions10. The formation of adipocere, therefore, hinders grave reuse in countries where cemeteries na 43 44 carry out this practice11. Graves are generally occupied by body remains for 15-25 years, during ur 45 which they should undergo complete decomposition12. After this time, the grave is reused. However, Jo 46 this practice cannot take place in cases where there is formation of adipocere, as at the opening of the 47 grave the bodies are partially or fully preserved 13. 48 In a forensic context, knowledge about adipocere formation is important because of this property of 49 inhibiting decomposition14. As a consequence of this, on one hand adipocere can cause issues in 50 estimating the PMI but, on the other hand, it can aid investigations, by facilitating the personal 51 identification of an individual (due to preserved facial or body features), and the determination of the 52 cause and manner of death (due to preserved injuries)15. Furthermore, finding adipocere in a body can 53 help the investigators to backtrack the type of environment it has formed in, shedding light on the 54 remains post-mortem period2, 15-17. 55 The process of the formation of adipocere, the type of adipocere formed, and the distribution of 56 adipocere within a body varies case-by-case and environment-by-environment, making every 57 investigation unique. Research into adipocere formation is pivotal to identify the various 58 environments adipocere forms in, the factors that influence adipocere formation, and the timing of its 59 formation – as all of these factors can affect the estimation of the minPMI. Besides this, an overview 60 of previous cases in which adipocere was found may aid future investigations. 61 The present review of the literature will provide pathologists and investigators with an update on the 62 present knowledge of the chemical composition of adipocere, the mechanism of its formation, the 63 numerous factors affecting its formation, and its formation throughout the body. Furthermore, a table 64 detailing a large number of case-reports has been compiled to provide forensic professionals with easy of 65 access to several reference scenarios that could be referred to in relation to current case work. If the ro 66 features of a new case and case-report are similar, the processes and techniques – as well as possible 67 mistakes – reported in the literature could be used as a reference and guideline, providing a greater 68 -p chance to succeed in the current case. Lastly, a list of recommendations is provided in case human re 69 remains recovery activities are taking place or are planned to take place in an environment favourable lP 70 for adipocere formation. na 71 72 2. Adipocere composition ur 73 At the end of the 1700s, Fourcroy described the composition of human adipocere taken from cemetery Jo 74 samples as consisting of “soap of ammonia and phosphate of lime”9. More than a century later, in 75 1917, Ruttan and Marshall described the adipocere formed on an almost 50-year-old pig as composed 76 predominantly of palmitic acid, followed by hydroxystearic acids, oleic acid, stearic acid, and calcium 77 soaps18. 78 As analytical tools and techniques advanced, GC-MS and various infrared spectroscopy analyses were 79 used to further investigate adipocere composition. These studies confirmed the previous findings, but 80 were also able to identify several new components. The confirmed components included, saturated 81 fatty acids including palmitic, myristic and stearic acid, hydroxy fatty acids, fatty acid salts of 82 magnesium, calcium, sodium and potassium, unsaturated fatty acids including palmitoleic, oleic and 83 linoleic acid, along with triglycerides and oxo fatty acids14, 19-22. Hydroxystearic acids were thought to 84 be derived from oleic acid through hydration18; oxo fatty acids, instead, were thought to be formed 85 from hydroxy fatty acids, because 3-month old adipocere was found containing only hydroxy fatty 86 acids, while 6-month old adipocere contained both hydroxy fatty acids and oxo fatty acids19, 20. 87 As newly reported or further investigated, Takatori and Yamaoka (1977) confirmed the position of the 88 hydroxyl groups on the hydroxy fatty acids previously identified23. Two hydroxy acids and their 89 structures were determined following the GC-MS analysis of 3 to 6-month-old human adipocere19. 90 The hydroxy acids were determined to be 10-hydroxyhexadecanoic acid (10-hydroxypalmitic acid) 91 and 10-hydroxyoctadecanoic acid (10-hydroxystearic acid) and comprised around 2-30% of the total 92 adipocere composition. Furthermore, they discovered a new component – oxo (or keto) fatty acids – of 93 which were thought to be produced from the oxidation of hydroxy fatty acids23. The two oxo fatty ro 94 acids were 10-oxohexadecanoic acid (10-oxopalmitic acid) and 10-oxo-octadecanoic acid (10- 95 oxostearic acid), and together comprised around 1-2% of the total adipocere composition23. Other 96 -p components found in some adipocere samples were 10-hydroxy-12-octadecenoic acid and cis-12- re 97 octadecenoic acid, derivatives of linoleic acid24, 25 , and epicoprostanol, a reduced compound of lP 98 cholesterol20. Takatori (2001) also found several fatty acids in tissue from a newborn, specifically 9- na 99 chloro-10-methoxy palmitic (9-methoxy-10-chloro palmitic) and 9-chloro-10-methoxy stearic (9- 100 methoxy-10-chloro stearic) acid26. ur 101 Analysis of volatile organic compounds (VOCs) associated with adipocere found that adipocere Jo 102 contains 18 VOCs 27. 103 The importance of research into the composition of adipocere lies in the fact that the different 104 chemical components and their proportion in a corpse or carcass can be used in both research and case 105 studies to confirm the presence of adipocere28-30, to develop theories as to the mechanism of its 106 formation28, 31, 32 and to classify it into the various stages of its formation14. 107 108 3. Formation of adipocere 109 The current knowledge on the mechanism of adipocere formation is still limited and there is no single 110 accepted model of formation. It is known that adipocere formation is the result of the post-mortem 111 decomposition of adipose tissue and is facilitated by intrinsic lipases as well as the enzymatic activity 112 of micro-organisms that originate from within the intestines and respiratory system33. Aerobic 113 organisms initially utilise the oxygen present in the tissues which causes an anaerobic environment to 114 form and favours the survival of anaerobic organisms that are associated with adipocere formation33. 115 In the present review the mechanism of formation is divided into chemical-based and 116 microbiological-based. However, adipocere is the result of a combination of these two processes. 117 118 3.1 Chemical factors affecting adipocere formation 119 There has been extensive research into the chemistry of adipocere formation, in particular regarding 120 its chemical composition and the mechanism of formation. Due to restrictions on the use of human of 121 remains in research, in many countries pigs (Sus scrofa L.) have been used as the next best model of ro 122 human decomposition. A study conducted by Notter et al. (2009) found that pigs were suitable to be 123 used as human analogues in the investigation of adipocere although considerations needed to be taken 124 -p regarding differences between the composition of adipose tissue and the process of adipocere re 125 formation in pigs compared to humans34. These differences cause a margin of error which affects the lP 126 application of the pig study findings to human adipocere formation. na 127 Adipose tissue consists mainly of lipids, of which 90-99% are triglycerides. Triglycerides are 128 composed of a glycerol molecule attached to three fatty acid molecules 35. The fatty acids contained ur 129 within adipose tissue are mainly unsaturated fatty acids that make up 65% of the total fatty acid Jo 130 content of human adipose tissue. Overall, oleic acid (~45%) is the most abundant unsaturated fatty 131 acid followed by linoleic acid (~15%), while palmitic acid (~20%) is the most abundant saturated 132 fatty acid36. In contrast with the content of human adipose tissue, the main components of adipocere 133 are saturated fatty acids, where palmitic acid is the most abundant28. As confirmation of this, hydroxy 134 acids are only detected in adipocere samples and not adipose tissue samples19. 135 The decomposition of adipose tissue and the main process of adipocere formation involves both the 136 hydrolysis of triglycerides into the saturated and unsaturated fatty acids described previously, and the 137 hydrogenation of the unsaturated fatty acids into saturated fatty acids 14. Lipases within cells initiate 138 the hydrolysis of the triglycerides for a brief period after which bacterial enzymes facilitate more 139 extensive hydrolysis. If there is a sufficient concentration of enzymes and water, the process will 140 continue until most or all triglycerides are depleted. Water may be derived from the environment or 141 from the body itself, either within the fatty tissue or drawn from surrounding tissue by diffusion33. 142 Furthermore, in addition to unsaturated and saturated fatty acids, adipocere also contains fatty acid 143 soaps, hydroxy fatty acids and oxo fatty acids that are produced as by-products of the process of 144 adipocere formation 14. Sodium ions in the interstitial fluid and potassium in cell water may interact 145 with the fatty acids released from the adipose tissue to form their respective fatty acid soaps14. 146 According to Vass (2001), adipocere will be hard and crumbly if sodium soaps are formed, or soft 147 with a paste-like consistency if potassium soaps are formed instead37. However, Powers (2005) states 148 the opposite, with adipocere being hard and crumbly if potassium soaps are formed and soft and of 149 paste-like if sodium salts are formed, but that if sodium is displaced by calcium it will produce a more brittle substance38. To note, the environment in which a body is placed in will also contain metal ions, ro 150 151 such as calcium and magnesium, that can displace the sodium or potassium ions35. The overall process 152 of adipocere formation is shown in Fig. 1. -p re 153 Overall, with regards to the chemical mechanism of adipocere formation, three main theories have lP 154 been suggested. Notably, all three theories explain some observations of adipocere formation but none of them explains all aspects of it 19, 28: na 155 156 • The fat migration theory states that fat spreads throughout the surrounding tissue as ur 157 decomposition of adipose tissue occurs. Triglycerides contained within the fat are broken Jo 158 down and the resulting free fatty acids, e.g. oleic acid, and glycerol diffuse or separate out due 159 to gravity. 160 • The saponification theory states that the main reaction of formation is the hydrolysis of fat 161 into fatty acids and glycerol. Insoluble soaps of the fatty acids are then formed. These soaps 162 only constitute 2-35% of the total adipocere composition and saponification is not thought to 163 play a major role in adipocere formation19. 164 • The hydrogenation theory states that fatty acids are released and spread into adjacent tissues. 165 Unsaturated fatty acids are the main component and are hydrogenated into saturated fatty 166 acids. 167 The saponification theory is not thought to play a significant role in formation as soaps generally 168 make up only a partial component of adipocere, around 2-35% 19, 28. 169 The hydrogenation theory is more widely supported by research, although results from several studies 170 have shown contradictions. In their experiment with adipocere tissue samples from the Tyrolean 171 Iceman Ötzi, Bereuter et al. (1996)39 noted that the tissue was in a high oxidational state while the 172 theory requires a reductive state. Adachi et al. (1997)20 also noted that the presence of epicoprostanol 173 in adipocere indicated that both oxidation and reduction had occurred in the formation process due to 174 the nature of epicoprostanol production from cholesterol. of 175 ro 176 3.2 Microbiological factors affecting adipocere formation 177 Following the death event, microorganisms from the intestines and respiratory system tend to migrate 178 -p into surrounding (adipose) tissues, and both aerobic and anaerobic bacteria are thought to be involved re 179 in adipocere formation40. The metabolism of aerobic fauna causes tissue oxygen depletion and the lP 180 development of an anaerobic environment, causing a decline in the aerobes. Anaerobic organisms then have the opportunity to flourish, and aid in the development of adipocere33. na 181 182 Many microorganisms including Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, some ur 183 Pseudomonas species and Clostridium perfringens are able to convert oleic to hydroxy fatty acids. Jo 184 Micrococcus luteus, Flavobacterium meningosepticum and other Pseudomonas species can also 185 convert hydroxy fatty acids to oxo fatty acids. This suggests that such bacteria have both cis-9-enoic 186 acid hydrase and 10-hydroxy fatty acid dehydrogenase26, 41. Fatty acids with a cis-9-unsaturation, such 187 as myristoleic, palmitoleic and oleic fatty acids, can be converted into both hydroxy and oxo fatty 188 acids. Linoleic can only be converted to hydroxy fatty acids. Oxo fatty acids are not converted to 189 hydroxy fatty acids by any of the bacteria. 190 It has been hypothesised that hydroxy and oxo fatty acids, along with fatty acid soaps, may play an 191 important part in the formation and stabilisation of adipocere23, 30, 40. The high melting point of the 192 hydroxy fatty acids (78.5-79°C) and oxo fatty acids (73.5-74°C and 80-81°C) were thought to be the 193 reason behind such stabilisation, although oxo fatty acids only constitute a small percentage of the 194 composition and do not appear in all samples of adipocere23, 40. However, other authors suggested that 195 palmitic and stearic acid also contribute to stability, alongside other fatty acids with high melting 196 points30. In disagreement regarding the role of hydroxy fatty acids in adipocere stability, Schoenen 197 and Schoenen (2013) point out that such studies have not taken microbial activity into account, and 198 suggest that the lack of oxygen is the true cause of adipocere formation and preservation 42. 199 In environments conducive to adipocere the amount of oxygen necessary for complete decomposition 200 by respiration is not available; in such environments, bodies undergo an incomplete decomposition 201 process through fermentation instead of respiration. In the respiration process, glycerol cleaved from 202 triglycerides is degraded into carbon dioxide and water, while in the fermentation process it results in of 203 soluble alcoholic components that may wash away in the environment. Respiration degrades fatty ro 204 acids through beta-oxidation into carbon dioxide and water, but since fermentation of fatty acids 205 cannot occur, fatty acids are converted to polyhydroxy acids, that are a form of energy storage. These 206 -p polyhydroxy acids are insoluble in water, and along with fat and long-chain fatty acids, they cannot be re 207 washed away and they will remain where they first originated in the body42. lP 208 na 209 3.3. Models of adipocere formation 210 The process of adipocere formation is sometimes referred to as “saponification”. However, ur 211 “saponification” specifically refers to the formation of soaps within the process of adipocere Jo 212 formation. Despite that, “saponification” is often used interchangeably with the terms “adipocere 213 formation”, “saponified tissue” and “adipocere”16. 214 In this paragraph two main models of adipocere formation are reported, while discussion regarding 215 intrinsic and extrinsic factors affecting adipocere formation, timing and distribution of adipocere 216 formation within the body and case studies involving adipocere formation will be discussed in the 217 following sections. 218 Yan et al. (2001)32 produced a preliminary model for adipocere formation using pigs immersed in 219 several water environments. The goal of this experiment was to observe the adipocere formation from 220 the beginning, as compared to the majority of the previous studies that were focused on later stage 221 formation. Oleic acid was detected a few hours after each pig was immersed in water, and the 222 formation of adipocere followed the general trend of a decrease in oleic acid with a related increase in 223 the level of hydroxystearic acid as time since immersion increased. The authors suggested that the 224 ratio of oleic to hydroxystearic may be able to be used to improve the PMI estimation, but that 225 validation studies with human samples need to be carried out. These authors also propose a theory as 226 to the formation of hydroxystearic acid from oleic acid32. Oleic acid was observed to decrease as 227 hydroxystearic acid increased and this suggested that there was conversion of oleic acid into other 228 derivatives, though the method of conversion is not clear. They suggested that adipocere formation 229 may be due to partial hydrogenation where some double bonds are saturated, and some undergo 230 stereomutation or bond migration. In the case of oleic acid, hydrogenation would produce stearic acid of 231 and stereomutation would produce R-(Z)-12-hydroxycis-9-octadecanoic, while hydration produces ro 232 hydroxystearic acid and dehydrogenation of hydroxystearic results in oxostearic acid. 233 Another model of adipocere formation was proposed by Forbes et al. (2004)14, who based their 234 -p findings on pigs buried in soil. They classified adipocere into three stages of development: re 235 • Early stage formation indicates that only a small amount of tissue is undergoing lP 236 saponification; its composition is still similar to adipose tissue, but there is a decrease of na 237 unsaturated fatty acid content and increased saturated fatty acid content. Triglycerides are still 238 detectable at this stage. ur 239 • Intermediate stage formation shows a further increase in palmitic acid and decrease in oleic Jo 240 acid, with the addition of increased stearic acid content. Triglycerides are also still detectable 241 at this stage. 242 • Advanced stage adipocere is indicated by palmitic acid comprising more than 50% of the total 243 adipocere composition. Oleic acid is even more reduced and other unsaturated acids and 244 triglycerides are no longer present. 245 Notter and Stuart (2012)43 considered the total percentage of saturated fatty acids to monitor the 246 different stages. Early stage was 40-60%, intermediate was 70-90% and advanced was more than 247 90%. 248 It should be noted that the stages are not completely distinct, and one sample may fit into more than 249 one stage, depending on which component is assessed. An interesting finding of the Forbes et al. 250 (2004) study was that classification into each stage was not dependent on the length of 251 decomposition14. Stuart et al. (2000)21 also found similar results with a 26-month-old sample having a 252 comparable composition to a 27-year-old sample. 253 Further discussion of adipocere formation in regard to the timing and distribution of adipocere 254 formation within the body as well as the factors affecting adipocere formation and case studies 255 involving adipocere formation are contained in the following sections. 256 257 4. Degradation of adipocere of 258 Once adipocere is formed, it may remain stable and unchanged for thousands of years39. However, ro 259 adipocere is not an indestructible substance, and if exposed to certain environmental conditions, it will 260 degrade. To note, at present there is a paucity of research and reports on the degradation of adipocere, 261 and more research is needed in this area. -p re 262 lP 263 4.1. Chemical factors affecting adipocere degradation na 264 In general, adipocere degradation takes a longer time if the environment is characterised by a poor 265 oxygen exchange. In such cases, the degradation can only occur on the surface of the adipocere ur 266 tissue42. A similar conclusion – that exposure to aerobic conditions facilitates adipocere degradation – Jo 267 was reached by Fründ and Schoenen (2009)44 after examining the degradation of adipocere under 268 various conditions. These authors determined that the half-life of adipocere is 11-82 years under 269 anaerobic conditions, while exposure to aerobic conditions resulted in a half-life of only 0.7-10 years. 270 Adipocere formed in burials in active soil was observed to have a half-life of 1.2-2.1 years. 271 Comparing fresh adipocere with suspected degraded adipocere, Fiedler et al. (2015)45 noted the first 272 contains higher carbon and hydrogen levels, while the degraded material is higher in sulphur, nitrogen 273 and oxygen content. These authors postulated that poikiloaerobic conditions (= alternating aerobic 274 and anaerobic environment) are also favourable for the degradation of adipocere within burial 275 environments. Under poikiloaerobic conditions, adipocere undergoes the following reactions: 1) 276 autoxidation of fatty acids; 2) use of alternative electron acceptors to produce iron sulphide; and 3) the 277 Maillard reaction. 278 279 4.2 Microbiological factors affecting adipocere degradation 280 Pfeiffer, Milne and Stevenson (1998)46 examined microorganisms associated with adipocere and 281 found Gram-negative bacteria – Pseudomonas, Serratia, Alcaligenes, Enterobacter – and Gram- 282 positive bacteria – Bacillus, Nocardia, Cellulomonas. It was observed that the Gram-positive bacteria 283 hydrolysed the adipocere and that the resistance of adipocere to degradation was due to the exclusion 284 of Gram-positive bacteria from the environment of adipocere formation. 285 of 286 4.3 Microfauna affecting adipocere degradation ro 287 Carrion insects are attracted by corpses and carcasses in different stages of decomposition, arriving in 288 sequences known as successional waves47. Flies of the genus Piophila sp. (Diptera: Piophilidae) and 289 -p beetles like Omosita colon (L.) (Coleoptera: Nitulidae) are part of the fourth wave of insects re 290 colonising an exposed corpse, attracted by the ‘caseic’ fermentation – when a cheesy odour develops lP 291 – that generally happens 3-6 months after death47. However, not all bodies with adipocere tissue colonised by insects present the typical insects listed in such wave48. na 292 293 Piophila casei (L.) is known as the cheese-skipper for its habit of arching its body, grasping its anal ur 294 papillae with its mouthparts, and then releasing its grip so it is flung into the air. In doing this, the Jo 295 small larva can rise several cm, and this becomes an effective escape mechanism. These larvae tend 296 to colonise bodies late, typically when saponification of the fat is well marked and after the primary 297 waves of insects attracted to fresh remains have had access to the body49. However, they are also 298 found to colonise the body when the primary cadaver flies are for some reason unable to have access 299 to it50. 300 Studies by Motter (1898)51 found P. casei colonising bodies in graves from 3 to 10 years old and up 301 to approximately 2 metres deep. In a study looking at insect succession on pigs in Canada, Piophila 302 species were found feeding on the adipocere and were observed to cause the adipocere to become 303 foamy. They appeared from 90-92 days after death to 336 days after death and were observed on both 304 pigs placed exposed to the sun and in the shade52. 305 A similar study performed in China, showed instead the presence of O. colon, a saprophagous beetle, 306 in association with adipocere. O. colon is widely distributed throughout China and found in bodies in 307 an advanced stage of decomposition. The beetle was observed to break down the adipocere slowly, 308 and the authors suggested that while not a primary carrion insect, it may represent an important 309 forensic indicator in relation to adipocerous bodies53. 310 In a case investigated by Magni et al. (2013)48, several insects were found colonizing a body 311 recovered from a well, including species of the family Calliphoridae, Fanniidae, Muscidae, 312 Trichoceridae, Sphaerocerdidae, Psychodidae and Nematocera (Fig. 4). of 313 ro 314 5. Environmental factors affecting adipocere formation and degradation 315 Bodies containing adipocere may be found in any environment, both terrestrial (e.g. vaults, and 316 -p burials) and aquatic (either natural body waters or artificial enclosures). Each environment can re 317 influence adipocere in differing ways, enhancing or inhibiting its formation and/or degradation. lP 318 Besides this, environmental factors (also known as extrinsic factors) can influence the chemical na 319 composition of the adipocere, and must be taken into consideration when an estimation of the minPMI 320 is required. Based on the environment, adipocere formation has been described as “typical” or ur 321 “atypical”25. Typical conditions are considered to be water immersion, wet graves and damp vaults, Jo 322 while atypical conditions are dry conditions, where adipocere generally forms more than 10 times 323 slower than in typical conditions. 324 Overall, in the presence of a sufficient amount of adipose tissue, the factors that the majority of 325 authors agree upon as being the most conducive to adipocere formation in any environment are: 326 mildly alkaline pH, moisture, anaerobic conditions, warm temperatures and presence of bacteria16, 54. 327 However, there are a great number of different factors that may affect the formation of adipocere on a 328 body. The following discussion will consider each factor as a single factor, for ease of reading, 329 however it needs to be underlined that such factors never appear in isolation. 330 331 5.1 Aquatic environments 332 Formation of adipocere may occur in a range of different water sources including natural 333 environments e.g. seawater, rivers and lakes20, 55, as well as man-made set ups, e.g. bathtubs and 334 wells48. Furthermore, the process has been described to happen in warm, tropical, and cold waters and 335 even in melting glaciers31, 48, 54, 56. The main factors proposed as enhancing and/or inhibiting adipocere 336 formation in an aquatic environment are discussed in depth in the following sections, and summarised 337 in Table 1. 338 339 5.1.1 Water temperature of 340 The factor that influences adipocere formation the most in an aquatic environment is the water temperature55. Adipocere can form in a variety of different temperatures, but depending on the ro 341 342 temperature range, the process will happen at a different rate54. 343 -p Overall, the formation of adipocere is enhanced by warm temperatures between 21°C and 45°C, while re 344 the process happens at a reduced rate in colder waters56. In 4˚C waters adipocere can be formed in 12- lP 345 18 months, while in 15-22°C waters the process happens faster. In 10-12°C seawater adipocere has been found to form in 38 days, while it takes 3 months at 13°C54, 56. Temperatures between 7°C and na 346 347 16°C promote adipocere formation into early or intermediate stages, but if already formed adipocere ur 348 is exposed to colder temperatures, it will not develop into an advanced stage56. Jo 349 Warm temperatures are thought to enhance formation because the activity of Clostridium perfringens, 350 a major bacteria associated with adipocere formation, has shown to be optimal between these 351 values57. However, formation of adipocere is possible even at temperatures below the C. perfringens 352 optimum, because the enzymes released by C. perfringens remain present in the water even after 353 temperatures drop below the optimum range. While the low temperatures negatively affect the 354 bacterial growth, the enzymes in the water will continue to facilitate the adipocere formation, at a 355 slower rate57. 356 357 5.1.2 Type of water 358 Yan et al. (2001)32 investigated the effects of different waters on adipocere formation. Specifically, 359 they considered the effect of distilled, chlorinated and saline water on the degradation of oleic acid 360 and on the formation of hydroxystearic acid. Distilled water showed the highest degradation rate, 361 while saline was the slowest. Conversion to hydroxystearic was most rapid in distilled water, and the 362 least in chlorine. Saturation of oleic degradation, the point where the rate of degradation did not 363 increase further, was rapid in distilled and slow in saline. 364 Stuart et al. (2016)58 found that seawater inhibits adipocere formation, while river water enhances it. 365 Chlorine was also studied and found to enhance formation, however, these results are complicated by 366 the fact that there may have been a competing reaction of chlorohydrin formation that induced the 367 appearance of enhanced adipocere formation. The different results were suggested to have developed of 368 due to the bacterial activity in each environment. In this experiment, the chemical characterisation of ro 369 adipocere was also carried out and showed that only seawater significantly affects its elemental 370 composition. As a result of such findings, the authors suggested that there is a potential to utilise the 371 -p chemical analysis approach on adipocere, to determine in what type of environment a body has been re 372 immersed58. lP 373 na 374 5.1.3 Submersion 375 Complete submersion in an aquatic environment is conducive to adipocere formation as the ur 376 underwater placement prevents contact between the body and carrion insects that facilitate the fast Jo 377 consumption of the body. However, aquatic scavengers may be attracted to the body instead59. 378 Forbes et al. (2011) found that the depth of submersion does not influence the formation of 379 adipocere56. Insects and terrestrial scavengers are more likely to have access to the body if it is 380 floating, and in open waters (e.g. sea) the body will be swept along by currents. These circumstances 381 tend to inhibit adipocere formation, while in a body totally surrounded by water at any depth, 382 adipocere formation is highly facilitated34, 60, 61. 383 However, complete submergence, while favourable, is not necessary for adipocere formation. 384 O’Brien and Kuehner (2007)54 placed three human bodies in water-filled pits containing water from 385 an underground pipeline. Liquid and tissue samples from each body were collected and analysed for 386 fatty acids and microbes. One of the bodies had an open trauma and remained submerged for the 387 entire duration of the experiment. Adipocere did not form on it, and C. perfringens was not found in 388 the body. The other two bodies remained floating the entire duration of the experiment and adipocere 389 formed on both bodies after 3 months. Signs of adipocere formation appeared within the water-level 390 zone, approximately 6 weeks into the experiment. This experiment shows that the absence of certain 391 bacteria may affect the formation of adipocere more than the position of the body in the water 392 column. The temperature varied throughout the experiment and this indicated that stable temperatures 393 are also not necessary for adipocere formation. Following this experiment, the authors compiled a 394 table of adipocere development stages54. Early formation of adipocere was noted as 6-8 weeks, 395 increased formation at 8-10 weeks, and advanced formation at 10-12 weeks. of 396 ro 397 5.1.4 Bacteria 398 Ueland et al. (2014)61 investigated lake environments in relation to the effect of aquatic bacteria in 399 -p early adipocere formation. An experiment was carried out with pigs and involved collection of re 400 bacteria samples from both the water and tissue. It was discovered that the presence of Gram-positive lP 401 bacteria is essential in the early stages of adipocere formation, as they provide the means to break na 402 down the tissue and release fatty acids through lipolysis. Most Gram-negative bacteria are not capable 403 of this. At later stages, Gram-negative bacteria exclude Gram-positive bacteria. Tissue submerged in ur 404 deionised water, acting as the control, did not develop adipocere and bacterial concentrations of the Jo 405 lake tissue declined as adipocere formed. 406 407 5.1.5. Skin slippage 408 Skin slippage occurs during the decomposition process and is caused by the epidermal layer of the 409 skin separating from the dermal layer and sloughing off the body62. 410 Direct exposure of adipose tissue to water via skin slippage was shown to promote adipocere 411 formation in rabbits63. This was also seen by Ueland et. al (2014)61. As skin slippage is already used 412 as an indicator for PMI estimations, this should be further explored in aquatic/adipocere contexts, as a 413 factor that could potentially improve such an estimation when adipocere is present63. 414 415 5.1.6 Clothing and other materials covering bodies found in aquatic environments 416 The presence of clothing is generally considered to enhance adipocere formation54. However, the type 417 of material, as well as the way it is wrapped around the body, has different effects on adipocere 418 formation. 419 Dix (1987)64, however, observed that in bodies disposed in lake waters, areas of the body with absent 420 or loose clothing formed more advanced adipocere compared to clothing-covered areas. Notter and 421 Stuart (2012) studied several materials in which a body may commonly be covered or wrapped when 422 found in an aquatic environment, e.g. polyester, wool and cotton clothing, wool and acrylic carpets. In 423 all the samples wrapped in such materials, adipocere formation was enhanced, as compared to of 424 unwrapped control samples. The wool and cotton materials made of natural fibre were better at ro 425 enhancing adipocere formation than the synthetic polyester and acrylic carpet materials. Formation to 426 an advanced stage of adipocere was faster in the natural fibre material. Overall, the wool carpet and 427 -p the cotton clothing were the most efficient, followed by the wool clothing, while the acrylic carpet re 428 enhanced formation better than polyester. To note, natural fibres are more absorbent than synthetic lP 429 fibres, and in this experiment the natural fabrics were also thicker, therefore with an overall greater na 430 capability in absorbing and retaining moisture and body fluids. Comparing wool fabric and wool 431 carpet, the latter was better at enhancing adipocere formation, through higher absorbency and an ur 432 insulating effect. As a further note, a recent experiment in the sea by Tingey et al.65, showed that Jo 433 natural fabrics like cotton – especially if thin – tend to disintegrate in water, causing the exposition of 434 the body after a certain time. In their experiment, performed using inorganic substrates, the first holes 435 in the fabric were seen after 3 months, while after 6 months more than half of the fabric was lost65. 436 The effect of plastic on adipocere formation is less clear. Pakosh and Rodgers (2009)66 placed 437 dismembered pig limbs into plastic bags and submerged them along with uncovered controls. The 438 uncovered limbs lost soft tissue significantly more than the plastic enclosed limbs, however, 439 adipocere rarely formed within the plastic. This may have been due to low temperatures, low 440 submersion time or low fat content as the plastic was expected to enable adipocere to form. The limbs 441 were preserved by the plastic but did not form adipocere. 442 443 5.2 Terrestrial environments 444 Soil is composed of various minerals, salts, organic components and micro- and macroscopic 445 organisms which all together have an effect on the decomposition process and on the potential 446 formation of adipocere. When soil is removed from the ground and replaced during the burial of a 447 body the chemical, physical and biological nature of the soil changes. Compared to undisturbed soil, 448 there is greater porosity and permeability of the soil. Pollens, seeds and fungal hyphae can find a 449 better environment to germinate, but insects and other scavengers are restricted in their access to the 450 body67-69. The passage of water and gases into the soil is also disrupted. If a body is decomposing in 451 the soil, such an environment becomes anaerobic quite quickly and oxygen diffusion into the soil is of 452 blocked by gases from decomposition diffusing in and out of the soil35. These features create an ro 453 environment conducive to adipocere formation. The main factors proposed as enhancing and/or 454 inhibiting adipocere formation in a terrestrial environment are discussed in depth in the following 455 sections, and summarised in Table 2. -p re 456 lP 457 5.2.1 Soil type na 458 Experiments on adipocere formation have been performed in many different types of soils. Several 459 have been shown to promote adipocere formation, such as loam-rich, clay-rich, sandy loam, clay, ur 460 clay-gravel, sterilised loamy sand, and loamy sand. On the contrary, soils that have been found to Jo 461 inhibit formation are those with low clay content and high organic matter70, 71. Generally, addition of 462 sand to the soil is suggested as good practice in cemeteries to limit the adipocere formation11. 463 However, in some cases it remains unclear whether soils such as sand and silty sand promote or 464 inhibit formation. These are both well-draining soils and would not be expected to maintain much 465 moisture72. Adipocere is not likely to form in a dry, desert sand environment but may form with burial 466 in a sandy beach where there is constant moisture29. Fiedler and Graw (2003)12 considered the parent 467 material (= the underlying geological material in which soil horizons form) as the most important 468 characteristic of soil, whereas Durães et al. (2010)71 and Carter et al (2010)73 state that the most 469 crucial factor is the water content of the soil. Soils that retain moisture will generally promote 470 adipocere formation. These soils would have poor drainage or be largely composed of fine particles12. 471 Strong, lateral water flow caused by soil such as clay also leads to high moisture12. 472 473 5.2.2 Soil characteristics 474 Forbes et al. (2005)29 studied the effects of soil pH, temperature, moisture and oxygen content. They 475 found that mildly alkaline pH, warm temperatures, anaerobic conditions and both dry and wet soil 476 promoted adipocere. Acidic pH, lime, cold temperatures and aerobic conditions inhibited it. They 477 found that optimal parameters for adipocere formation are a pH between 5 and 9 and a temperature 478 between 22-40°C. At lower temperatures, adipocere formation will be inhibited, while higher 479 temperatures will promote the desiccation and mummification process. Either very low or very high of 480 temperatures will inhibit bacterial growth and therefore adipocere formation29 as well as biologically and microbially active soils74. Excess moisture was not necessary, but enhanced the rate of the ro 481 482 adipocere formation29. These authors found that acidic pH inhibits adipocere formation29, but on the 483 -p contrary, Schotsmans et al. (2011)72 found extensive adipocere on a body buried in a well-draining re 484 soil with a pH between 2.6 to 4. Guareschi et al. (2019)11 analysed more than 600 bodies from two lP 485 burial grounds and found that an important variable affecting the decomposition process was the na 486 presence or the absence of stone slabs above the graves. In a cemetery set up, gravel and sand are 487 added to grave soils in order to improve drainage, as their presence increases pore spaces available for ur 488 water and air movement. However, the presence and the weight of the slabs tend to compact the soil Jo 489 underneath and reduce the soil pore spaces, the drainage, and the air movement, causing the 490 stagnation of water and promoting adipocere formation11. 491 Evans (1963)33 observed that weather at the time of burial may have an influence on the soil 492 characteristics. In his observations, fog and misty conditions at the time of burial enhanced formation 493 of adipocere. 494 495 5.2.3. Method and depth of burial 496 People of different cultures bury their dead in different ways, based on religious beliefs, historical 497 rituals, or public health requirements11. Besides this, bodies can be buried in an attempt to conceal a 498 crime. The method of burial can therefore range from direct burial into the soil, burial within a coffin 499 in the soil or in a tomb, burial within a mass grave and burial with or without clothing or material 500 covering the body 11, 33, 35. 501 Mass graves more commonly form adipocere than isolated, single graves33. If the grave contains 502 bodies piled on top of each other the layers tend to trap moisture and the middle of the grave will tend 503 to contain bodies with the most adipocere formation. Bodies in mass graves that form only a single 504 layer will exhibit decomposition closer to bodies that are buried in isolated, individual graves75. Vane 505 and Trick (2005)76 carried out research on a mass grave of pigs and cows. They found that there was a 506 mixture of early and late stage adipocere. At a depth of 50-70 cm adipocere was found to be at an of 507 advanced stage along with samples of subcutaneous fat taken from the bodies. At 220 cm, however, ro 508 samples were only observed to be in the early stage of formation. This observation may be explained 509 by mass graves containing variations in moisture resulting from infill that isolates various parts of the 510 -p grave. This would result in a mixture of early and advanced stage adipocere. re 511 Coffins inhibit formation due to slight aerobic conditions inside and will also prevent cation exchange lP 512 compared to direct soil burial. Oak, lead and zinc coffins enhance formation while pine and spruce cause rapid decomposition along with coffins lined with a straw bedding12. na 513 514 A study considering the analysis of 408 human bodies exhumed from two cemeteries in the north of ur 515 Italy – 338 placed in grave pits and 70 from stone tombs and buried in different types of coffins Jo 516 between 1920 and 1989 – showed that 98.5% of the bodies placed in the grave pits were 517 skeletonised, while only 11.4% of the bodies placed in the stone tombs were (Fig. 2). Nearly 90% of 518 the bodies placed in the stone tombs were preserved thanks to the formation of adipocere, and only 519 bodies placed in coffins where multiple breach points were produced, were able to skeletonise11. 520 The depth of burial and the soil compactness can have an effect on adipocere formation due to the 521 possibility of insects and other scavengers reaching the body68, 69. A body that is buried at a depth of 522 90 cm or below is generally safe from scavengers and will be exposed to a reduced number of insects 523 and microorganisms12. 524 525 5.2.4 Bacteria 526 Guareschi et al. (2019)11 analysed adipocere bodies recovered from double coffins (wood and zinc), 527 placed in stone tombs for a period of 19 or 40 years. The bodies showed the presence of aerobic and 528 facultative anaerobic bacteria (e.g. Pasteurella pneumotropica/haemolytica) as well as moulds (e.g. 529 Penicillium ss.pp. and Cladosporium ss.pp.) and a similar microbiological profile even if entombed 530 for a different amount of time. The entombment in such a coffin allows the environment surrounding 531 the body to remain chemically and (micro) biologically stable for decades, therefore the 532 decomposition of the body is mostly prevented, facilitating adipocere formation instead. A different 533 result was observed in bodies placed in lead coffins, as this metal is poison for the microorganisms, of 534 preventing decomposition occurring past the initial autolysis stage77. ro 535 536 5.2.5 Clothing and other materials covering bodies found in terrestrial environments 537 -p As previously stated, clothing can generally enhance the formation of adipocere, but its effect is based re 538 on the way the body is wrapped. In terrestrial environments, clothing is able to maintain a higher lP 539 humidity level with respect to the surrounding environment. Thanks to its ability in trapping the na 540 environmental moisture and the body fluids, it creates a micro-environment that can facilitate 541 adipocere formation in either inhumed or entombed bodies 11, 73. Such stagnation of liquid can also be ur 542 caused by coffin upholsteries11. Jo 543 In a forensic case-work by Guareschi et al. (2007)78, a body hanging from a tree showed the presence 544 of adipocere exclusively in the feet, that were found wearing shoes (Fig. 5). 545 In a case-work by Magni et al. (2013)48, a body found showed adipocere only in the brain (protected 546 by the skull) and on the feet (protected by the shoes) (Fig. 4). 547 Forbes et al. (2005)29 found that direct burial in soil, clothing, and plastic and clothing promoted 548 adipocere formation whereas burial in plastic and coffins was found to inhibit formation. Polyester 549 clothing allowed cation exchange which is thought to have allowed hardening of the adipocere to take 550 place. 551 552 5.3. Dry environments 553 Dry environments are an atypical place for adipocere25. Generally, the development of adipocere 554 requires at least a damp or moist environment, however, cases of adipocere in dry environments have 555 been reported, with adipocere found in combination with desiccation72. 556 Research by Evans (1963)33 on bodies in dry vaults where over half of the bodies in each vault had 557 formed extensive adipocere, suggests that the internal water of the bodies can be sufficient for 558 adipocere formation. 559 Green (2006)79 studied bodies contained in vaults consisting of an outer wooden shell and a sealed 560 lead coffin with inner untreated wooden lining inside. Inside, the bodies were laid on wood shavings. of 561 In earth burials, wood shavings or sawdust enhanced decomposition, however, the airtight condition ro 562 of the coffins was able to produce sufficient conditions to promote adipocere. 563 Zimmermann et al. (2008)80 described a case in which a suspected trace of human remains containing 564 -p adipocere was found on a concrete surface, in an exposed indoor environment. Nushida et al. (2008)25 re 25 565 reported a case of a female body sealed in a clothes box covered in plastic bags for 4 years . lP 566 Adipocere formed extensively throughout the body, within the subcutaneous and the visceral fat. A recent observation by Byard et al (2020)81 described focal adipocere on an almost completely na 567 568 skeletonized individual that was recovered from a cave located in an arid desert environment. The ur 569 body had been wrapped in a piece of leather fabric, which had created, in only some of its folds, an Jo 570 enclosed and moist microenvironment favourable to adipocere formation. 571 572 6. Timing of adipocere formation 573 Formation of adipocere is most commonly observed at three to six months after death and placement 574 in a suitable environment, with partial formation at six weeks. Extensive formation takes at least a 575 year, while complete formation requires around 2 years4, 28. 576 Initial adipocere development within the cutaneous areas of the body generally occurs after two 577 months. Muscles will start to form adipocere at least 6 months after the initial transformation process 578 begins and a complete transformation is reported to take around 2 years. Organs may remain 579 preserved in their natural state even after 2 years4. 580 However, several cases in which formation has been extremely fast have been reported in the 581 literature. Many of these cases all took place in areas of India which typically have a very hot and 582 humid climate (e.g. Delhi)82. Sikary and Murty (2015)83 reported cases in which adipocere formation 583 occurred in less than 2 days. Most of the bodies were located indoors, without air conditioning and the 584 extent of adipocere was similar between them. Mohan Kumar et al. (2009)15 observed formation on a 585 body within 3 days of submersion in a marshy pond. Powell (1917)84 observed adipocere formation 586 around 4 days in a male buried in gravel and shale, with heavy rainfall before and during the period of 587 burial. of 588 Generally, a body is rarely observed to be completely converted into adipocere, involving both the ro 589 subcutaneous tissue and the internal organs. Cases of complete transformation are rare, because 590 several factors need to be in play together. An example is the case of a male discovered after 7 years 591 -p at the bottom of a lake, trapped in a car85. The car was found relatively undamaged, and this may have re 592 created a restrictive micro-environment with low pH, that was maintained this way thanks to the lP 593 limited or absent water flow at the bottom of the lake. Besides these, the oligotrophic nature of the na 594 lake and the depth determined the absence of scavengers. Furthermore, the limited amount of oxygen 595 at the bottom of the lake created desirable conditions for anaerobic bacterial growth. All of these ur 596 factors together have contributed to the complete conversion of the body into adipocere. Jo 597 Cotton et al. (1987)57 presented a similar case of extensive subcutaneous adipocere but without 598 transformation of the organs. Two bodies were submerged in a vehicle in freshwater for 5 years. They 599 were thought to have entered the water when the temperature of the water was at its highest point. The 600 temperature subsequently dropped, slowing down the adipocere formation. 601 602 7. Distribution of adipocere within the body 603 The general description of adipocere varies considerably. It has been reported as a yellow, greyish 604 white, red, grey or grey-green substance and its odour has been associated with cheese and ammonia2, 16 605 . Adipocere can be formed on a small area of the body or the whole body could be turned into it85. It 606 was thought that only the subcutaneous tissues could form adipocere, however internal organs and 607 bone marrow cavities can exhibit adipocere formation if adequate fat is present. Even in areas with 608 low fat content, the pressure from putrefactive gases can cause liquefied fat to penetrate the tissue 609 (Evans, 1963)33. 610 The rate of adipocere formation varies between different bodies and even different areas of the same 611 body. Bereuter and Lorbeer et al. (1996)28 cited a case of a man and woman in the same car 612 submerged in a mountain lake. The man was skeletonised while the woman’s soft tissue was 613 preserved by adipocere, probably due to the different fat composition between men and women, with 614 women generally having a higher fat content. 615 Ferreira and Cunha (2013)86 looked at 25 cases in a cemetery, all with a similar PMI of approximately of 616 5 years, but showing different stages of decomposition and adipocere presence and distribution. ro 617 Desiccation and adipocere have also been shown to occur on the same body: a male buried in a 618 shallow grave with his right leg and arm and temporal bone exposed exhibited desiccation in the 619 -p exposed areas and adipocere in the areas covered by soil72. re 620 lP 621 7.1 Subcutaneous tissue na 622 Adipocere formation is initiated within the subcutaneous tissue and spreads outwards. The most 623 common areas of formation are the cheeks, buttocks, abdomen and breasts. Adipocere is not likely to ur 624 form on the hands or feet due to their relatively low fat content. Hands and feet tend to skeletonise Jo 625 and become disarticulated especially in water environments. However, if the feet are covered by 626 shoes, adipocere is likely to form (Fig. 4,5). 627 An increase in weight due to formation of adipocere may be experienced as well as a general increase 628 in the size of a body12, 87. 629 Adipocere is generally more common in women, newborn babies, obese, and well-nourished bodies, 630 due to their higher fat composition88. There has been some correlation between age and formation of 631 adipocere but not all studies support this observation12, 33, 83. 632 Adipocere distribution throughout the subcutaneous tissue has been visualised using multislice 633 computed tomography89. 634 Although adipocere causes problems with PMI estimation, adipocere formation within the soft tissue 635 may aid in preserving identifiable features of the body such as perimortem injuries and facial 636 features15, 90. 637 638 7.2 Organs 639 Within a body with complete or partial adipocere formation, organs may be found well preserved 640 because they are protected from putrefaction by the “shell” of adipocere developed over the body 641 (conversion of the subcutaneous fat into adipocere) or because they may themselves be converted of 642 into adipocere57. ro 643 The preservation of organs depends on the level of putrefaction at which adipocere begins, and organs 644 that have been well preserved indicate that the formation of adipocere occurred closely after death. If 645 -p the organs are only converted into adipocere on their surface, they are found in a dehydrated form re 646 below it 72. lP 647 Different organs will have different rates of decomposition and some tend to preserve better than na 648 others. The intestines and spleen are quickly putrefied followed by the brain. The heart is more 649 resistant, followed by kidneys, lungs and the bladder. The prostate and uterus are very resistant to ur 650 putrefaction2. Jo 651 Brain is rarely preserved by adipocere91, 92. O’Connor (2011)91 pointed out that while fats are a large 652 component of the brain tissue, they are mostly in the form of phospholipids or glycolipids. 653 Triglycerides and free fatty acids constitute only a tiny portion of total lipid content. They also noted 654 that all reported brains were shrunken, whereas adipocere usually increases the volume of the body. 655 Furthermore, the brain is the closest organ to the facial orifices that are the typical places for the 656 colonisation of carrion insects. From the nose, mouth, eyes and ears fly larvae have easy access to the 657 body cavities and they will be able to reach and consume the brain very fast93. 658 Most cases of suspected adipocere formation in brains have been observed in archaeological or 659 historical contexts with cold temperatures and acidic soil30, 91, 92, 94. Some of the most well-preserved 660 adipocere brains were those found in Spanish Civil War mass graves30, 94. The bodies were laid side 661 by side and the bodies with adipocere were spread throughout the area. The gross morphology of the 662 brains was present but could not be recognised histologically. It was proposed that the formation of 663 adipocere in the brains of the soldiers was due to the gunshot wounds to the head which caused blood 664 loss and inhibited brain decomposition. The climate at the time of burial was colder than usual with 665 heavy rainfall which would lead to waterlogged soil. The wounds allowed water to come into contact 666 with the brain and adipocere formed. 667 The other confirmed case of brain adipocere involved the preservation of the left cerebral hemisphere 668 of a 13th century infant92. The main brain structures were well preserved and could be identified 669 macroscopically and microscopically. The body was situated in a leather envelope within a wooden of 670 coffin buried in acidic, clay soil that contained fresh and saltwater. Continuous water immersion ro 671 inside the grave was thought to have aided adipocere formation. The analysis carried out on this brain 672 was only qualitative and it is difficult to determine to what extent adipocere conserved the brain. 673 -p Adipocere may have only partially played a role in preservation. It is possible that a red-brown re 674 deposit on the surface may have been adipocere but it was not analysed91. lP 675 In a case-report by Magni et al. (2013)48 regarding a man found in a well 8 months after his na 676 disappearance, both brain and bone marrow were found completely converted into adipocere. 677 Serrulla et al. (2015)95 found a heart preserved by adipocere within the same Spanish Civil War mass ur 678 grave as described above. Furthermore, a burial of 6 years resulted in the conversion of the brain and Jo 679 heart along with subcutaneous tissue96. 680 681 7.3 Bones 682 Even when a body has been skeletonised, adipocere may be found on the bone surfaces and within the 683 bone marrow cavities48, 97, 98 . Pokines and Higgs (2015)99 found adipocere in 20% of cases where 684 bones were recovered in or near the ocean. The adipocere was found adhered to the bone and 685 embedded in exposed cancellous bone. Adipocere within bones is protected from surface erosion and 686 can persist for some time. 687 Moses (2012)100 studied defleshed bovine bones buried in artificial soil, which developed adipocere 688 on the surface of the bone. Results of his studies show that for adipocere formation to occur the 689 presence of a large amount of adipose tissue is not necessary. Laboratory experiments on the 690 decomposition of bone found soft adipocere in bone marrow cavities submerged in pH 4 and pH 7 691 water while hard adipocere formed within the marrow cavities and surface of the bone in pH 10 692 water17. 693 Jopp-Van Well et al. (2016)101 states that adipocere formation within bone is common in conventional 694 burials and they present a case of an almost completely skeletonised corpse with extensive adipocere 695 in the medullary cavities of the femur and humerus. The authors attempted to determine the PMI 696 using radiocarbon dating in conjunction with knowledge on adipocere formation within bones. In this 697 case the radiocarbon dating pointed to the death occurring between 1667-1952, with adipocere not of 698 being useful in further narrowing down the estimation. ro 699 700 8. Analytical techniques for adipocere investigation 701 -p Throughout the study of adipocere, numerous techniques and methods have been developed with the re 702 aim of producing analytical methods that are simple, fast and reliable to effectively examine the lP 703 chemical composition and the mechanism of formation of adipocere. The technique used to analyse na 704 an adipocere sample will depend on the type of sample (e.g. adipocere in the soil or a tissue sample) 705 and the type of result required (e.g. quantitative or qualitative, specific component of adipocere to be ur 706 analysed/identified). Jo 707 A common technique used to study adipocere is gas chromatography-mass spectrometry (GC-MS)20, 28, 31, 102, 103 708 . 709 Bereuter et al. (1997)39 used attenuated total reflectance infrared spectroscopy (ATR-IR) to analyse a 710 tissue sample from Ötzi, the Tyrolean Iceman. ATR-IR has the advantage of being a less destructive 711 technique compared to GC-MS and absorption bands relating to triglyceride and C=O stretching that 712 are visualised using this technique may be utilised to determine the stage of adipocere formation22. 713 However, when analysed with ATR-IR, adipocere samples must either be extracted and cast as a film 714 to be analysed or made up into a solution for analysis21. 715 Stuart et al. (2000)21 used diffuse reflectance infrared spectroscopy. This technique can detect 716 structural isomers, whereas GC-MS cannot, and the sample preparation – allowing analysis of 717 powdered or soil samples containing adipocere – is easier than both GC-MS and ATR-IR. 718 Forbes et al. (2004)14 used Fourier transform infrared spectroscopy (FTIR) alongside GC-MS. FTIR is 719 able to detect and identify triglycerides and calcium salts in the adipocere samples in contrast to GC- 720 MS. 721 Forbes et al. (2005)70 and Stuart et al (2016)58 used inductively coupled plasma-mass spectrometry 722 (ICP-MS) to identify the type of cations and their percentage in the fatty acid salts of adipocere. 723 Algarra et al. (2010)104 used a liquid chromatography-mass spectrometry (LC-MS), method to detect 724 free fatty acids in soil. 725 Solid phase extraction has also been used alongside GC-MS as a quantitative technique for identifying of 726 free fatty acids105. ro 727 Adipocere has also been visualised using multislice computed tomography which produced an image 728 of the distribution of adipocere throughout a body whose subcutaneous tissue was almost completely 729 transformed into adipocere89. -p re 730 Finally, Liu et al. (2010)106 used lyophilization to convert the tissue being analysed into a fine powder lP 731 before being extracted with hexane. The lyophilization increased the amount of free fatty acids na 732 recovered from the sample and the sample was then analysed in conjunction with GC-MS. The 733 lyophilization increases the yield of fatty acids recovered from a sample. ur 734 A comparison of the techniques used for adipocere analysis is shown in Table 3. Jo 735 736 9. Case studies involving adipocere 737 Table 1 in supplementary material reports several case studies involving adipocere. These are 738 particularly useful for forensic investigators as a comparison to current cases. 739 Listed within the table for each case considered is the year in which the case took place, the 740 characteristics of the body (sex, weight, age), the presence/absence/type of clothes, the 741 presence/absence/type of insects associated with the remains, the PMI, the location and distribution of 742 adipocere, the environment the body was found in, the cause of death and anything else notable. 743 The table considers publications produced between 1939 and 2020, selected based on the amount of 744 information provided for each case. 745 The cases were investigated between 1950 and 2015. The PMI ranged from 21 hours up to an 746 estimated 30-150 years. The age range of the victims was between 4 years and 78 years with the 747 majority of the cases involving males. Most bodies exhibited subcutaneous formation of adipocere 748 while only one showed complete adipocere transformation where both subcutaneous and internal 749 organs were transformed into adipocere. The table reports cases from terrestrial environments and 750 aquatic environments, considering both salt and freshwater, open and closed water. The majority of 751 cases are of bodies found in water environments, where the cause of death was generally by 752 drowning. Other causes of death were due to gunshot injuries or sharp or blunt force trauma. Most of 753 bodies were clothed. Only seven cases reported the presence of insects, however, it is known that insects are often neglected in forensic cases and especially in aquatic environments107. ro 754 755 756 -p 10. Operational consideration in cases involving adipocere re 757 Complete or partial adipocere transformation represents a complicating factor in every forensic lP 758 procedure, by reason of both the environmental circumstances of adipocere formation and the na 759 physical difficulties in handling the adipocere connected with the remains. Specifically, adipocere 760 develops in environments which usually prove arduous to access and/or assess in death scene ur 761 investigations, such as aquatic settings or peculiar terrestrial locations (e.g. caves, woods, cemetery Jo 762 tombs and vaults). Furthermore, the greasy texture of adipocere, either crumbly or soft, can mask 763 critical findings on bodies or remains, and definitely increases the risk of injuries in performing 764 external examinations and autopsies. 765 766 10.1. Death scene investigation 767 Unfortunately, and not limited to adipocere cases, when forensic experts arrive at a death scene the 768 body or the remains have almost invariably been touched or moved by rescuers. This is unfortunately 769 not limited to adipocere cases and is usually justified by the need to obtain an identification as soon as 770 possible, through the recognition of facial features and clothing or through the search of personal 771 documents, potentially tucked within clothes pockets. The laborious and uncomfortable handling of 772 bodies and remains, alongside the slippery nature of adipocere, might cause the accidental 773 displacement of associated objects in the surrounding environment. Therefore, in open and/or 774 cluttered terrestrial environments, following the retrieval and removal of the body remains, a visual 775 search in the deposition area and its proximity is recommended. The aim of this search is to recover 776 objects and small body parts that might have detached from the subject as a consequence of the 108-111 777 progression of adipocere transformation or the action of carrion fauna . If possible, the search 778 should be strengthened by the use of a metal detector, to locate metallic objects. In cases of highly 779 decomposed and adipocere bodies, hair is the only biological matrix left for reliable toxicology 780 analyses, and scalp can be detached from the body because of the decomposition process. In aquatic of 781 environments, the use of an amphibious body bag is highly suggested, to prevent the loss of clues which commonly occurs when bodies or remains are pulled out of the water in tarps or nets112. ro 782 783 Amphibious body bags are resistant and reusable, hold a filtered water drainage and can be easily 784 hoisted by helicopters or crane winches. -p re 785 lP 786 10.2 External examination and autopsy na 787 A crucial guideline to follow with a body in complete or partial adipocere transformation is to obtain 788 a comprehensive, and possibly three-dimensional (3D) computed tomography (CT) scan before ur 789 performing any other investigation, such as external examination and autopsy 113-116. This means that Jo 790 the body, or the remains, must be scanned before being taken out of the body bag – either ordinary or 791 amphibious – in which they had been placed in the course of recovery. CT scanning involves X-rays 792 to take cross-sectional 360-degree images of objects. X-rays are blocked in different measures 793 depending upon the material they pass through, so that hyperdense materials, like metals and bone, 794 appear white on the film or on the computer screen, isodense materials present the same shade of grey 795 as the reference structure, and hypodense materials, such as blood and soft tissues appear in darker 796 shades of grey. Air appears very dark, close to black. Hence, CT scans not only detect foreign bodies 797 enclosed in remains 117, for example splinters, bullets and knife blades, but also reveal trauma 118, such 798 as haemorrhages, lacerations and fractures, and pathology, like tumours, surgical stitches and 799 prostheses 119. 800 Once CT scans have been completed, external examinations rarely hold relevant surprises. On the 801 contrary, the autopsy can still be challenging, due to the possible retention of objects within inner 802 body cavities, massive changes to organ and tissue colours, and overall soft tissue decay. Finally, the 803 increased risk of puncture/sharp injuries, connected to the greasy texture of adipocere, ought not to be 804 overlooked 120. 805 806 11. Conclusion 807 Adipocere is a transformative process of corpses and carcasses that has been observed and analysed of 808 for several hundred years16. The general chemical composition of adipocere is well-established and ro 809 has been used to carry out research into the mechanism of adipocere formation as well as attempting 810 to solve the problem of post-mortem interval estimation. 811 -p After the latest review by Ubelaker and Zarenko (2011)16 research has continued into the factors that re 812 influence the formation and the degradation of adipocere, along with the application of new analytical lP 813 techniques for adipocere investigation. na 814 Despite the large body of knowledge available on the topic, the PMI estimation of adipocerous bodies 815 remains difficult. Adipocere formation and the timing of that formation is highly variable throughout ur 816 the body, and is influenced by a wide variety of factors both environmental and body specific. Jo 817 Continued research into the elemental composition of adipocere may aid in further distinguishing 818 adipocere samples into the different stages of formation: early, intermediate and late stage, and may 819 also be useful to differentiate between different water environments in which adipocere has formed. 820 Chemical analysis of the environment in which adipocere forms is also necessary to further 821 understand the variables, such as pH, presence of oxygen, bacteria and water flow, that influence 822 formation58. Furthermore, research in the area of adipocere degradation is suggested, especially with 823 focus on the biochemical pathways and environmental influences that govern adipocere formation42. 824 Lastly, further studies on the bacteria associated with formation and degradation of adipocere are 825 needed, as research around this area is also limited. For example, DNA analysis of bacteria found in 826 association with adipocere could be utilised to determine species and their relative abundance61. 827 Further studies on all of the above areas will assist in a better understanding of the nature and the 828 evolution of adipocere. As per this work, a summary of the key findings so far, the development of 829 reference material outlining the characteristics of several cases and the proposal of guidelines for 830 crime scene and autopsy practice aim to help forensic professionals during present case-work 831 investigations. 832 833 834 References 835 836 1. Payne JA. A summer carrion study of the baby pig Sus scrofa Linnaeus. Ecology. of 837 1965;46:592-602. 838 2. Pinheiro J. Decay process of a cadaver. In: Schmitt A, Cunha E, Pinheiro J, editors. Forensic ro 839 Anthropology and Medicine. Totowa, NJ: Humana Press; 2006. p. 85-116. 840 3. Haglund WD, Sorg M. 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Postmortem abdominal 1107 CT: Assessing normal cadaveric modifications and pathological processes. European Journal of 1108 Radiology. 2011;81(4):639-47. 1109 120. Shaha K, Patra A, Das S, Sukumar S, Mohanty M. AWARENESS OF RISKS, HAZARDS of 1110 AND PREVENTIONS IN AUTOPSY PRACTICE: A REVIEW. Journal of Evolution of Medical and 1111 Dental Sciences. 2013 06/03;2. ro 1112 -p re lP na ur Jo 1 2 Table 1 Factors that inhibit and enhance adipocere formation in water environments. 3 Factor Enhance Inhibit Author (year) Forbes et al. Temperature 21˚C to 45˚C Less than 21˚C (2011)1 O’Brien and If present, can facilitate adipocere formation. This is Kuehner (2007)2; for both natural fibre material (wool, cotton) and Clothing/ If absent, can inhibit (slow Notter and Stuart synthetic fibre material (polyester, acrylic). However, material down) adipocere formation (2012)3; natural fibres can decompose faster and get lost in Tingey et al. water. (2020)4 Skin If absent, can inhibit (slow Widya et al. If present, can enhance/facilitate adipocere formation slippage down) adipocere formation (2012)5 Forbes et al. Scavengers/ If present, can inhibit If absent, can enhance (2011)1; Ueland insects adipocere formation et al. (2014)6 Seawater Yan et al. River Chlorine (2001)7; Stuart et of Water type Distilled Saline al. (2016)8 Deionised ro Partial submersion can potentially inhibit the Notter et al. Complete submersion can enhance, but floating Submersion formation of adipocere, (2009)9; Ueland bodies can also develop adipocere -p especially if floating and et al. (2014)6 exposed to currents O’Brien and re Presence of Gram-negative and Gram-positive (early Lack of Gram-positive Kuehner (2007)2; Bacteria stages) bacteria at early stages Ueland et al. (2014)6 lP 4 5 na ur Jo 6 7 Table 2 Factors that enhance and inhibit adipocere formation in soil environments 8 Factor Enhance Inhibit Author (year) Cold 10 Temperature Warm, 20-40°C Forbes et al. (2005) More than 40°C Scavengers Absent Present Fiedler and Graw (2003)11; Carter et al. (2007)12; Absent Clothing/body • Present Forbes et al. (2005)10; Schotsmans et al. (2011)13; Plastic coverings • Polyester Guareschi et al. (2019)14 coverings • Loam-rich • Clay-rich • Sandy loam • Loamy sand High organic matter • Clay Fiedler and Graw (2003)11; Forbes et al. (2005)15; Gravel Soil type • Clay gravel Fiedler et al. (2009)16; Durães et al. (2010)17; Active soil • Sand Low clay Guareschi et al. (2019)14 • Sterilised content • Silty sand of • Poor draining • Fine particles ro • High, in excess. • Lateral water flow. Evans (1963)18; Forbes et al. (2005)10; Fiedler et al. Moisture Low • Fog and mist at (2012)19 Oxygen time of burial. Low, anaerobic environment -p High, aerobic Forbes et al. (2005)10; Schotsmans et al. (2011)13 re • Directly in soil • Mass grave Pine and spruce Evans (1963)18; Dent et al. (2004)20; Fiedler and Graw lP • Oak, lead and zinc coffins (2003)11; Forbes et al. (2005)10; Green (2006)21; coffins Straw bedding Method of burial Schotsmans et al. (2011)13; Guareschi et al. (2019)14 • Clothing with Wood shavings plastic Lime na • Depth at or below Coffin 90cm Acidic Soil pH Mildly alkaline, pH 5-9 Forbes et al. (2005)10 ur Highly alkaline 9 10 Jo 11 12 Table 3 Comparison of techniques that have been used to analyse adipocere 13 Type of Technique Sample preparation Type of result Author (year) sample Extraction and conversion into methyl Bereuter et al. esters of fatty acids; (1996)22; Extraction and Adachi et al. conversion into (1997)23; Forbes Gas chromatography- Quantitative and Tissue, trimethylsilyl esters of et al. (2002)24; mass spectrometry (GC- qualitative detection of Soil fatty acids; Forbes et al. MS) fatty acids Lyophilization, (2003)25; Forbes extraction and et al. (2004)26; conversion into Liu et al. trimethylsilyl esters; (2010)27 Solid phase extraction Liquid chromatography- Extraction and Qualitative detection of Algarra et al. mass spectrometry (LC- Soil conversion into amide fatty acids (2010)28 of MS) derivatives of fatty acids Attenuated total Semiquantitative Bereuter et al. reflectance infrared Tissue, Cast into film or made detection of triglycerides, (1997)29; Stuart ro spectroscopy Soil up into solution fatty acids and fatty acid et al. (2005)30 (ATR-IR) salts Qualitative detection of Diffuse reflectance infrared spectroscopy Fourier transform Soil powdered KBr -p Sample ground with fatty acids, triglycerides and fatty acid salts Qualitative detection of Stuart et al. (2000)31 re Tissue, Sample ground with Forbes et al. infrared spectroscopy triglycerides, fatty acids Soil powdered KBr (2004)26 (FTIR) and fatty acid salts lP Image of adipocere Multislice computed Whole Jackowski et al. None distribution throughout a tomography body (2005)32 body Inductively coupled na HNO3, HCl and H2O2 Forbes et al. plasma-mass Type and abundance of Tissue added, diluted with (2005)10; Stuart spectrometry cations of fatty acid salts deionised water et al. (2016)8 (ICP-MS) ur 14 15 Jo 16 References 17 1. Forbes SL, Wilson MEA, Stuart BH. Examination of adipocere formation in a cold water 18 environment. International journal of legal medicine. 2011;125(5):643-50. 19 2. O'Brien TG, Kuehner AC. Waxing grave about adipocere: soft tissue change in an aquatic 20 context. J Forensic Sci. 2007;52(2):294-301. 21 3. Notter SJ, Stuart BH. The effect of body coverings on the formation of adipocere in an 22 aqueous environment. J Forensic Sci. 2012;57(1):120-5. 23 4. Tingey E, Verduin J, Dadour IR, Magni PA, editors. Colonization rates of barnacles 24 (Crustacea: Cirripedia) on different fabrics as a tool for forensic investigation of human remains in a 25 marine environment. Proceedings of the American Academy of Forensic Sciences, 72nd Annual 26 Scientific Meeting; 2020; Anaheim, CA. 27 5. Widya M, Moffatt C, Simmons T. The formation of early stage adipocere in submerged 28 remains: a preliminary experimental study. J Forensic Sci. 2012;57(2):328-33. 29 6. Ueland M, Breton HA, Forbes SL. Bacterial populations associated with early-stage 30 adipocere formation in lacustrine waters. International journal of legal medicine. 2014;128(2):379-87. 31 7. Yan F, McNally R, Kontanis EJ, Sadik OA. Preliminary quantitative investigation of of 32 postmortem adipocere formation. J Forensic Sci. 2001;46(3):609-14. 33 8. Stuart BH, Notter SJ, Dent B, Selvalatchmanan J, Fu S. The formation of adipocere in model ro 34 aquatic environments. International journal of legal medicine. 2016;130(1):281-6. 35 9. Notter SJ, Stuart BH, Rowe R, Langlois N. The initial changes of fat deposits during the 36 37 10. -p decomposition of human and pig remains. J Forensic Sci. 2009;54(1):195-201. Forbes SL, Stuart BH, Dent BB. The effect of the method of burial on adipocere formation. re 38 Forensic science international. 2005;154(1):44-52. 39 11. Fiedler S, Graw M. Decomposition of buried corpses, with special reference to the formation 40 of adipocere. Naturwissenschaften. 2003;90:291-300. lP 41 12. Carter DO, Yellowlees D, Tibbett M. Cadaver decomposition in terrestrial ecosystems. 42 Naturwissenschaften. 2007;94(1):12-24. 43 13. Schotsmans EMJ, Van de Voorde W, De Winne J, Wilson AS. The impact of shallow burial na 44 on differential decomposition to the body: a temperate case study. Forensic science international. 45 2011;206(1):e43-e8. 46 14. Guareschi E, Dadour IR, Magni PA. A taphonomic examination of inhumed and entombed ur 47 remains in Parma cemeteries, Italy. Glob J of Forensic Sci & Med. 2019;1:2-8. 48 15. Forbes SL, Dent BB, Stuart BH. The effect of soil type on adipocere formation. . Forensic Jo 49 science international. 2005;154(1):35-43. 50 16. Fiedler S, Buegger F, Klaubert B, Zipp K, Dohrmann R, Witteyer M, et al. Adipocere 51 withstands 1600 years of fluctuating groundwater levels in soil. J Archaeol Sci. 2009;36(7):1328-33. 52 17. Durães N, Cortez D, Algarra M, Sánchez FG, Rodríguez-Borges JE, Bobos I, et al. 53 Comparison of adipocere formation in four soil types of the Porto (Portugal) district. Forensic science 54 international. 2010;195(1-3):168.e1-6. 55 18. Evans WED. The chemistry of death: Springfield: Charles C. Thomas; 1963 56 19. Fiedler S, Breuer J, Pusch CM, Holley S, Wahl J, Ingwersen J, et al. Graveyards — Special 57 landfills. Sci Total Environ. 2012;419:90-7. 58 20. Dent BB, Forbes SL, Stuart BH. Review of human decomposition processes in soil. Environ 59 Geol. 2004;45(4):576-85. 60 21. Green MA. 19th-century pathology: The examination of 83 vault-interred bodies. Forensic 61 Sci Med Pathol. 2006;2(1):19-24. 62 22. Bereuter TL, Lorbeer E, Reiter C, Seidler H, Unterdorfer H. Post-mortem alterations of 63 human lipids — Part I: evaluation of adipocere formation and mummification by desiccation. In: 64 Spindler K, Wilfing H, Rastbichler-Zissernig E, zur Nedden D, Nothdurfter H, editors. Human 65 Mummies: A Global Survey of their Status and the Techniques of Conservation. Vienna: Springer 66 Vienna; 1996. p. 265-73. 67 23. Adachi J, Ueno Y, Miwa A, Asano M, Nishimura A, Tatsuno Y. Epicoprostanol found in 68 adipocere from five human autopsies. Lipids. 1997;32(11):1155-60. 69 24. Forbes SL, Stuart BH, Dent BB. The identification of adipocere in grave soils. Forensic 70 science international. 2002;127(3):225-30. 71 25. Forbes SL, Keegan J, Stuart BH, Dent BB. A gas chromatography-mass spectrometry method 72 for the detection of adipocere in grave soils. Eur J Lipid Sci Technol. 2003;105(12):761-8. 73 26. Forbes SL, Stuart BH, Dadour IR, Dent BB. A preliminary investigation of the stages of 74 adipocere formation. J Forensic Sci. 2004;49(3):566-74. 75 27. Liu C, Park HM, Monsalve MV, Chen DDY. Free fatty acids composition in adipocere of the 76 Kwäday Dän Ts'ìnchí ancient remains found in a glacier. Journal of forensic sciences, 55(4), 1039. 77 doi:10.1111/j.1556-4029.2010.01373.x. J Forensic Sci. 2010;554(4):1039-43. 78 28. Algarra M, Rodríguez-Borges JE, Esteves da Silva JCG. LC-MS identification of derivatized 79 free fatty acids from adipocere in soil samples. J Sep Sci. 2010;33(2):143-54. 80 29. Bereuter TL, Mikenda W, Reiter C. Iceman's mummification—Implications from infrared 81 spectroscopical and histological studies. Chem Eur J. 1997;3(7):1032-8. 82 30. Stuart BH, Craft L, Forbes SL, Dent BB. Studies of adipocere using attenuated total 83 reflectance infrared spectroscopy. Forensic Sci Med Pathol. 2005;1(3):197-201. 84 31. Stuart BH, Forbes S, Dent BB, Hodgson G. Studies of adipocere using diffuse reflectance of 85 infrared spectroscopy. Vib Spectrosc. 2000;24(2):233-42. 86 32. Jackowski C, Thali M, Sonnenschein M, Aghayev E, Yen K, Dirnhofer R. Adipocere in ro 87 postmortem imaging using multislice computed tomography (MSCT) and magnetic resonance 88 imaging (MRI). The American journal of forensic medicine and pathology. 2005;26(4):360-4. 89 -p re lP na ur Jo of ro -p re lP 1 2 Figure 1 Flow diagram of the process of adipocere formation 3 na ur Jo 4 of ro -p re lP 5 6 Figure 2. Particular of adipocere of the hands of a body of an adult male, exhumed from a double na 7 coffin buried in a cemetery ground of the North of Italy. The case is discussed by Guareschi et al. ur 8 (2019)1. Jo 9 of ro -p re 10 11 Figure 3 Body of an adult female recovered from a floodplain in the North of Italy, with PMI lP 12 estimated in 10-20 days days, showing the formation of subcutaneous adipocere. The case is 13 discussed by Guareschi and Magni (2020)3. na 14 ur Jo of ro -p re lP 15 16 Figure 4 Remains of an adult male recovered from a well in the North of Italy, 8 months after his na 17 disappearance. At the recovery, the body was partially dismembered due to the decomposition 18 process, (a) the trunk of the body handled by the forensic pathologist; (b) presence of insects within ur 19 the folds of the clothes; (c, d) adipocere of the left foot, found enclosed in a shoe. The case is Jo 20 discussed by Magni et al. (2013)4. 21 a b c d 22 of a b ro Figure 5. Remains of an adult male recovered from a woodland in the North of Italy, 4-5 months after his disappearance. The feet (a) were covered by socks and enclosed in leather boots (b), creating a -p microenvironment that facilitated the development of adipocere. The case is discussed by Guareschi et re al. (2007)2. lP 23 24 25 na 26 References ur 27 28 1. Guareschi E, Dadour IR, Magni PA. A taphonomic examination of inhumed and entombed Jo 29 remains in Parma cemeteries, Italy. Glob J of Forensic Sci & Med. 2019;1:2-8. 30 2. Guareschi E, Antonopolou C, Frati P, Gnetti L, Magni PA, Lanzi G, et al., editors. Case 31 Report – A suspect of identification: basic forensic anthropology and related techniques as useful, 32 quick and inexpensive procedures for identification of skeletal remains. 3rd Mediterranean Academy 33 of Forensic Sciences combined with 5th Latin-American Congress of Medical Law and 3rd Iberian 34 Congress of Legal Medicine; 2007 21-23 June 2007 Oporto (Portugal) 35 3. Guareschi E, Magni PA. After the flood: a multidisciplinary investigation of human remains 36 found in a floodplain and colonized by raft spiders. Australian and New Zealand Forensic Science 37 Society; Sydney (NSW, Australia)2020. 38 4. Magni PA, Borrini M, Dadour IR. Human remains found in two wells: a forensic entomology 39 perspective. Forensic Sci Med Pathol. 2013 Sep;9(3):413-7. PubMed PMID: 23568015. 40 1 Highlights 2 3 Adipocere is formed from the decomposition of adipose tissue 4 It disrupts the typical decomposition process and affects the PMI estimation 5 Intrinsic and extrinsic factors affect formation and degradation of adipocere 6 Several analytical methods have been developed to analyse an adipocere 7 Typical CSI and autopsy procedures must be adjusted in presence of adipocere of ro -p re lP na ur Jo CONFLICT OF INTEREST STATEMENT Manuscript title: A practical review of adipocere: key findings, case studies and operational considerations from crime scene to autopsy The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria, educational grants, participation in speakers’ bureau, membership, employment, consultancies, stock ownership or other equity interest, and expert testimony or patent- licensing arrangements) or non-financial interest (such as personal or professional relationships, knowledge or beliefs) in the subject matter or material discussed in this manuscript. of Author names ro PA Magni J Lawn EE Guareschi -p re The authors whose names are listed immediately below certify that they have affiliations with lP or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or material discussed in this manuscript. Please specify the nature of the conflict on a separate sheet of paper if the space below in inadequate. na Author names ur NIL Jo This statement is signed by all the authors to indicate agreement that the above information is true and correct: Author’s name (typed) Author’s signature Date PA Magni 16/12/2020 J Lawn 16/12/2020 EE Guareschi 16/12/2020