Australian Journal of Forensic Sciences

ISSN: 0045-0618 (Print) 1834-562X (Online) Journal homepage: http://www.tandfonline.com/loi/tajf20

Recent advances in the estimation of post-mortem

interval in forensic taphonomy

Mohammad Asif Iqbal, Maiken Ueland & Shari L. Forbes

To cite this article: Mohammad Asif Iqbal, Maiken Ueland & Shari L. Forbes (2018): Recent
advances in the estimation of post-mortem interval in forensic taphonomy, Australian Journal of
Forensic Sciences, DOI: 10.1080/00450618.2018.1459840

To link to this article: https://doi.org/10.1080/00450618.2018.1459840

Published online: 09 May 2018.

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Australian Journal of Forensic Sciences, 2018
https://doi.org/10.1080/00450618.2018.1459840

Recent advances in the estimation of post-mortem interval in

forensic taphonomy
Mohammad Asif Iqbal, Maiken Ueland  and Shari L. Forbes
Centre for Forensic Science, University of Technology Sydney, Broadway, Australia

ABSTRACT ARTICLE HISTORY

One of the key elements in a criminal death investigation is the Received 20 March 2018
estimation of time since death, as this information can assist with Accepted 22 March 2018
identifying the victim and prosecuting an offender. Estimating post-
KEYWORDS
mortem interval (PMI) is a challenging task given the many variables Decomposition scoring; bone
that act on the rate and process of decomposition. This review dating; botanical evidence;
presents current advances in estimating PMI in forensic taphonomy. taphonomic mycota;
The use of scoring systems based on visual observations and the decomposition chemistry
development of empirical formulas have been proposed for soft
tissue analysis. In the absence of soft tissue, the citrate content of
bone and radiometric dating are recommended for PMI estimations
of hard tissue. Recent studies have shown an increased focus on
associated evidence found on or near the remains. This includes
botanical and mycological evidence such as vegetation changes, the
presence or absence of certain plant species, and fungal succession.
Decomposition chemistry represents an emerging discipline where
biomarkers can be analysed within different mediums such as tissue,
soil associated with decomposing remains and decomposition fluid.
Although advances are being made in these many taphonomic
disciplines, the field still has a long way to go in terms of finding the
elusive formula for accurately estimating PMI of decomposed remains.

1. Introduction
Estimation of post-mortem interval (PMI; time since death) is an integral part of medico-legal
death investigations. Physical and anatomical changes such as algor, rigor, and livor mortis
can be used for PMI estimation within the first 72 h of death1. The chemical composition of
vitreous humour (i.e. hypoxanthine, K+, and free amino acids) has also been correlated with
a PMI of up to 120 h2,3. During the early post-mortem period and well beyond, insects can
be a powerful tool for PMI estimation. Forensic entomologists study the colonization and
succession of insect activity to estimate the PMI, and this is one of the preferred methods if
entomological evidence is available4. There are, however, scenarios where remains are skel-
etonized due to a lengthy recovery time or instances where insect colonization has been
restricted (i.e. burial decomposition)5. In such cases, the estimation of PMI using entomology
may not be possible4. As an alternative approach, Vass attempted to develop empirical for-
mulas based on three environmental parameters (i.e. temperature, moisture, and the partial

CONTACT  Shari L. Forbes  shari.forbes@uts.edu.au

© 2018 Australian Academy of Forensic Sciences
2   M. A. IQBAL ET AL.

pressure of oxygen) to determine the PMI of human remains for surface and burial decom-
position in Knoxville, Tennessee, USA6. However, a recent attempt to validate these formulas
in a Canadian environment reported large variation of PMI estimation7. According to this
study, the rate of decomposition was not consistent throughout all stages of decomposition.
Differences in temperature extremes and humidity levels between geographic regions along
with other environmental and intrinsic variables make it impractical to apply formulas devel-
oped in one region to any other region7.
Forensic taphonomy is an important field that can assist the estimation of PMI, especially
when forensic pathology is generally redundant (i.e. PMI exceeds 72 h). Forensic taphonomy
research focuses on studying the decomposition of human remains and its interaction with
the surrounding environment to understand the process and the rate of decomposition8,9.
In general, the decomposition of a body follows a typical progression and is divided into
five stages – fresh, bloat, active decay, advanced decay, and dry remains or skeletonization5,10.
Temperature plays a vital role in determining the length of each stage and the overall rate
of decomposition, as the rise in surrounding temperature increases cellular metabolism
within the soft tissue by affecting the reaction rates of enzyme catalysts11. This follows the
classical chemical principle known as the Arrhenius equation, which states that the reaction
rate doubles for every 10°C rise in temperature11,12. Besides temperature, the local biome,
especially vertebrate and invertebrate scavengers, plays a vital role in the process of decom-
position8,13. Geologic parameters such as the environment of deposition (e.g. surface versus
burial) and soil variables (e.g. type of soil, soil microbiology, soil pH, moisture and the oxygen
content within the grave site) also greatly influence decomposition processes13,14. Additionally,
there are numerous anthropogenic factors that may impact the decomposition of a body
based on the circumstances surrounding death, including ante/post-mortem injuries13;
criminal burning of a body15; absence/presence of clothing or wrappings16; burial type (ter-
restrial versus aquatic)17, and the burial depth (shallow versus deep)18.
This article reviews the recent advances in PMI estimation in the field of forensic tapho-
nomy. As this is a review of recent advances and future aspects, articles published before
2005 were excluded. Considering the large number of published articles in the literature,
only studies that have post-mortem interval/forensic taphonomy/decomposition in the title
or as a keyword or focus are included. Studies that focused on forensic entomology, in par-
ticular insect colonization, are not within the scope of this article as they have been detailed
extensively elsewhere.

2.  Taphonomic changes to human remains

The stages involved in cadaveric decomposition and applicable disciplines commonly called
upon to estimate PMI in each stage (e.g. forensic pathology, anthropology, entomology, and
taphonomy)19,20 are shown in Table 1. Autolysis is described as the first identifiable change
in a cadaver, and is the self-digestion of cells through the action of their own enzymes5,21.
This process occurs primarily in the most metabolically active cells and is believed to be
triggered by the decrease in intracellular pH as a result of anoxia11,22. The onset of autolysis
is indicated by the presence of fluid-filled blisters on the skin14,23. Marbling of the skin may
also be apparent in the fresh stage, and occurs due to the haemolysis of blood inside blood
vessels24. This early stage of decomposition ranges between one hour to two/three days after
death depending on the surrounding environment and other factors, and is said to end with
 AUSTRALIAN JOURNAL OF FORENSIC SCIENCES   3

Table 1. Stages involved in cadaveric decomposition and applicable methods to estimate PMI in each
stage.
Decomposition stage Description Forensic Methods References
[A] Fresh Autolysis Biochemistry 5,10,20,21,69
Time since death (1 h to 2/3 days: Fluid filled blisters on the skin Molecular Biology
From death to the first signs of Depletion of oxygen and Pathology  
bloating)a marbling of skin
[B] Bloated Breakdown of soft tissues by Biochemistry 10,20,21,69
microorganisms
Time since death (3 to 10 days) Greenish discoloration of the skin Entomology
Accumulation of fluids followed Pathology
by purging
  Anaerobic fermentation Taphonomy  
[C] Active decay End of bloating/deflation Entomology 5,10,20,21,69
Time since death (10 to 20 days) Black putrefaction Taphonomy
Breaking of the skin and rapid Anthropology
leaching
Increased biological activity
including insects and bacteria
Collapse of abdominal cavity
  Possible adipocere formation  
[D] Advanced decay Dehydration of corpse Entomology 5,10,20,21,69
Time since death (20 to 50 days) Only skin, cartilage, hair, bones, Taphonomy
and minimal flesh
  Butyric fermentation Anthropology
[E] Dry to skeletonization Diagenesis or mummification of Taphonomy 5,20,70,71
remaining skin
Time since death (50 to 365 days) Bone exposure and skeletoniza- Anthropology/Archaeology
tion
a
The time since death estimates are variable and typically based on warm conditions when biological activity is optimal6,35

the appearance of bloating10. Bloating results from the build-up of gases during autolysis
which causes distension of the torso, and represents the commencement of putrefaction,
i.e. the breakdown of the body by the action of micro-organisms (bacteria, fungi, and pro-
tozoa)5. This process is identified by green/purple discoloration of the lower abdomen, due
to the overgrowth of colonic bacteria5,23,25. Through putrefaction, the complex molecules
present in soft tissues (e.g. carbohydrates, proteins and lipids) that started to break down
during autolysis continue to be catabolized into gases and liquids in the form of simple
molecules11,14,23. This process builds pressure inside the body cavity, and may result in severe
purging from the orifices, which can be mistaken for post-mortem injuries to the head5,14,23.
Following the purging of gases, the body deflates and the active decay stage begins,
which is characterized by black putrefaction and the darkening of the skin5,10. At this stage,
the insect activity (e.g. feeding of Calliphoridae larvae) is optimal26. Muscle proteins are
broken down into volatile fatty acids and other reported malodourous decomposition prod-
ucts, such as polysulphides, indole, skatole, cadaverine and putrescine5,10,14,23,26. Putrefaction
continues during active decay (generally, days to weeks post-mortem), as chemical constit-
uents are still being degraded, which results in the strong odour of putrefaction associated
with tissue liquefaction5,10. The increased activities by Calliphoridae larvae and other families
of flies and beetles (e.g. Staphylinidae, Histeridae, Muscidae, Silphidae, Sarcophagidae) accel-
erate the process of soft tissue loss and the bones become exposed, indicating the end of
active decay10. This stage is followed by advanced decay which normally occurs weeks to
months after death, depending on the environment10. This stage is characterized by the
butyric fermentation of the body in an anaerobic condition, releasing the earthy-cheesy
4   M. A. IQBAL ET AL.

odour of butyric acid10,27. Later in this stage, any remaining soft tissue dries out and the
remains are visible as predominantly skin, cartilage, hair and bones, with little soft tissue10.
Another important indicator of this stage is an increase in beetle activity and a reduction of
the fly activity on the body5.
The final stage of decomposition is dry, where any remaining moist skin and tissue are
converted to a ‘leather-like’ covering that adheres to bones5,14,23. Skeletonization also occurs
later in this stage, which is characterized by the appearance of exposed bones in over 50%
of the body and proceeds until only the harder and more resistant bones, teeth and cartilage
remain5,25,28. Chemical weathering of these remains continues but occurs over a considerably
longer timeframe than previous stages (several months to years post-mortem)5,10. Finally,
diagenesis occurs, which alters the proportions of organic collagen and inorganic compo-
nents of bone (e.g. calcium, phosphate, and magnesium) and the surrounding soil14,29.

3.  Advances in the estimation of PMI using taphonomic approaches

3.1.  Empirical formulas based on visual scoring of decomposition
3.1.1.  Terrestrial decomposition
The state of decomposition of a body often provides valuable insight into the estimation of
PMI in human remains cases. Megyesi et al.30 were among the first to demonstrate an ancillary
technique of PMI estimation in cases of surface decomposition utilizing a point-based system
based on total body score (TBS). This scoring method was originally proposed by Galloway
et al.28 and adopted after some modification. In brief, decomposition was divided into four
major stages (fresh, early decomposition, advanced decomposition, and skeletonization)
and the degree of decomposition was scored according to separate categories for different
anatomical regions of the body (i.e. head and neck, trunk, and limbs). The point values for
each region were added to determine the TBS. The lowest score a case could receive was 3
(fresh in all regions) and the highest score was 35 (dry bone in all regions). Utilizing this
approach, a total of 68 human remains cases with a known date of death were scored for
decomposition. A regression equation was calculated to predict accumulated degree days
(ADD: heat-energy units that represent the accumulation of thermal energy required for the
chemical and biological reactions of decomposition to take place) from TBS values
(ADD=10(0.002×TBS×TBS+1.81) ± 388.16; where 388.16 is the standard error of the regression in
untransformed (non-logged) ADDs) 30. Among the 68 cases, 57 were outdoor cases and in
11 cases remains were found in indoor environments; the datasets were also geographically
diverse and covered most regions of the United States (a total of 19 states). The findings of
this study showed that ADD accounts for approximately 80% of the variation in decompo-
sition and the pattern of decomposition is best modelled as dependent on accumulated
temperature, not just time. Simmons et al.31 also described a similar approach based on a
linear equation to correlate surface decomposition scores with logADD values, which were
obtained from a total of 18 relevant articles available in the literature. The study reported
that the progression of decomposition can be recorded reliably using ADD and a simple
linear equation. The major factor dominating the decomposition rate was insect activity,
and was reported to be independent of depositional environment, season, or species.
Another important factor that contributes to the rate of decomposition was body size, espe-
cially when carcasses were accessed by insects. However, when insects were excluded, bodies
decomposed at similar rates regardless of size31.
 AUSTRALIAN JOURNAL OF FORENSIC SCIENCES   5

In an approach to validate the formula developed by Megyesi et al.30, Myburgh et al.32

recorded TBS and ADD from 30 pig carcasses for a period of 232 days. Surface decomposition
trials were conducted in an anthropology facility located in the central Highveld plateau of
South Africa32. Using the data obtained from these pig carcasses, the authors first developed
formulae to estimate PMI from ADD with a 95% prediction interval. Additional data obtained
from a subsequent set of pig carcasses (n=16) was used to validate the accuracy of this
method. The authors concluded that ADD had limited use in the prediction of PMI in a South
African setting, as only one of the 16 pig carcasses fell within the 95% prediction interval.
Another study from the same research group33 established the importance of body size on
the rate of decomposition, which confirmed the findings by Simmons et al.31. In this study,
a total of 30 pigs (15 large pigs and 15 piglets) were decomposed in the same facility during
the spring and early summer months33. Decomposition status was scored according to the
TBS method described by Megyesi et al.30. TBS, PMI and ADD values were analysed to assess
the pattern of decomposition and to compare decomposition rates between small and large
pigs. The findings indicated that rapid decomposition occurred during the early stages of
decomposition for both groups, while the overall rate of decomposition was significantly
higher (2.82 times faster) in small pigs compared with their larger counterparts33.
As an alternative to the ADD approach30, Fitzgerald and Oxenham34 proposed a method
based on time-since-death (TSD) to understand the pattern of decomposition in Australian
temperate conditions. The authors utilized pig carcasses (n 2: P1 in full sun and P2 in semi-
shade) as human analogues and applied a varied approach to score decomposition. Scores
were calculated using a degree of decomposition index (DDI) which provided a value from
0 to 5, by dividing the TBS observed on the whole body by the number of body elements
scored. Linear regression equations were developed for both P1 and P2 as follows: P1: TSD
= 10(0.0942 × 0.041) ± 15 days and P2: TSD = 10(0.0942 × 0.025) ± 14 days). The linear regression analysis
suggested that TSD accounts for the majority of variation in decomposition (using the DDI),
while the impact of macro-environment (sun versus shade) was not significant34. In a recent
study, Marhoff et al.35 evaluated the performance of both the ADD30 and TSD34 method in
an Australian climate. The study was conducted in the Hawkesbury region of New South
Wales, Australia, with adult pig carcasses (n 4) undergoing surface decomposition for three
months. The comparison between two approaches revealed superior performance of the
ADD method over time. However, both methods underestimated the PMI of all pig carcasses
and the authors proposed alternative predictive algorithms as follows: ADD = 85.8+39.7(TBS)
and TSD = –20.7+22.1(DDI). It should be noted that these equations are only applicable for
this particular study region due to the underlying factors affecting decomposition in a spe-
cific environment.
Vass13 attempted to develop universal empirical formulas based on environmental param-
eters (i.e. temperature, moisture and the partial pressure of oxygen) and decomposition
scores to determine the PMI of human remains for both surface and burial decomposition6.
Data collected over a period of 20 years at the University of Tennessee’s Anthropology
Research Facility, in Knoxville, Tennessee, USA (a warm humid temperate climate region)
were used to develop the formulas. The formulas for (I) surface and (II) burial decomposition
1285×(decomposition∕100)
are as follows: (I) PMIaerobic = 0.0103×temperature×humidity and (II)
1285×(decomposition∕100)×4.6×adipocere
PMIanaerobic = 0.0103×temperature×soilmoisture
. The details of these values and parameters are
described in the article and not repeated here to avoid redundancy. These formulas were
reported to be effective in areas that encompass the mid to eastern section of the United
6   M. A. IQBAL ET AL.

States where humidity, soil moisture, soil type and vegetation are similar to the study site.
However, the major limitation of these formulas is that the body under evaluation has to be
in the pre-skeletonization phase of decomposition (<1285 ADDs); otherwise, there is a very
high chance of error. In addition, the performance of formula II was relatively inferior to
formula I as there were several additional factors to further complicate the calculation (e.g.
variation of soil moisture content and the presence/absence of adipocere). Most importantly,
the successful application of both formulas was dependent on the accurate scoring of
decomposition, which required the involvement of experienced personnel6.
A study by Cockle et al.7 attempted to validate the two formulas developed by Vass6 using
a total of 64 Canadian cases with known PMIs (42 cases from a larger set of 96 cases for
formula I (bodies on the surface) and 22 cases for formula II (burial decomposition)). For
bodies exposed to warm temperatures, formula I consistently overestimated the known PMI
by a large and inconsistent margin, while formula II dramatically underestimated the PMIs
of bodies exposed to cold and freezing temperatures (< 4°C)7. According to this study, the
rate of decomposition was not consistent throughout all stages of decomposition. The study
concluded that differences in temperature extremes and humidity levels between geo-
graphic regions make it impractical to develop a universal formula applicable to all regions
of the globe7. The findings also indicated that apart from temperature and humidity there
are other region specific variables which may impact the rate of human decomposition and
must be considered for PMI estimation7.
The findings of a recent study indicate that conventional scoring systems28,36 are not
reliable in the estimation of PMI in buried human remains where the timespan can be sig-
nificantly longer (PMI: 12–19 years)37. This study analysed 86 human skeletons buried in
coffins and concluded that each burial creates a distinct micro taphonomic environment
that impacts the progression of decomposition37. Scoring older remains can be challenging
even in cases of surface decomposition. Nawrocka et al.38 assessed the inter-rater reliability
of TBS values obtained from 120 laymen trained to score decomposition of pig carcasses
from photographs. The performance of assessors was uniform irrespective of their qualifi-
cations. However, carcasses in the advanced decomposition stage received significantly
fewer accurate scores. Other factors impacting the inter-rater variation of scores included
the variable decomposition observed on the same carcass (e.g. mosaic or differential decom-
position) and the quality of photographs provided to the assessors. The authors recom-
mended a greater emphasis on these issues when teaching the TBS scale to improve the
performance of the assessors. In addition, they recommended the refinement of some com-
mon decomposition indicators (e.g. ‘heavy maggot activity’ or ‘some flesh relatively fresh’28,30)
to decrease inter-rater inconsistencies, and the addition of colour as an indicator of decom-
position status.

3.1.2.  Aquatic decomposition

The decomposition of a body in an aquatic environment differs significantly from a terrestrial
environment and may pose additional challenges in the visual evaluation of the degree of
decomposition. Heaton et al.39 applied the scoring method derived by Megyesi et al.30 to
predict the post-mortem submersion interval (PMSI) of individuals from 125 cases in the UK.
The bodies were recovered from three different waterways (the River Clyde, Scotland; the
River Mersey and canals in northwest England) between the years 1991 and 2006.
Decomposition was scored in terms of total aquatic decomposition score (TADS), and aquatic
 AUSTRALIAN JOURNAL OF FORENSIC SCIENCES   7

ADD values were also calculated from retrospective temperature data. The overall findings
of this study indicated that ADD had a significant effect on the decay process of submerged
bodies while no major differences in decomposition between the waterways were reported.
A single linear regression equation was also developed to predict PMSI by plotting the TADS
values against known logADD values from all three waterways: TADS = –3.706+7.778 log-
10
ADD. In subsequent studies, Humphreys et al.40 and De Donno et al.41 evaluated the equa-
tion developed by Heaton et al.39. In their study, piglet carcasses (n = 9) were submerged in
an agricultural reservoir located in Davis, CA, USA, and underwater in situ photographs were
taken to score decomposition in terms of TADS. The objective measurement of decomposi-
tion in this study required an in-depth examination of analogues using the TADS scoring
system rather than utilizing a carrion weight evaluation. The authors also developed an
equation for their system (TADS = – 13.746+4.8518 * LOG (ADD)). The PMSI equations devel-
oped by both Heaton et al.39 and Humphreys et al.40 are logarithmic in nature, which is similar
to the equations developed/evaluated by others in cases of terrestrial decomposition30−32,34.
However, the study by De Donno et al.41 reported a limitation of the TADS approach when
adipocere is formed in submerged bodies. A total of 68 human remains recovered from both
sequestered (n = 52) and non-sequestered (n = 16) marine environments were analysed. All
the bodies recovered from sequestered environments had a PMSI of more than seven months
and had initial adipocere formation which caused the preservation of some soft tissue. Such
formation of adipocere affected the accuracy of the TADS model41.
Estimation of aquatic decomposition was also reported in a multiple drowning accident
involving six victims at Meco Beach, south of Lisbon, Portugal42. ADD was calculated based
on the PMSI and sea surface temperature during this period using the following equation
∑n
ADD = i=1 (Ti × 24 hi
); where hi represents the number of hours in each day (i) during the
submersion period (n), and Ti the daily temperature]. The estimated PMSI was between 7.4
and 11.4 days, with an estimated ADD range between 115 and 174 (ADDmin) and 104 and
191 (ADDmax). The study found that there is a probable ADD range where it is expected that
a body will reach the bloating/floating stage and resurface. This was estimated to occur
somewhere in the ADD range of 100 to 140, with the PMSI depending on the ambient tem-
perature. The authors proposed a hypothetical ADD value of 130 for body resurfacing, which
may provide some information for search and rescue teams when locating drowned
victims42.
In a more recent study, Daalen et al.43 developed an aquatic decomposition scoring
method applying the TADS approach to colour photographs (n = 45) available from a total
of 38 bodies recovered within the borders of the North Sea during 1990 to 2013. These bodies
are part of a large dataset containing 703 bodies that were recovered in the study area during
this time frame; among them only 38 had colour photographs available. Twelve volunteers
participated in the study to score decomposition using a pictorial reference atlas containing
photographic examples of decomposition stages. The regression analysis performed in this
study demonstrated strong correlation (r = 0.91; p < 0.001) between PMSI and TADS values,
which indicates the potential of this method to estimate PMSI based on colour photographs
of bodies recovered from an aquatic environment43.
8   M. A. IQBAL ET AL.

3.2.  Dating skeletal remains

3.2.1.  Radiometric dating and spectroscopy of skeletal remains
In forensic anthropology, a major requirement is to distinguish between historical and recent
human remains, as a more recent PMI might merit judicial attention. Radiocarbon dating is
the most commonly used technique to distinguish between historical and recent artefacts.
Cappella et al.44 compared the performance of radiocarbon dating with two fast and inex-
pensive methods, luminol and the Oxford Histology Index, to estimate PMI of skeletal remains
from 20 forensic anthropological cases. Radiocarbon analysis dated 17 cases as pre-1950
while two were dated as post-1950 and analysis was not possible in one case. In comparison,
only four of the 20 cases yielded a positive response to luminol and a high Oxford Histology
Index score at the same time, which indicated that they were recent remains (post-1950). If
radiocarbon analysis is considered the gold standard, the accuracy obtained through the
combination of these methods is ~90% (only two false positives out of 20).
In an alternative radioisotope study, Schrag et al.45 measured 90Sr and 210Po incorporated
into bones as indicators of PMI (within 50 years). The authors utilized a 90Sr calibration curve
based on vertebrae that was established for the Swiss population between 1960 and 200146.
210
Po and 90Sr activity data were also collected from individuals deceased in 1990, during
the renovation of a cemetery near Milan. The application of their method to a real forensic
case (The Lutry remains) showed that, individually, both measurements (i.e. activities of 90Sr
and 210Po) are subject to major uncertainties. However, the combination of these measure-
ments was reported to yield a satisfactory estimation of PMI. In addition, the measurement
of 90Sr and 210Po in permanent teeth was recommended to improve PMI estimation as teeth
enamel is much less impacted by diagenesis.
Ramsthaler et al.47 compared the performance of four alternative approaches (i.e.
UV-fluorescence, luminol test, Hexagon-OBTI, and Combur tests) to estimate PMI of skeletal
remains. A lack of luminescence and reduced UV-fluorescence was reported to be indicative
of a long PMI and suggested the exclusion of forensic relevance, while positive luminol
reaction or strong UV-fluorescence warranted further estimation using additional confirm-
atory dating methods. The Hexagon-OBTI and Combur test were not successful in demon-
strating blood on skeletal remains and were thus not applicable to PMI estimation. The
application of UV-fluorescence has also been reported in several other studies48−50. For
instance, Hoke et al.49 evaluated the performance of UV-fluorescence for differentiating
between historical and recent skeletal remains. Over 213 bones with known PMI were ana-
lysed and classified into two major groups: (1) a recent and forensically relevant time period,
which consisted of 58 long bone samples from abandoned graves at a modern cemetery
(PMI ranged between 8 to 60 years); and (2) archaeological specimens comprising 79 human
bones and 76 horse bones (PMI ranged between 90 to 15,000 years). It was reported that
blue bone fluorescence, as well as blue fluorescence combined with other colours (mainly
yellow) was not a conclusive indicator of sample age. In contrast, overall yellow fluorescence
could indicate a historical specimen; however, UV-florescence falsely excluded 2% of all
forensically relevant samples, highlighting a limitation to its use as a sole criterion of screen-
ing skeletal remains.
The combination of UV-induced fluorescence and 490 nm-induced fluorescence has been
reported to distinguish between historical and recent human skeletal remains50. In this study,
the extent of fluorescence of 30 bone cross-sections with known PMI (within 50 years) was
 AUSTRALIAN JOURNAL OF FORENSIC SCIENCES   9

investigated. Bone samples were collected from abandoned graves at six different cemeteries
(for a total of 28 bone samples) and from a cadaver under investigation (two bones). Through
the combined application of these methods it was possible to distinguish between remains
with/without forensic relevance within a cut-off interval of 30 years. Moreover, the correlation
between UV-fluorescence colour and PMI reported in this study was similar to that observed
by Hoke et al.49. Boaks et al.48 also applied UV-florescence to quantify the degradation of
bone collagen with PMI estimation. Resin embedded cross-sections of pig bones were
stained using a histochemical reaction that differentially stains collagenous and non-colla-
genous proteins. The ratio of proteins (collagenous/non-collagenous) declined significantly
with increasing PMI. However, the moderate correlation obtained in this study (r = ̶ 0.563)
indicated that along with time there are several other possible factors (e.g. burial environ-
ment, soil chemistry, etc.) affecting the composition of skeletal remains48.
Raman spectroscopy has also been investigated as a tool for determining post-burial
interval (PBI) of skeletal remains51. McLaughlin et al.51 analysed the sections of a turkey bone
that had been buried for short intervals (12–62 days) using Raman spectroscopy. The findings
determined that the chemical changes in bone due to soil bacteria are time-dependent and
there is a correlation between bone spectra and burial duration51. A model that was sensitive
enough to track the changes in bone composition in a scale of days was created using peak
integration of Raman bands. The potential forensic application of Raman spectroscopy is
further demonstrated in a study by Delannoy et al.52 who investigated the effect of moisture
and temperature on bone mass as time progressed. The mass loss of partially buried human
ribs was monitored over a period of 90 days using a gravimetric method52. Raman spectro-
scopic analysis was performed to assess the post-mortem alteration of the bone matrix. The
bone mass loss was faster in a dry environment, which was also reflected by the spectra of
bone samples. The findings demonstrate the potential of Raman spectroscopy to provide
valuable insight into the bone diagenesis process52.

3.2.2.  Citrate content of bone

Several methods of PMI estimation based on the citrate content of bone have been pro-
posed53−55. An initial analysis of fresh rib bones from both human and pig determined that
the initial citrate content is relatively uniform, averaging 1.96±0.06 wt% of bone54,55. Pig ribs
(n = 6) that had undergone burial decomposition for up to 6 months were subsequently
investigated54. Bone citrate content was stable during the first month of burial and began
to decline linearly afterwards, losing ~50% of its initial value within six months of burial54.
In addition to pig samples, bone samples from six known forensic cases were also analysed.
The data from both pig samples (excluding the first month) and forensic cases were subse-
quently fitted to a logarithmic curve: C(t) = –0.647logt+20.971, r2 = 0.991; where C = citrate
in wt% and t = time in days54. Kanz et al.55 subsequently analysed temporal and femur bones
from 20 bodies buried in wooden coffins and body bags, applying the equation proposed
by Schwarcz et al.54. In the case of body bag samples, temporal and femur bones exhibited
significant differences in terms of known versus estimated PMI, while such differences were
insignificant for coffin samples. PMI values measured from femur bones of both groups were
lower than the actual PMI values. However, the authors recommended the use of femur
bones for citrate-based PMI estimations as their citrate content was consistently higher than
that of temporal bones54. The applicability of the citrate-based method has also been inves-
tigated in the estimation of PMI in aquatic cases53 whereby pig ribs were decomposed in
10   M. A. IQBAL ET AL.

two different environments (terrestrial and aquatic) for a period of 1–165 days. No significant
differences in citrate loss were identified between terrestrial and aquatic environments.
However, higher citrate variation, lower citrate recovery, and a weaker association with time
was reported, compared with studies conducted in terrestrial environments54,55.

3.3.  Taphonomic mycota

As reviewed by Carter and Tibett56, the fruiting structures of certain fungi (e.g. the ammonia
and the post-putrefaction fungi) have been recorded in association with decomposed bodies
in different regions of the world. In order to utilize these fungi as a tool for the estimation
of PBI, it is necessary to understand their fruiting behaviour as they undergo a succession
in which one set of fungi is later replaced by another56. Ishii et al.57 were the first to describe
the details of species of fungus obtained from two human cadavers. The first case involved
a body found on the concrete floor of an abandoned house in a forest and was confirmed
to be a man who had been missing for 10 months. Yellowish and white fungi were identified
on the front surfaces of the chest, abdomen, and thighs. In a second case, skeletal remains
clothed in a shirt and pants were found in a forest. The remains were of a man who had hung
himself at least 6 months earlier. Identical yellowish and white fungi were reported on the
surfaces of the right scapula, right pubic bone, and acetabulum. The main isolate colonized
on the surfaces of the skin and bones was determined to be Eurotium repens.
The same authors have also demonstrated the use of fungi for PMI estimation in a case
report involving an elderly clothed male who was found at the bottom of an open well58.
The face of the male victim was dotted with colonies of white fungi that were identified as
Penicillium sp. and Aspergillus terrous. These species widely inhabit organic matter and gen-
erally colonize 3–7 days after attaching to the subject. Visual observation and police inves-
tigation reported that the man had last been seen 12 days before discovery and the fungal
evidence indicated that the man had been dead for approximately 10 days. Based on these
observations, it was suggested that fungi can provide a useful means of estimating PMI
when forensic entomology is not applicable. However, further studies on the growth rate of
fungi on human cadavers would provide valuable insights into the applicability of forensic
mycota for PMI estimation.
The use of fungi to estimate PMI has also been reported in several cases from the UK59.
In the first case, the body of a man was found by a railway line and the medical examiner
estimated the PMI as more than 48 h. No insect activity was found but there was a large
circular fungal colony under the chin, suggesting the body had been in situ for 3 to 5 weeks.
It was later revealed that the PMI was 4 weeks 3 days. In a second case, two female bodies
were recovered from water covered in fungal growth. Several fungi were identified including
species of Fusarium, Geotrichum, Mucor and Pythium. Based on the decomposition status
and the fungal evidence, the PMI of the first and second bodies were estimated to be at least
2 and 5 weeks, respectively. These estimates also agreed with the times the victims were
reported missing.
Another case report outlines the importance of the growth rate of fungus moulds in an
indoor environment59. A stabbed victim was found in a closed flat with fungal colonies
developed on the body fluids that had soaked into the carpet and sofa. A study of fungal
growth rate on the same carpet using bovine body fluid allowed for the estimation of PMI
as 5 days, which was consistent with a subsequent confession of guilt. A further case study
 AUSTRALIAN JOURNAL OF FORENSIC SCIENCES   11

utilized the differential growth of fungus in the estimation of PMI. A broken twig with lichen
Xanthoria parietina was found on the ground under a plastic-wrapped body part. This species
grows deep orange sporophores in light, while the colour of sporophores becomes green
in dark and moist conditions. A simple experiment on the differential growth of this species
assisted in estimating the deposition time as less than a week. The use of taphonomic mycota
while still in its infancy has demonstrated potential in casework; however, controlled research
studies are required to further enhance the accuracy of forensic mycology as a tool for esti-
mating PMI.

3.4.  Botanical evidence

Plants can provide important clues in the estimation of PMI through the pattern of their life
cycle and physiology. Cardoso et al.60 describe the estimation of PMI of human skeletal
remains found in a wooded area of northern Portugal from the growth rate of mosses and
shrub roots. The recovered remains were highly disarticulated, the ribcage was bound by a
dense mass of roots, and several of the bones were covered by green algae and mosses. The
authors determined the age of the mosses (Bryum capillare Hedw. and Hypnum cupressiforme
Hedw.) and shrub roots (Cistaceae Juss) and concluded that the minimum PMI was 3 years,
to which several months or a few years error had to be added to account for the complete
decomposition of the body. The police investigation suggested the individual disappeared
~6 years prior to the recovery of the remains60.
Lancia et al.61 also reported the application of moss in the estimation of PMI of human
skeletal remains that were found in a wooded area near Perugia, Central Italy. A significant
bryophyte colony had developed in the lower part of the recovered cranium that reached
the inferior nuchal line. Part of the colony was observed with a stereomicroscope and was
identified as Leptodyctium riparium Hedw. Warnst, which grows in humid and muddy envi-
ronments61. The growth period of the identified moss was determined through the exami-
nation of annual segments on the stem of another similar well-documented species (Hypnum
cupressiforme). Applying this approach, the minimum PMI was estimated as 24 to 30 months
and assisted in narrowing the list of missing persons. Further investigation suggested that
the individual was an elderly woman who had disappeared around 3.5 years prior to the
recovery of the remains; a timeframe slightly longer than the estimated minimum PMI.

3.5.  Decomposition chemistry

3.5.1. Gravesoil
As a body decomposes, fluids will leach from the remains into the surrounding environment.
In the case of terrestrial deposition, whether surface or burial, the soil environment represents
the medium that traps these decomposition by-products. Analysis of these by-products can
provide information about the time since deposition, which may correlate closely with PMI.
Vass et al.26 were the first to report the analysis of soft tissue decomposition products found
in soil solutions for the estimation of PMI. In this study, the soil underneath decomposing
cadavers (n=7) was sampled up to ~5250 ADD and soil extracts were analysed for specific
volatile fatty acids (VFAs) and ions. Propionic, iso-butyric, n-butyric, iso-valeric, and n-valeric
were the five major VFAs reported in soil extracts, the concentrations of which changed with
ADD and provided important clues in the estimation of PMI. Sixteen ions were also measured
12   M. A. IQBAL ET AL.

in the soil extracts and seven of them (i.e. sodium, chloride, ammonium, potassium, calcium,
magnesium, and sulphate) were shown to be stable in the environment and to change with
ADD. In a subsequent study, Vass62 also reported the analysis of volatile organic compounds
(VOCs) in soil samples (n = 186) associated with older gravesites (10–60+ years). The con-
centration of cyclic and halogenated VOCs in gravesoil decreased with increasing PBI; how-
ever, the trend was reversed in the case of aldehydes, ketones and straight-chain alkanes,
becoming more prominent as time progressed. A total of 56 compounds were reported to
be associated with human decomposition. The study provided information concerning the
approximate time interval when a particular compound is most commonly detected and
the decomposition phase when the compound is most likely to be present. This information
could offer valuable insights into the estimation of PBI based on the analysis of VOCs presents
in gravesoil.
Benninger et al.63 investigated the dynamics of gravesoil nutrients (e.g. carbon, nitrogen,
and phosphorus-based compounds) in soil beneath pig carcasses for the estimation of PMI.
Five pig carcasses placed on a soil surface were investigated for a period of 100 days and
soil samples beneath the carcasses were collected in weekly to monthly intervals.
Decomposition was assessed through physical observation and the measurement of soil pH
and nutrients. Soil pH and nutrients increased significantly as decomposition progressed
and different nutrients reached their maxima at different PMIs: 43 days (lipid-phosphorus),
72 days (total nitrogen), and 100 days (soil extractable phosphorus). Further studies were
required to validate these PMI estimations in different soil types and regions, which to date
have not been carried out.
A number of studies also reported the potential of ninhydrin reactive nitrogen (NRN) in
gravesoil as an indicator of PMI64,65. The study by Carter et al.64 reported that the decompo-
sition of rat carcasses can almost double the concentration of NRN in gravesoil and may
remain constant well beyond the skeletonization stage. A further study from the same
research group measured NRN influx into gravesoil during both surface and burial decom-
position of pig carcasses65. The burial trial utilized six pig carcasses that were buried at ~40 cm
depth; soil samples were collected from the graves once per month for six months and again
after 12 months. The surface trial utilized five pig carcasses and soil samples were collected
more frequently; once every day for 10 days, every two days for a further six days, and once
per week until 97 days post-mortem. NRN concentrations increased significantly in the centre
(~20 cm depth) and the in the base (~40 cm depth) of the graves during the initial two to
six months of burial, respectively. However, surface decomposition exhibited higher NRN
concentrations during the initial 97 days of decomposition. The results of this study reflect
the potential of soil NRN as a presumptive tool for the estimation of PMI within two to three
months post-mortem following burial and surface decomposition, respectively65. The anal-
ysis of NRN from a surface decomposition trial was also reported in a study which utilized
pig carcasses of different body masses (~1, ~20, ~40, and ~50 kg) to investigate the use of
NRN in gravesoil to estimate early PMI (up to 15 days)66. Small (~1 kg) carcasses were found
to release NRN at a slower rate but at a greater concentration (in terms of NRN/kg carcass).
These findings suggest that the release of NRN is influenced by body mass and that smaller
bodies (e.g. neonatal carcasses) require a different equation from larger carcasses when
using gravesoil chemistry to estimate PMI66.
 AUSTRALIAN JOURNAL OF FORENSIC SCIENCES   13

3.5.2.  Decomposition fluid and tissue

During the earlier stages of decomposition when soft tissue remains, the analysis of decom-
position by-products can be conducted using tissue or fluid samples, rather than the sur-
rounding soil to estimate PMI. An earlier study by Vass et al.23 attempted to identify
time-dependent biomarkers in decomposing human tissue for the measurement of PMI. A
total of 18 cadavers underwent natural surface decomposition within a time span of four
years. Tissue samples were collected regularly from decomposing organs (liver, kidney, heart,
brain, and muscle) for up to a cumulative degree hour (CDH) range of ~1000 (3 weeks).
Biomarkers from amino acids, neurotransmitters, and decomposition by-products exhibited
distinct patterns and provided important clues in the determination of PMI based on CDH.
Eighteen compounds were reported as important tissue biomarkers for PMI estimations
while four compounds (gamma amino butyric acid, proline, methionine, and oxalic acid)
were further highlighted as the most important components. The progression of decompo-
sition was modelled based on the evolution of these biomarkers in different organs with
increasing CDH. PMI values in intervals as narrow as 5 CDH were achieved and the method
was reported to be limited only by the ability to obtain correct temperature data from a
crime scene23.
VFAs have also been reported by Swann et al.67 in the decomposition fluid from pig and
piglet carcasses as potential time-dependent biomarkers of decomposition. This study
involved tissue samples and carcasses decomposing in an outdoor environment without
the presence of a soil matrix. Collected samples were analysed for target VFAs as described
by Vass et al.26 and several long-chain fatty acids. Both short-chain and long-chain acids
followed an apparent cyclic trend changing with ADD. However, further studies in this area
have not been carried out to confirm these trends.
A method of estimating PBI based on the measurement of decomposition fluid conduc-
tivities in gravesoil was proposed by Pringle et al.68. The study involved regular in situ soil–wa-
ter conductivity analysis from a pig grave in a semi-rural environment. The gravesoil–water
conductivity increased rapidly up to one-year post-burial and slowly thereafter (until two
years). Multiple linear regression equations using the fluid conductivity (y-axis) and ADD
values (x-axis) were developed as follows: (1) first year: y = 9.16984x – 584.40866, R2 = 0.99315
and (2) second year: y = 0.71189x + 27,164.17258, R2 = 0.60713. The application of this
approach to a separate simulated burial estimated PBI with a 12% error, which indicated the
potential of this method as an auxiliary technique, although it requires further optimization
and extension to include human cadavers and varied environments68.

4.  Conclusion and future direction

While many advances have been made in the last decade, a formula to accurately estimate
soft or hard tissue decomposition remains elusive in the field of forensic taphonomy. During
the early post-mortem period, forensic pathology markers still remain the most commonly
used for estimating time since death due to an absence of alternative thanatology methods
that have come to light. Once putrefaction commences, forensic entomology represents
the more accurate method assuming insect evidence is available. In the absence of insect
activity, other methods must be investigated.
The area most commonly investigated for estimating soft tissue decomposition involves
body scoring systems based on visual observations and empirical formulas. While many of
these formulas are reported to be relatively accurate for their local environment, studies to
14   M. A. IQBAL ET AL.

test the formulas in varying environments are rarely successful, highlighting an inherent
limitation of scoring systems as well as a subjectivity in visual observations. Likewise, dating
of hard tissue (skeletal remains) often finds success in separating historical from recent
remains, but is less valuable in criminal death investigations where the PMI may be much
shorter and the error too large to be helpful.
Alternative methods have been investigated in the form of evidence associated with
remains such as taphonomic mycota, botanical evidence and soil biomarkers. All reported
studies have shown some potential but have limited validation to date. Similarly, studies to
identify biomarkers in soft tissue and decomposition fluid need further replication in order
to provide valuable information for both the early and later post-mortem period. Like all
forensic science research, we have come a long way in developing new and alternative
methods for estimating PMI; however, research must continue in these areas if we hope to
develop the elusive formula for time since death.

Disclosure statement
No potential conflict of interest was reported by the authors.

ORCID
Maiken Ueland   http://orcid.org/0000-0002-9155-3502

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