CHEMICAL ANALYSIS FOR THE DETERMINATION OF
BURNING TEMPERATURE OF HUMAN BONES
Course
:
Forensic Science
Submitted by:
Shambhavi Nataraj 11391596
INTRODUCTION:
Fire is considered to be one of the most detrimental forces in the world, causing damage to the properties and loss of life. Fire is formed by combustion. Combustion is a sequence of chemical processes accompanied by the liberation of light and heat. Since there is generation of heat, the process of combustion can be considered as an exothermic redox reaction. Most fires have molecular oxygen as oxidant. This oxidant contributes up to 20.95% of the reaction (Jackson et al., 2011). Certain other elements also act as oxidants under exceptional situations. The nitrate ion is one such oxidizing agent in fireworks. A fire is said to be self-sustaining only when it meets four conditions; the presence of fuel and suitable oxidant brought together in appropriate amounts, with a supplementation of sufficient energy for the process of ignition. In addition to this, the heat that is generated in the process helps in continuously reigniting the fire. The conditions of incineration also depend on the situation. And the temperature exposure also varies drastically. For example; A house fire might reach up to 700 °C while a petrol accelerated burning motor vehicle can reach up to 1100 °C. A simple campfire reaches an average of 400 °C to 800 °C depending on the fuel that has been used to start the fire. The most natural firestorms have the capacity to reach up to 2000 °C (S.T.D Ellingham et al., 2014).
The heat can be generated by two different processes known as flames and smolders. Flames can be described as the crest of burning gas while smolders are nothing but the production of heat energy in the absence of flames. Flames may emerge from a process known as pyrolysis. Pyrolysis results when heat has an impact on the breakdown of chemicals of a solid fuel, such as coal or wood. Pyrolysis might also result from the vaporization of a liquid fuel (ex. Petrol) or fuel itself being a gas such as methane. When solid fuels burn, they give rise to smolders (smoke). Pyrolysis of organic solid fuels like wood results in both flammable gas and char. The char, in turn, experience smoldering combustion (Jackson et al., 2013).
When it comes to incidents of fire under forensic relevance, the temperature at which the body or bones have been exposed play an important role in terms of even reconstruction. Fire modifies the human body as whole resulting in structural and physical deformities. Bohnert et al. have made some observations on the degree of destruction of human bodies relating to the duration of the fire. In their study, it is mentioned that the whole body might show “pugilistic attitude” after 10 minutes at a temperature between 670 and 810 °C. The calvaria becomes free from the soft tissue at 20 minutes, while at around 30 minutes the body cavities become visible with the exposure of organs. Around 40 minutes the internal organs shrink and show a net-like structure (Michael Bohnert et al., 1998). In fire accidents, when the body is completely burnt, bones are still present and are used for the investigations. The alterations related to bones after the body has totally burned away are studied thoroughly to know the condition of the person (dead or alive) and type of fire at the time of the incident. The investigation also helps in the determination of temperature to which the body was exposed. The incinerated human body is found to be in four different states namely, charred remains, partially burnt, incompletely, or completely burnt. The charred remains can be analyzed easily, without any exceptions, through serological tests or visual identification. Partially cremated remains will have an amorphous mass that consists of the organs and bony tissue. However, there is a greater problem in the taphonomic analysis and identification of an incompletely incinerated body as the bones become discolored, shrunk and unrecognizable (Pamela M. Correia, 1997). Generally, the bone undergoes many notable changes. Though these changes haven’t been fully understood, the morphological, mechanical and internal
changes of the bones due to extreme heat can be depicted as in Figure 1 (T.J.U Thompson.,
2005).
Figure 1: The changes occurring in bones at different heat-regimes (taken from T.J.U Thompson., 2005)
In order to understand the alterations occurring in bones due to rise in temperature, a wide range of observation and chemical research techniques have been applied. But these applications have produced results that are nonpareil with each other. Characterization of a burnt bone using chemical techniques and other methods is known to have come from various sources such as the medicolegal and anthropological communities. The characters of the burnt bone are classified into two sectors named, visual characters and histological characters. These characters are seen due to step by step stages of dehydration, decomposition, inversion, and fusion. These stages overlap at a certain temperature and they are connected with one another (Megan J. Richardson., 2017). Shipman et al. conducted a research on the study of color, morphology, crystal structure and shrinkage of bones and according to them, the morphological changes occur with five different heating stages. They are as follows:
➢ First stage: 20-<180 °C; Even though the bone texture is normal, the surface seem to surge a little.
➢ Second stage: 185-<285 °C; the surface is now distorted and become little, sharp granular. These irregular granules are divided by fissures.
➢ Third stage: 285-<440 °C; Disappearance of the asperities takes place followed by the bone turning glassy and smooth.
➢ Fourth stage: 440-<800 °C; the bone particulates in the beginning of this stage, but eventually becomes frothy.
➢ Fifth stage: 800-<940 °C; the particles of fourth stage melt and amalgamate to a large structure.
The visual and histological observations of a bone for the determination of the temperature at the time of burning depend on the following four examinations:
● Change of color: The study of change in color of the bone is one of the earliest studies in the analysis of burnt bones. It is considered as a benchmark analysis to determine the temperature range to which a bone has been exposed. Due to the incineration of the organic materials carbon and collagen (carbonization) of the bone elements, a fresh bone will have an ivory color
in the beginning and changes to brown and black gradually (S.T.D. Ellingham et al., 2014). According to Shipman, the burnt bones unveil color change gradually from brown -> gray-blue -> black -> gray -> gray-white -> chalk white (Shipman et al., 1984). The change in the color of burnt bones depend on other factors such as the positioning of the bone to the heat source, exposure time, availableness of oxygen and also the inorganic and organic material associated with the body. The observations made from the change of color can help one argue about the type of fire the bone has been exposed to and the maximum temperature that has been achieved. But the drawback of this analysis is that there might be different colors present on one single bone fragment or the distribution of all colors might be seen in one single incineration. Various methods have been suggested for colour analysis and Munsell Soil Colour Charts are one of the most commonly used charts for colour comparison of the burnt bones. Even though colorimetry could be used for the analysis of colour, there is no sufficient database to compare the findings for any of the colour models. And also, in general, these colour estimations are subjective and only a few researchers have studied the accuracy and precision of these analyses (T. Krap et al., 2017). According to Rebecca A. Nicholson and her research on the morphological investigation of burnt animal bone and an evaluation of its utility in archaeology, the results suggested that mammal and non-mammal bones might experience the same range of color changes with rising of temperature. But, there is a huge difference in temperature in both types of bones achieving the same stage (Nicholson A., 1992). This might indicate that there is variation in the organic content and bone chemistry between mammals and non-mammals. Table 1 represents the color observations made by 3 different groups of people with different designs of experiments. Since the effect of heat on human body depends on various factors, the color changes seen cannot decipher the exact temperature at which the bone was burnt. This might be quite difficult for forensic analysts to conclude anything on a burnt victim in an event involving fire.
(Table 1 of 1) Temperature
in °C
Color observation Temperature in °C Color observation 100 1. Yellow-white 2. Neutral white/yellow 3. Yellowish 600 1. Grey 2. Black/blue-grey/some reddish yellow patches 3. Black 200 1. Yellow-white 2. Neutral white/yellow 3. Yellowish 700 1. Milky-white
2. Mostly white/some grey-blue patches 3. Light grey 300 1. Brown-black 2. Reddish-brown/dark brown/dark grey/reddish yellow 3. Dark grey 800 1. White
2. Mostly white/some grey-blue patches 3. Light grey 400 1. Brown-black 2. Reddish-brown/dark brown/dark grey/reddish yellow 3. Grey-black 900 1. White
2. Mostly white/some grey-blue patches
3. White
2. Black/blue-grey/some reddish yellow patches 3. Black 2. White/some reddish-yellow patches 3. White
Table 1: Variations in color of bones under different temperature (taken from S.T.D. Ellingham et al., 2014) (Table 2 of 1)
The above table represents the color change in three different experimental designs. 1 indicates the color observation made by Wahl J., in the year 1981; 2 indicates the color observation made by Shipman et al., in the year 1984; and 3 indicates the color observation made by Quatrehomme G. et al., in the year 1998.
● Deformities and shrinkage: Reduction in length and width along with the weight of the bone also occurs simultaneously with the color change when the bone is under high temperatures. Color change and weight loss indicate a loss of organic matter and water leading to dehydration which eventually leads to the shrinkage in bones. Sometimes the shrinkage of bones may alter the decisions of forensic anthropologists about the sex, age, and stature of an individual. But, according to T.J.U. Thompson’s research about heat-induced dimensional changes in bone and their consequences for forensic anthropology, the results also indicated some kind of expansion, important for the analysts. The study finally concludes that experiments carried out under controlled environment resulted in the structural deformities of bones. These bones were either shrunk or expanded as a result of heating. This eventually affected the techniques used to examine the shrinking of bones and resulting in the reduced accuracy of the techniques themselves. The accuracy of this method is low as the deformities formed under heat (shrinkage or expansion) will definitely affect the uni-variate, bi-variate and multivariate methods. There is a lot of misclassification in the groups as they are confused with one another (T.J.U Thomson., 2005). Examination of bone deformities is hence not a straightforward analysis to conclude the burning temperature of the bone.
● Skeletal analysis: The first and foremost thing to do in the skeletal analysis is to verify whether the burnt bone is from animal or human origin, the minimum number of individuals constituting and how much fragment have been survived. When a body is incinerated, the compact bones will break into little fragments. At extremely high temperatures, the clavicles, carpals and sacral vertebrae are totally destroyed. These are less denser bones which have the least chances of survival. Apparently, the denser bones and the bones attached with muscle tissue can be used for the analysis. Since they are also deformed, the analysis wouldn’t be of much help for the determination of the burning temperature (Pamela M. Correia, 1997). But, before any analysis is carried out, the burnt fragmented bone should be identified as either animal or human bone, for further analysis. According to Catteneo et al., the burnt bones could be identified as animal or human using certain histological observation. The significant difference in animal and human bones are the mean size of osteon and the Haversian canal and also the lamellar pattern formed by primary osteons in animals. This lamellar pattern is absent in human bones (Cattaneo et al., 1999). Apart from the microscopic observations of these differences, various other methods have also been applied to differentiate the burnt bones. Immunological methods and DNA analysis might be considered to be of much importance. In the immunological methods, inhibition ELISA can be applied to detect the human albumin. Albumin is chosen for its extreme species-specificity and its survival till a temperature of 300 °C. For DNA analysis, mitochondrial DNA is used as a human-specific marker. The main drawback of these identification methods are
that if a bone is burnt at extreme temperatures, then it is hard to find both human albumin and mtDNA due to recrystallization (C. Cattaneo et al., 1999).
● Patterns of fracture: A fracture of bone or the alteration taking place depends on whether the bone is dry, semi-fleshed (when there is partial muscle tissue on the bones) or completely fleshed (bone completely covered by the muscle and other tissues) at the time of burning. While the dry bone shows serrated fractures at epiphyses and parallel-sided fractures in the shaft, the flesh bone show diagonal cracking, transverse and serrated fractures along with some twisting due to the muscle pull. According to Pamela M. Correia, methods for visual observation of fractures are not adequate to differentiate the conditions of bones. Even though there enough research done on this particular topic, none of the methods are standardized. And, there are also contradictions between researchers (Pamela M. Correia, 1997).
● Light microscopy: R.A. Nicholson worked with mammalian and non-mammalian (reptiles and other small animals) bones to establish the differences they possess after they have undergone heating. For interpreting the surface morphology, light microscopy was used in this study. Here the fresh bones were pre-treated by washing with detergent and brushed with alcohol and acetone. The specimens were then viewed through a light microscope with 10X to 40X magnifications. Unlike the scanning electron microscope, colors may be viewed in the light microscope. Nicholson recognized a number of temperature-related aspects. Below table (Table
2) is the representation of the microscopic appearance through the light microscope at different
temperatures.
(Table 1 of 2)
Temperature in °C
Macroscopic appearance Microscopic appearance at 40X
Stage 1
20 Gently undulating, continuous surface Gently undulating, continuous surface, penetrated only by vascular canals 200 Very similar to fresh bone, but with a
greasy, alsmost plastic surface on the articular ends
Where the glassy surface has been removed the articular surfaces are rougher and more granular than fresh bone, and the vascular canals are more prominent.
Stage 2 300 Large area are covered with glass-like, bubbly layer of char
Large areas are covered with a peeling, bubbly layer, beneath which is a granular surface with clear, regular vascular canals
Stage 3
400 The glass-like surface has largely combusted, revealing a flat, granular, occasionally cracked surface
The surface is flat and granular, and vascular canals are numerous and clearly visible. Polygonal cracking can be seen on and around the articular
surfaces 500 The articular surfaces show extensive
polygonal cracking and many areas appear powdery
The surfaces show extensive polygonal cracking, with the edges of the cracks beginning to curl upwards. The bone appears pitted and granular
600 The surface appears powdery and extensively polygonally cracked
The surfaces of the bone are similar to 500°C, with the edges of the cracks
curled upwards. Individual particles sit on the bone surface.
Stage ¾ 700 The bone surface is extensively cracked, but some cracks seems to be superficial. Some powderey areas.
The articular surfaces are extremely variable in color and appearance. Some surfaces appear pitted and very granular while others appear smooth; this seems to be related to the color of the bone
Stage 4 <800°C->900°C
The bone appears chalky and smoother than before. Much of the cracking has disappeared
The bone appears smooth, and most of the cracks have become infilled, making them shallower and more rounded at the edges. The vascular canals are less prominent than at lower temperatures.
Table 2: The heating stages of mammalian bones and the microscopic observations at each stage (adapted from R.A. Nicholson 1993) (Table 2 of 2)
Some researchers have also used light microscopy to differentiate between burnt and unburnt bones. According to them, the bones could not be distinguished at very low temperature but, some little changes were reported at medium or high temperatures. Especially, the presence of cracks extending out of Haversian canals was observed at medium temperature and loss of histological structures was seen at high temperature (David Goncalves, 2012).
R.F. Castillo et al. studied the effects of temperature on bone tissue using optical microscope at 100X. They used 165 samples of bone biopsy of left ilium from forensic cadaver autopsies for the study. The samples were exposed from 100-1100 °C with an interval of 100 °C. The samples were decalcified using dehydration in 96% alcohol and inclusion with methyl methacrylate. Samples were stained with hematoxylin-eosin, toluidine blue and Goldener’s trichrome. They recorded the histological changes occurring in both the cortical and spongy bone. They also have classified the heat-related changes into four categories. Table 3 represents these categories.
Table 3: Histological changes occurring at different temperatures (Taken from R.F Castillo et al., 2013)
Basically, this study talks about the changes occurring in the bone through collagen polymerization and hydroxyapatite crystallization. Though the above table represents the whole changes in between 200 °C, the study also shows the changes occurring in each and every 100 °C rise in temperature. Figures 2-12 shows the exact changes happening at the single rise in temperature (R.F Castillo et al., 2013).
Figure 2: 100°C; appearance of micro-factures and separation of collagen fibers due to the fractures (Taken from R.F Castillo et al., 2013)
Figure 3: 200°C; Bar
arrangement of the collagen fibers for the more
analogous arrangement (Taken from R.F Castillo et al., 2013)
Figure 4: 300°C; Partition of fibers due to actual fracture (Taken from R.F Castillo et al., 2013)
Figure 5: 400°C; Formation of uneven surface from closely bound fibers
(beginning of crystallization) (Taken from R.F Castillo et al., 2013)
Figure 6: 500°C; Deformed tissue turns crystalline (cubical in shape), arrival of macromolecular crystalline polymer phase (Taken from R.F Castillo et al., 2013)
Figure 7: 600°C;
Disappearance of cubical crystals and the matrix gains irregular crystalloid structure (Taken from R.F Castillo et al., 2013)
Figure 8: 700°C; Arrival of large round crystalline structures (Taken from R.F Castillo et al., 2013)
Figure 9: 800°C; Crystals expand and also starts melting with others (Taken from R.F Castillo et al., 2013)
Figure 10: 900°C; The melting results in little round crystals (Taken from R.F Castillo et al., 2013)
● Scanning Electron Microscopy (SEM): Scanning electron microscopy (SEM) is another observation based technique used to look for the heat-induced changes in the microstructure of the bones. SEM can give results about the morphological changes happening at the ultra-structural level. Some of the morphological changes include recrystallization of the crystals at the beginning of the bone mineralization.
J.L. Holden et al. conducted two different experiments on the incinerated human femoral bone. The bone was incinerated over the range of 200-1600 °C with different time durations of 2, 12, 18 and 24 hours at the rate of 50 °C/min. The samples were cooled and then observed under SEM. The results of SEM at a categorized range of temperature are given below:
● At the range of 200-600 °C for 2 hours: The original structural features were unaltered, but there were bundles of mineralized collagen fibers. The most change occurred in Haversian canal by the disintegration of the endosteum at around 200 °C. Flaking of the membrane was observed along with the process of shrinkage.
● At 600 °C: The endosteum was completely destroyed. The underlying skeleton was completely exposed. At higher magnifications like 10000X, chances of finding new crystals as the indication of recrystallization were observed, which might suggest that recrystallization started at around 600 °C.
● At 800-1600 °C for 2 hours: Various heat-induced changes were observed between 800-1400 °C. Mainly two crystal morphologies, one being spherical morphology as seen in 600 °C and the other one being a new hexagonal morphology, were observed. These hexagonal crystals were found to be increasing in its dimensions at around 1200 °C. But the size remained constant after the rise of temperature from 1200 °C and above. The hexagonal crystals tend to fuse within the same localized areas at a temperature rise between 1000-1400 °C.
● At 1000-1400 °C: Presence of new crystal morphologies was also observed. These crystals were spread randomly.
● All the above mentioned structural morphologies disappeared when the temperature was raised to 1600 °C. The bone became glass-like at this temperature.
When the bone is heated for a prolonged period, time would have a different effect on the bones and their ultrastructure. Most of the components observed at 2 hours would be destroyed with the same heat regimes for the extended periods (J.L. Holden et al., 1995).
Figure 11: 1000°C;
Microcrystals are seen with the removal of the previous round crystals (Taken from R.F Castillo et al., 2013)
Figure 12: 1100°C; Microcrystals combine together forming linear cord-like reticular shapes with large lacunae between them (Taken from R.F Castillo et al., 2013)
From the above mentioned studies on the observation of the burnt bones, it is pretty much clear that one cannot clearly report on the burning temperature of the bones based only on the observation and microscopic analysis. It might lead to more errors and false prediction of results. More standardized and accurate techniques must be used to provide an empirical data on the burning temperature of bones. This requires the study of the microstructural and histological aspects of the burnt bones using chemical techniques which brings us to our research question below:
“Is it possible to determine the temperature to which the bone was exposed, based on chemical analysis?”
In an attempt to answer this question, few analytical techniques have been discussed which were used to determine the exposure temperature of bones at the time of fire.
OVERVIEW:
An array of methods and techniques for analyzing the microstructural histological changes of burnt bones exist, including thermogravimetry, X-ray diffraction, Fourier transform infrared (FTIR) absorbance spectroscopy. In this review, the emphasis is given to the most commonly, regularly used techniques that give faster accurate results and also comparison study between them, since the main research question of this review is to find more accurate and precise techniques there is also a sub-question about the empirical data, accuracy and precision of these techniques.
VARIOUS CHEMICAL ANALYSIS FOR TEMPERATURE ESTIMATION OF BURNT BONES:
Burnt bones cause numerous problems for the identification. When the bone is severely damaged, finding sufficient DNA for profiling is not an option. In these situations, one has to rely only on the anthropological results for the identification. One must obtain possible information with the available frangible bone fragments that have undergone extreme heat regimes. An accurate result on determining the exposed temperature can be achieved by chemically analysing the histological aspects of the burnt bones since, visually used techniques for observation of both morphological and histological aspects might be not very reliable. Some of the most commonly used and successful chemical techniques are discussed below.
THERMOGRAVIMETRY (TGA) AND DIFFERENTIAL SCANNING COLORIMETRY (DSC)
When the bone tissue is heated, it experiences a change in weight due to loss of water, pyrolysis of organic components and the loss of carbonates and crystal fusion. The examination of the change in these three phases is done by thermogravimetry analysis. Differential scanning calorimetry is another thermally analyzing technique used for determining the temperature at the time of the burning of bones. According to studies, TGA with DSC gives essential information about the properties of bones when they are exposed to high temperatures (Lozano L.F. et al, 2003). Differential scanning calorimetry is also used to examine the properties of hydroxyapatites. Both TGA and DSC measure the effects of temperature and the corresponding chemical reactions with phase transitions in the bones.
The TGA and DSC are used for the determination of the chemical changes i.e., internal changes occurring in the bone as a result of external and mechanical alterations from heat exposure. The internal changes are categorized into four phases corresponding to the exposure to different heat regimes. The four phases are as follows:
● Dehydration- removal of water content in the bone. Occurs below 600 °C. ● Decomposition- removal of organic components. Occurs between 500-800 °C.
● Inversion- loss of carbonates and beginning of recrystallization. Occurs between 700-1100 °C. ● Fusion- melting of crystal. Occurs above 1200-1600 °C (C. de Becdelievre et al., 2015).
The four phases occur in both animal and human bones but with little changes in between the mammals. Comparison between bovine, porcine and human bone was done in one of the studies. The thermogravimetric analysis with an interval of 10 °C/min showed that both human and bovine bone exhibit similar water contents while porcine bone had higher water content in the first phase. In the second phase, there was a 4% difference between the species. But, the third phase showed the same results in all the three species (S.T.D Ellingham et al., 2015).
S.T.D Ellingham et al. did thermogravimetry analysis on domestic sheep rib bones. The samples were heated from 20-1100 °C in triplicates with heating rates of 6 °C/min, 12 °C/min and 24 °C/min to a maximum of 300 °C and 400 °C. The weight loss was seen in all the three different heating patterns.
Figure 13 shows the results of TGA curves of bones subjected to different temperatures. To calculate the
exact temperature changes with the weight change, they used the first derivative of the TGA curve (Δmass/Δtemp). The first derivative peak is the point where one can see the highest rate of change on the
weight loss curve (Figure 14).
Figure 13: TGA results of bone curves at different heating system (Taken from S.T.D Ellingham et al., 2015)
Figure 14: Bone curves from 1st derivative (point at which the weight loss is at its peak) at different heating system (Taken from S.T.D Ellingham et al., 2015)
The difference in peaks with the rate of heating the weight loss is given in the below table.
Rate of heating Peak in the weigh lo
At 6°C/min At 100°C, 350°C, 475°C and 750°C
At 12°C/min At 125°C, 375°C, 500°C and 775°C
At 24°C/min At 150°C, 400°C and 800°C
As for DSC, Figure 15 shows the flow curves with an approximation of 25°C delay for every temperature rise of the matrix phase changes. This can be reasoned by a heat exchange endothermic process that is taking place until a rise up to 350°C with a peak between 100-150°C. This endothermic reaction is suppressed by an exothermic reaction around 450°C. At 1100°C they saw three different weight losses with the largest being at the interval of 6°C/min (40.4%). The percentage of weight loss was 42.3% and 45.9% at 12°C/min and 24°C/min, respectively (S.T.D Ellingham et al., 2015).
The phase changes mentioned above mainly depends on the rate of the heat exposure and also the duration of the exposure. Some of the phase changes at certain prolonged exposure may be characteristical. But this time elongation doesn’t result in the entry of the next phase. So both, TGA and DSC are helpful in collecting the information and to better understand the behavioral and structural studies of the bone that are exposed to different heat regimes (S.T.D Ellingham et al., 2015).
L.F. Lozano et al. did thermal analysis study of human bone with different techniques including thermogravimetry. They u sed healthy human skull (S-bone) and adult human radius (R-bone) as their samples. From the fragments of skull, type I collagen was extracted by two different extraction techniques and named as EXT-collagen and EDTA-collagen. The powdered S-bone, R-bone, EXT-collagen, and EDTA-collagen were analyzed by TGA. For TGA experiments 20 mg of samples were used. In the results, the thermograms showed complete evaporation of water signaling 10% of weight loss before 200°C. In the next phase, there was 24% and 29% loss of collagen mass in R-bone and S-bone, respectively. There was also an increase in percentage up to 80% in EXT-collagen and 90% in sigma-collagen. There was 2% loss of mass for S- and R-bones at a temperature interval from 600-800°C. Figure
16 and 17 shows the results of the first derivative of TGA (Lozano L.F. et al, 2003).
Figure 15: DSC results of bone curves at different heating system (Taken from S.T.D Ellingham et al., 2015)
Figures 16 and 17: Thermograms with 1st derivatives (in the dotted plot) of R-bone, S-bone, EXT-Collagen, and Sigma-Collagen (taken from Lozano L.F. et al., 2003)
Coming to the results of DSC, the authors observed different exothermic transitions for different collagens. While the EXT-collagen shows peaks at 340°C, 480°C, and 530°C, the EDTA-collagen shows one change close to 500°C. Sigma-collagen shows the changes even before the EXT-collagen at around 185,300 and 490°C, respectively. Figure 18 represents the exothermic transitions occurring in each sample.
From the results of TGA and DSC experiments, it can be established that the extracted collagen samples are thermally stable than the collagen present inside the bone. This is due to the building up of cross-links between the denatured proteins giving rise to a whole new structure, which cannot be found in the bone due to lack of gap zone because of thermal exposure (Lozano L.F. et al, 2003). TGA is one of the valuable techniques to gain complete understanding of how bones act under thermal pressure. TGA shows the matrix change within a certain phase. But when the bone is exposed to certain temperature, there could be progression in the character within a phase and not the change in phase itself (S.T.D Ellingham et al., 2015). TGA and DSC could be used as lab techniques under conditions but is hard to use for the actual cases.
Figure 18: Representation of DSC curves of all the three collagen samples (taken from Lozano L.F. et al., 2003)
X-RAY DIFFRACTION (XRD)
The bone is a complex composition including mineral matrix. The mineral matrix is carbonated calcium hydroxyapatite having heteroionic substitutions. The hydroxyapatite mineral phase is the main inorganic phase of bones. The effects of extreme temperatures on the bone mineral matrix are studied using X-ray diffraction (XRD). The main aim of XRD is to study the characteristical changes of the microstructure of the bone due to crystallization as a result of exposure to extreme heat, meaning it measures the crystallinity index (J.C. Hiller et al., 2003). XRD is considered to be the ideal method for describing crystallinity parameter of bioinorganic phase. The XRD pattern involves 3D periodicity i.e., the degree is assembled in all the three dimensions of the cell (P Giampaola et al., 2016). This can be explained as the contact of the crystals and the monochromatic X-ray. The production of different waves in the results can be due to the diffraction angle and lattice spacing of the sample.
When a bone undergoes extreme heat treatment, a part of inorganic apatite component transforms to β-three-calcium-phosphate phase (β-TCP). The shape of the v4-v3 bands are affected by
the mineralogical phases (due to taphonomical changes). This, in turn, affects the CI calculation. CI is the profile of hexagonal apatite in an angular range of 15-120° in 2θ (S.T.D Ellingham et al., 2014).
K.D. Rogers et al. have studied the effects of heat treatment on cortical bone mineral microstructure or can also be described as a crystallographic study. The specimens were heated up to 1200°C and the mineral composition was studied using an XRD. Sections of cortical bone from the epiphysis of trauma victims were collected and washed in chloroform/methanol solution. The samples were heated from 200-1200°C with an interval of 200°C for 2 hours. The specimens were then air cooled to -20°C and pulverized carefully. After performing XRD, the diffractograms were then subjected to microstructural analysis. The crystalline phases were identified at each temperature (Table 4).
From the above table, we can see that most of the significant changes occur around 600-800°C. Most crystallinity changes happen in this region. There is a growth of crystals which might be due to lattice carbonate. At above 800°C, the size of the crystallite remains the same. At 1200°C, there are signs of calcium hydroxyapatite (CHA) decomposition.
The diffraction data of this experiment shows less peak width between 400-600°C. It steadily decreases around 1000°C. The only apatite shows significant diffraction but there is no indication of β-TCP. Ca2P2O7 has limited proof at 600-700°C. Figure 19 describes the diffraction data of the sample (K.D.
Rogers et al., 2002).
Table 4: The change in phase at each time interval (taken from K.D. Rogers et al., 2002) Table 4: The change in phase at each time
Another study was conducted by G. Piga et al for the analysis of burned human remains using powder XRD for the hydroxyapatite phase. But at very high temperatures, there might be the presence of other minerals too. This study was conducted to understand the mineralization of bones in high-temperature regimes. The samples underwent heat treatments at 200-1000°C. The time interval was 0, 18 and 60 min and the rate of heating was 20°C/min. As for the XRD, powdered samples were taken. From the quantitative Rietveld analysis points, it is considered that hydroxyapatite to be the main phase. But, both in untreated and heat treated bones, hydroxyapatite is not the only phase. Figure 20 gives the result of the XRD patterns with a scattering angle of 2θ.
According to the Rietveld analysis, there is a significant growth of hydroxyapatite crystallites due to an increase in temperature. Figure 21 shows the behavioral changes occurring in the samples that have been treated above 650°C. Even in XRD, there is a sudden increase in the size of the crystallite around 750°C.
Figure 20: The XRD patterns here shows both hydroxyapatite crystals and calcite. The Calcite is only seen after 700°C suggesting that there is de-carbonation (Calcium carbonate->Calcite) reaction occurring at around 775°C (taken from G. Piga et al., 2008)
Figure 19: Diffraction data of the bone treated at different heat regimes. CHA shows a corresponding increase in the peaks with each rise in temperature. CaO peaks are present at 700°C. The sharp peaks from 100 to 400°C is from the internal standard Al (taken from K.D. Rogers et al., 2002)
Tables 5 and 6 suggest that the average crystallite size of hydroxyapatite phase is almost the
same in both XRD and Rietveld investigations, respectively.
The above-mentioned results suggest that both XRD and Rietveld investigations can be combined to see the mineral changes occurring in the heated bones. This evidence is supported in another study performed by P. Giampolo et al. There is presence of β-TCP in the XRD patterns suggesting that with Rietveld investigations of XRD, the occurrence of new minerals after 700°C is common (Figure 22).
Figure 21: Evolution of the hydroxyapatite crystallite size corresponding to temperature rise. The sigmoid curves of the logistic type are fitted with the Rietveld analysis. In the figure, there is still some uncertainty about the heat treatment of the bone (taken from G. Piga et al., 2008)
Table 5: XRD results of the average size of the crystals of HA phase at different temperature regimes and time durations. Here 1 Å = 10-10 m (taken from G. Piga et al., 2008)
Table 6: Rietveld investigation on the average size of the crystals of HA phase at different temperature regimes and time durations. (Taken from G. Piga et al., 2008)
As mentioned earlier, β-TCP phase occurs as a transformation part of hydroxylapatite phase at higher temperature and it effects the shape of the bands. The calculation of crystallinity index due to the presence of β-TCP, by one single method may be inaccurate. Therefore, XRD might require a combination of other physicochemical techniques such as ATR-IR, TGA, XRF etc. to actually know the burning temperature of the bones (P Giampaola et al., 2016).
SMALL-ANGLE X-RAY SCATTERING (SAXS)
SAXS is considered to be the complementary method to XRD which gives more accurate results about the crystallite size and shape during heating. A study was conducted by J.C. Hiller et al. to see the mineral changes during heating of bones. The study was conducted using XRD with wide-angle X-ray scattering (WAXS) and complementary method small-angle X-ray scattering (SAXS). Cortical bone of sheep was used as the samples for the experiment. They also noted the temperature that the samples were treated at and the weight loss in percentage of each of the sample at different temperatures and duration (Table 7). Samples were put in a pre-heated furnace to avoid the impact of extreme heat regimes that influence the microstructure of the burnt bone. The samples were used for scattering measurements and the data was collected over 3 or 9 hours of exposure using 1.25 m as the sample to detector distance for SAXS, and 22.5 cm for WAXS.
Table 7: Percentage of weight loss measured in each sample at different temperature and heat regime (taken from J.C. Hiller et al., 2003)
Figure 22: XRD patterns suggesting the presence of β-TCP peaks at around 700-1100°C
As for the results, they observed peaks corresponding to the reflections of carbonate hydroxyapatite at the same temperature changes that as the XRD, both at 15 and 45 minutes. There is a slight shoulder on the 3.6-3.8 nm-1 narrow peak at 700 or 900°C (time interval 15 minutes) (Figure 23). The peaks corresponding to hydroxyapatite is much narrower when the time interval is 45 min (Figure
24) and the temperature was 900°C and which talks about the crystalline hydroxyapatite. WAXS shows
almost the same results for control samples that are unheated (Figure 25)
SAXS provides the crystal thickness up to 50 nm and here the results showed alterations in the morphology of the crystals along with heat treatment. SAXS provided complementing and more accurate results as that of XRD. It also gave size and shape that are independent of each other and also from the crystal lattice. Table 8 represents the thickness of crystals in each sample. In conclusion, SAXS shows fine-scale changes in the size of CHA at increasing temperatures. This can be used along with XRD for more accurate results.
Figure 23: XRD results of the samples at 500°C (lower), 700°C (middle) and 900°C (topmost) showing the peaks of crystalline hydroxyapatite. The samples are heated at respective temperature for 15 minutes (taken from J.C. Hiller et al., 2003)
Figure 24: XRD results of the samples at 500°C (lower), 700°C (middle) and 900°C (topmost) showing the peaks of crystalline hydroxyapatite. The samples are heated at respective temperature for 45 minutes (taken from J.C. Hiller et al., 2003)
Figure 25: Wide-angle XRD results of unheated control samples (taken from J.C. Hiller et al., 2003).
FOURIER TRANSFORM INFRARED SPECTROSCOPY (FTIR)
Most of the thermal experiments on bones are done using FTIR as it is one of the most accurate techniques that give accurate results about the burning temperature of the bones. Though XRD is considered to be the ideal method for the measurement of crystallinity index, FT-IR provides a fingerprint of the chemical environment. This is obtained by the excitement of the bond vibrations by IR beam. FT-IR is more advantageous in examining fresh bones. In addition to this, FT-IR is portable, require smaller samples and cheaper to use (P Giampaolo et al., 2016).
There are two types of FTIR methods to measure the burnt bones. They are as follows:
1. FTIR-KBr: This method uses potassium bromite pellets as a support medium. This type is used for absorption measurements. Handling KBr is very risky and also FTIR-KBr is more susceptible to human errors.
2. FTIR-ATR (attenuated total reflectance): This method is used to reflect the internal properties when the beam is in direct contact with the sample.
Though both methods give almost the same kind of results, they cannot be compared with each other. FTIR-ATR is the most suggested method amongst the two methods as the sample preparation is easier compared to FTIR-KBr (S.T.D. Ellingham et al., 2014). The change in the bone internal structure due to heat treatment, according to FTIR-ATR is given in Table 9.
(Table 1 of 9) Temperature in °C (±50°C) Temperature in °C (±50°C) Unburnt 600
Table 8: Hydroxyapatite crystal thickness at different temperature and time duration (taken from J.C. Hiller et al., 2003).
100 700
200 800
300 900
400 1000
500
T.J.U Thompson et al. conducted a statistical approach for determining the CI of the heat-treated bone mineral from FTIR spectra. In this research, a statistical study of the FTIR spectra was studied using statistical classification model (LDA). The CI was calculated using the formula; CI = (565 cm-1 + 605 cm-1)/595 cm-1, each value being the absorbance at given wavelength. 565 and 605 cm-1 corresponds to phosphate’s bending vibration band. Both of them are directly proportional to CI, whereas 595 cm-1 is inversely proportional to the CI. The following table (Table 10) suggests the relationship between the wavelength and structural composition of bone.
Table 9: The changes in bone is recorded using FTIR-ATR at different heating regimes (adapted from S.T.D. Ellingham et al., 2014)
The bone minerals were examined using FTIR-ATR method. The samples were heated from 100-1100°C with 100°C interval for 45 minutes. In this study, the Carbon/Phosphate (C/P) ratio was also studied along with the crystallinity index (CI). Figure 26 describes the 'within the variation of the C/P ratio' in the bone due to different heat regimes.
Not only the ratio of C/P was calculated but also others including CO/P, CO/CO3 and CO3/P at
wavelengths around 1650 cm-1/1035 cm-1, 1650 cm-1/1415 cm-1 and 900 cm-1/1035 cm-1, respectively. Line width and phosphate high temperature (PHT) was calculated as well and is represented in the table (Table 11) below.
Table 11: CI, with ratio variance, PHT and line width at different heating regimes (taken from T.J.U Thompson et al., 2013)
Table 10: Suggests the wavelength of FTIR spectrum and possible function group at the corresponding wavelength (taken from T.J.U Thompson et al., 2013)
Figure 26: The within-group changes of the C/P ratio at different heating regimes (taken from T.J.U Thompson et al., 2013)
Recent studies conducted by S.T.D Ellingham et al. showed the decomposition of amide II peak around 300°C and entire degradation of amide I and amide III peaks at 350 and 400°C, respectively. PHT lead to the production of v4PO4 at 650°C around 625 cm-1. Coming to the peak ratios, most important
change occurs around 350-450°C and 750-850°C. Increase in CO/P, CO/CO3 and C/C ratios were
observed at 400°C (Figure 27). CI values increased with increase in temperature up to 800°C and started declining above that temperature (S.T.D Ellingham et al., 2016).
Figure 27: Variation in the ratio, PHT and line width accordingly with rising in temperature and time (taken from S.T.D Ellingham et al., 2016)
L.F. Lozano et al. chose a different approach to study the burning temperature using FTIR-KBr for the samples. The samples were heated from 20-600°C for 5 minutes each. As for the results, both S-bone and R-S-bone had similar characteristics. But for EXT-collagen, they observed a decrease in OH signal at a wavenumber of 3400 cm-1 and complete disappearance at 400°C. CH wavenumbers decreased at 2960 cm-1 and decrease at 400°C. A change is seen from 200-400°C in the structural composition due to loss of water interacting with the protein (Figure 28).
There are many nitrogen oxide gases that are produced during the process of collagen combustion and it is important to know which of the gases leave early. The time at which there is zero nitrogen in the protein structure might suggest the temperture to which the bone has been exposed. To know this, gas chromatography was performed along with FTIR only for the S-bone samples to detect the evolution of oxygen and nitrous oxide in the first step using Carboxen 1000 column and to detect the evolution of nitrogen, nitrogen dioxide, carbon dioxide, oxygen, and nitrous oxide in the second step using Porapak N Porous Polymers column. Collection of data were done at 250-700°C. At step one, oxygen was low complementary to carbon-dioxide/nitrogen dioxide value at 400°C (Figure 29). The carbon-dioxide value was found to be more in the second step, at 350°C and 500°C. This is due to the degradation and combustion of collagen due to rise in temperature (Figure 30) (L.F. Lozano et al., 2003).
Figure 29: Chromatographic value of oxygen and carbon dioxide using Carboxen 1000 column (taken from L.F. Lozano et al., 2003)
Figure 30: Chromatographic value of oxygen and carbon dioxide using Porapak N Porous Polymers column (taken from L.F. Lozano et al., 2003)
Figure 28: FTIR spectra of EXT-Collagen (taken from L.F. Lozano et al., 2003)
DISCUSSION:
Fire is one of the exceptionally powerful tools accessible by humans. At the same time, it is one of the most destructive tool that can turn anything to ashes within seconds. In forensic science, destruction of human bodies through fire is of significance. Fire scenes pose to be one of the most chaotic and difficult scenes to investigate. When the human body is under extreme heat, the soft tissue undergoes thermal degradation, subjecting the bones to fire. The bones become fragmented under extreme heat. At such times, the investigators find it difficult to differentiate between the fragmented burnt bones with other burnt items such as wood. Sometimes, the burnt bones may not at all be recovered/identified due to extreme fire scenes (Megan J. Richardson., 2017). In forensic science and anthropology, the investigation of burnt bones and the changes that have occurred due to heat is studied extensively. This is done in order to solve a criminal case or as a part of disaster victim identification (DVI). Therefore, it is very important to identify a burnt bone.
Bones undergo certain heat-induced changes which can be investigated both visually and by histological analysis. The heat-induced transformation of the bone at a corresponding range of temperature is given in the table below (Table 12).
Table 12: The four stages of transformation occurring in bone at different temperature regimes (taken from T.J.U. Thompson, 2004)
Some of the heat-induced changes can influence both observer based and chemical techniques due to the damage occurring, both outside and inside the bone (Table 13).
Table 13: Anthropological techniques affected by heat-induced changes
The morphological changes in the burnt bones can be investigated by observer based techniques like the change in bone color, shrinkage, deformities and pattern of fractures due to high temperature. Though these investigations give an idea about the condition of the bones at the time of burning, it is difficult to accurately point to the temperature at which the bone has been exposed. Hence, the researchers use more chemical techniques, since they are more sensitive, specific, accurate and precise, to see the histological and microstructural changes that occur in the bones. These techniques almost determine the temperature at which the bones are burnt.
Our research question of this review would be: Is it possible to determine the temperature that bone has been exposed to based on chemical analysis?
To answer this question few techniques have been discussed above, that have been used to determine the exposure temperature. Though, these techniques sound to be promising, there is still a huge gap in the literature about the forensic case studies and also about the empirical data, accuracy and precision of each techniques. According to the results of each literature, there are few upsides and drawbacks for each technique in terms of application in forensic cases. They are discussed in the below table (Table 14).
Techniques Upsides Drawbacks
Thermogravimetry Measures the weight loss due to rise in temperature. Considered to be one of the valuable techniques in understanding the thermal behavior of the bones.
Highly dependent on the type of exposure the bone has been undergone. This might be actually difficult to apply in real forensic cases.
Differential scanning calorimetry This technique is also based on the weight loss in the bone due to high temperatures.
Always combined with Thermogravimetry.
X-ray diffraction Provides spatial arrangement of the hydroxyapatite crystal phase in the microstructure of the bones.
Sometimes CI of other compounds like CaO is also found. The main drawback is that the crystallite phase change is recorded only above 600°C. Complementary methods like SAXS must be used to provide the structural and elemental details below 600°C
Fourier transform infrared spectroscopy
One of the most widely used techniques till now for the determination of burning of bones. Portable to the actual crime scenes
Do not have any comparable data in the literature since each sample produces different results. CI can also not be compared with that of XRD (T.J.U. Thompson et al., 2009)
Table 14: Pros and Cons of each chemical technique in terms of application in real forensic cases
All these discussed techniques have empirical data but, most of the literature studies have been performed on non-human bones and a very few studies are on the human bones. The studies used constant temperature along with the duration time for the results. This is not the case when it comes to real forensic case settings. Though the techniques are accurate and precise in the lab set up, their accuracy and precision are still in question in real cases. Certainty of the methods is another factor that should be discussed. Since, TGA and DSC requires lab settings, their certainty is quite questionable. As for XRD, it requires a combination of another physicochemical technique. Though FTIR-ATR is the most used technique and is much advantageous compared to other techniques, the main drawback is the lack of comparable data which hinders the certainty of the technique. Keeping all these factors in mind, one could still work with XRD and FTIR techniques as they are considered to be the most reliable ones. Most importantly, the bone shows different behavior when it is ground or studied in large pieces. Therefore, it
is also important to know about the pre-treatment of the sample obtained in order to obtain maximum result.
The problem in determining the exact temperature is first determining the type of fire the bones were exposed to, because as previously mentioned, a campfire is different from house fire compared to those researchers’ uses in the lab for the experiments. During cremation, the conditions remain constant while the temperature curve shows different phase in car or house fires. And as previously mentioned, it also depends on what kind of fire there is (flames or smolders). When two chemical techniques are compatible with each other, it is always safer to consider both these techniques for accurate results. When the same sample is analyzed under two techniques, chances are that the result might be comparable to each other. Both XRD and FTIR measure the CI of hydroxyapatite crystals along with some minor crystals. When these methods are combined one could try to provide a near accurate result. Combining observation technique such as light microscopy and SEM along with chemical techniques like FTIR and XRD would help in the determination of the burning temperature. The data of the previously performed experiments on the burnt bones could be used as reference for the future experiments and one could expect what kind of results they would get with the research. Researchers must use all these temperature prediction methods with much care as there are numerous factors that affect the thermal decomposition of the bone. One has to take both qualitative (observation based methods) and quantitative data (microstructure and CI analysis) into account for the most accurate results of temperature prediction. In addition to this more of forensic casework must be conducted in order to have more realistic approach suggesting realistic results. Although the sample size might vary, the results will be much accurate.
CONCLUSION AND FUTURE RECOMMENDATIONS:
From the abovementioned review and discussions, it is quite evident that determining the exposure temperature of bones under forensic relevance could be complicated. Moreover, the thermal decomposition of bones with the presence of soft tissue needs to be further investigated. All the studies mentioned above solely concentrates on de-fleshed bone specimens which cannot be applied directly on some archeological and forensic samples. In majority of cases, the bones are incinerated along with soft tissues. There is a need of further analysis for the determination of the burning temperature of bones, both, in presence and absence of soft tissue in order to bridge the gap in the literature. In addition to this, more systematic studies for the analysis of both biochemical and structural changes occurring must also be conducted in future. As already mentioned in the discussions, the combination of FTIR, XRD (chemical techniques) with SEM (observation technique) promises to be the most accurate techniques to apply in the determination of burning temperature. Application of these methods in future studies in determining the biochemical, histological, microstructural and morphological changes occurring in bones (with or without soft-tissue) in a fire accident or under extreme temperature conditions could be recommended to future researchers for them to get to well-informed results.
REFERENCES:
1. Bohnert, M., Rost, T. and Pollak, S. (1998). The degree of destruction of human bodies in relation to the duration of the fire. Forensic Science International, 95(1), pp.11-21.
2. Castillo, R., Ubelaker, D., Acosta, J. and de la Fuente, G. (2013). Effects of temperature on bone tissue. Histological study of the changes in the bone matrix. Forensic Science International, 226(1-3), pp.33-37.
3. Cattaneo, C., DiMartino, S., Scali, S., Craig, O., Grandi, M. and Sokol, R. (1999). Determining the human origin of fragments of burnt bone: a comparative study of histological, immunological and DNA techniques. Forensic Science International, 102(2-3), pp.181-191.
4. Cattaneo, C., Gelsthorpe, K., Sokol, R. and Phillips, P. (1994). Immunological Detection of Albumin in Ancient Human Cremations using ELISA and Monoclonal Antibodies. Journal of Archaeological
Science, 21(4), pp.565-571.
5. de Becdelievre, C., Thiol, S., Santos, F. and Rottier, S. (2015). From fire-induced alterations on human bones to the original circumstances of the fire: An integrated approach of human cremains drawn from a Neolithic collective burial. Journal of Archaeological Science: Reports, 4, pp.210-225.
6. Ellingham, S., Thompson, T. and Islam, M. (2015). The Effect of Soft Tissue on Temperature Estimation from Burnt Bone Using Fourier Transform Infrared Spectroscopy. Journal of Forensic
Sciences, 61(1), pp.153-159.
7. Ellingham, S., Thompson, T. and Islam, M. (2015). Thermogravimetric analysis of property changes and weight loss in incinerated bone. Palaeogeography, Palaeoclimatology, Palaeoecology, 438, pp.239-244.
8. Ellingham, S., Thompson, T., Islam, M. and Taylor, G. (2014). Estimating temperature exposure of burnt bone — A methodological review. Science & Justice, 55(3), pp.181-188.
9. Glazewski, M. (2006). Experiments in Bone burning. Oshkosh Scholar, 1, pp.17-25.
10. Goncalves, D. (2012). The micro-analysis of human burned bones: some remarks. [ebook] Cadernos do GEEvH, pp.32-40. Available at: http://geevh.jimdo.com/cadernos-do-geevh/ [Accessed 8 Feb. 2018].
11. Gonçalves, D., Thompson, T. and Cunha, E. (2011). Implications of heat-induced changes in bone on the interpretation of funerary behaviour and practice. Journal of Archaeological Science, 38(6), pp.1308-1313.
12. Hiller, J., Thompson, T., Evison, M., Chamberlain, A. and Wess, T. (2003). Bone mineral change during experimental heating: an X-ray scattering investigation. Biomaterials, 24(28), pp.5091-5097. 13. Holden, J., Phakey, P. and Clement, J. (1995). Scanning electron microscope observations of
incinerated human femoral bone: a case study. Forensic Science International, 74(1-2), pp.17-28. 14. Holden, J., Phakey, P. and Clement, J. (1995). Scanning electron microscope observations of
heat-treated human bone. Forensic Science International, 74(1-2), pp.29-45.
15. Imaizumi, K. (2015). Forensic investigation of burnt human remains. Research and Reports in
Forensic Medical Science, p.67.
16. Jackson, A. and Jackson, J. (2011). Forensic science. Harlow: Pearson/Prentice Hall.
17. J. Richardson, M. (2017). Determining the Presence of Remains using the chemical composition of burned cadaver Ash. [online] Available at: http://scholarworks.umt.edu/etd/10955 [Accessed 8 Feb. 2018].
18. Krap, T., van de Goot, F., Oostra, R., Duijst, W. and Waters-Rist, A. (2017). Temperature estimations of heated bone: A questionnaire-based study of accuracy and precision of interpretation of bone colour by forensic and physical anthropologists. Legal Medicine, 29, pp.22-28.
19. LOZANO, L., PENA-RICO, M., HEREDIA, A., OCOTLAN-FLORES, J., GOMEZ-CORTES, A., VELAZQUEZ, R., BELIO, I. and BUCIO, L. (2003). Thermal analysis study of human bone. 38th ed. Kluwer Academic Publishers, pp.4777-4782.
20. M. Mayne Correia, P. (1997). Fire Modification of Bone: A Review of the Literature. [ebook] CRC Press LLC, pp.1-19. Available at: https://www.academia.edu [Accessed 8 Feb. 2018].
21. Nicholson, R. (1993). A Morphological Investigation of Burnt Animal Bone and an Evaluation of its Utility in Archaeology. Journal of Archaeological Science, 20(4), pp.411-428.
22. Piga, G., Gonçalves, D., Thompson, T., Brunetti, A., Malgosa, A. and Enzo, S. (2016). Understanding the Crystallinity Indices Behavior of Burned Bones and Teeth by ATR-IR and XRD in the Presence of Bioapatite Mixed with Other Phosphate and Carbonate Phases. International Journal of
Spectroscopy, 2016, pp.1-9.
23. Piga, G., Malgosa, A., Thompson, T. and Enzo, S. (2008). A new calibration of the XRD technique for the study of archaeological burned human remains. Journal of Archaeological Science, 35(8), pp.2171-2178.
24. Rogers, K. and Daniels, P. (2002). An X-ray diffraction study of the effects of heat treatment on bone mineral microstructure. Biomaterials, 23(12), pp.2577-2585.
25. Shipman, P., Foster, G. and Schoeninger, M. (1984). Burnt bones and teeth: an experimental study of color, morphology, crystal structure and shrinkage. Journal of Archaeological Science, 11(4), pp.307-325.
26. Snoeck, C., Lee-Thorp, J. and Schulting, R. (2014). From bone to ash: Compositional and structural changes in burned modern and archaeological bone. Palaeogeography, Palaeoclimatology,
Palaeoecology, 416, pp.55-68.
27. Squires, K., Thompson, T., Islam, M. and Chamberlain, A. (2011). The application of histomorphometry and Fourier Transform Infrared Spectroscopy to the analysis of early Anglo-Saxon burned bone. Journal of Archaeological Science, 38(9), pp.2399-2409.
28. Thompson, T. (2004). Recent advances in the study of burned bone and their implications for forensic anthropology. Forensic Science International, 146, pp.S203-S205.
29. Thompson, T. (2005). Heat-induced Dimensional Changes in Bone and their Consequences for Forensic Anthropology. Journal of Forensic Sciences, 50(5), pp.1-8.
30. Thompson, T., Gauthier, M. and Islam, M. (2009). The application of a new method of Fourier Transform Infrared Spectroscopy to the analysis of burned bone. Journal of Archaeological Science, 36(3), pp.910-914.
31. Thompson, T., Islam, M. and Bonniere, M. (2013). A new statistical approach for determining the crystallinity of heat-altered bone mineral from FTIR spectra. Journal of Archaeological Science, 40(1), pp.416-422.
32. Ubelaker, D. (2009). The forensic evaluation of burned skeletal remains: A synthesis. Forensic
