Forensic Science International
Volume 183, Issue 1 , Pages 6-13, 10 January 2009

Freezing skeletal muscle tissue does not affect its decomposition in soil: Evidence from temporal changes in tissue mass, microbial activity and soil chemistry based on excised samples

  • Kathryn L. Stokes

      Affiliations

    • Centre for Forensic Science, University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA 6009, Australia
    • Centre for Land Rehabilitation, School of Earth and Environment, University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA 6009, Australia
    • ,
  • Shari L. Forbes

      Affiliations

    • Faculty of Science, University of Ontario Institute of Technology, 2000 Simcoe Street North, Oshawa, ON L1H 7K4, Canada
    • ,
  • Mark Tibbett

      Affiliations

    • Centre for Land Rehabilitation, School of Earth and Environment, University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA 6009, Australia
    • Corresponding Author InformationCorresponding author at: Centre for Land Rehabilitation, School of Earth and Environment, Faculty of Natural and Agricultural Sciences, University of Western Australia, Crawley, WA 6009, Australia. Tel.: +61 8 6488 2635; fax: +61 8 6488 1050.

Received 28 February 2008; received in revised form 13 August 2008; accepted 14 August 2008. published online 17 December 2008.

Article Outline

Abstract 

The study of decaying organisms and death assemblages is referred to as forensic taphonomy, or more simply the study of graves. This field is dominated by the fields of entomology, anthropology and archaeology. Forensic taphonomy also includes the study of the ecology and chemistry of the burial environment. Studies in forensic taphonomy often require the use of analogues for human cadavers or their component parts. These might include animal cadavers or skeletal muscle tissue. However, sufficient supplies of cadavers or analogues may require periodic freezing of test material prior to experimental inhumation in the soil. This study was carried out to ascertain the effect of freezing on skeletal muscle tissue prior to inhumation and decomposition in a soil environment under controlled laboratory conditions. Changes in soil chemistry were also measured. In order to test the impact of freezing, skeletal muscle tissue (Sus scrofa) was frozen (−20°C) or refrigerated (4°C). Portions of skeletal muscle tissue (∼1.5g) were interred in microcosms (72mm diameter×120mm height) containing sieved (2mm) soil (sand) adjusted to 50% water holding capacity. The experiment had three treatments: control with no skeletal muscle tissue, microcosms containing frozen skeletal muscle tissue and those containing refrigerated tissue. The microcosms were destructively harvested at sequential periods of 2, 4, 6, 8, 12, 16, 23, 30 and 37 days after interment of skeletal muscle tissue. These harvests were replicated 6 times for each treatment. Microbial activity (carbon dioxide respiration) was monitored throughout the experiment. At harvest the skeletal muscle tissue was removed and the detritosphere soil was sampled for chemical analysis. Freezing was found to have no significant impact on decomposition or soil chemistry compared to unfrozen samples in the current study using skeletal muscle tissue. However, the interment of skeletal muscle tissue had a significant impact on the microbial activity (carbon dioxide respiration) and chemistry of the surrounding soil including: pH, electroconductivity, ammonium, nitrate, phosphate and potassium. This is the first laboratory controlled study to measure changes in inorganic chemistry in soil associated with the decomposition of skeletal muscle tissue in combination with microbial activity.

Keywords: Decomposition, Soil chemistry, Freezing, Skeletal muscle tissue, Forensic taphonomy

 

Back to Article Outline

1. Introduction 

The study of graves and death assemblages of bodies evolved from a crime or suspicious death (forensic taphonomy) might include investigation of site ecology and observed differences in the surrounding environment. For clandestine graves the body will typically be in intimate contact with soil, however data on the interaction of a cadaver with the surrounding soil environment is relatively limited and has been considered to be of little importance [1]. Cadavers or their components may be subject to freezing and thawing: either in a cold environment or due to deliberate preservation prior to burial or experimental procedures. Information about the effect of freezing on subsequent decomposition after thawing is limited and based on a few studies.

The process of freezing can lead to tissue damage that may affect tissue decomposition rate. During freezing the withdrawal of water from cells leads to a decrease in diffusion and material transfer between cells leading to inhibition of chemical reactions [2]. The damage caused by freezing skeletal muscle tissue (SMT) is not only morphologically destructive but it also leads to the denaturing of protein by dehydration and an incomplete re-absorption of water after thawing [2]. This can lead to water loss which depletes SMT of materials such as crystalloids, proteins, pigments and other breakdown products [3], [4]. Speed of freezing can affect the amount of destruction observed within SMT caused by immobilization of water as ice [3]. A slow freeze leads to the formation of ice crystals that are often a much larger size [3], [4]. This is related to ice crystals formation, slow freezing allows withdrawal of up to, or more than, 90% of the total water present within the cell excluding that which is chemically bound to cellular compounds [2], [3].

A single study exists that considers the effect of a freeze–thaw cycle on cadaver decomposition. Results suggested that freezing had a significant impact on the subsequent mode of decomposition observed. Cadavers frozen prior to being placed on a soil surface decayed aerobically while fresh cadavers decayed predominantly via anaerobic decomposition (putrefaction) [5]. This author suggested that this was caused by a biocidal effect on the enteric microbiota as a result of the temperatures associated with complete freezing of a cadaver [5], [6], [7]. This taphonomic study was a surface decomposition experiment rather than a burial event, limiting cadaver to soil contact. No analysis was done on the soil beneath the cadavers during the experiment.

Bacterial invasion after a freeze–thaw cycle has also been investigated, freezing led to more extensive bacterial proliferation in SMT in comparison to fresh SMT samples [8]. The results are thought to be a consequence of increased interstitial cavity sizes between cells caused by the formation of ice crystal artifacts [8]. By comparison another study found no increased bacterial invasion after freezing [9]. This raises the question about the behaviour of SMT frozen prior to inhumation in a microbially active soil environment and the effect it may have on the decomposition of the tissue and surrounding site ecology.

This study addresses the hypothesis that freezing SMT prior to interment in a soil environment will have a significant impact on its decomposition. In order to test the hypothesis three questions were addressed: (1) What effect does freezing SMT prior to interment have on the rate of decomposition? (2) What effect does the introduction of a nutrient rich substrate (SMT) to a soil environment have on the surrounding chemistry? and (3) Does freezing SMT prior to interment effect surrounding soil chemistry during decomposition? The results of the study will determine if the use of SMT which has been frozen prior to use in taphonomic studies affects the results. They may also be of use in interpreting case information from decomposition of body parts in cold climates.

Back to Article Outline

2. Materials and methods 

2.1. Experimental design 

The experimental protocol was based on an established method [10]. The design of the experiment had 3 treatments: SMT frozen prior to inhumation and SMT refrigerated prior to inhumation and a soil only control. Destructive sequential harvests were conducted at 2, 4, 6, 8, 12, 16, 23, 30 and 37 days and the experiment was replicated 6 times for each treatment and at each harvest, giving a total of 162 randomised soil microcosms. The microcosms for the last harvest (day 37) contained sodium hydroxide traps to monitor carbon dioxide (CO2) respiration for the duration of the incubation.

Soil was harvested from the top 10cm profile at Harry Waring Marsupial Reserve, Perth, WA. Prior to the microcosms being created the soil was sieved to 2mm to remove large aggregates, pieces of rock, vegetation and large arthropods. The sieved soil (100g, 40mm depth) was weighed into jars (72mm diameter×120mm height) and water holding capacity (WHC) was adjusted to 50% with the addition of deionised water. The WHC is close to the optimal water content for microbial activity in a soil system and was calculated after 100% WHC was obtained through soil capillary action. This was compared to 0% to obtain the volume required for 50%. After the addition of water the microcosms were placed in a constant temperature room (25°C) for 10 days to allow them to equilibrate.

Organic porcine (S. scrofa) SMT was cut into ∼1cm3 sized portions (∼1.50g) and weighed. 60 portions were frozen for 48h at −20°C and 60 portions were refrigerated at 4°C for 48h. On the day of burial the SMT from the freezer and refrigerator were allowed to equilibrate to room temperature. Each portion of muscle tissue was weighed and buried at a depth of 1cm, the control microcosms were disturbed at the same depth to simulate the effect that soil disturbance might have on microbial activity. After SMT interment the microcosms were sealed and placed in a constant temperature room (25°C) to incubate for the length of the experiment. Microcosms were randomised during incubation. The microcosms were removed from the constant temperature room daily to allow the CO2 trap to be changed and exchange of O2 and waste gases to take place in the head-spaces of the microcosms.

2.2. Sample collection 

At harvest, randomly selected microcosms were removed and destructively sampled, soil covering the SMT was carefully removed from the above the tissue surface. Remaining SMT was then removed for percentage tissue mass loss calculations. The 1cm of soil immediately surrounding the entire decomposition area (detritosphere) was removed with the soil from above the decomposing tissue (∼40g) and frozen (−20°C) prior to analysis.

2.3. Tissue mass loss 

At harvest any remaining SMT was removed from each soil microcosm and adhering soil was gently brushed off. The SMT was then frozen (−20°C) for 24h. When the soft tissue samples were frozen they were washed with deionised water to remove any remaining soil from the surface and were then dried with paper towel to absorb the excess water from the surface. The sample was then weighed to estimate fresh (or decomposed) mass. Any microcosms with no remaining SMT were designated at 100% tissue mass loss.

2.4. Carbon dioxide respiration 

CO2 respiration was estimated using alkali (NaOH) traps [11]. Traps held NaOH solution and CO2 released into the head-space was calculated after addition of barium chloride solution, phenolphthalein indicator and back titration with hydrochloric acid. Traps were changed every 24–96h (as appropriate) to avoid saturation and O2 depletion in microcosm headspaces. It is assumed that minimal CO2 is produced from calcium carbonate in the soil, as the soil used is an ancient soil and highly leached of minerals.

2.5. pH and electroconductivity 

pH and electroconductivity were estimated using a standard method [12]. Soil and deionised water were mixed in a 1:5 ratio and shaken end over end at 25°C for 1h. The suspension was allowed to settle for 30min prior to pH (Cyberscan20) and electroconductivity (TPS MC-84) measurements.

2.6. Nitrogen extraction for ammonium and nitrate analysis 

The method for extraction of mineral nitrogen was taken from Rayment and Higgins [12] using 2M potassium chloride at 1:10 solution ratio. The samples were shaken end over end at 25°C for 1h. The extracts were left to clear (∼30min) and were filtered prior to storage (−20°C) until analysis could be carried out. Aliquots of the extract were placed in vials and diluted as needed for colourimetric analysis (SKALAR autoanalyser).

2.7. Bicarbonate extractable phosphate and potassium extraction 

The method for extraction of phosphate and potassium through bicarbonate solution has been adapted from Rayment and Higginson method 9B1 [12] to measure soil of low PO4− and K+ extractability. Soil and sodium bicarbonate solution (1:100 ratio) were shaken end over end for 16h, samples were then centrifuged for 20min at 3000rpm.

2.7.1. Bicarbonate extractable phosphate 

The reagents for bicarbonate extractable PO4− are as follows: 2M sulfuric acid; reagent A [12g ammonium molybdate dissolved in ∼400mL deionised water, 140mL concentrated H2SO4 diluted in 300mL deionised water and added to the ammonium molybdate solution, 0.2669g potassium antimonyl tartrate added to the ammonium molybdate solution and made to 1L volume with deionised water]; mixed colour reagent (prepared fresh daily) [1.056g l-ascorbic acid dissolved in 200mL reagent A].

Sulfuric acid (0.7mL) was placed in a vial and 10mL of bicarbonate extract, blank or standard were added. Another 0.7mL aliquot H2SO4 was added and the samples were left ajar overnight to allow gas release. The following day 3.2mL of colour reagent was added to each sample and standard, while 3.2mL of reagent A was added to each reagent blank. All samples were made up to 23mL through addition of 8.4mL deionised water and shaken by hand. Samples were centrifuged for 5min at 2000rpm. Sample and standard absorbance was measured on a spectrophotometer (Hitachi U-1100) at 882nm.

2.7.2. Bicarbonate extractable potassium 

The reagents used for bicarbonate extractable K+ were as follows: 2M H2SO4; neutralised 0.5M NaHCO3 solution [500mL 0.5M NaHCO3, add 70mL 2M H2SO4 and leave uncovered to allow gas release overnight].

For K+ analysis 5mL bicarbonate extract was neutralised with 0.7mL H2SO4 and made to 10mL volume with 4.3mL deionised water. Vials were left ajar overnight for gas release. Extracted samples were analysed in absorption mode on an atomic absorbance spectrometer (Perkin Elmer AAnalyst 300, λ=766.5) calibrated with working standards.

2.8. Analysis of fresh soil 

The freshly seived (2mm) soil harvested from Harry Waring Marsupial Reserve, Perth, Western Australia was analysed using the above methods to ascertain basal values for: pH, electroconductivity, NH4+, NO3−, PO4− and K+ concentrations.

2.9. Data analysis 

Statistical analysis was carried out using SAS 9.1. Data which displayed normality was analysed using ANOVA. Carbon dioxide respiration was analysed using a repeated measured analysis with differential time periods taken into account. Correlation between pH and NH4+ was calculated with MS Excel. Multivariate analysis was conducted with Primer (version 6) and provided visualization with a non-metric multidimensional scaling (NM-MDS) [13]. NM-MDS were performed on all abiotic data (pH, electroconductivity, NH4+, NO3−, PO4− and K+) for all time periods. Resemblance matrix was based on normalised data and Euclidean distance measurement.

2.10. Histology of fresh tissue 

Portions of SMT were analysed through histology to identify the morphological changes in fresh, refrigerated and frozen samples. The fresh SMT cubes from each treatment were placed in formalin for 48h to dehydrate the tissue, each sample was then placed in an ethanol:0.9M saline (10:90, v/v) solution. Portions of each sample were processed (formalin, saline, 50% EtOH+1% detergent, 70% EtOH+1% detergent, 90% EtOH+1% detergent, 3×absolute EtOH, 2×toluene, 2×wax and wax vacuum embedder at 25mmHg) using a Shandon Citadell 1000. The samples were set within the block in longitudinal and transverse alignments. The 5μm thick sections cut from the wax block obtained using a Leica RM2255. Morphological variations were visualised using a haemotoxylin and eosin stain [14]. The stained samples were imaged using a Nikon 90i microscope, Nikon DFX1200 camera and ACT-1 capturing software. The images obtained were digitalised and interstitial space area (percentage) was calculated using ImageJ software (http://rsb.info.nih.gov/ij/).

Back to Article Outline

3. Results 

3.1. Soil chemical characteristics 

The soils were slightly acidic (pH 6.1) with low levels of salinity (26μS). Concentrations of major nutrients were: 7.8mgkg−1 dry soil for NH4+, 8mgkg−1 dry soil for NO3−, 10.6mgkg−1 dry soil for PO4− and 20.6mgkg−1 dry soil for K+.

3.2. Percentage tissue mass loss 

Freezing 1cm3 portions of SMT had no impact on the rate of percentage tissue mass loss in comparison to skeletal muscle tissue portions which were refrigerated. The frozen and refrigerated SMT treatments showed a rapid decomposition in the first 8 days, after this point the loss of mass was more gradual up until the final harvest at 37 days (Fig. 1). There was no significant difference between the 2 treatments. The SMT harvested at day 8 and after this period had a dehydrated appearance, with little apparent moisture on the surface.

  • View full-size image.
  • Fig. 1. 

    Tissue mass loss (%) of skeletal muscle tissue (Sus scrofa: 1.5g) frozen (●) or unfrozen (○) prior to burial in a sandy soil from Harry Waring Marsupial Reserve, Perth, WA. Bars±standard error, n=6.

3.3. Carbon dioxide respiration 

The introduction of SMT to soil led to significantly higher CO2 respiration than in control soils (basal respiration) (P<0.001) (Fig. 2(a)). Prior freezing on SMT had no effect. In both treatments there was an initial flush of CO2 after 3 days and the levels started to drop after 5 days incubation at 25°C. Notably in days 1 and 2, CO2 respiration was higher in the frozen treatment but this was not a significant increase.

  • View full-size image.
  • Fig. 2. 

    Microbial CO2 respiration (μgg−1 air dry soil h−1) over a period of 37 days following the addition of 1.5g of skeletal muscle tissue (S. scrofa) frozen (●), unfrozen (○) and control (▾) in 100g of sandy soil, Harry Waring Marsupial Reserve, Perth, WA. Incubation was conducted at 25°C. Bars±standard error, n=6. (a – top) time course microbial respiration and (b – bottom) cumulative microbial CO2 respiration.

Cumulative CO2 respiration was almost identical for soil with SMT regardless of freezing treatment with no significant variation. These levels were approximately 3 times higher than basal levels (Fig. 2(b)).

3.4. pH 

The introduction of SMT caused a significant impact on the measured pH in the soil surrounding it in comparison to control soil (P<0. 001). Freezing SMT prior to inhumation had no significant effect. Within two days of SMT being introduced to the soil microcosm the pH value had increased by 0.75–1 pH unit above that recorded for the control soil (Fig. 3(a)). A maximum pH value of 2 pH units above the control was reached within 6 days of SMT introduction, after which it began to decrease towards the value first recorded on day 2.

  • View full-size image.
  • Fig. 3. 

    Time course (a – top) pH values and (b – bottom) electroconductivity measured in the detritosphere soil surrounding skeletal muscle tissue (S. scrofa: 1.5g) frozen (●), unfrozen (○) and control (▾) in 100g sandy soil from Harry Waring Marsupial Reserve, Perth, WA. Bars±standard error, n=6.

3.5. Electroconductivity 

Microcosms containing decomposing SMT showed an increase in conductivity over the course of the experiment with a slight decrease in the last week of incubation (Fig. 3(b)). There was a significant effect caused by the introduction of SMT to the soil microcosm (P<0.001), but no significant differences were observed between frozen and unfrozen SMT samples.

3.6. KCl extractable nitrogen 

The introduction of SMT to a soil microcosm led to a significant increase in NH4+ concentrations in comparison to the control microcosms (P<0.001). Any variations between the frozen SMT and refrigerated SMT were not significant. The levels of NH4+ in the microcosms containing SMT increased rapidly over the first 2 weeks, then began to level, before decreasing (Fig. 4(a)). NH4+ concentration was strongly correlated with pH (R2=0.856).

  • View full-size image.
  • Fig. 4. 

    Time course (a – top) ammonium concentration (mgkg−1 dry soil) and (b – bottom) nitrate concentration (mgkg−1 dry soil) in detritosphere soil after the introduction of skeletal muscle tissue (S. scrofa, 1.5g) frozen (●), unfrozen (○) and control (▾) in 100g sandy soil from Harry Waring Marsupial Reserve, Perth, WA. Bars±standard error, n=6.

Microcosms containing SMT were significantly different to control microcosms (P<0.001). Freezing had no effect. NO3− concentrations increased slightly during the incubation in the control microcosms, while those containing frozen and unfrozen SMT portions had an initial lag period before a rapid increase in concentration (Fig. 4(b)).

3.7. Bicarbonate extractable phosphate 

SMT has a significant affect on the amount of bicarbonate extractable PO4− present in the soil (P<0.001) in comparison to soil sampled from the control. After introducing SMT, extractable PO4− concentration increased with a slight decrease by the final week of incubation (Fig. 5(a)). There was no significant effect of freezing. Control microcosm concentrations remained relatively constant with some minor variations over the course of the incubation.

  • View full-size image.
  • Fig. 5. 

    Time course (a – top) bicarbonate extractable phosphate concentration (mgkg−1 dry soil) and (b – bottom) bicarbonate extractable potassium concentration (mgkg−1 dry soil) in detritosphere soil after the introduction of skeletal muscle tissue (S. scrofa, 1.5g) frozen (●), unfrozen (○) and control (▾) in 100g sandy soil from Harry Waring Marsupial Reserve, Perth, WA. Bars±standard error, n=6.

3.8. Bicarbonate extractable potassium 

Bicarbonate extractable K+ showed that SMT introduction had a significant effect on the concentration present in the soil compared to control microcosms (P<0.001). Freezing had no significant effect on extractable K+ concentration. Soil containing SMT had increased K+ concentrations at the first harvest (2 days incubation), this increase continued until day 16 (160mgkg−1) for the frozen SMT samples before decreasing for the rest of the incubation. After day 12 the K+ concentration in soil containing refrigerated SMT steadied at approximately 110mgkg−1 dry soil and did not reach the concentration of the soil surrounding previously frozen SMT (Fig. 5(b)).

3.9. Multivariate analysis of soil chemistry 

NM-MDS showed a clear difference in the measurement made for soil chemistry between control and amended soils (pH, electroconductivity, NH4+, NO3−, PO4− and K+) (Fig. 6). These data were separated (distance 4) by cluster analysis. There was no effect of freezing SMT on the full data set. However, there were clear changes in the MDS plot with time in both control and amended soil.

  • View full-size image.
  • Fig. 6. 

    NM-MDS visualization of normalised abiotic data (pH, electroconductivity, NH4+, NO3−, PO4− and K+) for all time periods show 2 distinct clusters: control (right) and SMT amended soil (left). Changes over time are apparent in both clusters as indicated by the arrows.

3.10. Histology 

Fresh and refrigerated SMT portions are not visually different from each other. However, freezing led to increased size in interstitial channels and large zones containing no SMT material (Plate 1). Percent interstitial spaces for longitudinal sections were: 0.82% for control SMT, 11.49% for refrigerated and 24.02% after freezing. Interstitial spaces in transverse sections were as follows: 13.29% for control, 10.96% after refrigeration and 30.66% after freezing. There were noted increases in percent interstitial space after freezing.

  • View full-size image.
  • Plate 1. 

    20× magnification of longitudinal (left) and transverse (right) 5μm sections of SMT from (a) fresh, (b) refrigerated and (c) frozen portions. Dark grey spots indicate nuclei of muscle cells, large open spaces in the frozen sample indicate ice crystal artifacts and dehydration.

Back to Article Outline

4. Discussion 

4.1. Effect of freezing on skeletal muscle tissue decomposition 

The primary determinants of soft tissue decomposition (tissue mass loss and carbon dioxide respiration) indicate there is little effect of freezing on SMT decay in soil. The chemistry of the detritosphere soil showed no significant effect of freezing SMT. In the only similar taphonomic study, freezing was found to have a significant effect on the decomposition process [5]. Animal analogues which had been frozen previously decomposed predominantly from the outside, with good preservation of internal organs. Cadavers that decomposed in a fresh state decayed predominantly through anaerobic microbial activity, with rapid breakdown of internal organs [5]. This experimental finding was supported by a criminal murder case where the victim was frozen for over 2 years before his body was dumped. The pattern of decomposition appeared to progress from the outside in with relatively good preservation of internal organs [15]. It seems likely that freezing has at least a partial biocidal effect on the enteric flora [6], [7] and may have caused some enzymes to lose their functional conformation. However in both situations the cadavers were on a soil surface rather than buried in a soil environment, while the current study is conducted in a burial environment. Hence, caution must be applied to comparison of these studies [5], [15] with this study. There was also no enteric microbiota in this study due to the use of skeletal muscle portions, but internal organs are not the only source of microbes within a body. Microbial communities also inhabit other surface areas of the body such as skin [16]. Within muscle tissue there are also a range of enzymes which will breakdown cellular components and overall cell structure [17], [18].

The implications of the results from this study could be valuable in a forensic setting after discovery of dismembered body parts (i.e. arms, legs, feet). Under such circumstances, the affect of freezing on the decomposition of body-parts would not be expected to differ from unfrozen tissue. However, it should be borne in mind that data presented here are based on one soil type and one tissue type only. More work is needed to reach universal conclusions.

4.2. Effect of SMT on Soil chemistry 

Introduction of SMT to a soil environment under controlled environmental conditions leads to a significant alteration in the chemistry of the soil surrounding it and increased levels of microbial activity.

SMT mass decreased rapidly, probably caused by loss of water from the tissue to the surrounding environment. This was supported by the observation that the harvested SMT appeared dry after 8 days of interment. A body is composed of mainly water (64%) with protein (20%), fat (10%), carbohydrate (1%) and mineral salts (5%) making up the other major components [19]. Breakdown of a body has been described as competition between desiccation and decomposition [5], [6], [7], [20]. During freezing up to 80% of the water from within cells is drawn into the interstitial channels forming ice crystals. This water is not fully reabsorbed into the cell during thawing and remains in the interstitial channels, making the frozen SMT scenario more susceptible to water loss in the early stages of decomposition. It is thought this may explain the slightly higher mass loss observed in the frozen SMT harvest at day 2. Initial rapid tissue loss is also likely to be related to active decay and putrefaction. Gradual tissue mass loss in latter stages of the incubation may be related to late stage decay, when more resistant components are degraded.

When SMT was added to the soil microcosms a rapid and very large flush of CO2 was measured which was significantly higher than the levels observed in the basal respiration of control microcosms. The increased levels are likely to be due to a rapid increase in the size of the microbial population when a readily decomposable source of nutrients was introduced to the soil. Similar patterns have been observed in other laboratory based taphonomic experiments using SMT portions [21]. The natural anaerobic processes which occur in SMT during decomposition include the release of gases such as hydrogen, hydrogen sulfide, methane and CO2 as the macromolecules of the cells are broken into their smaller component units [18]. This is likely to also have some contribution to the carbon dioxide levels observed. However, in this experiment we assume that the majority of the carbon dioxide observed is due to microbial respiration rather than enzymatic and cellular decomposition processes in the SMT.

A nutrient poor sandy soil was chosen as the test soil in this experiment so that changes in nutrient concentrations due to SMT could be easily discriminated. Sand based soils have a tendency towards nutrient deficiency caused by leaching when high ion concentrations are introduced. All the nutrients analysed diffuse through sandy soils although their diffusion rates vary greatly [22]. When SMT was removed from the centre of the microcosms the zone (1cm) surrounding the SMT was sampled. Approximately 2cm of soil remained in the area surrounding the burial zone within the microcosm. The combination of inherent nutrient deficiency in sandy soils and tendency to diffuse readily when high nutrient levels are applied provides a possible explanation for the increase and subsequent decrease in most nutrients analysed. The small variations seen in the concentrations of nutrients for frozen and unfrozen skeletal muscle tissue may be linked to the damage which is caused to the tissue upon freezing. A known artifact is introduced during slow freezing due to the high water content of cells, leading to the formation of ice crystals which causes destruction to cells and their components [4].

Electrolytes, cations and anions, from the muscle tissue are released as it decomposes and its macromolecules are degraded by enzymes within the cells and the soil into soluble ions. This leads to an increase in electro-conductivity in the detritosphere soil, matched by increased concentrations of PO4−, K+, NH4+ and NO3− ions. Over the course of the incubation there were no obvious patterns in electroconductivity with fluctuations in the levels measured in all treatments.

A relationship between NH4+ and pH was suggested by Hopkins et al. [23] and our results support this hypothesis over a pH range of 2–3 units with a strong relationship between NH4+ and pH. The decrease in NH4+ is likely due to microbial immobilization and nitrification which outstripped ammonification after 16 days. This may have potential uses in gravesoil identification and postburial-interval (PBI)/postmortem-interval (PMI) estimation.

NO3− concentrations in the control microcosms slowly increased. This we ascribed to instability in the nitrogen cycle of the soil within the control microcosms and the breakdown of organic matter present in the soil caused by the disturbance of the soil during experimental set-up exposing new surfaces and substrates. This effect can clearly be seen as a temporal shift in control soil points in the multivariate ordination.

The introduction of SMT to the soil microcosm showed an initial decrease in NO3− concentration before a rapid increase. This pattern has previously been observed in soils amended with sewage sludge which has a high level of readily available carbon that can stimulate microbial immobilization of N [24]. There are several possible reasons for this observation such as inhibition of nitrification due to high soil pH [25], [26] or free ammonium [27], [28], [29], immobilization of nitrogen due to high levels of microbial activity during decomposition [24], or the slow rate of growth of nitrifying bacteria in comparison to other microbiota in the soil environment. Further investigation would be required to give a definitive explanation for the results, but they are likely caused by a combination of factors.

A living body contains large amounts of phosphorous in the form of enzymes, DNA and numerous other proteins. Within living SMT the main source of cellular energy is via ATP metabolism. After death ATP is metabolised by phosphorylases released from within the cell and it has been found, using 31P NMR, that tissue which has previously been frozen experienced a more rapid breakdown of ATP to free phosphate [30]. Fluctuations were observed in PO4− concentrations in the detritosphere with several possible explanations available such as diffusion of ions to areas where extraction is inefficient [31], increased bond strength of PO4− to adsorption zones or precipitation of less soluble compounds [31], [32]. Previously the analysis of phosphate was anticipated to be a suitable indicator of gravesoil however in clay based soil the ion became adsorbed and was difficult to accurately measure. Hence it was subsequently not analysed for graveyard groundwater pollution [19]. The soil type used in this study was a coarse sandy soil, which is known to have fewer exchange sites for the ions [33]. However, there was still no distinct pattern in the results obtained suggesting that it is not only absorption of PO4− that is important.

Histological analysis of SMT that had been refrigerated or frozen demonstrated the impact of freezing on gross morphology. The interstitial channel spaces were substantially enlarged in comparison to both fresh and refrigerated SMT. These variations were observed in both longitudinal and transverse sections. Within the images it was also possible to see the presence of solitary nuclei within interstitial channels with no other associated cellular material. This is likely to be due to the rupturing of cells during ice crystal formation, releasing cellular components to the interstitial cavity.

Back to Article Outline

5. Conclusions 

The interment of skeletal muscle tissue into a controlled soil environment has a significant impact on chemical properties of the soil surrounding the decomposing soft tissue that were tested during this taphonomic experiment. Current published research on soil chemistry is from a predominantly agricultural area after the introduction of nutrients, the results here illustrate the difficulty of adequately explaining the impact that introducing skeletal muscle tissue has on the surrounding environment. Decomposition of soft tissue caused significant changes to be observed in all the chemical aspects tested over the experiment but the exact interaction of these with each other is not readily identified.

Decomposing SMT which has been frozen showed no significant variation in the surrounding soil chemistry when compared statistically to the results obtained from SMT which has been refrigerated. However, there is a small (non-significant) indication of the effects of ice crystal cell damage on subsequent decomposition and chemical measurements.

Back to Article Outline

Acknowledgements 

We thank M. Smirk for assistance with chemical analysis, K. Murray for his advice and assistance with statistical analysis the results, B. Cooper for access to Harry Waring Marsupial Reserve, Perth, WA for bulk soil sampling and M. Lee for help with histological processing.

Back to Article Outline

References 

  1. Mant AK. Knowledge acquired from post-war exhumations. In:  Boddington A,  Garland AN,  Janaway RC editor. Death, Decay and Reconstruction: Approaches to Archaeology and Forensic Science. Manchester: Manchester University Press; 1987;p. 65–78
  2. Partmann W. Post-mortem changes in chilled and frozen muscle. Journal of Food Science. 1963;28(1):15–27
  3. Evans WED. The Chemistry of Death. Springfield, IL, USA: Charles C Thomas; 1963;
  4. Meryman HT. The mechanisms of freezing in biological systems. In:  Parkes AS editors. Recent Research in Freezing and Drying. Oxford: Blackwell Scientific Publications; 1960;23 pp
  5. Micozzi MS. Experimental study of postmortem change under field conditions: effects of freezing, thawing and mechanical injury. Journal of Forensic Sciences. 1986;31:953–961
  6. Micozzi MS. Postmortem Change in Human and Animal Remains: A Systematic Approach. Springfield, IL, USA: Charles C. Thomas; 1991;
  7. Micozzi MS, Pless J. Frozen environments and soft tissue preservation. In:  Haglund WD,  Sorg MH editor. Forensic Taphonomy: The Postmortem Fate of Human Remains. Boca Raton, FL, USA: CRC Press; 1997;p. 171–180
  8. Sikes A, Maxcy RB. Postmortem invasion of muscle food by a proteolytic bacterium. Journal of Food Science. 1980;45:293–296
  9. Gill CO, Penney N. Bacterial penetration of muscle tissue. Journal of Food Science. 1982;47:690–691
  10. Tibbett M, Carter DO, Haslam T, Major R, Haslam R. A laboratory incubation method for determining the rate of microbiological degradation of skeletal muscle tissue in soil. Journal of Forensic Sciences. 2004;49:560–565
  11. Rowell DL. Soil Science: Methods and Applications. Harlow, UK: Longman Group UK Limited; 1994;
  12. Rayment GE, Higginson FR. Australian Laboratory Handbook of Soil and Water Chemical Methods. Melbourne: Inkata Press; 1992;
  13. Clarke KR. Non-parametric multivariate analyses of changes in community structure. Australian Journal of Ecology. 1993;18(1):117–143
  14. Bancroft JD, Cook HC. Manual of Histological Techniques. Edinburgh, London, Melbourne and New York: Churchill Livingstone; 1984;
  15. Zugibe FT, Costello JT. The Iceman Murder: One of a Series of Contract Murders. Journal of Forensic Sciences. 1993;38(6):1404–1408
  16. Luckey TD. Introduction to intestinal microecology. The American Journal of Clinical Nutrition. 1972;25(12):1292–1294
  17. Clark MA, Worrell MBE, Pless J. Postmortem changes in soft tissue. In:  Haglund WD,  Sorg MH editor. Forensic Taphonomy: The Postmortem Fate of Human Remains. Boca Raton, FL, USA: CRC Press; 1997;p. 151–164
  18. Gill-King H. Chemical and ultrastructural aspects of decomposition. In:  Haglund WD,  Sorg MH editor. Forensic Taphonomy: the Postmortem Fate of Human Remains. Boca Raton, FL, USA: CRC Press; 1997;p. 93–108
  19. van Haaren FWJ. Churchyards as sources for water pollution. Moorman’s Periodieke Pers. 1951;35(16):167–172(in Dutch)
  20. Carter DO, Yellowlees D, Tibbett M. Cadaver decomposition in terrestrial ecosystems. Naturwissenschaften. 2007;94:12–24
  21. Carter DO, Tibbett M. Microbial decomposition of skeletal muscle tissue (Ovis aries) in a sandy loam soil at different temperatures. Soil Biology and Biochemistry. 2006;38:1139–1145
  22. Brady NC, Weil RR. The Nature and Properties of Soils. Upper Saddle River, NJ: Prentice Hall; 1996;
  23. Hopkins DW, Wiltshire PEJ, Turner BD. Microbial characteristics of soils from graves: an investigation at the interface of soil microbiology and forensic science. Applied Soil Ecology. 2000;14:283–288
  24. Smith MTE, Tibbett M. Nitrogen dynamics under Lolium perenne after a single application of three different sewage sludge types from the same treatment stream. Bioresource Technology. 2004;91(3):233–241
  25. Nugroho RA, Roling WFM, Laverman A, Verhoef HA. Low nitrification rates in acid scots pine forest soils are due to pH-related factors. Microbial Ecology. 2007;53(1):89–97
  26. Kyveryga PM, Blackmer AM, Ellsworth JW, Isla R. Soil pH Effects on Nitrification of Fall-Applied Anhydrous Ammonia. Soil Science Society America Journal. 2004;68:545–551
  27. Balmelle B, Nguyen KM, Capdeville B, Cornier JC, Deguin A. Study of factors controlling nitrite buildup in biological processes for water nitrification. Water Science and Technology. 1992;26(5–6):1017–1025
  28. Philips S, Laanbroek HJ, Verstraete W. Origin, causes and effects of increased nitrite concentrations in aquatic environments. Environmental Science and Biotechnology. 2002;1:115–141
  29. Vadivelu VM, Keller J, Yuan Z. Effect of free ammonia on the respiration and growth processes of an enriched Nitrobacter culture. Water Research. 2007;4:826–834
  30. Honikel KO, Hamm R. Influence of cooling and freezing of minced pre-rigor muscle on the breakdown of ATP and glycogen. Meat Science. 1978;2:181–188
  31. Freeman JS, Rowell DL. The adsorption and precipitation of phosphate onto calcite. Journal of Soil Science. 1981;32:75–84
  32. Javid S, Rowell DL. A laboratory study of the effect of time and temperature on the decline in Olsen P following phosphate addition to calcareous soil. Soil Use and Management. 2002;18:127–134
  33. He JZ, Gilkes RJ, Dimmock GM. Mineralogical properties of sandy podzols on the Swan Coastal Plain, south-west Australia, and the effects of drying on their phosphate sorption characteristics. Australian Journal of Soil Research. 1998;36(3):395–409

PII: S0379-0738(08)00334-4

doi:10.1016/j.forsciint.2008.08.013

Forensic Science International
Volume 183, Issue 1 , Pages 6-13, 10 January 2009

Article Tools

* Image Usage & Resolution