Background: Skeletal remains have become the mainstay in forensic investigations. Hence, it is imperative to study bone degradation and some influencers as to guide forensic practices. The purpose of this study was to determine the effect of soil pH on bone degradation. Methods: One-centimeter diameter sections of the mid-shaft of the femur of a male cadaver were buried for the duration of 6 weeks in soils of different pH: 2.98 for the acidic soil, 7.10 for the neutral soil, and 11.58 for the alkaline soil. Histological sections of the exhumed bones were prepared using Frost’s rapid manual method. Four fields of view at 12 o’clock, 3 o’clock, 6 o’clock, and 9 o’clock positions were obtained for each section. Results: Quantitative analysis showed that there was a reduction in the mean Haversian canal area (HCA) and Haversian canal diameter (HCD) in the samples of bone fragments buried in the acidic and alkaline soil when compared with control. The acidic soil sample showed a mean HCA and HCD of 130.58 μm and 12.24 μm as against 136.83 μm and 12.48 μm of the control, whereas the alkaline soil sample showed a mean HCA and HCD of 122.70 μm and 11.70 μm, respectively. Statistical analysis showed a significant difference in the HCA and HCD (P ≤ 0.05). Conclusion: The result of this study suggests that the extreme of soil pH can cause the weathering of bone, which can distort the histomorphometry. The study focused on the extremes of pH and not various scales. This should help guide forensic investigations.
Keywords: Bone, degradation, forensic science, Haversian system, soil pH
| Background|| |
A soil type is one of the most important factors that affects the decomposition of any organic material. Soils have different textures: particles that constitute the soil are called as sand (2–0.02 mm), silt (0.02–0.002 mm), and clay (<0.002 mm). Biochemical compounds also vary from one location to another, which helps define the soil composition of that location . Also 12 major textural classes are defined in several combinations according to the size of particles by United States Department of Agriculture textural triangle . It is known that the particle size of soil could affect the rate of decomposition. In this context, decomposition can be completely inhibited or decreased to the least in coarse-textured soil because of the diffusion of gasses through the soil matrix . It is also known that chemical reactions in soil, such as acidic environments, are some of the most destructive agents when it comes to organic material ,,,,.
Soil pH is a very important factor of bone degradation. Natural soil pH reflects the combined effects of the soil-forming factors (parent material, time, relief or topography, climate, and organisms). The pH of newly formed soils is determined by the minerals in the parent material. Temperature and rainfall affect the intensity of leaching and the weathering of soil minerals [1,5]. In warm, humid environments, soil pH decreases over time through acidification due to leaching from high amounts of rainfall. In dry environments where weathering and leaching are less intense, soil pH may be neutral or alkaline. Soils that have a high content of clay and organic matter are more resistant to changes in pH (higher buffering capacity) than are sandy soils. Although clay content cannot be altered, organic matter content can be altered by management practices and human manipulation. Sandy soils commonly have a low content of organic matter, resulting in a low buffering capacity and a high rate of water percolation and infiltration. Thus, they are prone to increased acidification [1,5]. Our study therefore aims at determining the effect of different soil pH on bone degradation as the effect of soil pH on bone degradation has not been extensively studied in the Niger Delta region of Nigeria.
| Materials and methods|| |
This experimental study was carried out with bone fragments buried in soils of different pH for a period of 6 weeks. The samples used for this study comprised of sections taken from the femoral mid-shaft of a male cadaver. Two 1-cm diameter bone sections were buried in soils that had their pH chemically altered to obtain acidic, alkaline, and neutral soils. After 6 weeks, the bones were exhumed and analyzed microscopically to determine the effect of the different soil pH on bone degradation. The bone samples included nontraumatized bones as well as those not previously distorted by natural or physical means.
The right thigh region of the male cadaver was carefully dissected using a surgical blade, and the muscles and soft tissues removed manually. The femur bone was detached from the hip bone at the hip joint and the tibia at the knee joint. The shaft of the femur was sawed transversely using a hack saw into 1 cm thick pieces. Each piece was then sawed into four equal fragments. Each type of soil had two fragments buried in it. Bone fragments were exhumed and prepared into histological sections using modified Frost’s manual method of bone preparation . The prepared slides were viewed with Leica ICC 50E photomicroscope (Germany). The micrographs were analyzed using the Image J software, and the following parameters were taken for four various fields per slide: total osteon count, Haversian diameter, Haversian area, number of primary osteons, number of secondary osteons, and number of osteon fragments.
| Results|| |
The results are shown in [Figure 1] and [Figure 2] as well as in [Table 1][Table 2][Table 3][Table 4]. [Figure 1] shows the bone sections obtained from acidic soil and neutral soil, whereas [Figure 2] shows bone sections obtained from alkaline soil. Qualitative analysis of the photomicrographs shows an obvious variation in the histomorphometry of the micrographs. Also quantitative analysis shows variations as well in the primary osteons, secondary osteons, as well as in the Haversian canal diameter (HCD). The samples obtained from control, neutral, and alkaline soils appear to have more number of osteons than those from acidic soils. It is therefore imperative to affirm that acidic soil has enhanced effect on bone degradation.
|Figure 1: (A) Photomicrograph showing bone section from acidic soil (pH 2.96), ×100. (B) Photomicrograph showing bone section from neutral soil (pH 7.0), ×100. f = osteon fragments, S = secondary osteons, v = Volkmann’s canal.|
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|Figure 2: (A) Photomicrograph showing bone section from alkaline soil (pH 11.58), ×100. (B) Photomicrograph showing the control section, ×100. H = Haversian canal, P = primary osteon.|
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|Table 1: Descriptive statistics for Haversian canal area and Haversian canal diameter.|
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|Table 2: Descriptive statistics for total osteon count, primary osteon, secondary osteon, and osteon fragments.|
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|Table 3: Test for differences in Haversian canal area and Haversian diameter.|
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| Discussion|| |
This study has investigated bone Degradation in various soil types. Qualitative analysis showed that the micrographs of the acidic and alkaline soil sample when compared with the micrograph of the control bone showed some kind of washing effect. The micrograph looked as though it was smeared, the borders of the osteons were not well defined, and the interstitial lamellae looked wavy. Also, around few of the Haversian canals, there appeared to be deterioration of the innermost concentric lamellae evidenced by bright spots around the Haversian canal. This was more conspicuous in the acidic sample micrograph than in the alkaline sample, but it was present in the both micrographs.
These differences shown were in contrast to those of the control bone micrograph whose osteons had clear borders and their interstitial lamella was not wavy. Also, their concentric lamella was well defined and did not show any deterioration on the innermost parts. The micrograph of the neutral sample was similar to that of the control bone, micrograph showing osteons with clear borders and well-defined lamellae.
Our findings suggest that the smearing effect and deterioration of concentric lamellae shown on the acidic and alkaline micrograph could have been caused by the chemical weathering of bone due to extreme pH. White and Hannus (1983)  reported that carbonic and organic acids in the soil could cause the weathering of bone causing deterioration of the bone hydroxyapatite. A study on the effects of acidity on bones revealed a significant correlation between the pH of the soil and the preservation of bone for both the adult and children’s bones . Watson (1967)  in his study documented that from the excavation of a grave site, it was discovered that there were no bones found outside of the termite mound (pH = 4.8), but there were inside (pH = 6.2). According to Nielsen et al. (2000) , an increased soil temperature leads to increased soil pH as a result of organic acid denaturation, which increases at high temperatures. This is further reinforced by the study by Xiao et al. (2012) , which showed that an increased winter temperature led to significantly increased soil pH. These findings are therefore in consent with our study as to the effect of high pH on bone degradation.
The mean Haversian canal area (HCA) and HCD were of smaller sizes for bone samples obtained from acidic soil as compared with the control bones [Table 1]. The same could be noticed with those of the alkaline sample except for a very few of the Haversian canals. Thus, there was a reduction in the mean HCA and HCD in the samples of bone fragments buried in the acidic soil when compared with control bone with a mean HCA of 130.58 μm against 136.83 μm and mean HCD of 12.24 against 12.48 [Table 1]. These variations can be appreciated statistically with a significant difference (P < 0.05) [Table 3]. The result of this study postulates that extreme pH levels (acidic and alkaline) tend to encourage the chemical hydrolysis of bone collagen because of the high levels of chemicals such as sodium and sulfur. These chemicals cause the deterioration of bone hydroxyapatite.
The findings show minimal variations in the various histomorphometric parameters for the controls and those buried in varying soil pH [Table 2]. However, these variations were not statistically significant (P > 0.05) except for those buried in the neutral soil [Table 4]. The reasons for this finding cannot be ascertained however. Our study has shown that soil pH may have little or no effect on total osteon count, primary and secondary osteon counts, as well as the count of osteon fragments. Some studies reported that the histological features of bones could stand the test of time even after it has been subjected to natural and human distortion . This could as well buttress the fact why bone histology for forensic investigations may yield much better outcomes than only gross morphological findings. Also Tumer et al. (2013)  buried his bones in four different types of soil (loamy, clayey, sandy, and organic) for a period of 3 months and 6 months and performed weight analysis to determine his results, which showed that the mass loss was greater in loamy and organic soil at both intervals. Our present study shows that soil pH affected the HCA and HCD more as compared with the osteon parameters. This is in consent with the study by Tumer and other co-researchers , , ,,as bone mass is determined by the bone density and consequently determined by the area of the Haversian system.
| Conclusion|| |
This study has shown that soil pH caused bone degradation. The mechanism could be through chemical hydrolysis of bone collagen causing the deterioration of bone hydroxyapatite. Acidic and alkaline soil may have encouraged this process evidenced by the smearing effect on the micrograph, undefined osteon borders, deterioration of innermost concentric lamellae, and reducing HCA and HCD. The result of this study suggests that the soil factor should be considered in analyzing skeletal remains in forensic investigation.
There is a paucity of data on similar studies and yet little or none from our environment. This could have aided our study design however. There is also little or no reference base for forensic investigations in our environment. Hence, there is a dire need for our research to focus on such areas. The availability of grant could have aided this study by testing the soil effect for various topographic regions. However, this study can stand as reference guide for future work.
The researchers would appreciate the staff of the Department of Anatomy, University of Port Harcourt for granting the enabling environment and leasing the anatomical garden so as to carry out this research.
LES: conceptualization, formal analysis, project administration, supervision and validation, and software as well as final proofreading and approval of the article. CDO: formal analysis, methodology, review and editing, and visualization and microscopy as well as final proofreading and approval of the article. TJH: resources, original draft, and funding acquisition as well as final proofreading and approval of the article.
This study was approved by the University Research and Ethics Committee (UPH/RDU/CHS/FBMS/ANA/3422), University of Port Harcourt.
Financial support and sponsorship
Conflict of interest
There are no conflicts of interest.
Data availability statement
The data from this study shall be made available to the editors whenever called upon to do so for proper assessment and analysis where required.
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Clinton David Orupabo,
Department of Human Anatomy, Faculty of Basic Medical Sciences, Rivers State University, PMB 5080, Nkpolu-Oroworukwo, Port Harcourt
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]