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Forensic Science International 195 (2010) 168.e1168.e6

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Forensic Science International

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Forensic Anthropology Population Data

Comparison of adipocere formation in four soil types of the Porto (Portugal) district
Nuno Duraes a, Debora Cortez a,c, Manuel Algarra a, Francisco G. Sanchez b, Jose E. Rodrguez-Borges c, Iulius Bobos a, Joaquim C.G. Esteves da Silva c,*
a b c

ncias da Universidade do Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal Centro de Geologia do Porto, Faculdade de Cie laga, 29071 Ma laga, Spain Departamento de Qumica Analtica, Facultad de Ciencias, Universidad de Ma ncias da Universidade do Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal Centro de Investigaca em Qumica [CIQ(UP)], Departamento de Qumica, Faculdade de Cie o

A R T I C L E I N F O

A B S T R A C T

Article history: Received 11 May 2009 Received in revised form 22 September 2009 Accepted 15 November 2009 Available online 14 December 2009 Keywords: Adipocere Free fatty acids Water content Soil types Forensic Anthropopulation Data

Four typical soils of the Porto (Portugal) area were characterized and used to study the decomposition of buried pieces of pork meat under controlled laboratory experiments (an 8 month experiment with a relatively high soil moisture and a 1 month experiment with relatively low soil moisture). The soils types were: organic, sandy, gravel and clay-gravel soils. Soils were characterized for their grain size distribution, pH, water content, organic matter percentage and mineral composition. Four free fatty acids (myristic, palmitic, oleic and stearic) were analysed (using a methodology based on an extraction step followed by a derivatization reaction and high performance liquid chromatography analysis) in soil samples as a sign of adipocere formation. The direct sensorial analysis of the buried sample residues and the free fatty acids proles of the sampled soils showed that sandy and clay-gravel soils (in a low moisture environment) slowed the normal decomposition process promoting the formation of adipocere. Nevertheless, this apparent soil effect is indirect and a consequence of the different water retention and permeability of the soils. Thus, the water content of the soils is a crucial factor for adipocere formation. 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The Directorate of the municipal cemeteries of the Porto city reports the existence of sections where corpses do not decompose after the regular resting time, some of them for more than 50 years, which causes management problems, due to the impossibility of grave reuse and deactivation of these sections as burial grounds. The visual inspection of the unskeletalized corpses suggests that the cause for these problems is related to an inhibition of postmortem changes due to adipocere formation [13]. In one of the cemeteries of Porto, the Agramonte cemetery, a possible cause for the problematic sections is the existence of an aquifer that passes underneath the cemetery. Indeed, the most problematic section contains an active well, and has already been deactivated as a burial site. This observation agrees with previous reported results that show that the presence of water is one of the most important factors for adipocere formation [4,5]. However, in other cemeteries without aquifers the apparent inhibition of postmortem changes due to adipocere formation is also detected in some sections and, in these cases, the soil type characteristics must

* Corresponding author. Tel.: +351 220402569; fax: +351 220402659. E-mail address: jcsilva@fc.up.pt (Joaquim C.G. Esteves da Silva). 0379-0738/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2009.11.010

be the critical factor. Previous literature reports showed that the soil characteristics affect the process of adipocere formation [4,6]. However, other factors such as burial environment, soil pH, temperature, moisture and oxygen content have been investigated [7,8]. The parent material of the soil was considered the most important characteristic to assess its suitability as a graveyard [1 3]. The following characteristics have been positively correlated with adipocere formation or degradation problems: loam-enriched moraine material; poorly permeable materials such as schist; sediments from the weathering of red sandstones; blackcoloured soils; soils with poor drainage (clay and loamy enriched soils); and soils with a high proportion of ne particles (<6.3 mm) (clay enriched soils). Adipocere is a post-mortem anaerobic decomposition product that is composed mainly of saturated fatty acids myristic, palmitic and stearic acids [812]. Other unsaturated fatty acids (for example, oleic acid), salts of fatty acids (mainly calcium salts) and hydroxyl fatty acids (for example, 10-hydroxy stearic acid) have been identied as constituents of adipocere [11,12]. This paper presents the results of a study about the effect of four soil types characteristic of the Porto district, namely organic (OS), sandy (SS), gravel (GS) and clay-gravel (CGS) soils, on the decomposition of pork meat under controlled experimental

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168.e2 es N. Dura et al. / Forensic Science International 195 (2010) 168.e1168.e6 Table 1 Soil sample macroscopic characterization. Soil classication Organic (OS) Sandy (SS) Gravel (GS) Clay-gravel (CGS) Macroscopic characterization Dark coloured soil with high percentage of organic matter. Brownish soil with a high percentage of sand collected near maritime coast. Greyish-white soil with a relatively high weathering of primary minerals. Reddish soil with a relatively high weathering of primary minerals.

conditions. Pork meat must be used in these types of studies due to ethical criteria. Two experiments, under semi-anaerobic conditions, were made using different soil moisture conditions (wet and dry) by burying pork meat in a soil sample placed inside an air tight plastic container for 1 month in the dark. Other wet experiments were done that took 4 and 8 months. The soil samples were characterized and four free fatty acids were monitored as an indication of adipocere formation in the soil samples: myristic, palmitic, oleic and stearic acids.
2. Materials and methods 2.1. Reagents Free fatty acids standards (myristic, palmitic, stearic and oleic acid) were supplied by SigmaAldrich Qumica SA (Spain). The derivatizing chiral agent, (R)(+)-1-phenylethylamine, dichloromethane, methanol (HPLC grade) and ethylic ether were purchased from Merck (Darmstadt, Germany) and Panreac (Barcelona, Spain), respectively. All compounds were analytical-reagent grade and the purities were stated to be higher than 99%. Stock standard solutions in methanol were used for HPLC measurements. 2.2. Synthesis of the corresponding standard fatty acid amide derivatives (FAA) Myristic, palmitic, oleic and stearic ceramides (FAA) were prepared from the corresponding fatty carboxylic acids (FFA). These acids were converted into the corresponding carboxyliccarbonic anhydride intermediate by treatment with ethyl chloroformate (2 equiv.) and triethylamine (2 equiv.) under argon using dried dichloromethane as a solvent (10 mL/4 mmol of fatty acid) following the methodology described in the literature [13]. The corresponding anhydride intermediate, which was not isolated, was concentrated by vacuum and was reacted with (R)-(+)-1-phenylethylamine (1.2 equiv.) under argon using dried dichloromethane as a solvent (20 mL/4 mmol of initial fatty acid), giving the corresponding fatty amide. The resulting organic mixture solution was washed with 1 M HCl (4 10 mL), the organic layer was washed with brine (20 mL) and dried with anhydrous sodium sulphate. Removal of the solvent using a rotary evaporator yielded the corresponding pure ceramide quantitatively (Scheme 1). The structure of the pure ceramides was checked by 1H NMR-400 MHz (CD3Cl) and molecular mass analysis. Standard FAA solutions were prepared by dissolving 10 mg of synthesized derivatized FAA in 10 mL methanol, stored at 4 8C and in absence of light. Working solutions were prepared by dilution with methanol. 2.3. Soil samples Four soil types representative of the Porto district were collected. These soils have a similar genesis from granite and/or metamorphic rocks. Their macroscopic characterization easily discriminated the four types of soils (Table 1) as organic (OS); sandy soil (SS); gravel (GS); and clay-gravel (CGS). The discriminating properties are mainly related to weathering minerals (clays) and organic matter content, which will affect the pH and permeability (water and air circulation) of the soils. 2.4. Soil characterization methods Raw soil samples were analysed for their grain size distribution using a standard sieve series in a mechanical shaker. Mineralogy was carried out by X-ray diffraction (XRD) using a Rigaku Miniex D/max. C series automated diffraction system equipped with a Cu Ka radiation. Samples were analysed in the range 4708 2u, using a 18 divergence slit, a step increment of 0.058 2u and a counting time of 10 s/ step. The pH of all soil samples were measured by mixing soil with deionised water (1:5 of Vsoil:Vwater) after 10 min stirring followed by a resting period of 2 h. pH was measured using a HI255 HANNA millivoltmeter, equipped with a HI1131B HANNA combined glass electrode, previously calibrated with two buffer solutions (4.01 and 7.01) from HANNA. The percentage of organic matter was determined by rigorous weighing by incinerating 2 g of dry soil with the following temperature program:

105 8C for 48 h at 375 8C for 1 h; 600 8C for 6 h. The moisture percentage was determined by weighing the soil before and after a 24-h period at 105 8C. 2.5. Pork decomposition experiments Two sets of experiments were performed that correspond to: (i) relatively high moisture in the soil (simulating winter weather conditions); and (ii) relatively low moisture in the soil (simulating low rainy seasons). The rst experiment (relatively high moisture) was performed during an 8-month period and the second experiment (relatively low moisture) during a 1-month period. The experiment with relatively high moisture was performed since the average precipitation in the Porto area is about 100 mm, or higher, in the fall, winter and spring months. The four soils were used in the experiments and triplicate samples were always assembled. The rst experiment consisted of the burial of small pieces (a parallelepiped of about 1 cm high) of pork loin with no visually detected fat (10.9 0.5 g), followed by the addition of deionised water (about 50 mL), in a plastic hermetic box (15 cm 15 cm 5 cm) the box was closed and kept inside a closed black plastic bag protected from light for the pre-selected time of 1, 4 and 8 months period. The volume of the soil occupied about 80% of the volume of the box and the piece of meat was placed in the centre of the box with about 1.5 cm of soil above it. Consequently, at the beginning of the experiments, the environment inside the containers contain some oxygen these conditions somewhat simulate burial under about 1 m soil of a dead body inside a cofn, which is a normal practice in Portugal. Also, in Portugal, it is not normal practice to treat the corpses prior to burial (e.g. embalming). After a period of time after burial, which is highly dependent of the environmental conditions, the unsealed wood cofn degrades and soil mixes with the decomposing body affecting the subsequent decomposition pathways. The second experiment followed a similar design as the rst but no water was added. In addition to pork loin with no visible fat, the second set used pork fat and skin in this case only a 1-month long experiment was performed. The pork samples were obtained from a local butcher and were from the same animal. Consequently any observed differences in the decomposition results of the pork samples were due to factors other than the samples alone. After the pre-selected resting time the boxes (three boxes for each soil) were opened and a visual inspection was performed. Also, a randomly selected portion of the soil inside the plastic box was removed for the soil analysis. The rest of the sample present inside the box was stored inside a 20 8C freezer until free fatty acid analysis could be performed. 2.6. Free fatty acids analysis Soxhlet extractions were performed using variable amounts of analytically weighed soils, without previous treatment. Samples (3075 g) were extracted under reux with 1:1 dichloromethaneether solution (100 mL) for 3 h. The samples were cooled to room temperature, mixed with anhydrous sodium sulphate and ltered. The extracts were dried using a rotary evaporator at 40 8C and reduced pressure. The extracts were transferred into a Schlenk tube reactor, equipped with a magnetic stirrer and maintained in an inert atmosphere (argon) during the reaction after the addition of anhydrous dichloromethane (2 mL/50 mg of isolated sample), ethyl chloroformate (40 mL/50 mg of sample) and triethylamine (50 mL/50 mg of sample), for 3 h at room temperature. After that, a gentle vacuum was applied, to evaporate all solvents and excess reactants. (R)-(+)-1-phenylethylamine was added

Scheme 1. Reaction scheme of the transformation of free fatty acids into ceramides.

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es N. Dura et al. / Forensic Science International 195 (2010) 168.e1168.e6 (30 mL/50 mg of sample) in anhydrous dichloromethane (2 mL/50 mg of isolated sample) to form derivatized compounds and left overnight. Dichloromethane (10 mL) was then added to the mixture. The resulting organic mixture was washed with HCl 1 M (4 20 mL) and, nally washed with saturated brine (1 30 mL). The organic extract was dried over anhydrous sodium sulphate and ltered. The solvent was removed under vacuum and the solid residual corresponded to the derivatized FAA. Extracts were dissolved in 4 mL of methanol and transferred into injection vials for liquid chromatography with mass spectrometry (LCMS) and high performance liquid chromatography with a UV detector (HPLC-UV) analysis. 2.7. Liquid chromatography method The UV measurements were performed with a MerckHitachi (Darmstad, Germany) liquid chromatograph consisting of an L-6200 pump, an AS-4000 autosampler, an L-4250 UVvisible detector, and a D-6000 interface. Integration was carried out with a PC computer and instrument parameters were controlled by HitachiMerck HM software. A 215 nm wavelength was used in all measurements. A mobile phase (methanol:water, 90:10) with a ow rate of 0.8 mL min1 was used in the chromatographic separations with a 10 mL injection volume. For the preparation of the mobile phases, the solvents were ltered through 0.2 mm nylon membrane lters and degassed for at least 1 h. 168.e3

3. Results and discussion 3.1. Raw soils characterization Besides a visual macroscopic characterization (Table 1 and Fig. 1), the four soils used in this work were analysed for their grain size distribution (Fig. 2), mineralogy (Fig. 3), pH (Table 2) and organic matter content (Table 2). The macroscopic characterization of the four soils shown in Table 1, together with the physical and chemical properties described below, led us to the following classication of the soils: organic (OS); sandy (SS); gravel (GS); and clay-gravel (CGS). A global analysis of the grain size distribution curves of the four soils (Fig. 2) shows that all can be classied as sand [14]. Indeed, particle sizes fall almost in the range of 0.063 up to 2 mm. OS shows the highest amount of greater than 2 mm sized grains and fewer amount of particles with sizes smaller than 0.063 mm. SS shows the highest amount of <0.063 mm sized particles followed by GS. According to Fig. 1 the four soils are poorly graded [SSSA], with the gravel soil being the least. Indeed, the Uniformity Coefcient (Uc), dened as the diameter ratio of the soil particles corresponding to 60% and 10% (D60/D10) [15], provides a comparative indication of the range of grain size and if Uc is less than 5 the soil is considered uniform. In this study the four soils were considered poorly calibrated because Uc was higher than 5 (OS, 7; SS, 7; GS, 14; and GCS, 6). The analysis of Fig. 3 shows that the mineralogy of the soils is not markedly variable, being composed of quartz as the principal mineral and some secondary mineral resulting from the weathering of granite, which is the main lithology of the area under research. The most weathered soils, GS and CGS, are characterized by a relatively high proportion of clay minerals, such as biotite, muscovite and kaolinite. The relatively high proportion of clay mineral in these two last soils may contribute to relatively low water permeability. Table 2 shows that the pH of the soils suggests the classication of very strongly acid (SS, GS and CGS) and slightly acid to neutral (OS) [16]. The soil organic matter content of the four soils is typical of surface mineral soils (<5%) [16] with OS showing an expected content of nearly 10%. 3.2. Soil properties modications Tables 2 and 3 show the pH, organic matter percentages and water content of the soils used in all the decomposition experiments. The analysis of Table 2 shows that there is an increasing trend in the pH with time, particularly for OS, GS and CGS samples. Indeed, for the high moisture experiments, the pH of the raw OS is 6.26 and the pH varies with time as 6.84, 7.07 and 7.21; the pH of the raw GS is 4.93 and the pH varies with time as 5.90, 7.03 and 6.66; the pH of the raw CGS is 4.53 and the pH varies with time as 5.50, 6.88 and 7.04; the pH of the raw SS is 4.50 and the pH varies with time as 5.85, 5.20 and 5.07.

Fig. 1. Picture of the four soils after 8 months under semi-anaerobic conditions (high moisture pork meat experiments): (a) organic; (b) sandy; (c) gravel; and (d) clay-gravel.

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168.e4 es N. Dura et al. / Forensic Science International 195 (2010) 168.e1168.e6

Fig. 2. Size particle distribution of the four soils.

The analysis of Table 2 shows a decreasing trend in the percentage of organic matter of the raw soils SS shows the highest and OS the smallest variations (in this soil, a slight increase of the organic matter percentage is observed in the 8-month experiment). The analysis of Table 3 shows that the water content of the four soils always has the following decreasing trend: SS > CGS > GS > OS. Also, for each soil, the water content within each experiment was almost constant as a function of time showing that experiments performed in hermetic boxes allowed no mass transfer with the exterior. 3.3. Pork meat decomposition experiments The results obtained in the pork meat decomposition experiments showed that the decomposition of the meat was different for the four types of soil (Fig. 1). The main differences were: (i) Macroscopic pieces of meat were observed mainly in the sandy soil experiments. Also, sandy soil samples were characterized by a black spot above the buried meat (Fig. 1b). In the other soil types experiments no macroscopic pieces of meat were observed. (ii) The samples of gravel soil were particularly bad smelling, releasing a cadaveric odour when the containers were opened and when the soil was heated during moisture percentage determination. This shows a high degree of decomposition of the pork meat in these samples. Indeed, the evolution of bad smelling gases, like hydrogen sulphide, ammonia, and the expected putrecine and cadaverine [3], is characteristic of later stage decomposition of proteins by proteolytic bacteria, which is only achieved when there are no conditions for adipocere formation [3]. Concerning the detection of free fatty acids, the results were different for the four types of soil and for the two moisture percentage levels (average trend values shown in Fig. 4). In the high level moisture experiments, relatively high quantities of free fatty acids, mainly palmitic and myristic acids, were detected in all samples but particularly high in the sandy soil. In the low level moisture experiments, less relative quantities of free fatty acids were detected and the sandy soil showed the highest quantities of palmitic and myristic acids, followed by the clay-gravel soil no marked differences in fatty acid compositions were detected for the meat and skin experiments. The comparison of the sensorial analysis (visual inspection and smell) of the samples with the relative quantities of free fatty acids shows that when pieces of meat were detected, there was a corresponding large quantity of free fatty acids. This was observed
Fig. 3. XRD patterns of randomly powdered oriented samples of the four soils under study: (a) organic; (b) sandy; (c) gravel; and (d) clay-gravel. Each peak corresponds to the mineral represented by the following letter: Q, quartz; Bt, biotite; M, muscovite; K, kaolinite.

for the sandy soil samples and suggests that the meat decomposition was stopped by adipocere formation. In the low moisture experiments for the clay-gravel soil samples, relatively large quantities of free fatty were detected, suggesting the existence of conditions for adipocere formation. This result, which cannot be easily explained, could be related to the characteristic clay-gravel soil microbiology. Indeed, when the containers were opened, the four soil types under investigation showed different quantities of, and apparently different qualities, of mould. Although, sandy and clay-gravel soils are different, they promote the formation of free fatty acids and they have one

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es N. Dura et al. / Forensic Science International 195 (2010) 168.e1168.e6 Table 2 Soil sample pH values and organic matter percentages. Soil Raw soil Organic Sandy Gravel Clay-gravel Soil 1st month pH Organic matter (%) pH 6.26 4.50 4.93 4.53 4th month pH Organic matter (%) 10.75 5.19 4.07 4.50 Organic Matter (%) 9.84 4.33 5.05 4.92 8th month pH Organic matter (%) 11.27 3.81 3.94 4.41 168.e5

High moisture experiments (pork meat) Organic 6.84 9.67 7.07 Sandy 5.85 3.72 5.20 Gravel 5.90 4.44 7.03 Clay-gravel 5.50 4.64 6.88 Pork meat (1 month) Soil Ph Organic matter (%) 9.80 0.28 4.08 4.54

7.21 5.07 6.66 7.04

Pork fat (1 month) pH Organic matter (%) 8.27 1.65 3.90 4.34

common property, both show the highest moisture percentage (Table 3). The comparison of average fatty acid compositions between the high and low moisture experiments, with the discussed exception for the clay-gravel soil, shows relatively high amounts for the high moisture experiments. This result agrees with the suggested adipocere mechanism formation from the hydrolysis of adipose tissue triglycerides molecules [3]. Consequently, water content is indeed a key factor in adipocere formation in soil. In the case of gravel soil samples, signicant amounts of free fatty acid quantities were not detected and all the samples were characterized by a particularly bad smell, which suggest that decomposition proceeded to late stages as expected [3]. Also, the organic soil samples did not show signicant amounts of free fatty acids, but were not characterized by the intense bad smell as were the gravel soil samples. This result may be a consequence of the characteristic microbiology of the two soils and of the reactivity of the organic matter molecules (humic substances) towards the decomposition products. 4. Conclusions The decomposition of pork loin in four typical soil types of the Porto (Portugal) area, under two different moisture levels and under semi-anaerobic conditions, allowed the observation of the effect of the soil type in the decomposition of meat buried in those soils. Four free fatty acids (myristic, palmitic, oleic and stearic) were analysed in the soil and used as an indication of the extent of decomposition the higher the amount of free fatty acids detected, indicating adipocere formation, the lower the decomposition rate. Sandy soil showed the highest amount of adipocere formation. Clay-gravel soil, in the low moisture experiments, also showed a relatively high amount of adipocere formation. Organic and gravel soils did not show relatively high amounts of adipocere formation and apparently promoted meat decomposition. The results obtained in this work suggest that the soil type has a marked effect on adipocere formation. Nevertheless, this soil effect is indirect and a consequence of the different water retention and permeability of the soils and of the characteristic soil microbiology. Also, the effect of the soil type and environmental factors on the cofn degradation and collapse is a factor that deserves research because this promotes the mixture of the soil with the decomposition corpses which will affect adipocere formation. The results observed in this study, together with literature descriptions, allow us to recommend the use of soils with relatively high organic matter and low clay percentages in cemeteries to promote decomposition. Acknowledgements The Directorate of the Divisao Municipal de Higiene Publica of the Municipally of Porto is acknowledged to authorize soil sample collection inside the cemeteries of Agramonte and Prado Repouso. Financial support from Fundac o para a Ciencia e Tecnologia a (Lisboa) (FSE-FEDER) (Project PTDC/QUI/71001/2006) is acknowledged. Routine fatty acids soil extraction laboratory work is acknowledge to Marcela J.C. Oliveira, Sandra C.C. Monteiro and Vania M.P. Vieira working under PEEC (Programa de Estagios Extra Curricular da Faculdade de Ciencias da UP). The authors would like to thanks the Fundac o para a Ciencia e Tecnologia (Lisboa, a Portugal) under the frame of the Ciencia 2007 program. References

Low moisture experiments Organic 6.38 Sandy 6.35 Gravel 6.65 Clay-gravel 6.12

6.58 6.47 6.57 6.04

Table 3 Soil samples water contents (percentages)a. Organic High moisture experiments 1 month 27.3(2) 4 month 29(3) 8 month 28(1) Low moisture experiments Pork meat (1 month) Pork fat (1 month) Sandy 58.5(6) 58(1) 55.9(5) Gravel 31(2) 31(5) 31(2) Clay-gravel 43(1) 43(3) 40(1)

6.9(7) 6.2(2)

28.9(5) 27(1)

16.0(9) 13.8(6)

21.6(6) 20(1)

a Averages and standard deviations under parenthesis of three independent samples.

Fig. 4. Average concentrations of free fatty acids in the high (a) and low (b) moisture experiments.

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