Micro-sampling of human bones for mobility studies: diagenetic impacts and potentials for elemental...

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Journal of Archaeological Science 40 (2013) 4509e4527

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Journal of Archaeological Science

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Micro-sampling of human bones for mobility studies: diageneticimpacts and potentials for elemental and isotopic research

Ian Scharlotta a,*, Olga I. Goriunova b, Andrzej Weber a

aDepartment of Anthropology, University of Alberta, Edmonton, AB T6G 2H4, CanadabDepartment of Archaeology and Ethnography, Irkutsk State University, Karl Marx Street 1, Irkutsk 664003, Russia

a r t i c l e i n f o

Article history:Received 20 September 2012Received in revised form10 July 2013Accepted 13 July 2013

Keywords:Bone microstructureMicro-samplingMobilityStrontium isotope analysisDiagenesis

* Corresponding author. Tel.: þ1 780 492 9269.E-mail address: [email protected] (I. Scharlotta

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a b s t r a c t

The nature of long bone formation and the pathways of interaction between bone samples and the burialenvironment suggest that portions of the bones disconnected from the arterial system are resistant todiagenetic alteration. Preliminary work on femurs from Early Bronze Age hunter-gatherers in Cis-Baikal,Siberia shows that the nature and progression of chemical changes in the bone matrix due to microbialattack can be analyzed using laser ablation inductively coupled plasma mass spectrometry. Intra-osteonvariability in elemental concentrations and strontium isotope ratios (87Sr/86Sr) indicate the presence ofunaltered portions of bone within diagenetically modified bone and suggest that useful data remainaccessible. These biogenic signals can potentially be useful for mobility research in broad terms and thesmaller timescales within an individual’s lifetime (months, years), accessible therein. Laser ablationmicro-sampling of femur specimens showed that intra-osteon elemental composition of Ba, Re, and Csvaried within and was correlated between multiple osteons of a single bone. Portions of chemicallyunaffected bone were identified within, and effectively discriminated from diagenetically altered bonetissue. Areas showing visual alterations and erratic or uncorrelated Ca and Sr elemental results also hadanomalous Sr isotope ratios, suggesting diagenetic alteration in those places. Compositional and isotopicanalysis of intact portions of bone supports the hypothesis that hunter-gatherer groups in Cis-Baikalmade numerous major movements during their lives. Microscopic analysis of long bones clarifies as-pects of biodeterioration and correlations between trace elemental results and diagenetic alteration.Micro-sampling of intact portions of bone expands the scope of available materials for research onmobility and other aspects of human past behavior.

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1. Introduction

The goals of this study are: 1) to examine the applicability oflaser ablation as a sample introduction method for trace elementaland strontium isotope analysis using ICP-MS on human skeletalmaterials; 2) to assess its potential to access unaltered portions ofarchaeological bone; and 3) to provide useful insights on humanbehavior in general and mobility specifically. For mobility studies,tooth enamel is the preferred material due to its resistance todiagenetic alterations, leaving bone samples either neglected orregarded with suspicions of providing spurious data. In the currentstudy we used femur samples from individuals from Khuzhir-NugeXIV (KN XIV), an Early Bronze Age hunter-gatherer cemetery on thewest coast of Lake Baikal, Siberia (Fig. 1). Both TIMS and ICP-MStechniques have been previously applied to human teeth and

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bones from KN XIV (e.g., Haverkort, et al., 2010, Haverkort et al.,2008, Weber et al., 2003, Weber et al., 2007) and these data areuseful in assessing the efficacy of laser-ablation trace element andSr isotopic analyses of bones. More precisely, the study addressesthe following two main questions: 1) Are there intact portions ofbones, specifically intact osteons, which can be effectively analyzedfor trace element concentrations and strontium isotope ratios bylaser-ablation? and 2) How effective is micro-sampling of individ-ual osteons for tracking mobility within the context of Cis-Baikalgeology and human interactions with different plant and animalresources?

Sampling and analytical protocols are always integral part ofgeochemical tests performed on archaeological materials andinvolve a series of deliberate decisions. For geological specimens,these choices often reflect the specific research question(s) beingpursued. The situation is rather more involved with complex andorganic mineral structures as there are frequently difficulties inaccessing the desired target data. Geochemical analysis of archae-ological human skeletal materials includes such considerations as

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Fig. 1. Lake Baikal, Siberia showing the location of the KN XIV cemetery, cultural micro regions, and the age of the dominant bedrock formations.

I. Scharlotta et al. / Journal of Archaeological Science 40 (2013) 4509e45274510

the analytical technique, the kind and extent of diagenetic alter-ation of the samples, and the formation of the skeletal materialitself. For skeletal materials, the relationship between the forma-tion event of the physical sample and the chemical interactionsoccurring during life being reconstructed based on the obtainedresults can be complicated and open to interpretation. Strontiumisotope analysis has traditionally relied on thermal ionization massspectrometry (TIMS) due to its reliability and analytical precision.The advent of multi-collector inductively-coupled-plasma mass-spectrometry (MC-ICP-MS) as an alternative to TIMS, coupledwith either a laser micro-drill or a laser ablation (LA) unit for micro-sampling, greatly expanded the possibilities for application of Srisotope tracers in archaeological research. Both laser micro-drillsand laser ablation are far less destructive and enable higher

spatial resolution for analysis than the TIMS and MC-ICP-MStechniques. However, micro-sampling for solution preparationstill requires significant laboratory processing whereas laser abla-tion requires virtually no such special handling (Copeland et al.,2008; Nowell and Horstwood, 2009; Prohaska et al., 2002;Scharlotta, 2010). In spite of several studies using LA on humanteeth, bone and high-Sr apatites (Prohaska et al., 2002, Weber et al.,2007), analysis of phosphate minerals by LA has not been testedextensively for strontiumwork until quite recently (Copeland et al.,2010; Horstwood et al., 2006; Pye, 2004; Scharlotta, 2010, 2012;Simonetti et al., 2008; Vroon et al., 2008; Weber et al., 2008,Weber et al., 1998; Woodhead et al., 2005).

A significant amount of research has been devoted to the subjectof diagenesis of archaeological bone materials, highlighting both

I. Scharlotta et al. / Journal of Archaeological Science 40 (2013) 4509e4527 4511

the importance and the complexity of the matter. The majority ofarchaeological bone is likely to be biologically altered to someextent (Jans et al., 2004). Microbial attack is a significant factor ofdiagenetic alteration with a growing body of work focused on thespecific causes, processes and effects of this alteration (e.g., Jans,2008; Jans et al., 2004; Smith et al., 2007). This work has shedlight on changes in the porosity, bone mineral (i.e., crystallinitychanges), the decreases and ultimate loss of bone collagen and therelationship between many agents involved in biodeterioration,although the question regarding the potential for utilizing unal-tered portions of bone for geochemical research has been largelyleft unaddressed (Jans, 2008; Smith et al., 2007; Trueman et al.,2008).

Previous work (Copeland et al., 2008; Scharlotta, 2010;Simonetti et al., 2008; Woodhead et al., 2005) has indicated anumber of potential problems in gathering accurate strontiumisotopic data from calcium phosphate matrices using LA-MC-ICP-MS. In addition to interference from rubidium (87Rb), doublycharged rare earth elements (Paton et al., 2007), and calcium di-mers (Woodhead et al., 2005), there is the production of a poly-atomic species of CaPO that interferes with the 87Sr (Scharlotta,2010, 2012, Scharlotta et al., 2011, Simonetti et al., 2008, Vroonet al., 2008). This polyatomic species appears to be unique to thelaser ablation technique as it is not apparent with solution mode(SM) MC-ICP-MS.

The problem of CaPO polyatomic interference is limited tocertainmatrix types. For archaeological teeth, method to correct forthis interference is necessary in order to obtain accurate data fromsamples with low concentrations of Sr (Scharlotta et al., 2011).However, for bone samples, there does not appear to be any sig-nificant offset in isotopic values resulting from this polyatomicspecies as the ratios measured previously by (SM) MC-ICP-MS(Haverkort et al., 2008) and those measured by LA-MC-ICP-MSand reported here are quite compatible (Figs. 26e28, Table 1).Since, similar results were noted by Radloff et al. (2010), confir-mation that this problem is not applicable to bone tissue wouldease the pathways of analysis and interpretation.

Bones themselves provide enough ambiguity regardingstructural formation, reformation, and subsequent alteration tocause concerns about sampling methodology without additionalconcerns regarding the use of micro-sampling. A long bone canreflect well over 20 years of an individual’s life. Individualosteons generally have a lifespan of no more than 20e25 yearsand form themselves by cutting through and replacing old bonematerial (Frost, 1963). However, this does not necessarily trans-late to a simple age of the bone as equivalent to that of thecurrent osteons for remnants of the old bone are frequentlyvisible in histological analysis. These old portions are also rep-resented in the homogenized powders and solutions of tradi-tional macro- or bulk-sampling methods. The advantage of directmicro-sampling is that it can alleviate this aspect of uncertaintyby providing a clear link between what is being sampled and thetarget analytical data.

Table 1Averaged laser ablation and solution mode 87Sr/86Sr ratios, demographic and dietary innumber, and the individual number when more than one interment was present. GFS ¼

Burial Master ID Sample ID Age Sex Avg. laser87Sr/86Sr

2s er

B.27-1 K14_1998.027.01 1998.304 35e50 Male 0.71027 0.000B.35-1 K14_1998.035.01 1998.312 18e20 Probable male 0.71088 0.001B.35-2 K14_1998.035.02 2003.597 8e10 Undetermined 0.71018 0.001B.39 K14_1999.039 2003.591 9e11 Undetermined 0.71031 0.001B.45 K14_1999.045 2003.623 8e10 Undetermined 0.71095 0.001B.57-2 K14_1999.057.02 1999.175 35e50 Probable male 0.71127 0.001

Previous Sr isotopic research has focused primarily on sedentaryagrarian groups (e.g., Bentley and Knipper, 2005; Grupe et al., 1997;Price et al., 2002, Price et al., 2008; Price et al., 2007, Price et al.,1994). With such groups, the goal of research is to identify thelocal signature so that organisms of nonlocal origin (people andanimals) can be recognized. Such an approach is of only limitedutility for the study of hunter-gatherers, as many groups utilizedlarge ranges of territory and would thus have less localized (i.e.,averaged over larger areas) isotopic signatures reflecting theirlifetime mobility. It is this circumstance that drives interest inmicro-sampling of skeletal materials to access insights into mobileindividuals with greater chronological resolution. However, furtherresearch is needed to understand better the dynamic interactionbetween direct chemical contact of living organisms with the bio-logically available strontium, the formation of skeletal tissues, anddata recovery from these tissues. This study is focused on the datarecovery side of this problem, examining the range of variability instrontium isotope ratios and trace element composition foundwithin human femur samples.

2. Cis-Baikal resources and geology

The Cis-Baikal region of Siberia refers to the area including thewestern coast of Lake Baikal, the upper sections of the Angara andLena river drainages, and the Tunka region adjacent to the south-western tip of Lake Baikal (approximately between 52� and 58� Nand 101� and 110� E; Fig. 1). The topographic complexity of the riftvalley that formed Lake Baikal led to the formation of a largenumber of micro-habitats, with a variety of seasonally availableresources (Galazii, 1993; Weber, 2003; Weber et al., 2002). Thethermal capacity of Lake Baikal itself moderates the local climateimmediately along its coastline, resulting in generally mildertemperatures during the winter and cooler temperatures duringthe summer. As a result, the Angara River Valley remains relativelyfree of snow during the long winter which attracts various speciesof ungulates looking for forage and less restricted mobility(Formozov, 1964). There is a variety of large andmedium-size gamefound in the region including moose (Alces alces), red deer (Cervuselaphus), roe deer (Capreolus capreolus pygargus), reindeer (Rangifertarandus), mountain goat (Capra sibirica), and wild boar (Sus scrofa).Smaller species such as hare (Lepus sp.), marmot (Marmota sibirica),suslik (Spermophilus citellus), and waterfowl such as geese are alsoabundant in many areas around the lake. During the summer, largeruns of black grayling (Thymallus arcticus baicalensis) are found inthe uppermost section of the Angara River near its source at LakeBaikal, and several fish species enter the tributaries of the Angara inlarge numbers to spawn. The shallow coves and bays in the LittleSea region of Lake Baikal, between Ol’khon Island and the westcoast of the lake, also provide excellent opportunities for fishingand during the late winter and early spring when the lake is frozen,nerpa, the Lake Baikal seal (Phoca sibirica) can be hunted (Levin andPotapov, 1964; Losey et al., 2008; Marchiafava et al., 1974; Weber,1995). Ethnographic studies of boreal forest populations highlight

formation. Master_ID: cemetery name in abbreviated form, excavation year, gravegame-fish-seal diet type. GF ¼ game-fish diet type.

ror Soln.87Sr/86Sr

2s error Birth C/N d13C d13N Diet 14C age BP

74 0.71037 0.00001 Non-local 3.5 �19.7 11.8 GF 4060 � 12026 0.71012 0.00002 Non-local 3.3 �19.6 11.7 GF 4030 � 7075 0.71036 0.00003 Non-local 3.3 �19.4 11.4 GF 3770 � 14016 0.71066 0.00002 Local 3.5 �18.4 15.6 GFS 3930 � 18033 0.71056 0.00002 Local 3.4 �18.5 14.3 GFS 4820 � 9062 0.71096 0.00002 Non-local 3.4 �18.7 13.7 GFS 4080 � 550


the use of mushrooms, berries, and pine nuts as other food re-sources (Haverkort et al., 2010; Katzenberg and Weber, 1999; Lam,1994; Marles, 2000). While archaeological evidence for plant useduring the Baikal’s middle Holocene is very limited, there is suffi-cient ethnographic information about the use of plants in borealforager subsistence systems around the world to speculate upontheir usage as diverse but limited in overall energetic contribution(e.g., Kelly, 1983; Marles, 2000; Winterhalder, 1981, 1982).

Within the Cis-Baikal region there are four main geologicalzones that roughly correspond to archaeological micro-regions(Fig. 1). The main zones are 1) the Baikal basin, including the lakeitself, the coastal areas as well as the Little Sea area enclosed byOl’khon Island; 2) the drainage of the upper and middle AngaraRiver bounded by the Eastern Sayan Mountains to the west and theCentral Siberian Plateau to the east and extending north towardsBratsk; 3) the upper Lena river basin cutting through the CentralSiberian Plateau as it heads northwards; and 4) the Tunka regioncovering a sizeable valley running south of the Eastern SayanMountains and broadly connecting the southwestern tip of LakeBaikal to Lake Khovsgol inMongolia. The upper andmiddle sectionsof the Angara River flow through Mesozoic and Quaternary de-posits, with expected 87Sr/86Sr values in the range of 0.705e0.712.The upper Lena watershed and the surrounding Central SiberianPlateau are dominated by Cambrian and Precambrian limestones,with expected values fairly tightly clustered around 0.709(Haverkort et al., 2008, Huh et al., 1994). The Baikal basin includesthe Primorskii and Baikalskii mountain ranges and is characterizedby relatively high 87Sr/86Sr (w0.720e0.735) due to the presenceof Archean and Proterozoic granites (Galazii, 1993). Bedrock ofsimilar ages occurs around the southwestern shores of Lake Baikaland drainages adjacent to the Eastern Sayan Mountains, howeverour preliminary data on distribution of biologically availablestrontium isotopes in the Cis-Baikal region, provided by examina-tion of modern plant and water samples, indicate that these tworegions have quite different 87Sr/86Sr values (Scharlotta, 2012).Both Archean and Proterozoic zones (Little Sea and Tunka Region)overlap 87Sr/86Sr ranges of neighboring regions (e.g., Angara

Fig. 2. Site plan of Khuzhi

Drainage), while only the Little Sea area exhibits values above0.720. Further clarifications of the distinction between these twozones of similar age will be possible upon completion of regionalsampling efforts. Overall values for Lake Baikal water are reportedas 0.7085 (Kenison Falkner et al., 1992).

3. Khuzhir-Nuge Xiv cemetery

The KN XIV cemetery is located on the west coast of the LittleSea micro-region of the Lake Baikal basin, near the southern end ofOl’khon Island and c. 3 km southwest of the mouth of the SarmaRiver (53�0405800 N,106�4802100 E). It occupies the southeast slope ofa hill rising from a shallow bay (Fig. 2). With 79 graves and a total of89 individuals unearthed, KN XIV is the largest Early Bronze Agehunter-gatherer cemetery ever excavated in the entire Cis-Baikalregion (Weber et al., 2008, Weber et al., 2007). All the graveswere only c. 30e60 cm deep sub-rectangular pits filled with rocksand loamy sand, and covered by surface structures built of stoneslabs still visible on the surface prior to archaeological excavation.Most graves contained single inhumations, sevenwere double, andtwo were triple interments.

The north-south orientation of Grave 7 is consistent with theLate Neolithic Serovo culture of the Ol’khon region, while all theother graves show clear similarities with the mortuary tradition ofthe Early Bronze Age Glazkovo culture (Goriunova and Mamonova,1994; Konopatskii, 1982; Weber et al., 2002). The most diagnosticGlazkovo characteristics include the generally west-east orienta-tion of the burials and such grave goods as copper or bronze objects(rings, knives, needles, and bracelets), kaolinite beads, and ringsand discs made of white nephrite or calcite (Weber et al., 2008).Analysis of approximately 80 14C dates indicates that the KN XIVcemetery was used continuously by Glazkovo peoples for amaximum of 700 years between w4650 and 3950 cal. BP but themajority of the burials (70%) date to between w4450 and 4250 cal.BP (Weber et al., 2005). However, more recent reassessment of theradiocarbon evidence suggests much shorter use of the cemetery,perhaps not exceeding 100 years (Weber, 2011). Since the analysis

r Nuge XIV cemetery.


did not reveal any obvious temporal trends in mortuary attributes,it seems justified to treat the cemetery, with the exception of themuch earlier Grave 7, as one analytical unit (McKenzie, 2006;Weber et al., 2005).

To date, a sample of 25 individuals from KN XIV were analyzedfor strontium isotope ratios and compared with 79 faunal speci-mens collected throughout Lake Baikal and the Cis-Baikal region(Haverkort et al., 2010, Haverkort et al., 2008, Weber et al., 2003).The human materials included 20 adult individuals for which threemolars (M1, M2 and M3) and a femur sample were available and 5subadult burials with only M1 and M2 crowns completed. The sixfemur samples used for the current study came from this pool ofpreviously tested individuals (Table 1).

This past work allowed to expand the possible applications ofstrontium isotope research and to identify an interesting generalpattern present in several mobility profiles within the sample of KNXIV individuals. Broadly speaking, it appears that there was a sig-nificant amount of movement of individuals during their lifetime,whereby people buried at KN XIV were frequently not born in theLittle Sea region, but only migrated there as subadults or adults(Haverkort et al., 2010, Haverkort et al., 2008; Weber andGoriunova, 2012). There appears to be significant variabilitywithin the cemetery itself as to the place of origin, the timing ofmigration to the Little Sea, dietary preferences, mortuary protocols,and cemetery spatial organization that is the subject of ongoingresearch (cf. Weber and Goriunova, 2012; Weber et al., 2011).

4. Microbial biodeterioration

Microbial attacks on buried bone are either fungal or bacterial innature which only rarely co-occur in the same bone (Jans et al.,2004). Marchiafava et al. (1974) noted that invading fungal hy-phae contained mineral in solution, whilst the tunnels they createdheld no redeposited minerals, suggesting that the dissolved min-eral was likely transported out of the bone. Fungal alteration ofbones forms tunnels up to 250 mm in diameter, dissolving andremoving mineral from the bone, and crossing cement lineswithout apparent difficulty. Yet, it is unclear whether this is theprocess of harvesting nutrients directly from the bone or the fungiare using the bone as amedium (Hackett,1981; Jans, 2008). Cementlines have a high sulfur content and low calcium and phosphorouscontent, though a high Ca/P ratio, making them different enough incontent and structure to inhibit bacterial progression (de Ricqleset al., 1991; Jackes et al., 2001; Martin and Burr, 1989). Bacteriaappear to produce pores in bone, causing substantial degradationas they spread from blood vessels and fill osteons until they reach acement line or another area of bacteria (Bell, 1990; Hackett, 1981;Katzenberg et al., 2009). This produces biodeterioration with acomplex morphology while mineral is dissolved to expose collagenand redeposited as a hypermineralized rim at the edge of bacterialfoci, reorganized rather than removed (Jans, 2008). It is unknownwhether the rarity of co-occurrence of fungal and bacterial attacksis due to competition between the organisms, a lack of remainingnutrients after an initial alteration, or whether such alterationcreates environmental incompatibility for further alteration (e.g.,intrusive phosphate minerals such as francolite or brushitedeposited by bacteria cannot support fungi as a nutrient source oras a medium) (Child, 1995; Jackes et al., 2001; Jans, 2008; Jans et al.,2004; Katzenberg et al., 2009; Smith et al., 2007).

Trace element assay of archaeological bone has long been animportant aspect of research on bone diagenesis (e.g., Gilbert, 1975;Katzenberg et al., 2009; Nielsen-Marsh and Hedges, 2000b; Pate,et al., 1989; Price, 1989; Pye, 2004; Radosevich, 1993; Truemanet al., 2008; Tuross et al., 1989). Much of this work has focused onwhat are the expected “normal” and “diagenetic” values for human

skeletal tissues in a fashion similar to the use of C/N ratios as ameasure of data integrity in dietary isotope studies (e.g., Burton,et al., 2003; Cucina et al., 2007, Cucina et al., 2011; Grupe, 1998;Hedges, 2002; Koenig et al., 2009; Lappalainen et al., 1981;Maurer et al., 2011; Molleson, 1988; Vrbic et al., 1987). Clear re-lationships in chemical composition exist between the biogeo-chemical environment of an individual’s lifetime on the one sideand their skeletal remains on the other. However, many researchershave written off the potential utility of trace element data to pro-vide any useful information about human behavior (e.g., mobility,see Cucina et al., 2007; Cucina et al., 2004, Cucina et al., 2011;Knudson and Price, 2007) and favor the use of such data strictlyfor identification and removal of possibly diagenetically alteredtissues. Debates over whether trace element concentrations relateto diagenetic or anthropogenic processes have quieted in recentyears as improved laboratory techniques (e.g., Garvie-Lok et al.,2004; Koch et al., 1997; Nielsen-Marsh and Hedges, 2000b; Pate,et al., 1989) have enabled researchers to understand the nature ofdiagenetic changes and remove any mineral structures resultingfrom interaction with the burial environment. Two caveats, how-ever, remain to be addressed: first, sample pretreatments may notremove all diagenetic alterations or may overcorrect for hypothe-sized alterations and thus bias the resultant data; and second, suchapproaches operate on the assumption, typically implicit, thatsample homogenization is an appropriate means to acquireanthropogenic and behaviorally meaningful geochemical infor-mation. One possible approach to alleviate both of these potentialproblems is to micro-sample bone structure, thus avoiding alto-gether bone structures that exhibit diagenetic effects.

5. Materials and methods

As mentioned, femur samples analyzed in this study representsix individuals from the KN XIV cemetery (Table 1) all of which havebeen previously examined by Haverkort et al. (2008). Samples weremounted in epoxy, thin-sectioned, mounted on slides and groundto a thickness of w100 mm to enable observation of internalstructures. All samples were analyzed for elemental compositionusing LA-ICP-MS and for 87Sr/86Sr ratios using LA-MC-ICP-MS. Allsample preparation and analyses were conducted at the RadiogenicIsotope Facility of the Department of Earth and Atmospheric Sci-ences at the University of Alberta, Edmonton.

5.1. Trace element analysis

Laser ablation for elemental concentrations was conducted us-ing the Perkin Elmer Elan6000 quadrupole ICP-MS coupled to aUP213 nm laser ablation system (New Wave Research, USA). Theinstrument was optimized using the NIST SRM 612 internationalglass standard reference material (RF power 1200 W, peak hoppingacquisition, 50 ms dwell time). Individual osteons were sampled ina serial fashion using a 6 mm beam, with a repetition rate of 20 Hzand an energy density of w13 J cm�2, beginning at the Haversiancanal and progressing outwards towards the cement line toexamine the nature and extent of useful intra-osteon geochemicalvariability (Fig. 3).

Experiments were conducted in a mixed He/Ar atmosphere(ratio of 0.5:0.1 Lmin�1) within the ablation cell, andmixed with Ar(1.03 L min�1) prior to entering the torch assembly. The laserablation cell was flushed with a higher flow rate of He (up to0.9 Lmin�1) for approximately 1min between laser ablation runs toensure adequate particle washout. The NIST SRM 612 glass standardwas used as the external calibration standard. Quantitative resultsfor 58 elements (Li, Be, B, Na, Al, Si, P, K, Ca, Sc, Ti, V, Cr, Mn, Fe, Ni,Co, Cu, Zn, Ga, Ge, As, Rb, Sr, Y, 90Zr, 91Zr, Nb, Mo, Ag, Cd, In, Sn, Sb,

Fig. 3. Laser ablation scars from analysis. Biodeterioration (whitish and cloudy discoloration) appears to progress from cracks and hypermineralized areas as has been noted in SEMstudies (e.g., Bell, 1990; Jackes et al., 2001). Letters denote osteon and data series. Scale ¼ 100 mm. Series C (top right) shows how, while the majority of the osteon is intact, wherethe biodeterioration begins (whitish or cloudy discoloration) an intrusive phosphate mineral is already being formed and the cement line is no longer visible in the compositionaldata, though the laser’s entry into the hypermineralized intrusive phosphate looks very similar to the composition of the cement line. The presence of intrusive material is clearlyshown in the Ca content of series D (Fig. 4).


Cs, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hf, Ta, Re,Au, Tl, Pb, Bi, Th, and U) were obtained and normalized to 24Mg, asthe internal standard using the GLITTER� (XP version, MacquarieUniversity) laser ablation software (SupplementaryMaterial 1). Mg,measured by solution analysis, was used instead of Ca in order toassess variation in calcium concentrations in these samples.

5.2. Osteon formation and sampling

As noted, individual osteons generally have a lifespan of nomore than 20e25 years and form themselves by cutting throughand replacing old bone material (Frost, 1963). Osteons appearingintact and showing variable levels of alteration were sampled inthis manner to observe the compositional changes associated withmicrobial deterioration. During bone remodeling, osteoid will bedeposited at a rate of approximately 0.3e1.0 mm per day (Frost,1963; Parfitt, 1979, 1984). This is followed by a minimum lag timeof one week before mineralization will begin. Thus, each laserablation line (represented by a single data point within each series)could reflect less than a month during the individual’s life. This,however, is difficult to gauge precisely given several factors, such asthe age and sex of the individual, their state of health, and nutrition,that can impact the growth and size of individual osteons (Parfitt,1979, 1984). It is more likely that a given ablation line willapproximate closer to a span of six months to one year of the in-dividual’s life. Individual elements have different residence times inthe body and will vary in their sensitivity to changes resulting frommovements or other external factors (Beard and Johnson, 2000;Britton et al., 2009; Schweissing and Grupe, 2003).

Progressive lines were spaced roughly 5e10 mmapart in order toensure that individual laser lines did not represent overlappingperiods of time (Fig. 3). The size and geometry of osteons is

correlated with the age, sex, and weight of individuals during life;however, many researchers have noted broad temporal trendsrather than refined spans of time (e.g., Currey, 1964; Iwamoto et al.,1978; Kerley, 1965, 1969; Kerley and Ubelaker, 1978; Koenig et al.,2009; Landerso and Frost, 1964; Pye, 2004; Scharlotta, 2012;Yoshino et al., 1994). The external lamella of an osteon forms firstand the growth progresses inward from the periphery to the Ha-versian canal, with additional lamellae visible as an osteon ages.Histological analysis is necessary to confirm visually that osteonsare indeed of similar age; however, estimates can bemade based onvisible voids left from osteocytes.

Osteons with similar geometry and visible lamellae are likely tobe of similar age but, as mentioned, this can be only be verifiedthrough separate histological or geochemical analysis. Chemicalcomposition of bones produced and mineralized during the sameinterval will be the same, thus forming patterns in the micro-sampled data, reflective of different environments or periods ofspecific chemical interactions that will result from intra-osteonalanalysis. The age of individual osteons can be externally verified,but this may not be necessary. Using a series of osteons, anygeochemical patterning present will become apparent and therelative age of specific osteons can be determined by wigglematching these patterns. In this way, the goal of finding usefulpatterning from intra-osteonmicro-sampling also serves to providea buffer against potential time gaps between individual osteonsbeing sampled.

5.3. Strontium isotope analysis

Laser ablation for isotopic analysis was conducted using aUP213 nm laser system coupled to the Nu Plasma HR MC-ICP-MSwith the sample-out line from the desolvating nebulizing


introduction system (DSN-100 from Nu Instruments) to allow forsimultaneous aspiration of a 2% HNO3 solution. At the beginning ofeach analytical session, parameters for the introduction system andthe ion optics were optimized by aspirating a 100 ppb solution ofthe NIST SRM 987 Sr isotope standard. Experiments with the lasersize and power quickly highlighted a problem with a replication ofthe analyses conducted for elemental composition. Similar spotsizes were unable to yield high enough Sr signals to gain useable87Sr/86Sr ratios.

In order to determine the minimum spot size that could beeffectively used on these bone samples, a size range experimentwas conducted on Durango Apatite. The results showed that a spotsize as small as 60 mm, yielding signal strengths of 1e1.5 V on88Sr (100% laser power; 20 Hz repetition rate; w15 J cm�2 energydensity) could be effectively used, however, more reliable signals(3 þ V) were gained from analyses using sizes greater than 100 mm.Strontium levels are lower in most human samples than in DurangoApatite, so stable signals as low as 0.5 V were accepted. Analyseswith questionable signals were repeated to ensure stability. Not allosteons are large enough to be able to hold a 100þ mm laser beam,so the majority of analyses were conducted using an 80 mm beam,with 100 mm applied to a few particularly large osteons. This largebeam size limits temporal assessment within the bone to the age ofa given osteon, thus to the last years of life of a given individual.More sensitive equipment than available for this study would berequired to conduct intra-osteon micro-sampling for measuring Srisotope ratios.

5.4. Determination of diagenetic alteration

Altered portions can be visually identified, but determining theextent of alteration, or the factors impacting the osteon, can only beassessed via chemical analysis. Diagenetic alteration was assessedby visual inspection of changes in the osteon’s physical structure.Such changes include discolorations, changes in material structure,cracking, and the deposition of intrusive materials. Burial 45 wasused to experiment with different number of ablation lines. Someof these lines intentionally crossed the cement line and into areas ofbone that were identified visually as altered. The progression ofchemical compositional differences between intact and diageneti-cally altered bone was previously unknown. Specific attributes ofdifferent diagenetic processes have been discussed in the literature,but the exact history of diagenetic alteration for the current sam-ples had to be determined. Sequential sampling of materialsappearing intact and grading into areas that visually appear to bealtered provides some insights into the chemical changes thatcorresponded with the changes in appearance. Based on theseguidelines, trace element analysis of the remaining specimensemployed five ablation lines for each of four intact osteons(Supplementary Material I). Furthermore, within each bone sam-ple, nine intact osteons were analyzed for strontium isotope ratiosalong with five osteons that were suspected to have been impactedby diagenetic processes. This allowed assessment of the directionand potential extent of diagenetic alteration of intact osteons(Supplementary Material II).

6. Results and discussion

Our method of bone micro-sampling and the resulting traceelement and strontium isotope data provide new and importantinsights on the following three points: middle Holocene hunteregatherer mobility in Cis-Baikal, the process of bone diagenesis inarchaeological settings, and applicability of laser ablation as anintroduction method for examination of trace elements andstrontium isotope ratios.

6.1. Hunteregatherer mobility

A number of conclusions can be drawn from the elemental andisotopic results. Areas of bone visually assessed as structurallydeteriorated or altered showed changes in trace element and iso-topic profiles as compared to intact areas. That obvious signs ofstructural deterioration are coupled with skewed chemical data isless surprising than the documentation of this process progress. Itappears that chemical changes actually precede the visual changesand that the chemical data are a better indicator of the bone min-eral integrity as well as the nature and extent of diagenetic alter-ation in process in any given sample than the visual characteristicsalone. Foremost, the femur samples appear to suffer primarily frombacterial attack as opposed to fungal attack. Biodeteriorated regionsare visible and show the characteristics described for bacterialattack in the literature such as mineral redistribution and rede-posited phosphate with significantly lower Ca levels (e.g., Jackes,et al., 2001; Jans, 2008; Jans et al., 2004). Also note that analysesbegin at the Haversian canal with lower Ca values and that wherethe laser approaches or crosses the cement line, Ca values spikedown (Fig. 4). For Burial 45, data series BeF (each reflecting sam-pling within a single osteon) correspond with osteons shown inFig. 3. Six osteons within a single femur sample from Burial 45 wereanalyzed for trace elements to examine intra- versus inter-osteonalcompositional differences (Fig. 4). The results from Burial 45showed that trace elemental composition was strongly correlatedbetween intact portions of osteons while diagenetically alteredportions inform primarily on localized microscopic changes to thebone structures. The remaining burials were analyzed for fourosteons each after it was determined that similar patterning waspresent and additional osteonal sampling would provide redun-dant data (Supplementary Material I). The six osteons of Burial 45were of similar size, but, as noted, factors such as age and health canimpact the formation, size, and mineralization of osteons. Threeosteons were more extensively sampled to investigate the boundsof useful data, and provide guidelines for what to expect inchemical compositional data when either altered portions of boneare encountered or features such as the cement line are crossed.Series B, C, and D, in Fig. 4 represent sampling beyond intact areas,into deteriorated areas, and past the cement lines (B, C). Series C(top right in Fig. 3) shows how, while the majority of the osteon isintact, where the biodeterioration begins (whitish or cloudydiscoloration) an intrusive phosphate mineral is already beingformed and the cement line is no longer visible in the composi-tional data. In this case, the laser’s entry into the hypermineralizedintrusive phosphate looks very similar to the composition of thecement line of other osteons. The presence of intrusive material isclearly shown in the Ca content of series D (Fig. 4). It is important tobe able to identify the type of microbial action and/or otherdiagenetic processes in action in any given sample to ensure thatappropriate measures are taken into consideration prior to inter-pretation of the results.

Sr values display an interesting pattern, being present as astructural component and a trace element (i.e., floating in mineraltraps within the matrix). Fully biodeteriorated portions demon-strate a loss of Sr in addition to a redistribution of the remainingportion present in the intrusive phosphates (Fig. 5). Along with thestable Sr:Ca values (Fig. 6), this suggests that the form of theintrusive phosphate is more akin to brushite than to francolite(Garvie-Lok et al., 2004, Prohaska et al., 2002), though this isdifficult to assess conclusively from a limited sampling area. Prior tocomplete dissolution, Sr does appear to be resistant to diageneticeffects (cf. Radosevich, 1993). Ba shows an interesting aspect oftrace element uptake within the osteon (Fig. 7). Ba can act as amineral replacement for Ca or Sr, yet those points closest to the

Fig. 4. Calcium distribution (in ppm. concentrations divided by 1000) of six osteons of Burial 45 beginning at the Haversian canal and ending at or past the cement line. The series orpath names (BeF) correspond with the osteons shown in Fig. 3. Shorter series came close to, but did not cross the cement line at their ends. Longer series crossed the cement lineapproximately where noted. Osteon C was specifically chosen as a partially impacted osteon to investigate the relationship between visual and chemical compositional changes. Thesix osteons were of similar size, but three osteons were more extensively sampled to investigate the bounds of useful data. Series B, C, and D, represents sampling beyond intactareas, into deteriorated areas, and past the cement lines but likely do not represent greater spans of time in terms of useful data.


Haversian canal suggest diagenetic Ba values and that trace ele-ments were not secured into the mineral matrix at the time ofdeath and that the Ba of the most recently deposited bone matrixremained soluble upon burial.

Burials 35-2 and 39 (Figs. 8 and 9) show an interesting divisionin barium values. Recent research (e.g., Swanston, et al., 2012), hassuggested that differences in trace elements values relate to the ageof individual osteons, providing insight into the chemical envi-ronment of the individual closer to the time of death. The rate andextent of mineralization likely plays an important role in this dif-ferential uptake of trace elements, with older osteons still con-nected to the Haversian system, but less likely to incorporateelements such as barium in a structural capacity, limiting uptake ofthese older and more fully mineralized structures to flaws withinthe mineral matrix. The transitional values noted in Burial 35-2

Fig. 5. Strontium distribution (in ppm. concentrations) of six osteons of Burial 45 beginning(AeF) correspond with the osteons shown in Fig. 3. Shorter series came close to, but did not cwhere noted. Osteon C was specifically chosen as a partially impacted osteon to investigat

(Fig. 8, series A) indicate such differences are progressive, as thepathway to full mineralization and reduced incorporation of higherBa concentrations trends from high values at the Haversian Canal tolower values closer to the cement line. Such a difference could be auseful discriminator when attempting to identify older versusyounger osteons within a sample and also provide additional evi-dence for movements in the last few years of life.

Remaining three burials, i.e., 27, 35 and 57 (Figs.10e12), indicatemore stable levels of Ba throughout the lives of the sampledosteons. This would suggest that these individuals remained in thesame biogeochemical area for many years. Nonstructural elements(i.e., Fe) show significant variations in concentrations, markedredistribution associated with biodeterioration and reduced pre-dictability in terms of uptake (Fig. 13). As an essential nutrient, thiscould relate to episodes of nutritional abundance and stress, could

at the Haversian canal and ending at or past the cement line. The series or path namesross the cement line at their ends. Longer series crossed the cement line approximatelye the relationship between visual and chemical compositional changes.

Fig. 6. Strontium to calcium ratio distribution of six osteons of Burial 45 beginning at the Haversian canal and ending at or past the cement line. The series or path names (AeF)correspond with the osteons shown in Fig. 3. Shorter series came close to, but did not cross the cement line at their ends. Longer series crossed the cement line approximately wherenoted. Osteon C was specifically chosen as a partially impacted osteon to investigate the relationship between visual and chemical compositional changes.


indicate interactions with the biogeochemical environment, orcould be an artifact of diagenetic changes, but are difficult toseparate at present.

It is difficult to tell whether trace elements were deposited inthe bone from the soil or if alteration lowered theMg values used tocalibrate the data. Analysis of trace elements and 87Sr/86Sr on teethfrom the six individuals analyzed herein and several other in-dividuals from KN XIV demonstrated a relationship betweencertain trace elements and the mobility data traditionally providedby isotope analysis (Haverkort et al., 2008; Scharlotta, 2012;Scharlotta et al., 2011).

Scharlotta et al. (2011) demonstrated that within this popula-tion, Re replicated Sr isotope data, with higher Re concentrations inareas with higher Sr isotopic ratios. Thus, variability, as well as

Fig. 7. Barium distribution (in ppm. concentrations) of six osteons of Burial 45beginning at the Haversian canal and ending at or past the cement line. The series orpath names (AeF) correspond with the osteons shown in Fig. 3. Shorter series cameclose to, but did not cross the cement line at their ends. Longer series crossed thecement line approximately where noted. Osteon C was specifically chosen as a partiallyimpacted osteon to investigate the relationship between visual and chemical compo-sitional changes.

repeated patterns between osteons, in Re values for Burial 45suggest that there were at least three major movement events (thepatterning of peaks and valleys) in this individual’s life within theLittle Sea region. When the sampled osteons were initially formed,the individual lived in an area of low Re values, shown by samplingpoints near the cement line (Fig. 14). This was followed by amovement from this area into a significantly different Re area (PeakX, Fig. 14). Then there was movement out of the high Re area,producing Valley Y (Fig. 14). The third movement showed a returnto higher Re values indicated by sampling nearer to the HaversianCanal. It is possible that this final movement represents a return toa higher Re area of the Little Sea near the time of death or trans-portation of the body after death, if the elevated Re values in theHaversian Canal represent local soil conditions and/or if the cem-etery was used to inter more than just individuals who died nearthe location. That similar patterning, of peaks and valleys, is seenacross multiple osteons of the same individual (Burial 45) suggestthis to be anthropogenic and not diagenetic alteration. To see such

Fig. 8. Barium distribution (in ppm. concentrations) of four osteons of Burial 35-2beginning at the Haversian canal and ending inside the cement line, each data series(AeD) reflects one osteon. Note the divisions between groups of osteons.

Fig. 11. Barium distribution (in ppm. concentrations) of four osteons of Burial 35-1beginning at the Haversian canal and ending inside the cement line each data series(AeD) reflects one osteon.

Fig. 9. Barium distribution (in ppm. concentrations) of four osteons of Burial 39beginning at the Haversian canal and ending inside the cement line, each data series(AeD) reflects one osteon. Note the divisions between groups of osteons.


duplication in diagenetic processes in multiple locations, each withunique history of preservation and potential alteration would behighly unlikely.

The preservation of Re signatures is somewhat easier to see inother individuals. Burial 35-2 (Fig. 15) has three series that are fairlywell correlated and one that appears to be offset. Series C has avalley at ablation line 2, while the other series show a small peak.Then at ablation line 3, there is a peak while the others have avalley. The beginning and ending of this series may have beenslightly offset relative to the span of time represented by otherosteons. The placement of laser ablation lines relative to the Ha-versian Canal and spacing was consistent within each bone sample,likely indicating that this osteon is either older or younger than theother osteons sampled. The pattern of beginning in a low Re area,moving to a higher Re area, and then back again would suggestthree or possibly four movements. Intriguingly, whereas Burial 45showed elevated Re values near the Haversian Canal, Burial 35-2shows a decrease. This raises that possibility that either extremely


localized changes in the soil chemistry are impacting diageneticalterations within the KN XIV cemetery, or that one of these twoindividuals was transported to the cemetery after death. The rela-tively smaller peaks and valleys could indicate that these move-ments are taking place across less geologically diverse areas of theLittle Sea, or that these movements are part of a multi-year patternof relocation and return to the same or similar areas.

Burial 39 (Fig. 16) also appears to have osteons of two differentages. Series A andD showelevated Re values at ablation line 4,whileseries B and C show reduced values. Again at ablation line 3, series Ais quite lowwhile series C is high. Together, there still appears to be acombination of peak and valley, or valley and peak, followed by afinal reduction in Re values near the Haversian Canal, suggestingthat the individual likely made three movements during life.

Burial 57-2 (Fig. 17) also likely shows three movements. Revalues decrease from near the cement line at ablation line 5 toablation line 3. This is followed by a peak at ablation line 2 andreturn to lower Re values at ablation line 1 near the Haversian


Fig. 13. Iron distribution (in ppm. concentrations) of Burial 45 beginning at the Haversian canal and ending at or past the cement line. The series or path names (AeF) correspondwith the osteons shown in Fig. 3. Shorter series came close to, but did not cross the cement line at their ends. Longer series crossed the cement line approximately where noted.Osteon C was specifically chosen as a partially impacted osteon to investigate the relationship between visual and chemical compositional changes.


Canal. There is some divergence at ablation lines 3 and 4 that couldsuggest differences between the mineralization rate and Re intake,or perhaps shorter termmovements in between areas of higher andlower Re that are not equally represented by the ablation lines in allosteons.

Fig. 14. Rhenium distribution (in ppm. concentrations) of Burial 45 beginning at theHaversian canal and ending at or past the cement line. The series or path names (AeF)correspond with the osteons shown in Fig. 3. Shorter series came close to, but did notcross the cement line at their ends. Longer series crossed the cement line approxi-mately where noted. Osteon C was specifically chosen as a partially impacted osteon toinvestigate the relationship between visual and chemical compositional changes. Revalues for Burial 45 suggest that there were at least three major movement events inthis individual’s life within the Little Sea region. When the sampled osteons wereinitially formed, the individual lived in an area of low Re values, shown by samplingpoints near the cement line (Fig. 14). This was followed by a movement from this areainto a significantly different Re area (Peak X, Fig. 14). Then there was movement out ofthe high Re area, producing Valley Y (Fig. 14). The third movement showed a return tohigher Re values indicated by sampling nearer to the Haversian Canal. It is possible thatthis final movement represents a return to a higher Re area of the Little Sea near thetime of death or transportation of the body after death, if the elevated Re values in theHaversian Canal represent local soil conditions and/or if the cemetery was used to intermore than just individuals who died near the location.

Burial 35-1 (Fig. 18) shows two major movements during theirlife. There are possible small peaks or valleys present at ablationline 3 and 4, though the clearest trend is a reduction in Re valuesfrom ablation line 5 to ablation line 2, with a subsequent increase at

Fig. 15. Rhenium distribution (in ppm. concentrations) of four osteons of Burial 35-2beginning at the Haversian canal and ending inside the cement line each data series(AeD) reflects one osteon. Three series (A, B, D) are fairly well correlated and oneappears to be offset (series C). Series C has a valley at ablation line 2, while the otherseries show a small peak. Then at ablation line 3, there is a peak while the others havea valley. The beginning and ending of this series may have been slightly offset relativeto the span of time represented by other osteons. The placement of laser ablation linesrelative to the Haversian Canal and spacing was consistent within each bone sample,likely indicating that this osteon is either older or younger than the other osteonssampled. The pattern of beginning in a low Re area, moving to a higher Re area, andthen back again would suggest four movements. Intriguingly, whereas Burial 45showed elevated Re values near the Haversian Canal, Burial 35-2 shows a decrease.This raises that possibility that either extremely localized changes in the soil chemistryare impacting diagenetic alterations within the KN XIV cemetery, or that one of thesetwo individuals was transported to the cemetery after death. The relatively smallerpeaks and valleys could indicate that these movements are taking place across lessgeologically diverse areas of the Little Sea, or that these movements are part of a multi-year pattern of relocation and return to the same or similar areas.

Fig. 16. Rhenium distribution (in ppm. concentrations) of four osteons of Burial 39beginning at the Haversian canal and ending inside the cement line each data series(AeD) reflects one osteon. Burial 39 appears to have osteons of two different ages.Series A and D show elevated Re values at ablation line 4, while series B and C showreduced values. Again at ablation line 3, series A is quite low while series C is high.Together, there still appears to be a combination of peak and valley, or valley and peak,followed by a final reduction in Re values near the Haversian Canal, suggesting that theindividual likely made three movements during life.

Fig. 18. Rhenium distribution (in ppm. concentrations) of four osteons of Burial 35-1beginning at the Haversian canal and ending inside the cement line each data series(AeD) reflects one osteon. Burial 35-1 shows two major movement events during theirlife. There are possible small peaks or valleys present at ablation points 3 and 4, thoughthe clearest trend is a reduction in Re values from ablation line 5 to ablation line 2,with a subsequent increase at ablation line 1. Multiple smaller movements may haveoccurred, but the data only show two clear movements between significantly differentareas of Re concentration.


ablation line 1. Multiple smaller movements may have occurred,but the data only show two clear movements between significantlydifferent areas of Re concentration.

Individual 27-1 (Fig. 19) may not have moved between geologiczones, or was possibly more contaminated than other samples.

Fig. 17. Rhenium distribution (in ppm. concentrations) of four osteons of Burial 57-2beginning at the Haversian canal and ending inside the cement line each data series(AeD) reflects one osteon. Burial 57-2 also likely shows three movements. Re valuesdecrease from near the cement line at ablation line 5 to ablation line 3. This is followedby a peak at ablation line 2 and return to lower Re values at ablation line 1 near theHaversian Canal. There is some divergence at ablation lines 3 and 4 that could suggestdifferences between the mineralization rate and Re intake, or perhaps shorter termmovements in between areas of higher and lower Re that are not equally representedby the ablation lines in all osteons.

Fig. 19. Rhenium distribution (in ppm. concentrations) of four osteons of Burial 27-1beginning at the Haversian canal and ending inside the cement line each data series(AeD) reflects one osteon. Individual 27-1 does not appear to have moved betweengeologic zones, or was possibly more contaminated than other samples. Values nearthe cement line and Haversian Canal are similar. There are clear peaks and valleys inthe Re values, but there is not a consistent pattern between multiple osteons, making itlikely that localized microscopic changes could be influencing the data, and wouldrequire further analysis to clarify the matter based on Re data alone. Analysis of cesiumsupports multiple movement events as opposed to diagenetic changes. There are clearpeaks and valleys in the Re data, with points of relative convergence at ablation lines 2and 4. These points could indicate low Re values with higher Re values along both thecement line and the Haversian Canal. Ablation line 3 presents the real mystery in thisinstance, indicating either stable, low values of Re, or significantly elevated Re values. Itis possible that a shorter duration movement occurred in this period of time and thatdifferences in the mineralization of different osteons altered the incorporation of Reduring this time, with some osteons readily accepting Re, while others only registeredthe relatively lower values.

Fig. 21. Cesium distribution (in ppm. concentrations) of four osteons of Burial 35-1beginning at the Haversian canal and ending inside the cement line each data series(AeD) reflects one osteon. Cs values show a general trend from high values near thecement line to low values at ablation line 2. Intermediary peaks and valleys mayrepresent smaller scale movements or other environmental changes, but are notcorrelated between all sampled osteons. There was a second movement, back to a highCs area indicated by ablation line 1.


Values near the cement line and Haversian Canal are similar. Thereare clear peaks and valleys in the Re values, but there is not aconsistent pattern between multiple osteons, making it likely thatlocalized microscopic changes could be influencing the data, andwould require further analysis to clarify the matter based on Redata alone.

Another element that showed strong promise for anthropogenicdata was cesium, with similar correlations between strontiumisotope ratios and Cs concentrations (Scharlotta et al., 2011). Indi-vidual 27-1 appears to have made three major movements in theirlife, in contrast with the Re results (Fig. 20). This inference is basedon transitions between low and high concentrations with twopeaks, and one trough clearly observed in two osteons, and verysimilar in a third. The initial peak at ablation line 2 shows higherconcentrations than ablation line 1, closest to the Haversian canaland most likely to reflect the composition of the burial environ-ment. Using Re alone, the data did not clearly indicate threemovements as multiple ablation lines produced divergent data.With Cs supporting multiple movement events in an unambiguousfashion, a re-examination of the Re data is in order. As noted, thereare clear peaks and valleys in the Re data, with points of relativeconvergence at ablation lines 2 and 4. These points could indicatelow Re values with higher Re values along both the cement line andthe Haversian Canal. Ablation line 3 presents the real mystery inthis instance, indicating either stable, low values of Re, or signifi-cantly elevated Re values. It is possible that a shorter durationmovement occurred in this period of time and that differences inthe mineralization of different osteons altered the incorporation ofRe during this time, with some osteons readily accepting Re, whileothers only registered the relatively lower values. Ultimately,multiple lines of evidence would tend to reinforce one another andsupport that movement events have occurred with all uncertaintyin the nature or locations of these events.

Based on Cs data, individuals 35-1 (Fig. 21), and 57-2 (Fig. 22)appear to have only made two major movements and so likely

Fig. 20. Cesium distribution (in ppm. concentrations) of four osteons of Burial 27-1beginning at the Haversian canal and ending inside the cement line each data series(AeD) reflects one osteon. Individual 27-1 appears to have made three major move-ments in their life. This inference is based on transitions between low and high con-centrations with two peaks, and one trough clearly observed in two osteons, and verysimilar in a third. The initial peak at point 2 shows higher concentrations than point 1,closest to the Haversian canal and most likely to reflect the composition of the burialenvironment.

occupied area with a more even geochemical gradient as opposedto the sharp shifts noted in Re. The results for Burial 35-2 (Fig. 23),Burial 39 (Fig. 24), and Burial 45 (Fig. 25) also suggest three majormovements, though with varying degrees of clarity. Individual 45showed concentrations of cesium close to the Haversian canal thatwere higher than the other individuals examined. It is possible that

Fig. 22. Cesium distribution (in ppm. concentrations) of four osteons of Burial 57-2beginning at the Haversian canal and ending inside the cement line each data series(AeD) reflects one osteon. Burial 57-2 shows a gradual trend of increasing Cs valuesbetween ablation line 4 and ablation line 2. There is a small decrease between ablationlines 5 and 4, but it not clear if such a small change should be inferred as a clearmovement, or if this could be explained by other factors. The sharp decrease betweenablation lines 2 and 1, on the other hand, more clearly indicates that a significantmovement event has occurred.

Fig. 23. Cesium distribution (in ppm. concentrations) of four osteons of Burial 35-2beginning at the Haversian canal and ending inside the cement line each data series(AeD) reflects one osteon. Sampled osteons for this individual begin in an area withhigh Cs, but reduce sharply by ablation line 4. This is followed by a return to higher Csvalues at ablation line 3. Cs values drop for some series by ablation line 2, but all showreduced Cs values by ablation line 1. This indicates a total of three movements.

Fig. 25. Cesium distribution (in ppm. concentrations) of four osteons of Burial 45beginning at the Haversian canal and ending inside the cement line each data series(AeD) reflects one osteon. Similar to Re values, the Cs data are slightly offset, but showa general trend of beginning in an area with low Cs near the cement line. Movementinto a high Cs area occurs around ablation lines 4 and 5. Ablation lines 2-3 show avalley or movement into an area of low Cs values. A final movement occurred atablation line 1, returning to a high Cs area.


this is a diagenetic signal indicating an extremely localized differ-ence in soil chemistry. It is also possible that this individual arrivedin the Little Sea region near their time of death and did not inhabitthe region long enough for new bone to mineralize. Cesium doesnot precisely replicate strontium isotopic values, but rather

Fig. 24. Cesium distribution (in ppm. concentrations) of four osteons of Burial 39beginning at the Haversian canal and ending inside the cement line each data series(AeD) reflects one osteon. Burial 39 is less clear, with Cs data showing divergence atablation lines 2 and 4. Ablation line 4 shows a reduction in Cs values for three of fourdata series, with series C possibly representing a different span of time or diageneticalteration. Values return to slightly higher Cs at ablation line 3, followed by either anincrease or decrease in Cs values at ablation line 2. The combination presents a smallvalley, small peak, and larger valley, or small valley, return to beginning values and alarger valley. This suggests at least three movement events have occurred, thoughpossibly more.

suggests greater degrees of mobility within geological areas withsimilar signatures.

Overall, these results indicate a lifestyle involving frequentmovement by these six individuals between areas with muchdifferent geochemical signatures. Since three of the examined in-dividuals were c. 8e10 years old (Burials A, B and C), one 18e20years old (Bural D), and two 35e50 years old (Burials E and F), it ispretty much safe to say that such travel characterized any stage oflife of these six persons and whoever else traveled with them. Forthe younger individuals the age of osteons cannot be greater thanthe age of the individual, thus analysis of individual osteons rep-resents small spans of time in the last years of life. Patterning inmovements between geochemical regions suggested that 2e3movements were the norm, not for example 1 or 5 movements.This suggests a consistent strategy of movement every other year,though the meanings of these movements could be differentdepending on the age of the individuals and not represent thepatterns of a cohesive group.

Individuals in this study reflect two dietary patterns, three in-dividuals with a game-fish (GF) and three with a game-fish-seal(GFS) diet (Table 1; see Weber et al., 2011). Furthermore, onlytwo of these six individuals (Burial A and B) had been identified asborn locally (Weber and Goriunova, 2012). As a result, consistentpatterning of movement suggests that dietary differences are notsimply the result of location, or resource availability during life.Perhaps individuals in the region were closely knit into a largernetwork that permitted the fluid exchange of people betweensmaller groups in different areas. Since, in a different study(Scharlotta, 2012) we documented a substantial amount of mosaicin distribution of various geochemical traces across the Little Sea,west coast of Baikal, and Upper Lena micro-regions, it is impossibleat this stage of our research to pinpoint more precise locations ofthese areas. In other words, it is too early to relate this informationto the scale of movement which could be anything from relativelyshort distances to quite substantial. Further work on geochemicalmapping of Cis-Baikal is in progress.


6.2. Bone diagenesis

Elemental compositional data also provide insights into thenature and extent of diagenetic alteration within samples that mayappear visually intact and/or identical in preservation as well asengage in discussion as to the role and manifested nature ofdifferent elements within primary bone structure and diageneticmineral counterparts in archaeological bone samples. Experimen-tation regarding the correlation between visible physical changesand chemical alteration suggested that portions of the bone thatwould be identified as altered or deteriorated in histological anal-ysis also showed chemical changes related to the nature and extentof diagenetic factors. Perhaps most importantly from the perspec-tive of this study, the elemental results can provide additional in-formation on provenance and mobility of individuals during thelatter portions of their lives as well as locality and dietary contri-butions in adult individuals during the years immediately prior todeath, both aspects generally extending beyond the accessible re-cord of molar formation. Further work with bone samples of youngadults could help to elucidate problems or uncertainties withresidence time of different elements within the body system as awhole and within specific tissues as there would be overlappingrecords of bone and teeth, accessible in micro-scale.

One additional point of interest with micro-sampling bones isthat with the progression of microbial alterations, based uponobservation of slides prepared for this research and locating intactosteons therein, there appears to be strong evidence that theperiosteum harbors a disproportionate number of intact osteons inspite of the seemingly more direct contact with the burial envi-ronment. Consideration of this matter highlights one importantaspect of bones, that they contain water themselves, and that theirpost-depositional alteration progresses with water contact.Regardless of the form of diagenesis, there are a limited number ofways in which the various effects can alter the composition of thebone matrix.

Microbial attacks are generally discrete intrusions that may ormay not target collagen (Child, 1995; Hedges, 2002; Hedges andMillard, 1995; Jans, 2008; Jans et al., 2004; Landerso and Frost,1964). Organic diagenesis regards the processes of collagen decayand loss, though the ability of certain humic substances to pene-trate bone and attach to collagen is somewhat worrisome. Diffusionreactions and internal remodeling of the bone matrix controlinorganic diagenesis. Newer lamellae are present in all livingosteons, and will likely not be fully mineralized, still highly solubleand closest to the cavities that were Haversian canals until theorganisms’ death. With specific note that the internal bone water isgenerally sufficient to maintain a closed system that requires thediffusion of ions either in or out; the remaining liquid-phase min-erals on free-floating collagen fibrils will dissolve the partiallymineralized structures of the young lamellae to create the satu-rated solution and the base material for any recrystallization(Hedges, 2002).

The patterning or layout of each individual osteons is unique,the result of cellular life history, as will be the progression ofdiagenetic factors. Caniculi connect all living osteocytes, howeverthe diffusion capabilities, or liquid penetration, through thesecaniculi will be limited (Tuross et al., 1988). Caniculi generally leadto secluded and fully mineralized bioapatite portions of olderlamellae. Saturated or partially saturated solutions will have diffi-culty in dissolving a largely insoluble mineral crystal through suchlimited pathways (Katzenberg, 2008). As such, the liquid penetra-tion of diagenetic solutions will be limited within the complexmicrostructure of bones and there will be preferential dissolutionof decaying mineral structures. Thus, even if in diffusion equilib-rium with the external environment, it will be difficult for

diagenetic ions to reach the mature mineral structures until mi-crobial attack can break down the matrix and eliminate thecollagen fibrils holding everything together (Hedges, 2002;Katzenberg, 2008; Katzenberg and Harrison, 1997; Nielsen-Marshand Hedges, 2000a).

Furthermore, no materials cross the cement lines of osteonseven when the systems are alive, thus penetration of the cementline will be difficult even if the protein structure does not grantany buffering effects (Kerley, 1965). Osteocytes have a limitedlifespan (w25 years), and their mineralized structures are replacedas new osteons cut through the existing bone (Frost, 1963;Maximov and Bloom, 1957). As a result, the interstitial lamellae(bone matrix between the cement lines of living osteons) ismineralized material from dead osteons, thus any remaining ma-trix is not connected to any active caniculi and will be very hardpressed to be in dissolution contact with bone fluids post-mortem.Given the nature of the post-mortem bone liquid solution, it isunlikely that the ideal balance of ionic raw materials will be pre-sent to enable the precipitation of largely insoluble bioapatitemineral crystals rather than the precipitation of more solublemineral forms, especially in the absence of guidance from nucle-ation or any of the protein/substance signals that control boneformation and mineralization during life (Hedges, 2002; Millard,2001; Sillen, 1989).

Finally, proportions of available poorly mineralized bioapatitecrystals for dissolution (i.e., hypercalfied bright lines and laminarbone) will be different for specific bone structures depending onhow they are in contact with the bone liquid and depend on the ageand species of the organism (Evershed et al., 1995; Glimcher, 1976).Thus there are number of reasons to believe that there will besignificant variability and/or inequities within the bone matrix as itrelates to the impact and effects of diagenesis, regardless of theform(s) in operation over any given period of time.

6.3. Laser ablation and strontium isotope analysis

There is an ongoing debate in archaeology and geochemistryregarding problems with isotopic analysis of strontium in calciumphosphate matrices using laser ablation as an introduction method,for some aspect of the laser ablation sample introduction leads tothe formation of a polyatomic species that interferes with mass 87and thus interpretation of strontium isotope ratios (Prohaska et al.,2002, Scharlotta, 2010, 2012, Scharlotta et al., 2011, Simonetti et al.,2008, Woodhead et al., 2005).

Strangely enough, even though there is great similarity in themineral matrices that form teeth and long bones, there is no evi-dence for significant alteration of isotopic signals from bones.Looking at the elemental data, there appear to be similar forma-tions of the related polyatomic species at mass 91, though withfairly high error terms and low concentrations all around, yet thereis no clear interference as a result. This can be seen in Fig. 25; foreach individual, the average 87Sr/86Sr value for the nine unalteredosteons falls close to that obtained for the same individual usingsolution-mode analysis. The values for the diagenetically alteredosteons are, in contrast, clearly aberrant and cover a wide range ofvalues (Figs. 26 and 27, Supplementary Material II).

As these samples were recovered from a high-87Sr/86Sr region, itis entirely possible that diagenetic contaminants were not fullyremoved from samples during solution preparation and purifica-tion, thus leaving an elevated result that couldmask the presence ofinterference resulting from laser ablation. This seems unlikely inthis particular case as several of the samples’ diagenetic portionsyielded lower Sr isotope ratios, and only one sample (Burial 35-1)was suspected of having partial alteration on even intact osteons,and thus was likely to be almost completely compromised.

Fig. 26. Solution mode and laser ablation 87Sr/86Sr ratios compared withdiagenetically-impacted portions of bone. Fig. 28. Solution mode and laser ablation 87Sr/86Sr ratios compared with

diagenetically-impacted portions of bone.


There are a few possible explanations for this, and likely othersnot considered here (see e.g., Radloff, et al., 2010). First, the problemmay bewith the solution data masking the presence of interferencein the laser data. Second, perhaps there is enough difference in thestructure of the mineral matrices that the laser interaction does notactually create the polyatomic interference noted in analyzing teethand the elemental data are simply the result of large error factorson low concentrations. Third, what is being seen is actually a largecoincidence, with a homogenized complex life history coming upwith similar numbers as an altered laser averaging.

There are a few solution preparation procedures intended toremove diagenetic overprinting by eliminating the most solubleportions of the bone structure, namely those that weremobilized inthe burial environment rather than life. However, there remains adegree of uncertainty as to what is being removed, what the truetarget data are (i.e., remaining solids after n leaches, or the nthleachate) and whether an efficient leaching has been achieved. Thisdifficulty is further confounded by the complexity of bone structureand formation processes. Any long bone is a combination of ma-terial from potentially well over 30 years of an individual’s life. The

Fig. 27. Solution mode and laser ablation 87Sr/86Sr ratios compared withdiagenetically-impacted portions of bone.

lifespan of any given Haversian system is generally no more than20e25 years; however, remnant portions of old Haversian systems,not fully removed by the cutting actions of visible osteons,frequently remain visible in histological slides. The true age of theseremnants is often uncertain and they could thus contribute intact,diagenetically resistant bone material to the analytical resultwithout much regard as to what its contribution is truly telling usabout the individual as we do not have a good fix on the chronologyof such portions. This matter is the focus of continued investigationto assess the intriguing source of this confusion.

7. Conclusion

Numerous laboratory procedures and a series of checks havebeen implemented to assess the quality of a sample and to coun-teract or remove the effects of biodeterioration on bone sections orpowders (e.g., Garvie-Lok et al., 2004; Koch et al., 1997; Nielsen-Marsh and Hedges, 2000b). Such methods have proven effectiveand necessary, given the prevalence of biodeterioration, in access-ing bio- and geochemical data obtained from archaeological bone.The downside of these techniques is that they all rely on homog-enized sample tissue which makes it impossible to identifyimportant movement or dietary events within individual lifehistories.

While microscopic observations of biodeterioration are not new(e.g., Bell, 1990; Jackes et al., 2001; Nielsen-Marsh and Hedges,2000b), our data show that many chemical alterations reported inthe literature can be avoided and/or monitored in tandem withchemical analyses of bones. Micro-sampling (i.e., laser ablation,micro-drilling) provides an avenue towards obtaining anthropo-genic chemical data and, most importantly, individual life historyevents. Areas of biodeterioration can be avoided or corrections forspecific alterations observed can be applied with greater finessethan bulk sampling approaches and potentially enable further useof archaeological materials that hold detailed data on diet, mobility,and health at different intervals of the entire individual life history.

In the Cis-Baikal region, micro-sampling of individual osteonsstrongly supports the hypothesis that these hunter-gatherers madenumerous major movements during their lives. Without moredetailed geochemical mapping of the region it is difficult to narrowdown their exact locations during life, however, it certainly doesappear that many individuals weremoving into and out of the LittleSea region of Lake Baikal. Furthermore, individuals were making


major movements either shortly before their death, or weretransported to the Little Sea post-mortem, both suggesting a strongcultural importance of either the area, or the Khuzhir-Nuge XIVcemetery in particular.

In terms of general application, the tandem use of strontiumisotope and elemental compositional analysis using LA-ICP-MSseems very feasible and effective. At present, the isotopic signa-tures of whole osteons can be observed and micro-sampling can beconducted using elemental data for further enhancement of prov-enance determination. Perhaps with future improvements inequipment, a more direct approach may become possible, but withthe equipment available today, major progress in the amount andnature of information available from bone samples is alreadyfeasible.

Acknowledgments

The authors would like to thank Robert Creaser, S. AndyDuFrane, Guangcheng Chen, and Krystle Moore (Radiogenic IsotopeFacility, Department of Earth and Atmospheric Sciences, Universityof Alberta) for use of laboratory facilities during sample prepara-tions and assistance with analyses conducted at the University ofAlberta. This research is part of the Baikal Archaeology Projectbased at the University of Alberta and funded by the MajorCollaborative Research Initiatives grant No. 412-2005-1014 fromthe Social Sciences and Humanities Research Council of Canada.Special thanks go to Dr. Sandra Garvie-Lok for her constructivecomments and perceptive suggestions.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jas.2013.07.014.

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Micro-sampling of human bones for mobility studies: diagenetic impacts and potentials for elemental...

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