An Electron Microprobe Evaluation of Diagenetic Alteration in

Chapter 24

An Electron Microprobe Evaluation of Diagenetic Alteration in Archaeological Bone Diana M. Greenlee

Downloaded by COLUMBIA UNIV on July 1, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0625.ch024

Department of Anthropology, University of Washington, Box 353100, Seattle, WA 98195-3100 Backscattered electron imaging and wavelength dispersive spectrometry documented variability in the structure and composition of archaeological human bone samples from the central Ohio and Mississippi River valleys. Structural alterations to bone histomorphology are consistent with those known to reflect microorganismic activities, with hydroxyapatite dissolution followed by remineralization involving ions from both dissolved bone mineral and the soil solution. Patterns in the distribution of structurally intact bone were observed to vary with the local post-depositional environment. Differences in the mean concentration and variability of elements (Ca, P, Sr, Ba, Mn, Fe, Zn, Cu and V) between structurally intact and structurally altered areas were examined; structurally altered areas frequently displayed higher elemental concentrations and greater variability than structurally intact areas. The structure and chemistry of bone virtually guarantee that it will interact with most geochemical environments. Bone's porous histological structure (Figure 1) allows easy invasion by soil microorganisms and weathering-induced cracks aid in penetration by ground water. Additionally, its microstructure, with structurally imperfect nonstoichiometric hydroxyapatite [Caio(P04>6(OH)2] microcrystals embedded in an organicfibrousmatrix, gives bone a huge surface area and highreactivity(7), and thus great potential for chemical interaction with the burial environment Indeed, bone is recognized as being quite susceptible to diagenesis, the physical and chemical alteration of materials resulting from interaction with the post-depositional environment The exact nature of diagenetic alterations to bone will depend on local factors such as pH, temperature, moisture conditions, geochemistry, and microbiology. Despite these potential modifications,researcherswho use archaeological bone chemistry to make inferences about the age, diet or environment of prehistoric organisms must assume that the elemental and isotopic levels they measure correspond 0097-6156/96/0625-0334$12.25A) © 1996 American Chemical Society

In Archaeological Chemistry; Orna, M.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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Figure 1. Backscattered electron (BSE) micrograph of an undecalcified transverse section of a modern human femur, illustrating histomorphological structure and variable mineralization surrounding Haversian canals. The lighter areas have a higher mean atomic density than darker areas. The cracks are artifacts of sample preparation.


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to those at death. Given the near certainty and complexity of chemical interaction between bone and the post-depositional environment, this assumption is problematic, especially for trace element studies of the mineral hydroxyapatite fraction of bone. The challenges, then, are to determine what manner of post-depositional alterations has occurred, how such alterations might affect our conclusions and how the effects might be minimized.


Strategies for Evaluating Diagenetic Alteration Histomorphological analysis using microscopic, radiographic, and electronic imaging has shown that many kinds of diagenetic alteration can be identified visually (2-10). Areas of structurally intact bone are often present, even in bones showing extensive damage, with the distribution of diagenetic alterations apparently determined by both bone structure and post-depositional context. Because the diagenetic processes that alter the structure of bone probably also alter its chemical composition, histomorphological analysis would seem a useful way to ascertain the likelihood of some kinds of chemical alteration. However, bone chemists seldom use histomorphological examination to identify diagenetic alteration; they choose instead to focus on patterns in elemental concentrations in the bone. If patterning in elemental concentrations meets certain expectations, the elemental levels are assumed to reflect dietary consumption; if concentrations do not conform to expected patterns, those elements are considered to be diagenetically altered (11-16). Most compositional analyses rely on analytical techniques which employ powdered, homogenized bulk samples of bone. While researchers have shown that various pre treatments designed to remove contaminants, e.g., cleaning with ultrasonic baths, physical removal of 1-3 mm of the periosteal and endosteal surfaces of the bone (17-19) and chemical washes (19-26), often change elemental concentrations in anticipated directions, they have yet to show conclusively that the diagenetically altered, and only the diagenetically altered, bone has been removed. Indeed, experimental data suggest that, with the chemical wash approach, at least some biogenic bone may be removed (19, 27) and some diagenetically altered bone may remain (28). The results of bulk sample chemical analyses, then, are "average" concentrations that are assumed to reflect primarily diagenetically unaltered bone, but may potentially include diagenetically altered bone, as well. Yet, successful documentation of prehistoric diets requires certain knowledge that diagenesis has not altered significantly the elemental concentrations measured. The Electron Microprobe Approach The potential contribution of electron microprobe technology to studies of archaeological bone chemistry lies in its ability to identify the structural, if not chemical, integrity of the bone from which compositional information is obtained. The electron microprobe (a.k.a. electron probe microanalyzer) uses a focused beam of electrons to irradiate a sample. As the beam strikes the specimen, elements in the sample gain energy and produce several types of secondary signals. Of particular importance to this study are the backscattered electron (BSE) and characteristic X-ray


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signals. The BSE signal permits the sample to be imaged, while X-rays permit characterization of its elemental composition. Thus, the electron microprobe allows documentation of the relationship between the microstructure of archaeological bone and its elemental composition. Backscattered Electrons. Backscattered electrons are former primary beam electrons that have collided with electrons in the sample and bounced back through the sample surface. BSE generation is dependent on the mean atomic number of the sample; as the atomic number of the sample increases, the number of electrons in the sample with which the primary beam electrons can potentially collide increases, and thus, the number of emitted BSEs increases. In BSE images, areas of higher mean atomic number will appear brighter than areas of lower mean atomic number. Images generated by using the BSE signal provide nonspecific compositional information to a depth of 1-2 μπι in the sample, with differences in mean atomic number as low as 0.1% being distinguishable in topographicallyflatspecimens (29). BSE imaging has been used successfully byresearchersstudying mineralization and histological structure of both modern and prehistoric bone (2, 30-34). BSE images are particularly useful in studies of diagenetic change in archaeological bones because they allow the difference in atomic densityresultingfrom microorganismic destruction and mineralreplacementto be evaluated with respect to sample histomorphology. BSE images do not, however, allow particular elements to be identified, nor their concentrations to be specified. X-rays. Characteristic X-rays are produced when the primary electron beam dislodges an electron from an inner shell of an atom and an electron from an outer shell of that atom moves in to fill the vacancy. Tofillthe vacancy, the outer shell electron must lose energy in an amount equal to the difference in Coulomb force between the two orbitals, the emitted energy being within the X-ray portion of the electromagnetic spectrum. Because each element has a unique number of electrons and protons, the Coulombic field is unique to each element and each orbital of each element Therefore, all X-rays emitted have energies (and wavelengths) characteristic of that element and the particular orbital-vacancy transition. At the same time, there are primary beam electrons which fail to collide with other electrons and instead randomly lose variable amounts of energy proportional to the distance from atomic nuclei that they pass by. This lost energy forms a continuous range of X-ray wavelengths, known as bremsstrahlung or background radiation, which must be subtracted from the intensity of characteristic X-rays before quantitative analysis can be obtained (29). X-rays are generated within a small interaction volume (usually ~5 μπι ), the exact size and shape of which is determined by characteristics of the target sample. Even though the number of characteristic X-rays generated is proportional to the concentration of the element in the sample, the number of X-rays actually emitted will differ because of sample matrix effects. Sample composition and density influence both X-ray generation and emission and hence corrections to the X-ray intensities due to the effects of Ζ (mean atomic number), A (X-ray absorption) and F (secondary Xrayfluorescence)must be included in each quantitative determination (29). Thus, quantitative analyses depend on our ability to account for matrix differences and ZAF 3



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iterative correction procedures have been developed to do that One aspect of the sample that ZAF corrections cannot account for, though, is sample topography. Topographic variation in a sample significantly influences both X-ray generation and absorption; therefore, flat samples are imperative for quantitative analyses (29). The composition of biological specimens also introduces additional considerations during microprobe analyses as compared to analyses of most inorganic samples. As the electron beam deposits a very large amount of energy into a relatively small analytical volume, a significant increase in temperature occurs. This can lead to vaporization of components such as water, carbon-containing compounds (e.g., proteins and lipids), chlorine and fluorine, as well as migration of elements such as sodium, potassium and phosphorus (29). Vaporization and migration of some sample components will affect the relative concentration of other materials in the sample. Thus, microprobe studies of biological tissues must be sensitive to, and minimize the effects of, beam damage. Fortunately, in this regard, archaeological bone is more similar to geological specimens than fresh bone; archaeological bone is more stable under the beam due to the diagenetic loss of water, lipids and other organic components. My own beam/sample interaction studies indicate that, unlike fresh specimens, archaeological bones require no unusual analytical conditions to ensure their stability under the beam. Wavelength Dispersive and Energy Dispersive Spectrometry. By measuring the net intensity (counts/second) of characteristic X-ray emissions (known as X-ray lines), the relative abundance of each element can be determined. This determination can be made in two ways, by wavelength dispersive spectrometry (WDS) or by energy dispersive spectrometry (EDS). Both compare the intensity of characteristic X-rays in the unknown sample with that of a standard of "known" composition; their differences lie in which aspect of the X-rays they measure, their wavelength or their energy. In the case of WDS, diffracting crystals are arranged so as to selectively diffract X-rays of a particular wavelength to a detector, where they are subsequently counted. Consequently, a WDS spectrometer counts only one specified element at a time; however, instruments may have one or more detectors that can be used simultaneously. In contrast, EDS, which uses a solid state semiconductor detector to measure X-ray energy, receives and counts X-rays of all energies simultaneously. WDS is superior to EDS for trace element analysis because it has greater resolution and lower detection limits (under favorable conditions, on the order of 10 ppm vs. 1000 ppm for EDS), even if counting times are considerably longer (29). The advantage of using quantitative electron microprobe analysis in the study of materials is that precise measurements of elemental concentrations can be obtained from small volumes at known locations. Surprisingly, the use of the microprobe to examine variability in elemental concentrations within bones by medical researchers (35-38) has been somewhat limited. No studies of trace element chemistry in archaeological bone have used the microprobe in a systematic quantitative manner, microprobe analyses are typically presented as secondary investigations (7, 39-41) and generally lack sufficient descriptive detail to evaluate the results.


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X-ray Maps. X-ray maps display the distribution and concentration of a particular element across an area of the sample. Images are produced when the primary electron beam, rastering over an area of the sample, generates characteristic X-rays which are detected through either WDS or EDS spectrometry. The location of each X-ray is displayed on a monitor as a dot; after multiple raster passes, an image of dots appears that records distributional information about the element of interest Such maps have been used to document the distribution of elements incorporated into reprecipitated hydroxyapatite matrix and appearing in voids and along fractures in archaeological and paleontological bones (77, 52, 42-45). Digital X-ray images, like those collected here, provide quantitative information about the concentration, as well as the spatial distribution, of particular elements. Thus, elemental composition can be directly linked with the histomorphological structure of the bone. Implications. Structurally intact areas of bone have greater potential for retaining biological elemental concentrations than areas altered by post-depositional processes. From this, it follows that we should concentrate on determining the compositional integrity of structurally intact bone. If structurally intact bone is also chemically intact, analyses of these specific areas would provide secure dietary information. If structurally intact areas are determined to be chemically altered, the alterations may be less severe than in visibly altered areas; thus, chemical pretreatments might be more effective on structurally intact areas. Electron microprobe technology, because it permits both visual inspection of bone histomorphology and precise elemental analysis of very small volumes of material, may be useful in evaluating the chemical integrity of structurally intact archaeological bone. The remainder of this paper describes preliminary work toward identifying and analyzing both structurally intact and altered areas of archaeological bone, with considerations of the diagenetic processes that have influenced them and the kinds of data necessary to evaluate fully aie potential of this approach. Archaeological Application The archaeological research described here is part of a larger project aimed at documenting dietary variability through time and across different environments in the central Ohio and Mississippi River valleys. For this preliminary study, samples of archaeological human bone representing eight individuals were selected randomly from a range of archaeological contexts and post-depositional environments (Table I). The goal is to explore the structural and compositional variability in archaeological bones, establishing that the electron microprobe is an appropriate instrument for this kind of study and determining what kinds of additional information are needed to explain patterns in trace element chemistry. Sample Preparation. Transverse sections approximately 1 cm thick were removed from archaeological bones with a steel hacksaw. All sections originate from the mid shaftregionof long bones; several individuals wererepresentedby multiple samples, in some casesfromdifferent bones and in othersfromdifferent locations on the shaft of the same bone. Beyond a brief sanding of the cut surfaces with 240-grit silicon carbide



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Table L Archaeological Contexts and Post-Depositional Environments Represented Archaeological Permeability References Approximate Archaeological Sample SoilpH Deposit Age* Context Size Index (ίηΛή 46-47 Chilton (CHLT) 1 0.06 - 0.6 LW Limestone mound 6.6 - 8.4b 48-49 2 0.6 - 6.3 C.L. Lewis (CLL) MW 5.6 - 7.3 Limestone mound 50-51 Cleek-McCabe (CMC) ELP Earthen mound 1 0.6 - 2.0 6.1-7.3 32, 52-54 0.06 - 0.2 Midden 1 Poverty Point Object (PPO) LA 5.0 - 7.0 LA 1 0.06 - 0.2 32, 52, 54 Hankins (HAN) Midden 5.0 - 7.0 55-56 5.4-6.2 0.2 - 10.0 Childers (CHLD) LW 1 Pit burial MLP 57-58 Slone (SLO) Pit burial 1 0.8 - 5.0 4.0 - 5.0 Deposits have been partitioned into the following age groups: LA = Late Archaic (4000 B.C. - 500 B.C.); MW = Middle Woodland (0 - A.D. 400); LW = Late Woodland (A.D. 400 - A.D. 1000); ELP = Early Late Prehistoric (A.D. 1000 - A.D. 1200); MLP = Middle Late Prehistoric (A.D. 1200 - A.D. 1400); bthe pH of these archaeological deposits is probably neutral to alkaline due to dissolution of overlying limestone slabs; through time, the dissolution of bone and shell in these deposits has changed the soil chemistry to allow preservation of these materials by altering the pH of the local microenvironment from acidic to neutral to slightly alkaline.



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grinding paper to remove any contamination from the saw, no efforts (e.g., ultrasonic cleaning, surficial abrasion) were made to clean the bone prior to impregnation with Epo-thin resin. After curing, the resin blocks were sanded flat, mounted to a pétrographie slide and cut, leaving a thick section of bone approximately 250 μπι thick. Each sample was polished with a series of silicon carbide and levigated alumina grits to 5 μπι on a lap wheel and to 0.3 μπι with a Buehler Petropol automated polishing system. Between grits, samples were carefully rinsed in distilled water and examined microscopically to determine if they required ultrasonic cleaning before proceeding to the next grit Prior to analysis, samples were wiped with ethanol and a 25 μπι-thick conductive coating of carbon was sputtered onto the polished sections. To ensure proper conduction, lines of carbon paint were extended across each slide from the sample holder to the bone. Analysis. Each transverse section was examined with BSE imaging to firstly, identify qualitatively any patterning in the nature and location of diagenetic alterations to the histomorphology and secondly, to locate structurally intact and altered areas of bone. Compositional information was obtained for elements (Ca, P, Sr, Ba, Cu, V, Mn, Fe and Zn) often assumed to reflect bone preservation and dietary consumption (11-16). Both structurally intact and diagenetically altered areas along radially oriented (periosteal to endosteal) transects were analyzed. X-ray maps were acquired to examine the spatial distribution of elements of high concentration. Analytic parameters (beam current, beam diameter and counting time) for the quantitative WDS analyses were established through a series of beam/sample interaction experiments. X-ray counts and absorbed current were monitored at different beam currents and beam diameters to determine under which conditions there were no significant X-ray intensity variations through time resulting from beam damage to the archaeological specimens. BSE, X-ray imaging and WDS analyses were conducted on a JEOL 733 SuperProbe in the Department of Geological Sciences at the University of Washington using the following settings: 15 kV accelerating voltage; 40° take-off angle; e

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Figure 6. Results of the WDS analyses for (a) Mn, (b) Fe and (c) Zn. See Figure 4 caption for explanation of symbols. The absence of symbols in any graph indicates that elemental levels were below minimum detection limits in bones from that deposit.



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Conclusion In this paper, I have argued that electron microprobe analysis holds promise for studying the trace element chemistry of archaeological bones. The combination of BSE imaging and compositional analysis allows the characterization of individual structural features, both diagenetically altered and intact, thus allowing us to know precisely what is being measured. The results of compositional analyses comparing structurally intact and structurally altered areas of archaeological bone suggest the approach merits further consideration. In general, morphologically altered areas show greater compositional variability and higher elemental concentrations than areas that appear structurally intact, but this depends on the element and post-depositional environment involved. While this observation does not guarantee that structurally intact areas are chemically unaltered, they appear, at least, to have been exposed to less severe diagenetic processes. Clearly, to actually explain the effect of these specific post-depositional environments and diagenetic processes on the distribution of trace elements will require larger samples from these contexts. More information about the composition of the soil solution,reflectingthe composition of the soil parent material and the pH/redox potential of the local soil environment (27, 70-77), isrequiredin order to determine the productsresultingfrom its interaction with the calcium phosphate of bone. Additionally, future research is needed to characterize the distribution of elements in modern unaltered bone, particularly from organisms raised on controlled diets. Microstructural variability in the composition of bone is likely to be documented, given the temporal variability in bloodstream levels of elements, varying distance of mineralizationfrontsfromthe blood supply and the abilities of different hydroxyapatite crystal sizes to incorporate or adsorb ions. Once this information is obtained, the variability in elemental composition of archaeological bone can be evaluated relative to documented biological levels of variability and the utility of analyzing structurally intact areas of bone can be better assessed. Acknowledgments R. C. Dunnell, F. E. Hamilton, N. Justice and M. L. Powell made bone specimens available for this analysis. S. M. Kuehner advised on many aspects of the microprobe analysis. A. King and B. J. Carter kindly provided a sample of modern human femur for comparative purposes. This paper benefitted from the comments of B. J. Carter, R. C. Dunnell, S. M. Kuehner, M. V. Orna and two anonymous reviewers. Thin section equipment was available through a GSRF grant awarded to R. C. Dunnell; the Buehler Petropol polishing system was possible through NSF grant, SBR-9319278, awarded to L. Newell and R. C. Dunnell. Portions of thisresearchwere supported by NSF Dissertation Improvement Grant, SBR-9310137. Literature Cited (1) (2)

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