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DATE DUE DATE DUE DATE DUE 5/08 K:/Proj/Acc&Pres/CIRCIDateDue.indd THE EFFECTS OF SOIL CHEMISTRY ON SKELETAL PRESERVATION AT THE VOEGTLY CEMETERY By Timothy Bryan Lange A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Forensic Science 2008 ABSTRACT THE EFFECTS OF SOIL CHEMISTRY ON SKELETAL PRESERVATION AT THE VOEGTLY CEMETERY By Timothy Bryan Lange Soil factors played an important role in preservation of skeletal remains at the Voegtly Cemetery. While soil pH was found to have a direct relationship with increased bone degradation (R2 = 0.2064) it is believed the true causative agent, responsible for the acidic pH and vast degree of skeletal preservation were soil microorganisms. The remains from the Voegtly Cemetery were ideal for the current research because many factors contributing to skeletal weathering were controlled. For example, the skeletons were in the ground approximately the same length of time (150 years old), were of the same ethnic group, were exposed to same environmental conditions, and had similar burial methods. Despite these conditions it was noted that many skeletons weathered at different rates with some remains being in excellent condition while others were extremely decomposed. The thorough understanding of how soil contributes to degradation of bone including its component parts (collagen, hydroxyapatite, and DNA) is a worthwhile endeavor. If it is found that soil chemistry has correlations with organic (collagen) and mineral (hydroxyapatite) degradation this may allow forensic scientists to predict the length of time skeletal remains have been buried, which can be used to estimate the approximate time or date of death, providing circumstantial evidence in a criminal investigation. Soils were analyzed for pH, organic matter, exchangeable cations, total elemental concentrations of heavy metals, extractable phosphorous, and soil biomass. ACKNOWLEDGEMENTS This work would have not beenpossible without the help of my colleagues, friends, and family. I would like to express my sincere thanks and appreciation to Dr. David Foran for his attention to detail, constructive comments, and his drive for excellance in the writing of my thesis. In addition I would like to thank Dr. Delbert Mokma for his wise insight, guidance, and support in the writing and defense of my thesis. Special thanks are also due to Jon Dahl and other employees at the Michigan State University Soil and Plant Nutrient Laboratory for their time and assistance with the research involved in this project. Thanks are also given to Lisa Misner, Lina Patino, Shirley Owens, Nicholas Lange, Patricia Schabel, Dr. Abudu, Cheryl Helvey, Duane Lange, Phyllis Lange, Dennis Cowper, and Debbie Cowper for their help, comments, and suggestions. I cannot end without thanking Holly whose love and encouragement I have relied on while writing my thesis. I am very grateful for her constant support and it is to her that I dedicate my thesis and MS. degree. iii TABLE OF CONTENTS LIST OF TABLES .................................................................................. v LIST OF FIGURES ................................................................................. vi INTRODUCTION ................................................................................... 1 MATERIAL AND METHODS .................................................................. 17 RESULTS .......................................................................................... 30 DISCUSSION ...................................................................................... 52 CONCLUSION .................................................................................... 59 REFERENCES .................................................................................... 61 iv Table 1. Table 2. Table 3. Table 4. Table 5. Table 6. Table 7. Table 8. Table 9. LIST OF TABLES Summary of Voegtly soil samples ............................................... 17 — 19 Summary of particle size analysis ..................................................... 30 Summary of total elemental analyses without an aluminum filter at various skeletal weathering stages ............................................................... 42 Summary of total elemental analyses with an aluminum filter at various skeletal weathering stages ............................................................... 43 Statistical comparison of mean test results from VC soil samples and Voegtly samples associated with skeletal weathering stage of one (VS-l) . .. 44 Statistical comparison of VC soil samples and Voegtly soil samples associated with skeletal weathering stage two (VS—2) ........................................... 45 Statistical comparison of VC soil samples and Voegtly soil samples associated with skeletons of weathering stage three (VS-3) .................................... 46 Statistical comparison of VC soil samples and Voegtly soil samples associated with skeletons of weathering stage four (VS-4) ..................................... 47 Statistical comparison of VC soil samples and Voegtly soil samples associated with skeletons of weathering stage five (VS-5) ...................................... 48 LIST OF FIGURES Figure 1. Mercury intrusion porosimetry ....................................................... 11 Figure 2. Excavation site of the Voegtly Cemetery ............................................ 14 Figure 3. Layout of the Voegtly Cemetery ...................................................... 15 Figure 4. Map of Voegtly controls samples in relation to Voegtly Cemetery . 20 Figure 5. Description of the Voegtly control soil collected near the cemetery ............... 21 Figure 6. Location of samples tested for particle size analysis ............................... 26 Figure 7. Location of samples tested for elemental analysis ................................. 27 Figure 8. pH as a function of weathering stage ................................................ 31 Figure 9. pH mean values for VC and VS-l through VS-S ................................... 32 Figure 10. Extractable phosphorous as a function of weathering stage ..................... 33 Figure 11. Extractable phosphorous mean values for VC and VS-l through VS-S 34 Figure 12. Exchangeable calcium as a function of weathering stage ........................ 35 Figure 13. Exchangeable calcium mean values for VC and VS-l through VS-5 .. . 35 Figure 14. Exchangeable magnesium as a function of weathering stage ................... 36 Figure 15. Exchangeable potassium as a function of weathering stage . 36 Figure 16. Total organic carbon as a function of weathering stage .......................... 37 Figure 17. Total organic carbon mean percentage values for VC and VS-l through VS-5 ...................................................................................... 38 Figure 18. Total elemental analysis of sample B-27 with no aluminum filter .. . . . . . . . 39 Figure 19. Total elemental analysis of sample B-27 with an aluminum filter ............. 40 Figure 20. Fluorescent image of soil sample B-583 ............................................ 49 Figure 21. Fluorescent image of soil sample B-583 ............................................ 50 Figure 22. Fluorescent image of soil sample B-583 ............................................. 51 vi Introduction Forensic geology is an established field of investigation in the United States, as soil analysis has been used in numerous forensic investigations (Murray, 2001). Soil is an important form of trace evidence and is often found as deposits on shoes, surfaces, clothing or skin. It can easily get trapped on footwear and vehicle tires, allowing investigators to use soil data to help determine if a suspect had been at a crime scene. Forensic examination of soil consists of the analysis of natural occurring rocks, minerals, and vegetation. It can also include detection of manufactured materials such as ions from fertilizers and other environmental artifacts, including: lead, glass, paint chips, or asphalt (Cengiz et al., 2004). Today, a typical soil investigation can involve color comparisons, microscopic evaluation, total elemental analysis, soil density, and mineral identification. Soils by nature are diverse and constantly changing, and as such provide forensic scientists with a large amount of potential evidence. Forensic scientists commonly encounter skeletal remains or ancient skeletons that have been unearthed at a crime scene or burial environment, and a thorough understanding of how soil contributes to degradation of bone including its component parts (collagen, hydroxyapatite, and DNA) is a worthwhile endeavor. If it is found that soil chemistry has correlations with organic (collagen) and mineral (hydroxyapatite) degradation this may allow forensic scientists to predict the length of time skeletal remains have been buried, which can be used to estimate the approximate time or date of death, providing circumstantial evidence in a criminal investigation. Previous research has shown that skeletal degradation is a complicated process (Garland and Janaway, 1989; Nielsen-Marsh et al., 2000; Hedges, 2002; Collins et al., 2002), focusing on bone parameters including collagen content, histological integrity, porosity, and crystallinity. However, there is a surprising lack of research centering on soil and its contributions to explain skeletal preservation and destruction. The current research was unique because it involved investigating how soil characteristics influenced the wide degree of skeletal weathering seen at the Voegtly Cemetery, located in north Pittsburgh, Pennsylvania. Soil Composition Soil is a biologically active matrix composed of air, water, mineral matter, organic matter, and organisms (Cengiz et al., 2004). By volume, soil typically consists of about 45% minerals, 25% water, 25% air, and 5% organic matter. There are two general types of soils, mineral and organic (Garcia, 2000). Mineral soils are formed from decomposed rocks and sediments, while organic soils are formed from the accumulation of plant and animal materials. The latter can be further classified as containing humic and non-humic substances (Petchey, 2005). Primary among humic substances are humic acids, which are ubiquitous, high molecular weight polymers in organic soils (Barancikova et al., 1997). They are a heterogeneous mixture of partially decomposed organic substances that have a range of properties depending on size and attached functional groups. Humus, and in particular humic acids, can influence the water storage capacity, pH, and microbial activity of soil (Garcia, 2000). Non-humic substances are composed of biologic organic matter such as carbohydrates, lipids, and amino acids (Weber, 2007). Carbohydrates make up roughly 5 — 25% of the organic matter in soil. Soil lipids occur in 2 — 6% of the organic portion of soil and exist mostly as remnants of plant and microbial tissues. Amino acids are readily utilized by microorganisms, thus having a very short existence in soil. Ritter (2006) noted five important factors that influence the particular development of soil, including: 1. Parent material: Minerals and organic materials present at formation. 2. Climate: Parent material is broken down into smaller pieces, through a process called weathering. 3. Living organisms: Plants and animals contribute to the formation of soil because as they die, organic matter mixes with parent material and becomes soil. Living, they enrich the soil as they move through the earth or digest food. 4. Topography: The slope of the land affects soil because it can influence moisture content and erosion, which can influence whether soil is well drained or not. 5. Time: Weathering continues to act upon parent materials in the soil. Soils may also be classified based on different particle size, soil textures, and water- holding capacities. Ball (2001) stated that water-holding capacity is determined primarily by soil type and organic matter, with soil containing small particles such as silts and clays being capable of holding more water. There is also an affinity between organic matter and water such that the higher the percentage of organic matter the larger the water- holding capacity of a soil. History of Soil Chemistry Sonon et a1. (2005) presented a detailed description of the history of soil science. Analysis of soil chemistry began in 1819, when the Italian chemist Gazzeri observed that liquid manure became discolored without losing its soluble substances, once passed over clay particles. Later, the father of soil chemistry, J. Thomas Way, discovered that soils could retain cations like ammonium, potassium, and sodium in exchange for equivalent amounts of calcium ions. In 1859, a tool was created called the absorption isotherm, which was utilized to prove that soil organic matter can absorb ammonium ions. Furthermore, it was found that absorption of ions was reversible in soils, coining the familiar term ‘exchange of bases’. In 1888 it was discovered that cation exchange could involve calcium ions as well as sodium ions, which is critical in understanding the destruction of bone in the burial environment. In 1932 an ion exchange equation was created by Albert Vanselow that incorporated principles of mass action to describe exchange. This lead to the creation of a simple exchange equation that expressed soil exchanged phases in terms of milli-equivalents per 100 grams of soil. Soil chemistry was introduced to forensic science in 1908 by George Popp, a chemist and geologist in Frankfort, Germany (Murray, 2001). While a man was under investigation for murdering Margaret Filbert, Popp identified three layers of soil adhering to the leather on the suspect’s shoes. He reasoned that the innermost layer was the oldest, which contained earth materials, such as goose droppings. This layer proved similar to samples that were taken from outside the suspect’s house. The second layer contained red sandstone that was similar to samples taken adjacent to where Filbert’s body was discovered. The last and youngest layer contained brick, coal dust, cement and a series of other materials that were the same as samples collected at the crime scene. Similar methodology is still used today in criminal cases although new methods have been introduced as well. One such technique involves the use of scanning electron microscopy (SEM), which has provided help in identification of earth materials such as soil and minerals, and has also proven valuable in examining particles magnified over 100,000 times their original size (Spector, 2001). Another method that is used in forensic laboratories is capillary electrophoresis to determine anionic compositions of soil. Additionally, laser scanning confocal microscopy, fluorescence microscopy, and transmission electron microscopy may all be utilized to help identify soil. Bone Structure and Composition Human remains found in soil can be of particular forensic interest because skeletal material will degrade in various ways and rates based on characteristics of the soil. Bone is a connective tissue made of organic and inorganic components (Marieb, 2001). Its organic content includes cells (osteoblasts, osteocytes, and osteoclasts) and osteoid, which are approximately one-third of the non-mineralized bone matrix. Bone structure consists of compact (cortical) bone and spongy bone (Marieb, 2001). Compact bone looks dense and solid, but viewed through a microscope reveals passageways that serve as conduits for nerves, blood vessels, and lymphatic vessels. Spongy bone consists of trabeculae, containing irregularly arranged lamellae, and osteocytes interconnected by canaliculi, which lay between two thin layers of compact bone. Around 30% of bone is composed of organic matter, of which 95% is collagen and 5% non-collagenous proteins. Collagen is a fibrous protein that gives the bone strength and flexibility. The majority of bone, approximately 70%, consists of inorganic hydroxyapatite, which has chemical composition of Ca10(PO4)6(OH)2 (Petchey, 2005). Calcium salts also occur, in the form of tiny crystals that surround the collagen fibers. The crystals are tightly packed, giving the bone its exceptional hardness. Bone Diagenesis Bone diagenesis is defined as the physical, chemical, and biological changes in bone after it has been buried or deposited in the environment (Hedges, 2002). There are intrinsic and extrinsic factors that influence bone preservation. Intrinsic factors include bone chemistry (e. g. collagen content, collagen orientation, and hydroxyapatite), size, shape, and the density of bone, while extrinsic factors include groundwater, soil type, and temperature (Henderson et al., 1987). The makeup of the soil surrounding bone is considered the most influential extrinsic factor to affect its diagenesis (Garland and Janaway, 1989). Many conditions can influence the course of bone diagenesis including geochemistry, dissolution, collagen loss, microbiological attack, degree of porosity, and crystallinity alterations. In different burial sites, one or more of the pathways may dominate; which of these occurs first, and how much each influences the others is largely unknown. Geochemistry of Bone Diagenesis Quattropani et a]. (1999) suggested that the geochemistry of soil is responsible for the subtle changes in the degree of bone diagenesis, with soil pH having one of the greatest influences on the breakdown of bone. Preservation is generally better in soils with neutral to slightly alkaline pH, while an acidic pH, often found in sand or gravel, tends to result in poor preservation of bone (Henderson et al., 1987). Marx et a1. (1996) delineated ranges of soil pH as follows: 0 Strongly acidic soil has a pH below 5.1. 0 Moderately acidic soil has a pH 5.2 — 6.0. 0 Slightly acidic soil has a pH 6.1 — 6.5. 0 Neutral soil has a pH of 6.6 — 7.3. 0 Moderately alkaline soil has a pH of 7.4 — 8.4. o Strongly alkaline soil has a pH above 8.5. Gordon and Buikstra (1981) demonstrated a significant correlation between acidic soil and bone degradation. Their study involved seven burial sites, two of which were overlooking the Mississippi River near Hamburg, Illinois and the remaining five located in Woodville Township in Illinois. The sites near Hamburg were excavated between 1970 and 1971 while the others were in the process of excavation at that time. The remains had been buried for at least 700 years but no more than 1,200 years. The authors found that as soil pH decreased, the destruction of bone increased, resulting in poor bone preservation. Pate and Hutton (1988) discussed how exchangeable ions, the soluble portion of ions, influence bone degradation and are available for chemical interaction between bone and soil. In an acidic environment hydroxyapatite is less stable, producing exchangeable calcium and promoting bone degradation. The following equation demonstrates the breakdown of hydroxyapatite in the presence of acidic soil (Nielson- Marsh et al., 2000). C85(PO4)3OH + 7H+ " 5Ca2+ + 3H2PO4- + H20 Hydroxyapatite + Acid -> Exchangeable Calcium Pate and Hutton (1988) addressed chemical exchange between soil and bone mineral by comparing exchangeable ionic concentrations to total elemental concentrations in soils associated with archaeological bone. Total elemental concentrations of ions (Ca, K, Mg, K, Al, Fe, S, and P) in the soil were not found to be indicative of the soluble ionic concentrations because elements have different solubilities, thus only a certain percentage of the total quantity of elements are available to interact with bone. Likewise, the calcium and phosphorous components within hydroxyapatite must be in solution before they are available to interact with the surrounding soil. For this reason, when analyzing soil to predict survival of bone, the use of soluble ion concentration versus total elemental concentration is suggested (Pate and Hutton 1988). Dissolution of Bone Dissolution can also affect bone degradation (Hedges, 2002). Dissolution is the process of minerals being taken from bone by means of ground water, and is common in sites where water movement is prevalent. Soils of low pH and low calcium and phosphate concentrations allow dissolution to occur faster because the protons in the soil replace the calcium ions in hydroxyapatite. In contrast, neutral pH soils usually have calcium concentrations close to saturation, which leads to a much slower dissolution rate. Bone buried in a soil where water movement is limited and calcium and phosphorous concentrations are relatively high can survive for a long period, since the mineral structure is likely to remain unchanged (N ielsen-Marsh et al., 2000). Therefore, most dissolution takes place when conditions change (e. g. hydrology) and when concentrations of calcium and phosphate become low, either due to a reduction in pH or through replacement of water. Collagen Loss Collagen interacts with bone mineral giving bone its characteristic histology and strength while living (Petchey, 2005). It is thought that collagen can degrade within bone, leaving behind a brittle mineral portion. The dynamic relationship among collagen, hydroxyapatite, and the burial environment may also be affected by soil pH, site hydrology, temperature, and microbial attack, resulting in alterations to collagen. In a study by Hedges (2002) bone that showed a loss of collagen resulted in poor histological preservation while bones with high levels of collagen were associated with good histological preservation. Likewise, high or low soil pH can cause collagen swelling and promote its hydrolysis (Collins et al., 2002). The porosity of bone, as measured by water retention, decreases and the crystallinity of bone increases when collagen is lost (Hedges, 2002). Finally, in certain unique situations collagen content is maintained and bones are preserved, such as in abnormally cold, dry, wet, or anoxic (e.g., a bog) environments which inhibit microbial attack (Hedges, 2002). Microbial Attack Microbial attack is a complicated process that is affected by many parameters similar to those influencing the loss of collagen, including temperature, site hydrology, presence of inhibitors, and pH. For instance, burial sites exposed to very low temperatures demonstrate little microbial activity (Hedges, 2002). Likewise, permanently waterlogged burial sites and bogs have been found to inhibit microbial attack (Bocherens et al., 1997). Hedges (2002) noted that bones surrounded by soils containing large amounts of humic acids are less likely to be degraded by microbes, while a neutral pH prevents dissolution of bone, but alternatively promotes microbial invasion. An important point to consider is whether microbes attack bone first or if certain conditions have to be established within the bone for attack to occur. Neilson- Marsh et al. (2000) stated that dissolution of bone mineral and removal or rearrangement of the inorganic component of bone must occur before microbial invasion can begin. This is particularly intriguing considering that low soil pH or low soil cationic concentration may lead to dissolution and leaching of the bone’s inorganic matrix into surrounding soil, ultimately allowing microbial attack, while the low pH could itself inhibit microbes. Given this, the exact process of bone degradation by microbes is difficult to determine because there is such variability in bones and burial sites. Hedges (2002) postulated that if microbial attack is to take place it will be a relatively early event in bone diagenesis, occurring in the first 500 years, with little to no change seen in bones after 4000 years. Bone Porosity The porosity of bone is another major factor that can influence its degradation (Nielson-Marsh et al., 2000). As stated above, spongy bone is very porous, and the spaces influence the extent of diagenetic alterations that occur, resulting in increased porosity and a higher rate of mineral dissolution. One technique used to measure the porosity of archaeological bone is mercury intrusion porosimetry (Nielson-Marsh et al., 10 2000). This technique can help demonstrate the effects of bone degradation as a result of porosity alterations. Figure 1 illustrates the differences in porosity between modern bone and poorly preserved bone using this technique. Figure 1. Mercury intrusion porosimetry in e x -Archaeological Bone 0- Modern Bone :— u: _ —<—.P——.—..»o-,- -«--:~.-- psd cm‘ig'l um" O 100 100000 pore radius nm Mercury intrusion porosimetry traces for modern and diagenetically altered bone. Pore size distribution (psd) is compared with pore radius between archaeological bone and modern bone. It was noted that there is an increase in bone porosity of archaeological bone versus modern bone (Nielson-Marsh et al., 2000). Bone C rystallinity Crystallinity is the degree of structural order within bone (Nielson-Marsh et al., 2000). This feature is most often represented by a percentage of molecules that are arranged in a regular pattern (e. g. crystal). Anything that breaks the pattern of repeating molecules will ultimately reduce crystallinity. Bone dissolution and bone recrystallization are two general processes that contribute to crystallinity and may lead to incorporation of ions from the burial environment, which can affect the mineral structure and the size of bone apatite crystals (Nielson-Marsh et al., 2000). The level of 11 crystallinity can be determined by measuring the amount of transformation of hydroxyapatite to brushite in acidic environments. It can also be detected through infra- red spectrometry or x-ray diffraction as well (Hedges, 2002). Bone crystallinity is affected by chemical changes such as the uptake of fluoride and carbonate ions, and generally increases when there is a loss of porosity and collagen, as seen in burial sites located in warmer climates. Anthropological Assay of Skeletal Weathering Throughout bone degradation the protein component (collagen) undergoes slow hydrolysis to peptides, in turn breaking down into amino acids. During this breakdown a rearrangement of the inorganic crystalline matrix creates a weakened protein-mineral bond, and the bone is considered weathered. The following six stages of skeletal weathering were outlined by Behrensmeyer, (1978) to represent whole skeleton weathering. 1. Stage 0: No signs of cracking or flaking on the bone surface due to weathering. 2. Stage 1: Bone cracking, usually parallel to the fiber structure. 3. Stage 2: The outermost concentric thin layers of bone shows flaking. 4. Stage 3: The bone surface has patches of rough, homogenously weathered compact bone resulting in fibrous texture. 5. Stage 4: The bone surface is coarsely fibrous and rough in texture. 6. Stage 5: The bone is falling apart with large splintering. 12 History of the Voegtly Cemetery Ubelaker et al. (2003) described the history of the Voegtly Cemetery. In 1822, a German, Nicholas Voegtly Sr., and his wife Elizabeth purchased 161 acres of land in Old Allegheny Town of Pennsylvania, near Pittsburgh. Three-quarters of an acre was donated in 1833 by Voegtly to begin construction of the First German Protestant Evangelical Church of Allegheny (the Voegtly Church). As time passed the church membership grew and all associated with the Voegtly church had the privilege of being buried in the Voegtly Cemetery. Nicholas Voegtly died in 1852 and was buried in the Voegtly Cemetery with his wife. In 1865, the entire cemetery was reportedly moved to Troy Hill, which was a larger parcel of land beyond the church grounds. In 1959, the church was informed that it was in the right-of-way of planned construction for a new expressway linking Pittsburgh to Interstate 79. In November 1984 the Pennsylvania Department of Transportation acquired ownership of the property and started preparation for the new highway. The following year the church was razed, and the yard appeared as an open lot. Excavation started in the summer of 1987, which led to the unexpected discovery of 724 burials. It was later revealed that only two bodies had been moved to Troy Hill, Nicholas Voegtly and his wife. Church burial records were found handwritten in German script, and were illegible, having deteriorated with age. Former church members thought that the entire Voegtly Cemetery had been moved in the 1860’s to Troy Hill Cemetery. 13 Archeological Excavation of the Voegtly Cemetery Ubelaker et a1. (2003) detailed the archeological excavations and interpretations of the cemetery. Archeologists excavated the cemetery to define cemetery boundaries and began examination of its content. Technicians and osteologists worked June to September of 1987 to excavate all of the graves (Figures 2 and 3). Hand excavation began at the skull and continued towards the feet. Skeletal data were recorded by the field osteologists, and bones were individually foil-wrapped, bagged and labeled. The remains were transported to the Smithsonian Institution in 1988. In the Smithsonian laboratory, the bones were cleaned to prepare them for analysis. Some bones where in excellent condition and were cleaned with running water, while others were easily broken by movement and needed to be dry-brushed to remove soil. Some skeletons were well preserved, still having hair and soft tissue, while others were extremely decomposed. Figure 2. Excavation site of the Voegtly Cemetery This image is presented in color. Excavation of the Voegtly Cemetery began in 1987 (taken from Ubelaker et al., 2003). Figure 3. Layout of the Voegtly Cemetery 7“ T “25217 millf‘ i 77! -/i- if :1: m Mi! Hull/fl" ’7}! 'Ifi], _ r ‘ .. I 22:? p fifiiyflpfi D Willa/fl??? ,—,~7;‘r i" “ We " 1’29 [735?st r/GDUUW WWII-fl" .‘ Ifl. ’lUQQZZfi Lg fl W717 -57; iii” will i ' I dwlfiunszv #7::‘5‘ :—:— ~~ / “74*! iéflmttl‘l g Gil fl ll 0 if”. “\i l l l l l “ " ~~~~~ ,+’ mm ‘\ /Z_:‘ , um / l” EI‘I . IIfiTfi - as v .3,“ awn-3; _%,',’ [Nil . TM u'H' .ll‘lrirrili ' c Pictured is a map of the Voegtly Cemetery demonstrating the layout and location of gravesites relative to the foundation of the Voegtly Church (taken from Ubelaker et al., 2003). ' Goals of the Current Research The primary goal of the research presented here was to examine any association between skeletal weathering and the surrounding soil environment. Identification of such associations is dependent on eliminating confounding factors, such as bone age and the environment they existed in. Earlier researchers compared skeletal remains from different sites, but their work included many confounders. For example, Buckberry (2000) examined bone preservation based on skeletons from many different European countries that had different soil types and shallower children's graves. Gordan and Buikstra (1981) focused primarily on soil pH with samples collected from several different locations in Illinois. In both bodies of research the above mentioned variables were included, making interpreting true causative agents for bone degradation difficult. 15 In the present research microbial attack, dissolution, and environmental conditions were addressed, but there was also an intense focus on soil chemistry as a contributing factor for the variability in bone diagenesis. The skeletal remains from the Voegtly Cemetery were ideal in helping to understand bone degradation, because many confusing variables were eliminated as contributing factors to skeletal weathering. For example, the skeletons were in the ground approximately the same length of time (approximately 150 years), were of the same ethnic group, had corresponding large scale environmental conditions including temperature and rainfall, and had the same burial methods (remains were buried at equal soil depth and in plain wooden coffins). Despite these similarities it was clear that many skeletons weathered at very different rates leading to a set of tests being performed to determine which soil parameters may have affected bone preservation. Soils were analyzed for soil pH, extractable phosphorous concentration, exchangeable cation concentration, total organic carbon concentration, particle size , elemental concentrations including aluminum, arsenic, calcium, copper, iron, lead, magnesium, nickel, phosphorous and zinc, and soil biomass. Soils were collected at the Smithsonian Institution directly from the Voegtly remains and were sent to Michigan State University for analysis. The samples contained between 0.5 g and 137 g of soil. Previously, a soil survey was conducted by the United States Department of Agriculture in cooperation with Pennsylvania State University and Pennsylvania Department of Environmental Resources and found a mixture of 75% Urban Land soil, 15% Rainsboro soil, and 10% other soil types where the Voegtly Evangelical Church-Cemetery had been previously located. Urban Land was used to 16 describe original soils that were unable to be identified. Rainsboro soil is a silt loam with a slope of three to eight percent (USDA Soil Survey, 1981). Materials and Methods Voegtly Soil Samples Table 1 illustrates twenty-five soil samples which were collected from individuals that were anthropologically estimated to be less than 20 years of age; twenty-seven between 20 and 40 years of age, fourteen over 40 years of age, and four of unknown age. Of the seventy samples, thirty were associated with male burials, fourteen were female burials, and twenty-six were undetermined. Table 1. Summary of Voegtly soil samples Burial Amount of Soil Weathering I.D. Received (grams) Stage Sex Age B-027 7 5 M 22—32 B-O3O 9 5 M 45—55 B-O32 8 3 M 50—85 B-034 15 4 M 35—45 B-O49 52 4 F 20—30 B-054 1 l 2 M 55—75 B-1 1 l 10 2 M 30—35 B- 124 6 3 M 28—35 B- 128 55 2 NA 3 B- 147 9 3 NA 6—8 B- 164 58 3 M 22—26 B-167 20 2 M/F 15-16 B-169 77 NA NA NA B- 192 39 2 M 60—80 B-220 2 4 NA 2.8 Mo. B-232 1 10 4 NA 5 B-256 l 1 3 M 35-45 B-259 21 2 M 50—70 17 Table l (cont’d). B-26O 18 4 NA 2.5—3.5 B-28l 13 1 F 19—23 B-32O 83 5 M 23—28 B-321 107 2 F 45—50 B-325 45 5 WF 40—55 B-326 50 5 F 18.5 B-327 28 5 NA 13 B-328 72 2 M 40—45 B-330 7 3 F 40—50 B-345 8 3 M 30 B-346 15 4 M 23—27 B-348 45 3 M 27—35 B-350 6O 3 NA 8—10 B-356 23 5 NA 5—6 B-357 72 4 F 12 B-358 67 5 F 16—18 B-36l 45 5 NA 3-5 B-363 1 l6 2 M 28—35 B-379 l 1 5 NA 55—75 B-380 81 2 M 35—45 B-381 63 2 M 25-30 B-3 82 34 5 M 30—45 B-383 137 4 NA 14—16 B-389 6O 5 NA 4-7 B-392 52 4 M 20—27 B-402 41 5 F 25—40 B-409 7 3 M 30—40 B—437 21 3 M 35-40 B-449 14 3 F 27—33 B-450 5 1 NA 1—2 Mo. B-459 7 1 NA 2-3 Mo 8463 7 4 M 35—50 B-465 42 4 NA 3.5 B-489 43 2 NA 1.5 B498 15 3 M 27—40 B-519 42 3 NA 4.5-5 B-545 34 l M 25-32 B-583 67 3 M 30—45 B-612 14 4 NA 6.5 B-6 16 6O 4 NA 14—15 B-624 55 4 M 25—35 l8 Table l (oont’d). B-625 5 5 NA NA B-629 19 4 F NA B-632 20 5 NA 2 B-635 21 4 NA 7-8 B-639 45 3 F 17—24 B-641 92 3 F NA B-698 15 2 M 65-80 B-699 7 4 NA 2.5—3 B-704 9 5 M 22—25 B-717 28 4 F 28—38 B-718 4 4 F 17—23 The burial identification number given during excavation by Smithsonian workers is displayed, as well as the amount of soil available for examination, the weathering stage, and an anthropological estimate of the sex and age of the skeletons associated with the soil. Control Soil Samples Control soil samples were collected in May of 2004 from the ground near Phineas Way (Troy Hill Rd.), which was a border of the Voegtly Cemetery (Figure 4). Inspection of the soil (Figure 5) demonstrated an assortment of soil content at different depths for each of the Voegtly controls. The layers were found with distinct lines of separation. The gravel fill, red brick, and fine brown sand were evidence of human activity. The following control samples were taken from areas located near Voegtly Cemetery, but not associated with burial or excavation (Figure 4). 1. One hundred feet from the comer of Wettach St. and Peralta St. at a depth of 3 ft. 2. One hundred twenty-five feet from the corner of Wettach St. and Peralta St. at a depth of 3 ft. 3. One hundred fifty feet from the comer of Wettach St. and Peralta St. at a depth of 4ft. l9 Figure 4. Map of Voegtly control samples in relation to Voegtly Cemetery \a Y5 The landmark for reference during sampling was the comer of Wettach and Peralta Streets. The first sample (location ‘1’) was collected 100 ft from the comer at a depth of 3 ft. Location ‘2’ was 125 ft from the comer at a depth of 3 ft. Location ‘3’ was 150 ft from the comer of Wettach and Peralta Street at a depth of 4 ft. A ‘star’ marks the location of the Voegtly Cemetery. Chemical Analysis of Soils from Voegtly Cemetery Soil samples were analyzed at Michigan State University in the Plant and Soil Science Building, and Giltner Hall, testing pH, Bray P1 extractable phosphorous, exchangeable cations, total organic carbon, particle size, and elemental composition. Many of the samples did not have enough soil for complete analysis, in which case a subset of tests were preformed, typically including pH, extractable phosphorous, exchangeable cations, and total organic carbon. Testing for pH, extractable phosphorous, exchangeable calcium, exchangeable potassium, exchangeable magnesium, and total organic carbon were also completed on the Voegtly control samples. 20 Figure 5. Description of the Voegtly control soil collected near the cemetery Surface laver 1 ft Sand and rounded gravel fill Red brick 11/2 ft. Sand and gravel 2‘/2 ft. . Fine brown sand 3 ft. Clay and gravel 4 ft. The soil type at varying depths for control samples is noted. The deepest soil layer (3 - 4 ft) was composed of clay and gravel. Between 2% — 3 ft was fine brown sand. From l'lz— 21/2 ft sand and gravel were seen; above this mixture was a red brick layer. At a depth of 1 ft sand and round gravel fill was found. pH Analysis pH analysis was conducted on samples that contained at least 2 grams (g) of soil, using a pH meter calibrated with reference standards of pH 4.0 and 7.0 (Manual of Laboratory Procedures, 2004). Controls consisted of two soil types with a known pH, which were provided by the Michigan State University Soil and Plant Nutrient Laboratory. A control was measured after every twentieth sample. Two milliliters (mL) of de-ionized water was added into a 50 mL Erlenmeyer flask which contained soil measured with a NCR-13, 2 g organic matter scoop (Manual of Laboratory Procedures, 2004). The sample was then stirred thoroughly with a glass rod and allowed to sit for fifteen minutes. The sample was then mixed again and a pH reading was taken. 21 Extractable Phosphorous Analysis Extractable phosphorous was measured using a Bray P1 extraction procedure (Manual of Laboratory Procedures, 2004). Bray P1 concentrate was prepared by combining 200 mL of 3 N ammonium fluoride and 100 mL of 5 N HCL. A working solution of Bray P1 reagent was prepared by diluting the concentrate to 20 liters (L) with distilled water (this large batch of reagent was also used to test other soils at Michigan State University). The pH of the Bray P1 solution was 2.60. Two g of soil were added to 20 mL of Bray’s reagent in a plastic Erlenmeyer flask, which was placed on a mechanical shaker for five minutes at 180 to 200 oscillations per minute. The liquid/soil mixture was filtered into a plastic Erlenmeyer flask lined with Whatman # 1 filter paper. The filtered extract was poured into a plastic screw-cap vial. Two mL of the extract was combined with 18 mL of an acidic molybdate solution and analyzed for percent transmittance using a Brinkmann PC8OO fiber-optic probe colorimeter at a wavelength of 670 nanometers (nm). A five point calibration curve was established with phosphorus standards of 2, 4, 6, 8, and 10 parts per million (ppm). Exchangeable Cations Analyses Exchangeable cations, including calcium, magnesium, and potassium, were measured with an ammonia acetate extract (Manual of Laboratory Procedures, 2004). Two g of soil were added to 20 mL of ammonium acetate in a plastic Erlenmeyer flask and was shaken on a mechanical shaker for five minutes at 180 to 200 oscillations per minute. The solution was filtered into a plastic Erlenmeyer flask lined with Whatman #1 filter paper. Calcium and potassium analysis was preformed using a Technicon flame 22 photometer. Soil samples were automatically mixed with a lithium nitrate solution by the Technicon instrument and analyzed at 422 nm and 766 nm for calcium and potassium respectively. The lithium served as an internal reference standard for determination of calcium and potassium. The Technicon instrument was standardized using an ammonium acetate blank along with calcium and potassium standards. A calcium calibration curve was established using known standard concentrations of 100, 200, 300, 400, and 500 ppm. A potassium calibration curve was established using known standard concentrations of 10, 20, 30, 40, and 50 ppm. Potassium and calcium standards were prepared by the Michigan State University Soil and Plant Nutrient Laboratory by adding stock solution to 100 mL of ammonium acetate; potassium and calcium stock solutions were purchased commercially. Magnesium was measured using a Varian atomic absorption spectrophotometer (Manual of Laboratory Procedures, 2004). A magnesium blue solution was automatically added to each soil sample by the auto-analyzer and measured at a wavelength of 285 nm. The Varian instrument was standardized using an ammonium acetate blank and magnesium standards at 20, 20, 30, 40, and 50 ppm from a stock solution of 1000 ppm magnesium. Total Organic Carbon Analysis Total organic carbon was analyzed on a Leco Carbon Analyzer. The instrument was allowed to warm up for half an hour to one hour in order to reach operative temperatures of 40 0C. A cylinder of oxygen was set at a pressure of 3 — 5 psi. Two crucibles were prepared by adding one scoop (calibrated by the manufacturer of the 23 carbon standards) of iron and tin accelerator chips (Manual of Laboratory Procedures, 2004). Two crucibles were heated to 1600 oC, and the oxygen flow rate was adjusted to 1.5 L per minute. The instrument was then calibrated by running blanks, a high standard of 0.42% carbon and a low standard of 0.039% carbon, which were purchased commercially. A blank was then tested and during the last ten seconds of the heating cycle the volt meter reading was adjusted to zero. The high and low carbon standards were analyzed individually by adding a carbon standard "ring” and one scoop of tin accelerator chips into a ceramic crucible. Two blank crucibles were tested to calibrate the instrument. The soil samples were ground in a mortar and pestle, and passed through a # 25 mesh screen. The samples were dried for twelve hours at 75 0C. 0.1 g was added to a crucible containing one scoop of both iron and tin accelerator chips. Samples were heated to 1600 0C for approximately ten seconds and the amount of carbon dioxide produced was measured. After every twelfth sample either a low or high carbon standard was tested. The pre-filter dust trap was cleaned after every sixth sample. A moisture trap containing magnesium perchlorate was changed after every fortieth sample. Particle Size Analysis Fifty g of soil were required for particle size analysis so only selected samples were analyzed. Highlighted in Figure 6 are locations of the burial sites associated with the soils analyzed for particle size. Soil was added to a 500 mL plastic bottle with a screw cap containing 100 mL of 5% sodium hexametaphosphate. Distilled water was added so that the bottle was approximately half full. The solution was stirred using an 24 electric mixer for twelve hours, and transferred to a hydrometer cylinder (Bouyoucus cylinder). Distilled water was added to the suspension until it reached the 1000 mL mark. A plunger was inserted to thoroughly mix the solution. Temperature and hydrometer readings were recorded after forty seconds of mixing. A second reading was taken at seven hours. Temperature corrections were made to each of the hydrometer readings using the equation: adjusted readings = (temperature — 20 0C) (.36), which was used in the first two calculations below to determine the percentage of silt, clay, and sand. 0 (% silt and % clay) = corrected first hydrometer reading * 2 o % clay = corrected second hydrometer reading * 2 o % sand = 100 - (% silt and % clay) o % silt = 100 - (% silt and % clay) - % clay 25 Figure 6. Location of samples tested for particle size analysis \eggggggggfijg 11 111 111111 '"es / 7442171111317 WM; . 1 gags ”h" ‘1 4“ i’MM ' 1‘ ha (i \\\.\\ - ' 353;: . \\\\\\ ‘ .x 5‘. Q Elf: 5” {Iii I I '11:" a “1‘ “""":'l ‘I a I I -I‘ QB Highlighted are burial sites that had particle size analysis preformed and demonstrate their location within the Voegtly Cemetery. Elemental Analysis Elemental analysis was preformed by energy-dispersive x-ray fluorescence (XRF) on a $2 RANGER. Data were collected from elements ranging in atomic mass from sodium to uranium, over three regions according to the voltage, current, and the absence or presence of an aluminum filter. 0 Region one had a tube voltage of 20 kilovolts (kV), a tube current of 250 microamperes (pA), and no aluminum filter, with a sampling time of 100 seconds. 0 Region two had a tube voltage of 10 kV, a tube current of 500 uA, and no filter, with a sampling time of 100 seconds. 26 Region three had a tube voltage of 40 kV, a tube current of 250 pA, and a 500 um aluminum filter, with a sampling time of 100 seconds. The aluminum filter allowed detection of higher atomic weight elements. A copper disc was used to calibrate the instrument before sample analysis. Two g of soil were ground with a mortar and pestle. The sample was placed into a plastic sample cup. The soils tested were from burials: B-27, B—30, B-34, B-lll, B-124, B-164, B-167, B-192, B-260, B-328, B—345, B-348, B-38l, B—389, B-409, B-489, B-545, B-583, and B-358. Each sample’s location within the cemetery may be viewed in Figure 7. Figure 7. Location of samples tested for elemental analysis IRWW ”wit? I11g"’l’.M’fr 1 .177»: Law “""' W" . ”h I Highlighted are burial sites that had total elemental analysis preformed and demonstrate their location within the Voegtly Cemetery. Biomass Estimation Two modified techniques (Schmidt and Paul, 1982) were attempted in determining biomass. In the first, 2 milligrams (mg) of acridine orange was added to 10 27 mL of distilled water (prepared fresh). One g of soil was ground and mixed with a mortar and pestle. Two mg of soil was added to a fluorescent antibody slide (Erie Scientific), which was cured with a black ceramic background, containing ten wells measuring 6 mm in size. The acridine orange solution was added with a glass Pasteur pipette to flood the wells of the slide. The second method for determining biomass began by preparing 0.01 M phosphate buffer by adding 0.082 g of dibasic phosphate to 100 mL of deionized water. A staining solution was prepared by adding 2 mg of acridine orange to 10 mL of phosphate buffer. Two g of soil were added to 38 ml of de-ionized water in a 50 mL plastic bottle with a screw cap. The solution was mixed by vigorous hand-shaking for five minutes. Course particles were allowed to settle for thirty seconds, and 1 mL of the soil solution was removed with a pipette and transferred to a 16 x 100 mm glass test tube. One hundred uL of 40% formalin was added as a preservative. The soil solution was vortexed and four drops were placed onto each of the ten wells of the slide with a glass Pasteur pipette, without touching the surface of the slide. The slide was then air dried, and the stain was pipetted onto the entire slide and allowed to sit for thirty minutes. Excess stain was removed by carefully rinsing the slide with 0.01 M phosphate buffer twice, thirty minutes each, and then a final wash with distilled water. For both techniques a cover slip was placed onto the slide and immersion oil was added, which was drawn under the cover slip by capillary action. Bacteria were visualized on a Zeiss Pascal fluorescent confocal microscope. Images were taken with a frame size of 1024 by 1024 pixels, and a pixel dwell time of 1.28 microseconds, using a 63x, plan-apochromatic objective. Two dichroic beam splitters of 488 nm and 545 nm 28 were used along with a band pass 505 — 530 nm filter and a long pass 650 nm filter. Data were collected using a triple-line argon ion laser set at 488 nm. Non-specific fluorescence was seen as red, yellow, and green objects. Determining if objects were bacteria or fluorescent material was based upon size, color, intensity, image sharpness, and clear boundaries. Inductively Coupled Plasma Mass Spectrometry (ICPMS) ICP/MS analysis for qualitative/quantitative elemental analysis was prepared by Lina Patina at the ICP/MS laboratory at Michigan State University. The five samples tested were B-34, B-l24, B-328, B-363, and B-389. These were chosen because each represented different pH values, different degrees of bone preservation, and differences in elemental analysis by XRF. Additional ICP/MS analysis and time-of-flight mass spectrometry were not preformed. Statistical Methods A coefficient of correlation was calculated for each analysis (pH, extractable phosphorous, exchangeable calcium, exchangeable potassium, exchangeable magnesium, and total organic carbon) to determine if there was any relationship with respect to skeletal weathering stages. Statistical analyses were preformed using Microsoft Excel. A Student’s two-tailed t-distribution test was chosen to analyze the effects of pH, extractable phosphorous, exchangeable calcium, exchangeable magnesium, exchangeable potassium, and total organic carbon at all weathering stages between Voegtly soil samples and controls. The Student’s t-distribution analyses involved comparing 29 population means and standard deviations between two sample sets to determine if there was a significant difference between the two groups compared. Results were considered significant at a level of p < 0.05. Results Particle Size Analysis Ten skeletal soil samples, from weathering stages 2, 3, and 4 were analyzed for particle size (Table 2). All samples were classified as clay loams. Each had percentages of sand, clay, and silt ranging from 28 — 43%, 28 — 36%, and 23 — 38% respectively. Table 2. Summary of particle size analysis M _ ' Weathering §it I.D. soanypg °/o and % Clay % Ilt §tagegf Rgmalng B-049 Clay loam 28 34 38 4 B-128 Clay loam 41 36 23 2 B-164 Clay loam 41 33 26 3 B-328 Clay loam 29 34 37 2 8-357 Clay loam 39 31 30 4 B-363 Clay loam 39 31 30 2 B-380 Clay loam 39 31 30 2 B-61 6 Clay loam 43 28 29 4 B-624 Clay loam 31 35 34 4 8-641 Clay loam 34 33 33 3 Burial identification number is displayed as well as the soil type, percentage of sand, clay, silt, and weathering stage of the skeletal remains associated with the soil. pH Analysis Figure 8 represents the correlation between soil pH and skeletal bone preservation. Soil pH ranged from 4.48 — 7.41. A relationship with skeletal weathering 30 was seen; the pH decreased as the skeletal weathering stage increased. The correlation coefficient (R2) was 0.2064, showing a negative slope. Figure 8. pH as a function of weathering stage 8 7 5 ~ y = -0.228‘7x + 6.547 74 ‘ , ‘ R2=0.2064 6.5 ~ pH 62 5.5 - 5—1 4.5 s o Weathering Stage The pH of the sample was plotted against the skeletal weathering stage for each soil analyzed. A best fit line was added to help visualize the results. A negative slope was seen with a correlation coefficient of 0.2064. Mean pH values for soils and p-values for each weathering stage are shown in Figure 9. pH values for both the VC (7.56) and VS-l (6.75) were similar with a p—value of 0.1 129. The pH values for VS-2, VS-3, VS-4, and VS-5 were 5.95, 5.87, 5.54, and 5.51 respectively. The p-values between VC and VS—2, VS-3, VS-4, and VS-S were 0.0018, 0.0002, <0.0001, and <0.0001 respectively. 31 Figure 9. pH mean values for VC and VS-l through VS-S 7.5 - (.1129) a 6.75 - :1. 6 (.0013) (.0002) (<-0001) (<.0001) 5.25 r 1 u . . VC VS-l VS-2 VS—3 VS-4 VS-S VC / Voegtly Sample Group Displayed are pH mean values and with p-values given in parenthesis. The means of VC compared to VS-2, VS-3, VS—4, and VS-S are all significantly different. Extractable Phosphorous Analysis Quantified extractable phosphorous values were compared to skeletal remains of weathering stages 1 — 5 (Figure 10). Results ranged fi'om 152 — 5510 ppm. The data show a weak positive relationship between skeletal weathering and extractable phosphorous concentrations (R2 = 0.0335). 32 Figure 10. Extractable phosphorous as a function of weathering stage 5000 ° y = 137.46x + 1085.8 8 R2 = 0.0335 9 . ‘5 4000 a “E. 8 E 3000 . . : ° : 3 o 8 z '3 2000 ~ 3 s ’ 2 ° 4 1:; ° 2 ° ‘1’ ; FI-l -—' ° 8 1000 — . . s . o O 0 0 O I i . 0 l 2 3 4 5 6 Weathering Stage Extractable phosphorous of each sample was plotted as a function of skeletal weathering stage. A best fit line was added that demonstrated a positive trend with a correlation coefficient of 0.0335. Figure 11 details mean extractable phosphorous results between the Voegtly controls and Voegtly soil samples of all of skeletal weathering stages. The VC had a mean concentration of 8.67 ppm. Phosphorous values for VS-l (796) and VS-2 (1407) were not significantly different from the VC, while VS-3, VS-4, and VS—S were. It was noticed that mean phosphorous levels for VS-2 and VS-3 were similar with very different p-values of 0.1314 and 0.0126 respectively. This is due to VS-2 having a standard deviation of 960, while VS-3 had a standard deviation of 1350. Means phosphorous levels of VS-3, VS-4, and VS-5 appeared to level off just below 1700 ppm. 33 Figure 11. Extractable phosphorous mean values for VC and VS-l through VS-S 1800 , (.0126) (.0008) ((0001) (.1314) 1200 600 Extractable Phosphorous VC VS-l VS-2 VS-3 VS-4 VS-5 VC / Voegtly Sample Group Displayed are the extractable phosphorous mean values with p-values given in parenthesis. Differences between VC and VS-3, VS—4, and VS-S were significant with p-values of 0.0126, 0.0008, and <0.0001 respectively. Exchangeable Cations Analyses Figures 12, 14, and 15 represent comparisons between exchangeable cations (calcium, magnesium, and potassium) and skeletal weathering. Average values for each cation varied little among the five weathering stages. Calcium values ranged from 781 — 3422 ppm, had the largest correlation coefficient (R2 = 0.0108), and represented a slight negative trend. Potassium and magnesium values showed virtually no trend with values ranging fi'om 41 — 328 ppm and 22 — 155 ppm respectively. Mean exchangeable calcium concentration for the VC was 2122 ppm (Figure 13). Voegtly soil calcium concentrations varied throughout the weathering process. A statistical difference between the control soil and Voegtly soil was not seen for VS-l through VS-4; however, the p-value for VS-5 compared to VC was 0.0015. 34 Figure 12. Exchangeable calcium as a function of weathering stage 3500 O 3000 _ y = -42.046x + 1848.9 2 ‘ R =0.0108 g 2500 ° 0 on: O i- 2 U 0 i 2000 1 . . . . a O n W 5 1500 - . z . . .fl 0 o O I t 0 H O O 1000 - 3 . O 500 a 1 0 1 2 3 4 5 6 Weathering Stage A best fit line was added with a slight negative slope. Exchangeable calcium results ranged from 781 — 3422 ppm and had a R2 value of 0.0108. Figure 13. Exchangeable calcium mean values for VC and VS-l through VS-S (.4623) (.1550) (.1544) Exchangeable Calcium VC VS—l VS—2 VS-3 VS-4 VS-5 VC / Voegtly Sample Group Displayed are the exchangeable calcium mean values with p-values given in parenthesis. VS-5 exchangeable calcium was statistically different than VC. 35 Figure 14. Exchangeable magnesium as a function of weathering stage 350 O 300 _ y = 2.35941: + 125.65 R =0.0033 5 250 ~ , ’ E . z i . it} 150 7 . : . ; a O O 0 g g a 100 ~ ° 9 .3 z o ’ 0 3 . ’ a: 50 ~ , O 1 l f 7 l 0 l 2 3 4 5 6 Weathering Stage Exchangeable magnesium concentrations ranged from 41 - 328 ppm. A slight positive slope was observed with a correlation coefficient of 0.0033, which suggested there was little to no relationship between skeletal weathering and exchangeable magnesium concentrations. Figure 15. Exchangeable potassium as a function of weathering stage O 150 - y = -0.6087x + 63.852 R2=0.0007 130 a . O E 110 - . a 90 ‘ ° ° 2 a c '5 70 - , i . a g 0 3 Q ; b 3 50 — ° 3 :2 ° 0 ill 0 . O : O 30 a 0 ‘ ° 0 O 10 . . . 0 1 3 4 5 6 WeatheringStage No trend between exchangeable potassium concentrations and skeletal weathering was observed with a R2 value of 0.0007; a slight negative slope was seen. 36 Total Organic Carbon Analysis Organic carbon content was quantified on sixty-four Voegtly soils. Total organic carbon results ranged from 0.38 — 6.69%; values were compared to skeletal weathering and resulted in a R2 value of 0.0018, showing no substantial trend (Figure 16). Figure 17 displays percentages of total organic carbon for VC and the Voegtly soil samples. VC had a mean total organic carbon value of 0.36%. VS-3 had the highest percentage of total organic carbon at 3.32%, while VS-S had the lowest percentage at 1.89%. When compared to the VC mean only the difference of VS-2 was not statistically significant. Figure 16. Total organic carbon as a function of weathering stage 8 4 = 0.0521! + 2.6344 7 . R = 0.0018 6 J = 8 é ° . a 5 7 U c U - .3 4 . 3 c E” 3 .1 ° ° . 2 O i _._ 2 a: 3 2 — 3 : g e f E z t l “ . . : o g o . . . . . 0 l 2 3 4 5 6 Weathering Stage A slight negative trend was observed when a best fit line was added. The correlation coefficient of 0.0018 added little weight as to a relationship between total organic carbon and skeletal weathering. 37 Figure 17. Total organic carbon mean percentage values for VC and VS-l through VS-S 4.25 7 : § (.0147) a 3.25 - U 0 E 2.25 _ (.0494) (.0548) E” O [-I 0.25 1 1 1 1 a 4 VC VS-l VS-2 VS-3 VS-4 VS-5 VC / Voegtly Sample Group Displayed are the mean total organic carbon values with p-values given in parenthesis. Differences in total organic carbon between VS—2 and VC were not significant with a p-value of 0.0548. Elemental Analysis Elemental data were collected from seventeen soil samples using x-ray fluorescence. Due to the large amount of elemental data collected; Figures 18 and 19 are given as examples of typical spectra collected. Figure 18 depicts elemental data from sample B-27 using no aluminum filter, which allowed for detection of lower atomic weight elements. Calcium was the most abundant element, with other prominent elements being phosphorous, manganese, and iron. Trace amounts of several other elements were also observed. Figure 19 displays elements that were detected using x-ray fluorescence from sample B-27 with an aluminum filter, which allowed detection of high atomic weight elements. The spectrum was dominated by manganese, iron, and calcium. Smaller amounts of phosphorous, titanium, potassium, copper, zinc and arsenic were observed. 38 €895 203 ass is .8353: .8235?“ 553 .E:EE=_~ he £5.0an ~88: .59. 203 :9: 98 £956 .msoconamona .828ng .8323 no 355:3 ems .393: 2.88 .5; 35823 5 888.23% macaw £an 2E. .93: 33> Shoo—8.3 me £5. E 35:0 8:80.52 weak 2F 605:5 ma? >25“ 05 558.0 .8582“. some: Soc 23 862% «5:53 05 3:805»: aqua :80 30% 985. 8:80: 25. SNA— =8 .8 $9228 a8 “.8: we? 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For example, in Table 3, fluorescence due to calcium associated with stage 1 skeletal weathering was 300 KCps and two soil samples with stage 4 bone preservation demonstrated fluorescence levels of 1400 KCps and <100 KCps respectively; these inconsistent calcium results occurred over the entire range of skeletal weathering. Results in Table 4 added little additional information with respect to higher atomic weight elements. Fluorescent levels for phosphorous, sulfur, potassium, titanium, manganese, nickel, copper, zinc, and arsenic were consistently low over the entire range of skeletal weathering, presenting insufficient evidence for any relationship between element concentration and skeletal preservation. 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