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Evaluation of elemental status of ancient human bone samples from Northeastern Hungary dated to the 10th century AD by XRF
Nuclear Instruments and Methods in Physics Research B 269 (2011) 2593–2599
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Evaluation of elemental status of ancient human bone samples from Northeastern Hungary dated to the 10th century AD by XRF I. János a,⇑, L. Szathmáry b, E. Nádas a, A. Béni c, Z. Dinya a, E. Máthé a a
Agricultural and Molecular Research Institute, College of Nyíregyháza, H-4400 Nyíregyháza, Hungary Department of Evolutionary Zoology and Human Biology, University of Debrecen, Debrecen H-4010, Hungary c Institute of Environmental Science, College of Nyíregyháza, H-4400 Nyíregyháza, Hungary b
a r t i c l e
i n f o
Article history: Received 17 February 2011 Received in revised form 11 July 2011 Available online 23 July 2011 Keywords: Elemental XRF analysis Human bone Ancient life Diagenesis Tenth century AD
a b s t r a c t The present study is a multielemental analysis of bone samples belonging to skeletal individuals originating from two contemporaneous (10th century AD) cemeteries (Tiszavasvári Nagy–Gyepáros and Nagycserkesz–Nádasibokor sites) in Northeastern Hungary, using the XRF analytical technique. Emitted X-rays were detected in order to determine the elemental composition of bones and to appreciate the possible influence of the burial environment on the elemental content of the human skeletal remains. Lumbar vertebral bodies were used for analysis. Applying the ED(P)XRF technique concentration of the following elements were determined: P, Ca, K, Na, Mg, Al, Cl, Mn, Fe, Zn, Br and Sr. The results indicated post mortem mineral exchange between the burial environment (soil) and bones (e.g. the enhanced levels of Fe and Mn) and referred to diagenetic alteration processes during burials. However, other elements such as Zn, Sr and Br seemed to be accumulated during the past life. On the basis of statistical analysis, clear separation could not be observed between the two excavation sites in their bone elemental concentrations which denoted similar diagenetic influences, environmental conditions. The enhanced levels of Sr might be connected with the past dietary habits, especially consumption of plant food. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Bone is one of the most complex structures in the human body, which consists of organic and inorganic components. It is not only important to support and move the body but also constitutes a substantial reservoir of the mineral materials in the human organism. To obtain information about the ancient life (paleodiet, health status or even living condition) of human population after death one can analyse the elemental status preserved in bones. For decades it has been a widely accepted technique to get information about the past life [1–7], but nowadays paleodiet studies have shifted from simple measurements of elemental concentrations to analyses of stable isotopes [8,9]. The applicability of bone strontium and zinc are firmly established as dietary indicators. It is well known that the Sr is taken in by the plants directly from the environment (soil). Therefore, the mammals (including humans) obtain this element secondarily through plants and other animals. It follows that the Sr is reducing in the trophic levels from the bottom to the top. Hence, plant tissue contains higher concentration of this element than the animal tissue and within mammalians, carnivores represent the least
⇑ Corresponding author. Tel.: +36 42 599400/2503; fax: +36 42 402485. E-mail address: [email protected] (I. János). 0168-583X/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2011.07.016
amount of Sr, herbivores show the highest level, whilst omnivores should demonstrate intermediate concentration of this element. It is also known that, there is a positive correlation between the Zn accumulated in bones and the protein diet connected with the consumption of animal foods. Thus in mammals the concentration of bone Sr is increasing through the consumption of plant foods and decreasing by animal protein intake while the bone Zn represents reverse fluctuation in concentration with the Sr according to the main diet components. The typical ranges of Zn and Sr in mammalian bones by different mode of nutrition are listed as a followings: (a) herbivores – Sr: 400–500 lg/g; Zn: 90–150 lg/g; (b) omnivores – Sr: 150–400 lg/g; Zn: 120–200 lg/g; (c) carnivores – Sr: 100– 300 lg/g; Zn: 175–250 lg/g. These elemental concentration levels can be typical for human bones in cases of herbivorous, omnivorous or even carnivorous nutritional habits [9–12]. The elemental status of buried bones are not only affected by the biological factors of ancient life (age at death, diseases, prevalent nutrition and metabolism), but the environmental circumstances such as soil composition, precipitation, pH and human land use, which can appear after burial. Such processes, termed diagenetic collectively, may cause changes in the original structure of osseous material [6,13]. The different types of bones respond to the remodelling processes in different way: spongy bone is more susceptible to diagenesis than compact bone from humerus and femur [14].
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In the present work two sets of human bone samples dating from the 10th century AD (the age of the Hungarian conquest) were analysed by using the high-performance energy-dispersive polarisation X-ray fluorescence (EDPXRF) spectrometry which permits fast multielement detection and the possibility to make precise screening analysis. The basic aims of this study were to obtain information about the elemental status and the inter-elemental relationships of these ancient bone samples of individuals as well as to demonstrate differences (or even correspondence) in bone elemental status between the two sites. Additionally, the study discusses the most appropriate reasons behind the results. 2. Materials and methods 2.1. Instruments and reagents For the analysis analytical grade reagents were used: methanol and Hoechest wax C micro powder for the sample preparation and deionised water for the cleaning procedure of the sample. An electric mixer mill (MM200 with a ZrO2 grinding vessel, Spectro Analytical Instrument, Kleve, Germany), manual press and a spectrometer model of SPECTRO XEPOS XEP01 (Spectro Analytical instruments, Kleve, Germany) were used for analysis. In the ED(P)XRF spectrometer the samples were irradiated by X-rays using Cartesian geometry (for polarisation of excited X-rays were used to reduce the spectral background) that means the X-rays exiting the tube are reflected or scattered by the target (Mo secondary target was used at voltage of 35 kV) at an angle of 90° with respect to the sample. Bottled helium was used to improve the sensitivity for light elements. The radiation typical of the elements present in the sample was detected by a PIN-diode semi-conductor detector. The measurement processes were controlled and stored by a PC computer using XLabPro software. The calibrations were made by using SPECTRO’S TURBOQUANT automatic calibration system. TURBOQUANT is able to analyse all elements between Na and U in real unknown samples. This means that all matrix effects, which will occur, are taken into account [15,16]. For the analysis of mobile Mg of the soil samples, atomic absorption spectrometry (FAAS) was applied by using the Varian SpectrAA-20 model. The pH of the soil samples were determined by an OP-211/1 type pH-meter. 2.2. Sites, samples and experimental The human skeletal material examined for this work was excavated in the Great Hungarian Plain, Northeastern Hungary, where two cemeteries (namely Tiszavasvári Nagy-Gyepáros and Nagycserkesz-Nádasibokor, both dated to the 10th century AD) were investigated (Fig. 1). The two cemeteries were located close to each other (the distance is only 13 km in between) and named after the neighbouring towns (Tiszavasvári and Nagycserkesz). Archaeological and historical evidence equally suggested that the graves belonged to early pagan Hungarian conquerors, who comprised various Finno-Ugric and Turkic ethnic groups with a seminomadic way of life. Excavations in the burial site of Tiszavasvári Nagy-Gyepáros were conducted in 1966, where 18 graves became known. The human skeletal remains belonging to 29 individuals of Nagycserkesz-Nádasibokor site were excavated in 1970 [17,18]. The samples were composed of lumbar vertebral bodies belonging to 35 skeletons (males, females and juveniles), since this type of bone was available from the highest number of graves (there were some fragmentary skeletons, especially in the case of juveniles). The ratio of the individuals analysed in the two excavation sites is 13:22 as five individuals from Tiszavasvári Nagy-Gyepáros and seven individuals from Nagycserkesz-Nádasibokor could not
be examined. Soil samples derived from the two excavation sites were also analysed in order to identify the possible compositional differences and elemental interactions between the bones and soil. The excavation area have temperate continental (moderately warm) climate with low humidity levels in both sites. The climate is also characterised by frequent rainshowers in spring, early summer and autumn as well as mildly cold, snowy winter. So the precipitation distribution is not even within a year. The majority of summer and winter is dry. January is the coldest, while the hottest month is July, as those in Hungary. Currently, there is no flooding on the sites. The further climate data relative to the two sites specifically on the basis of data on many-years observations are listed as a followings: (a) Tiszavasvári – average annual temperature: 9.8 °C, yearly absolute maximum temperature: 34 °C, yearly absolute minimum temperature: – 17 °C, average yearly rainfall: 550 mm, aridity index: 1.28, sunny hours: 1950, frostless period: from 12 April to 16 October. (b) Nagycserkesz – average annual temperature: 9.6 °C, yearly absolute maximum temperature: 34 °C, yearly absolute minimum temperature: – 17 °C, average yearly rainfall: 560 mm, aridity index: 1.21, sunny hours: 1950, frostless period: from 14 April to 16 October. The groundwater level generally is within the range of 2–4 m in both sites [19]. The osteological material was analysed in the Agricultural and Molecular Research Institute, College of Nyíregyháza. First, in order to remove macroscopic soil contamination, the bone samples were mechanically cleaned by flushing with tap – and washing with deionised water and dried in a clean environment at room temperature. After that, the bone samples were milled by using an electric mixer mill. From the powder obtained (<100 lm) several pellets (for three independent measurements) of each sample were prepared to reduce the errors of measuring. The experiment was carried out in the following way, according to the instrument documentation: 4 g of bone powder was homogenised with 0.9 g of Hoechst Wax and then pressed into a 32 mm pellet with 15 tons. The pressed pellet was put in a sample cup covered by Mylar film and analysed directly by applying high-performance energy-dispersive polarisation X-ray fluorescence (EDPXRF) spectrometry. The Mylar film and the Hoechest Wax do not contain measurable elements and the software of the instrument calculates the eventual concentration values taking the mass and the composition of these materials into consideration [15,16]. Homogenous sandy soils constituted the burial environments in both sites in the depth of the graves (60–120 cm). The soil samples derived directly from the burial environment of the skeletal remains at same depth. Several samples were collected from both sites (the means and the standard deviations are presented on the Table 1). The elemental compositions of soil samples were analysed in a way similar to that used for the bones by ED(P)XRF and here the pH values were also determined as a followings: 5 g of soil sample was mixed with 50 ml distilled water for 30 min and the pH was subsequently measured by the digital pH-meter. Only for the mobile magnesium of the soil samples FAAS analysis was performed on the following way: 5 g of soil samples was mixed with 25 ml distilled water for 30 min and the compound was centrifuged at 5000 rpm for 5 min. After that the supernatant was filtered on analytical filter paper and was measured by FAAS. The FAAS analysis was performed by using the following parameters: air-acetylene flame conditions: air: 4.6 L/min, acetylene gas: 1.7 L/min, sample introduction: manual by pneumatic nebuliser, observation height: 10 mm, lamp current: 4 mA, slit width: 0.5 nm, wavelength: 285.2 nm. 2.3. Statistical analysis Pearson correlations [20] were done on the entire data sets of all elements in the case of both sites to consider the inter-elemental
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Fig. 1. Location of the excavation sites described in the study. A = Tiszavasvári Nagy-Gyepáros site, B = Nagycserkesz-Nádasibokor site.
Table 1 Mean values and standard deviations for the elemental concentrations of the two examined bone series obtained by XRF and compared to published data of archaeological () and modern () bones as well as soil samples derived from the two sites. Bone P and Ca are given in mg/g all others in lg/g. Site/sample
Type of bone
Na
Mg
Al
P
Cl
K
Ca
Ca/P
TVN NAN Ref. [2]⁄ Ref. [3]⁄ Ref. [24]⁄ Ref. [14]⁄ Ref. [14]⁄⁄ Ref. [25]⁄⁄ Ref. [26]⁄⁄ Ref. [27]⁄⁄ TVN (soil) NAN (soil)
Vertebra Vertebra Not specified Tibia Vertebra Vertebra Vertebra Vertebra Ilium Not specified – –
1508 ± 315 1590 ± 337 – – 7716 ± 3190 2200 ± 100 6000 ± 300 – 4900 ± 900 –
385 ± 85 410 ± 90.2 – 5514 ± 1246.6 6704 ± 2672 64800 2200 ± 200 1560 ± 460 – – 47.055 ± 5147 6646 ± 470
135 ± 25.8 162 ± 88.6 – – – 410 ± 30 – – 19.5 ± 6.1 – 36.213 ± 1140 24.633 ± 8085
104 ± 14 110 ± 11.8 – – 158 ± 23.5 144 ± 0.8 97 ± 0.5 77.3 ± 14.3 98 ± 0.6 – 109 ± 132.7 19.6-21.4
150 ± 141 200 ± 126 – – – 6210 1300 ± 300 – – – 128 ± 118 103 ± 147
326 ± 239 592 ± 579 – – – – –
244 ± 25.7 242 ± 28.6 – 217.19 ± 34.3 347 ± 33.2 322 ± 15 254 ± 14 153 ± 25.9 213 ± 1.1 – 57.029 ± 8793 10.395 ± 2835
2.36 ± 0.13 2.20 ± 0.09 – – 2,23 ± 0.24 2.2 2.19 1.98 2.17 – – –
Site/sample
Type of bone
Mn
Fe
Zn
Br
Sr
TVN NAN Ref. [2]⁄ Ref. [3]⁄ Ref. [24]⁄ Ref. [14]⁄ Ref. [14]⁄⁄ Ref. [25]⁄⁄ Ref. [26]⁄⁄ Ref. [27]⁄⁄ TVN (soil) NAN (soil)
Vertebra Vertebra Not specified Tibia Vertebra Vertebra Vertebra Vertebra Ilium Not specified – –
115 ± 96.6 78 ± 68.2 55 ± 35.3 51.46 ± 43.41 1085 ± 1246 110 ± 10 <1.5 – – – 413.7 ± 37.8 407.7 ± 195.3
1506 ± 788 1957 ± 1628 269 ± 167.5 – 6822 ± 5868 5000 ± 1000 – 162 ± 60 183 ± 78 – 30523.3 ± 3040.8 26.150 ± 8738.4
89.2 ± 21.6 79.6.±17.3 90 ± 63.3 125.14 ± 34 250 ± 175.5 220 ± 10 – 120.1 ± 55.3 151 ± 22 117.5 ± 52 41.1 ± 0.6 28.9 ± 12
38.6 ± 18 33.2 ± 10.9 68.5 ± 9.2 – – 17.9 ± 1.1 110 ± 55 – – 8.9 ± 8.8 – –
488 ± 206 391 ± 143 152.5 ± 38.9 1515.6 ± 506.6 687 ± 403 6240 170 ± 30 – 51 ± 39.5 208 ± 83.4 325.8 ± 105.7 117.1 ± 5.4
– – 17.157 ± 481.9 12.550 ± 2972.6
TVN: Tiszavasvári Nagy-Gyepáros site; NAN: Nagycserkesz-Nádasibokor site.
relationships. In order to discover the relationships between the elemental status of the two groups of bone samples derived from the two contemporary sites and to integrate the information presented by the elemental contents of the individual bone samples,
principal component analysis (without Kaiser normalisation) was performed on the entire data set [21]. On the factor values of the first four principal components (PC) derived from the principal component analysis, Mann–Whitney (M–W) and Kolmogorov–
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Smirnov tests (K–S) [22] were carried out for comparison of the two sites. All the statistical analysis was done by using SPSS and Past software. All the tests were performed three times by using both softwares, in order to reduce the random error of statistical analysis.
Table 3 Eigenvalues, variance and cumulative for the four principal components.
3. Results and discussion During the examination, the following elements were determined by XRF analysis: P, Ca, K, Na, Mg, Al, Cl, Mn, Fe, Zn, Br and Sr. Descriptive statistics (means and standard deviation) of results together with published data (for archaeological and fresh bone samples) are presented in the Table 1. The bone samples of both sites are characterised by decreasing of certain elements such as Na, Mg and Cl as well as by increasing of other elements (Al, Mn and Fe) relative to values known from literature (Table 1). The Ca/P ratio is 2.15 with an SD value 0.17 in native hydroxiapatite [19]. Samples exhibiting a ratio with 2.36 ± 0.13 in Tiszavasvári Nagy-Gyepáros (TVN) site and 2.20 ± 0.09 in Nagycserkesz-Nádasibokor (NAN) site are presumed to be favourable theoretically to draw paleodietary conclusions [23,24]. Means of Br concentrations show sufficient approximations to data of modern bones therefore these are considered to accumulate during the past life while the Zn represents slightly lower levels as compared with modern reference data. The correlation matrix of the entire data set is presented in the Table 2 for the two sites (populations). Several significant correlations are similar to those known from the literature in both sites [4–8]: strong positive correlations are found in the cases of Ca– P (0.902 in TVN and 0.928 in NAN) which are the main constituents of bone mineral, and Mn–Fe (0.695 in TVN and 0.438 in NAN). Between Ca and Fe ( 0.869 in TVN and 0.53 in NAN) as well as Ca and Mn ( 0.521 in TVN and 0.261 in NAN) can be seen negative correlations but Ca–Mn correlations are not significant (Table 2). These negative correlations (Ca–Fe and Ca–Mn) can be explained as the consequence of diagenetic processes, since the Mn and Fe ions were incorporated in the bone apatite structure by ion ex-
Principal component
Eigenvalue
% of variance
Cumulative%
1 2 3 4
3.935 2.098 1.629 1.381
32.790 17.485 13.582 11.508
32.790 50.275 63.858 75.367
Table 4 Matrix of variable loadings. Highest loading absolute values are signed in bold face. Elements
PC1
Mg Na Mn Al K Fe Ca P Sr Zn Br Cl
PC2
0.801 0.794 0.786 0.763 0.709 0.644 0.5 0.18 0.466 0.153 0.119 0.096
PC3
0.088 0.411 0.415 0.585 0.07 0.014 0.272 0.886 0.574 0.043 0.456 0.067
PC4
0.34 0.065 0.06 0.042 0.277 0.076 0.461 0.112 0.379 0.798 0.49 0.421
0.35 0.284 0.278 0.045 0.514 0.189 0.163 0.109 0.359 0.06 0.184 0.77
changes with the Ca ions during the post-mortem period in the soil [4–6]. It may probably be confirmed by the elevated levels of Fe and Mn in soil samples (Table 1) as compared with those in vertebrae. The heightened levels of Al in the bone samples may possibly be the result of surface adsorption on the bone mineral because gross concentrations of this element were found in the soils of both sites. Elevated levels of Mg were also found in the soil samples (Table 1), but the mean levels of mobile Mg (determined by FAAS) in the aquatic phase of soil extracts (TVN – 33.6 ± 3.85 lg/g; NAN – 12.43 ± 3.19 lg/g were significantly lower than those in the bones (TVN – K–S: D = 1, p = 0.004; NAN – K–S: D = 1, p = 0.002). The pH
Table 2 The correlation coefficient matrix (according to Pearson) of the elements based on bone samples derived from the Tiszavasvári-Nagy Gyepáros (a) and Nagycserkesz-Nádasibokor (b) sites. Significant values which match in both sites are signed in bold face. Na
a b
Mg
Al
P
Cl
K
Ca
Mn
Fe
Zn
Br
(a) Na Mg Al P Cl K Ca Mn Fe Zn Br Sr
0.995b 0.968b 0.748b 0.16 0.647a 0.464 0.567a 0.705b 0.028 0.301 0.028
0.967b 0.758b 0.199 0.662a 0.478 0.538 0.695b 0.022 0.352 0.034
0.729b 0.189 0.55 0.429 0.507 0.616a 0.112 0.358 0.055
0.007 0.793b 0.902b 0.596a 0.886b 0.168 0.383 0.039
0.065 0.036 0.067 0.132 0.053 0.229 0.096
0.772b 0.321 0.885b 0.188 0.213 0.107
0.521 0.869b 0 0.196 0.012
0.695b 0.039 0.116 0.521
0.133 0.142 0.102
0.67a 0.421
0.467
(b) Na Mg Al P Cl K Ca Mn Fe Zn Br Sr
0.987b 0.085 0.334 0.276 0.205 0.29 0.343 0.376 0.023 0.471a 0.14
0.044 0.356 0.312 0.21 0.304 0.305 0.40 0.064 0.472a 0.103
0.201 0.308 0.617b 0.174 0.298 0.747b 0.018 0.105 0.085
0.073 0.008 0.928b 0.405 0.527a 0.161 0.165 0.06
0.032 0.021 0.018 0.211 0.109 0.382 0.385
0.049 0.335 0.647b 0.011 0.248 0.338
0.261 0.53b 0.412 0.212 0.088
0.438a 0.344 0.311 0.154
0.121 0.318 0.098
0.042 0.051
0.232
Correlation is significant at the 0.05 level (2-tailed). Correlation is significant at the 0.01 level (2-tailed).
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Fig. 2. Box plots of principal component values (PC1, PC2, PC3 and PC4) of the two past populations derived from the principal component analysis. Tiszavasvári NagyGyepáros (TVN) site and Nagycserkesz-Nádasibokor (NAN) site are compared with Kolmogorov–Smirnov (K–S) and Mann–Whitney (M–W) tests based on the first four principal components. (a) PC1 – K–S: D = 0.297, p = 0.397, M–W: U = 106, p = 0.213; (b) PC2 – K–S: D = 0.279, p = 0.474, M–W: U = 105, p = 0.2; (c) PC3 – K–S: D = 0.164, p = 0.966, M–W: U = 128, p = 0.620; (d) PC4 –K–S: D = 0.402, p = 0.105, M–W: U = 104, p = 0.188.
Fig. 3. 2D scatter plots of the individual values of the four principal components (PC1, PC2, PC3, and PC4).
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values of the soil samples coming from the burial environment were alkaline in both sites: 9.12–9.53 in TVN and 8.14–8.9 in NAN which are typical of the calcareous sandy soils. Comparing with data of modern bones, the Sr represents elevated levels which may be in accordance with past dietary habits (presumably consumption of plant foods). Knowing that Sr is also able to substitute Ca in the hydroxiapatite lattice, in case of Sr contamination strong negative correlation can be expected. Here, however, the former hypothesis cannot be supported since the Ca–Sr correlation coefficients are close to zero in both sites and the means of Sr concentrations of soil samples fall short of the mean values of bones (Tables 1 and 2). Although the soil Sr content was significantly higher in TVN than that in NAN (K–S: D = 1, p = 0.033), this element does not show significant differences in bones between the two sites (K–S: D = 0.283, p = 0.458). The correlation coefficient matrix represents quite good accordance between samples of the two populations regarding pairs of certain elements but do not in case of other elements. Matching values of significant correlation coefficients are marked by bold face in Table 2. Despite the fact that there are more significant correlation pairs in TVN than those in NAN, the main tendencies are similar in both cases. The minor dissimilarities might have been derived from population differences or from the random effects of the different sample size. In order to compare the two groups of samples (cemetery) on the basis of their elemental status, principal component analysis (without Kaiser normalisation) were carried out on the entire data set. Four principal components (with an eigenvalue higher than 1) could be extracted (Table 3). These principal components explain 75.4% of total variance. The results represented that P, Mg, Na, Fe, Ca, Mn and Br have similar loading on the first principal component (PC1). The second principal component (PC2) mainly correlates with Al and K as well as Mg, Na, Fe and Cl to a lesser degree. Sr and Cl have higher (and Zn smaller) loadings on the third principal component (PC3) and only one element, the Zn, can be classified to the fourth principal component (PC4) (Table 4). By fitting Kolmogorv–Smirnov and Mann–Whitney tests for PC1, PC2, PC3 and PC4, we found that there are no significant differences between the elemental statuses of bones belonging to the two contemporary populations (Fig. 2). This suggests that similar burial environment conditions (Table 1) have existed and similar changes have taken place during the post mortem period over time. However, looking at the scatter plots of the principal components, it is conspicuous that the factor values of NAN have higher heterogeneity than those of TVN which may derive from a minor population differences (Fig. 3). The results suggest that the elemental status of buried bones might be typical of a given ancient living ethnic group of humans and a certain archaeological period. Between the two populations, the K, Fe and Sr show minor (not significant) differences.
Secondly, the correlation coefficient matrix provided important information about the inter-elemental relationships: negative correlations were found between Fe–Ca and Mn–Ca in both populations that could probably be accounted for by the above mentioned ion exchange processes since the Mn and Fe might have incorporated in the bone apatite structure replacing Ca ions during the post-mortem period. The strong positive correlation between Mn and Fe in the bone samples can be explained by the similar ion radius of the Mn(II) as well as Fe(II) and Fe(III) ions which resulted in parallel mobility of these elements towards the bone tissue in accordance with their concentrations. The elevated levels of Al in the osseous material could be related to the attributes of Al ions which have gross affinity to surface adsorption on bone tissue in the soil. These observations can serve as evidence for diagenetic processes. Thirdly, the mean Zn concentrations denoted slightly lower levels than those of the reference data (Table 1) but fell within the typical range of diets high in foods of vegetable origin and low in meats. This element likely accumulated during the past life. Contrarily, the Sr indicated elevated levels in both sites which might probably also be connected with heightened consumption of plant foods (see the values listed in the Introduction chapter). The modern nutrition is usually more diverse and in most cases omnivorous, therefore the Zn and Sr concentrations in bone samples derived from healthy humans represent mainly moderate, intermediate levels. Depending on the main diet component the levels of these two elements can shift into opposites directions of each other. Lastly, comparing the two populations, it could be said that population of Nagycserkesz-Nádasibokor denoted higher heterogeneity in elemental values but there were no significant differences between the two populations regarding their bone elemental contents on the basis of statistical tests. In our opinion, a given archaeological period can presumably be characterised by typical levels of elements detected in human bones in similar environmental circumstances. On the basis of the elemental concentrations of bone samples, population (ethnic) differences will probably be detected between these series and samples derived from other contemporary cemeteries in the 10th century. Further examinations will be focused on analysing other type of bones within these series by different analytical methods and will be aimed at drawing conclusions regarding biological attributes such as age, sex and diet of these populations. Acknowledgements The authors thank all the colleagues who have helped the completion of this work. The anonymous reviewer also deserves thanks for his/her helpful comments and suggestions on an earlier version of the manuscript.
4. Conclusions References Taking it all round, by elemental XRF analysis of 10th century human bone samples from two excavation sites (Northeastern Hungary), at this stage of research, the following conclusions can be drawn: Firstly, seeing the elemental values as compared with those known from the literature, the data showed Na, Mg and Cl leaching which were probably due to the prevalent environmental circumstances over time. Despite the high concentrations of Mg in the soil samples (Table 1) the leaching seems to be feasible due to the significant rainfalls over time. Additionally, the mobile Mg, which was available for diffuse moving towards the bones in the aquatic soil extracts, showed significantly lower levels than the Mg in the solid soil and bone samples. The great part of soil Mg is appears to be bound in the soils in both sites.
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I. János et al. / Nuclear Instruments and Methods in Physics Research B 269 (2011) 2593–2599 [10] K. Szostek, H. Glab, A. Szczepanek, K. Kaczanowski, Homo 53 (2003) 235. [11] V. Smrcka, Trace Elements in Bone Tissue, The Karolinum Press, Charles University, Prague, 2005. [12] A.L. Rheingold, S. Hues, M.N. Cohen, J. Chem. Educ. 60 (1983) 233. [13] R.E.M. Hedges, Archaeometry 44 (2002) 319. [14] R.G.V. Hancock, M.D. Grynpas, K.P.H. Pritzker, Archaeometry 31 (1989) 169. [15] R. Schramm, Why using XRF for analysis? Instrument documentation, Spectro Analytical Instruments, Kleve, Germany, 2000. [16] R. Schramm, J. Heckel, J. Phys. IV France 8 (1998) 335. [17] P. Németh, Régészeti Füzetek 24 (1971) 59 (in Hungarian). [18] P. Németh, Tiszavasvári Nagyepáros cemetery from the age of Hungarian conquest, in: M. Wolf, L. Révész (Eds.), Archaelogical records from the age of Hungarian conquest, Hungary, Miskolc, 1996, pp. 7–21 (in Hungarian). [19] S. Marosi, S. Somogyi (Eds.), Inventory of microregions in Hungary, MTA FKI, Budapest, 1990.
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[20] P.J. Hubert, Robust Statistics, Wiley, New York, 2004. [21] I.T. Jolliffe, Principal Component Analysis, second ed., Springer, New York, 2002. [22] G.W. Corder, D.I. Foreman, Nonparametric Statistics for Non-Statisticians: A Step-by-Step Approach, Wiley, New York, 2009. [23] A. Fabig, B. Herrmann, Naturwissenschaften 89 (2002) 115. [24] L. Márk, Importance and application of bone chemical analyses in the physical and forensic anthropology, PhD Thesis, Universitiy of Pécs, Pécs, Hungary, 2006 (in Hungarian). [25] Y. Tohno, S. Tohno, M. Utsumi, T. Minami, Y. Okazaki, F. Nishwaki, Y. Moriwake, T. Naganuma, M. Yamada, T. Araki, Biol. Trace Elem. Res. 59 (1997) 167. [26] D. Gawlik, D. Behne, P. Bratter, W. Gatschke, H. Gessner, J. Clin. Chem. Clin. Biochem. 20 (1982) 499. [27] F.A. EI-Amri, M.A.R. EI-Kabroun, J. Radioanal. Nucl. Chem. 217 (1997) 205.
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Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
Evaluation of elemental status of ancient human bone samples from Northeastern Hungary dated to the 10th century AD by XRF I. János a,⇑, L. Szathmáry b, E. Nádas a, A. Béni c, Z. Dinya a, E. Máthé a a
Agricultural and Molecular Research Institute, College of Nyíregyháza, H-4400 Nyíregyháza, Hungary Department of Evolutionary Zoology and Human Biology, University of Debrecen, Debrecen H-4010, Hungary c Institute of Environmental Science, College of Nyíregyháza, H-4400 Nyíregyháza, Hungary b
a r t i c l e
i n f o
Article history: Received 17 February 2011 Received in revised form 11 July 2011 Available online 23 July 2011 Keywords: Elemental XRF analysis Human bone Ancient life Diagenesis Tenth century AD
a b s t r a c t The present study is a multielemental analysis of bone samples belonging to skeletal individuals originating from two contemporaneous (10th century AD) cemeteries (Tiszavasvári Nagy–Gyepáros and Nagycserkesz–Nádasibokor sites) in Northeastern Hungary, using the XRF analytical technique. Emitted X-rays were detected in order to determine the elemental composition of bones and to appreciate the possible influence of the burial environment on the elemental content of the human skeletal remains. Lumbar vertebral bodies were used for analysis. Applying the ED(P)XRF technique concentration of the following elements were determined: P, Ca, K, Na, Mg, Al, Cl, Mn, Fe, Zn, Br and Sr. The results indicated post mortem mineral exchange between the burial environment (soil) and bones (e.g. the enhanced levels of Fe and Mn) and referred to diagenetic alteration processes during burials. However, other elements such as Zn, Sr and Br seemed to be accumulated during the past life. On the basis of statistical analysis, clear separation could not be observed between the two excavation sites in their bone elemental concentrations which denoted similar diagenetic influences, environmental conditions. The enhanced levels of Sr might be connected with the past dietary habits, especially consumption of plant food. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Bone is one of the most complex structures in the human body, which consists of organic and inorganic components. It is not only important to support and move the body but also constitutes a substantial reservoir of the mineral materials in the human organism. To obtain information about the ancient life (paleodiet, health status or even living condition) of human population after death one can analyse the elemental status preserved in bones. For decades it has been a widely accepted technique to get information about the past life [1–7], but nowadays paleodiet studies have shifted from simple measurements of elemental concentrations to analyses of stable isotopes [8,9]. The applicability of bone strontium and zinc are firmly established as dietary indicators. It is well known that the Sr is taken in by the plants directly from the environment (soil). Therefore, the mammals (including humans) obtain this element secondarily through plants and other animals. It follows that the Sr is reducing in the trophic levels from the bottom to the top. Hence, plant tissue contains higher concentration of this element than the animal tissue and within mammalians, carnivores represent the least
⇑ Corresponding author. Tel.: +36 42 599400/2503; fax: +36 42 402485. E-mail address: [email protected] (I. János). 0168-583X/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2011.07.016
amount of Sr, herbivores show the highest level, whilst omnivores should demonstrate intermediate concentration of this element. It is also known that, there is a positive correlation between the Zn accumulated in bones and the protein diet connected with the consumption of animal foods. Thus in mammals the concentration of bone Sr is increasing through the consumption of plant foods and decreasing by animal protein intake while the bone Zn represents reverse fluctuation in concentration with the Sr according to the main diet components. The typical ranges of Zn and Sr in mammalian bones by different mode of nutrition are listed as a followings: (a) herbivores – Sr: 400–500 lg/g; Zn: 90–150 lg/g; (b) omnivores – Sr: 150–400 lg/g; Zn: 120–200 lg/g; (c) carnivores – Sr: 100– 300 lg/g; Zn: 175–250 lg/g. These elemental concentration levels can be typical for human bones in cases of herbivorous, omnivorous or even carnivorous nutritional habits [9–12]. The elemental status of buried bones are not only affected by the biological factors of ancient life (age at death, diseases, prevalent nutrition and metabolism), but the environmental circumstances such as soil composition, precipitation, pH and human land use, which can appear after burial. Such processes, termed diagenetic collectively, may cause changes in the original structure of osseous material [6,13]. The different types of bones respond to the remodelling processes in different way: spongy bone is more susceptible to diagenesis than compact bone from humerus and femur [14].
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In the present work two sets of human bone samples dating from the 10th century AD (the age of the Hungarian conquest) were analysed by using the high-performance energy-dispersive polarisation X-ray fluorescence (EDPXRF) spectrometry which permits fast multielement detection and the possibility to make precise screening analysis. The basic aims of this study were to obtain information about the elemental status and the inter-elemental relationships of these ancient bone samples of individuals as well as to demonstrate differences (or even correspondence) in bone elemental status between the two sites. Additionally, the study discusses the most appropriate reasons behind the results. 2. Materials and methods 2.1. Instruments and reagents For the analysis analytical grade reagents were used: methanol and Hoechest wax C micro powder for the sample preparation and deionised water for the cleaning procedure of the sample. An electric mixer mill (MM200 with a ZrO2 grinding vessel, Spectro Analytical Instrument, Kleve, Germany), manual press and a spectrometer model of SPECTRO XEPOS XEP01 (Spectro Analytical instruments, Kleve, Germany) were used for analysis. In the ED(P)XRF spectrometer the samples were irradiated by X-rays using Cartesian geometry (for polarisation of excited X-rays were used to reduce the spectral background) that means the X-rays exiting the tube are reflected or scattered by the target (Mo secondary target was used at voltage of 35 kV) at an angle of 90° with respect to the sample. Bottled helium was used to improve the sensitivity for light elements. The radiation typical of the elements present in the sample was detected by a PIN-diode semi-conductor detector. The measurement processes were controlled and stored by a PC computer using XLabPro software. The calibrations were made by using SPECTRO’S TURBOQUANT automatic calibration system. TURBOQUANT is able to analyse all elements between Na and U in real unknown samples. This means that all matrix effects, which will occur, are taken into account [15,16]. For the analysis of mobile Mg of the soil samples, atomic absorption spectrometry (FAAS) was applied by using the Varian SpectrAA-20 model. The pH of the soil samples were determined by an OP-211/1 type pH-meter. 2.2. Sites, samples and experimental The human skeletal material examined for this work was excavated in the Great Hungarian Plain, Northeastern Hungary, where two cemeteries (namely Tiszavasvári Nagy-Gyepáros and Nagycserkesz-Nádasibokor, both dated to the 10th century AD) were investigated (Fig. 1). The two cemeteries were located close to each other (the distance is only 13 km in between) and named after the neighbouring towns (Tiszavasvári and Nagycserkesz). Archaeological and historical evidence equally suggested that the graves belonged to early pagan Hungarian conquerors, who comprised various Finno-Ugric and Turkic ethnic groups with a seminomadic way of life. Excavations in the burial site of Tiszavasvári Nagy-Gyepáros were conducted in 1966, where 18 graves became known. The human skeletal remains belonging to 29 individuals of Nagycserkesz-Nádasibokor site were excavated in 1970 [17,18]. The samples were composed of lumbar vertebral bodies belonging to 35 skeletons (males, females and juveniles), since this type of bone was available from the highest number of graves (there were some fragmentary skeletons, especially in the case of juveniles). The ratio of the individuals analysed in the two excavation sites is 13:22 as five individuals from Tiszavasvári Nagy-Gyepáros and seven individuals from Nagycserkesz-Nádasibokor could not
be examined. Soil samples derived from the two excavation sites were also analysed in order to identify the possible compositional differences and elemental interactions between the bones and soil. The excavation area have temperate continental (moderately warm) climate with low humidity levels in both sites. The climate is also characterised by frequent rainshowers in spring, early summer and autumn as well as mildly cold, snowy winter. So the precipitation distribution is not even within a year. The majority of summer and winter is dry. January is the coldest, while the hottest month is July, as those in Hungary. Currently, there is no flooding on the sites. The further climate data relative to the two sites specifically on the basis of data on many-years observations are listed as a followings: (a) Tiszavasvári – average annual temperature: 9.8 °C, yearly absolute maximum temperature: 34 °C, yearly absolute minimum temperature: – 17 °C, average yearly rainfall: 550 mm, aridity index: 1.28, sunny hours: 1950, frostless period: from 12 April to 16 October. (b) Nagycserkesz – average annual temperature: 9.6 °C, yearly absolute maximum temperature: 34 °C, yearly absolute minimum temperature: – 17 °C, average yearly rainfall: 560 mm, aridity index: 1.21, sunny hours: 1950, frostless period: from 14 April to 16 October. The groundwater level generally is within the range of 2–4 m in both sites [19]. The osteological material was analysed in the Agricultural and Molecular Research Institute, College of Nyíregyháza. First, in order to remove macroscopic soil contamination, the bone samples were mechanically cleaned by flushing with tap – and washing with deionised water and dried in a clean environment at room temperature. After that, the bone samples were milled by using an electric mixer mill. From the powder obtained (<100 lm) several pellets (for three independent measurements) of each sample were prepared to reduce the errors of measuring. The experiment was carried out in the following way, according to the instrument documentation: 4 g of bone powder was homogenised with 0.9 g of Hoechst Wax and then pressed into a 32 mm pellet with 15 tons. The pressed pellet was put in a sample cup covered by Mylar film and analysed directly by applying high-performance energy-dispersive polarisation X-ray fluorescence (EDPXRF) spectrometry. The Mylar film and the Hoechest Wax do not contain measurable elements and the software of the instrument calculates the eventual concentration values taking the mass and the composition of these materials into consideration [15,16]. Homogenous sandy soils constituted the burial environments in both sites in the depth of the graves (60–120 cm). The soil samples derived directly from the burial environment of the skeletal remains at same depth. Several samples were collected from both sites (the means and the standard deviations are presented on the Table 1). The elemental compositions of soil samples were analysed in a way similar to that used for the bones by ED(P)XRF and here the pH values were also determined as a followings: 5 g of soil sample was mixed with 50 ml distilled water for 30 min and the pH was subsequently measured by the digital pH-meter. Only for the mobile magnesium of the soil samples FAAS analysis was performed on the following way: 5 g of soil samples was mixed with 25 ml distilled water for 30 min and the compound was centrifuged at 5000 rpm for 5 min. After that the supernatant was filtered on analytical filter paper and was measured by FAAS. The FAAS analysis was performed by using the following parameters: air-acetylene flame conditions: air: 4.6 L/min, acetylene gas: 1.7 L/min, sample introduction: manual by pneumatic nebuliser, observation height: 10 mm, lamp current: 4 mA, slit width: 0.5 nm, wavelength: 285.2 nm. 2.3. Statistical analysis Pearson correlations [20] were done on the entire data sets of all elements in the case of both sites to consider the inter-elemental
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Fig. 1. Location of the excavation sites described in the study. A = Tiszavasvári Nagy-Gyepáros site, B = Nagycserkesz-Nádasibokor site.
Table 1 Mean values and standard deviations for the elemental concentrations of the two examined bone series obtained by XRF and compared to published data of archaeological () and modern () bones as well as soil samples derived from the two sites. Bone P and Ca are given in mg/g all others in lg/g. Site/sample
Type of bone
Na
Mg
Al
P
Cl
K
Ca
Ca/P
TVN NAN Ref. [2]⁄ Ref. [3]⁄ Ref. [24]⁄ Ref. [14]⁄ Ref. [14]⁄⁄ Ref. [25]⁄⁄ Ref. [26]⁄⁄ Ref. [27]⁄⁄ TVN (soil) NAN (soil)
Vertebra Vertebra Not specified Tibia Vertebra Vertebra Vertebra Vertebra Ilium Not specified – –
1508 ± 315 1590 ± 337 – – 7716 ± 3190 2200 ± 100 6000 ± 300 – 4900 ± 900 –
385 ± 85 410 ± 90.2 – 5514 ± 1246.6 6704 ± 2672 64800 2200 ± 200 1560 ± 460 – – 47.055 ± 5147 6646 ± 470
135 ± 25.8 162 ± 88.6 – – – 410 ± 30 – – 19.5 ± 6.1 – 36.213 ± 1140 24.633 ± 8085
104 ± 14 110 ± 11.8 – – 158 ± 23.5 144 ± 0.8 97 ± 0.5 77.3 ± 14.3 98 ± 0.6 – 109 ± 132.7 19.6-21.4
150 ± 141 200 ± 126 – – – 6210 1300 ± 300 – – – 128 ± 118 103 ± 147
326 ± 239 592 ± 579 – – – – –
244 ± 25.7 242 ± 28.6 – 217.19 ± 34.3 347 ± 33.2 322 ± 15 254 ± 14 153 ± 25.9 213 ± 1.1 – 57.029 ± 8793 10.395 ± 2835
2.36 ± 0.13 2.20 ± 0.09 – – 2,23 ± 0.24 2.2 2.19 1.98 2.17 – – –
Site/sample
Type of bone
Mn
Fe
Zn
Br
Sr
TVN NAN Ref. [2]⁄ Ref. [3]⁄ Ref. [24]⁄ Ref. [14]⁄ Ref. [14]⁄⁄ Ref. [25]⁄⁄ Ref. [26]⁄⁄ Ref. [27]⁄⁄ TVN (soil) NAN (soil)
Vertebra Vertebra Not specified Tibia Vertebra Vertebra Vertebra Vertebra Ilium Not specified – –
115 ± 96.6 78 ± 68.2 55 ± 35.3 51.46 ± 43.41 1085 ± 1246 110 ± 10 <1.5 – – – 413.7 ± 37.8 407.7 ± 195.3
1506 ± 788 1957 ± 1628 269 ± 167.5 – 6822 ± 5868 5000 ± 1000 – 162 ± 60 183 ± 78 – 30523.3 ± 3040.8 26.150 ± 8738.4
89.2 ± 21.6 79.6.±17.3 90 ± 63.3 125.14 ± 34 250 ± 175.5 220 ± 10 – 120.1 ± 55.3 151 ± 22 117.5 ± 52 41.1 ± 0.6 28.9 ± 12
38.6 ± 18 33.2 ± 10.9 68.5 ± 9.2 – – 17.9 ± 1.1 110 ± 55 – – 8.9 ± 8.8 – –
488 ± 206 391 ± 143 152.5 ± 38.9 1515.6 ± 506.6 687 ± 403 6240 170 ± 30 – 51 ± 39.5 208 ± 83.4 325.8 ± 105.7 117.1 ± 5.4
– – 17.157 ± 481.9 12.550 ± 2972.6
TVN: Tiszavasvári Nagy-Gyepáros site; NAN: Nagycserkesz-Nádasibokor site.
relationships. In order to discover the relationships between the elemental status of the two groups of bone samples derived from the two contemporary sites and to integrate the information presented by the elemental contents of the individual bone samples,
principal component analysis (without Kaiser normalisation) was performed on the entire data set [21]. On the factor values of the first four principal components (PC) derived from the principal component analysis, Mann–Whitney (M–W) and Kolmogorov–
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Smirnov tests (K–S) [22] were carried out for comparison of the two sites. All the statistical analysis was done by using SPSS and Past software. All the tests were performed three times by using both softwares, in order to reduce the random error of statistical analysis.
Table 3 Eigenvalues, variance and cumulative for the four principal components.
3. Results and discussion During the examination, the following elements were determined by XRF analysis: P, Ca, K, Na, Mg, Al, Cl, Mn, Fe, Zn, Br and Sr. Descriptive statistics (means and standard deviation) of results together with published data (for archaeological and fresh bone samples) are presented in the Table 1. The bone samples of both sites are characterised by decreasing of certain elements such as Na, Mg and Cl as well as by increasing of other elements (Al, Mn and Fe) relative to values known from literature (Table 1). The Ca/P ratio is 2.15 with an SD value 0.17 in native hydroxiapatite [19]. Samples exhibiting a ratio with 2.36 ± 0.13 in Tiszavasvári Nagy-Gyepáros (TVN) site and 2.20 ± 0.09 in Nagycserkesz-Nádasibokor (NAN) site are presumed to be favourable theoretically to draw paleodietary conclusions [23,24]. Means of Br concentrations show sufficient approximations to data of modern bones therefore these are considered to accumulate during the past life while the Zn represents slightly lower levels as compared with modern reference data. The correlation matrix of the entire data set is presented in the Table 2 for the two sites (populations). Several significant correlations are similar to those known from the literature in both sites [4–8]: strong positive correlations are found in the cases of Ca– P (0.902 in TVN and 0.928 in NAN) which are the main constituents of bone mineral, and Mn–Fe (0.695 in TVN and 0.438 in NAN). Between Ca and Fe ( 0.869 in TVN and 0.53 in NAN) as well as Ca and Mn ( 0.521 in TVN and 0.261 in NAN) can be seen negative correlations but Ca–Mn correlations are not significant (Table 2). These negative correlations (Ca–Fe and Ca–Mn) can be explained as the consequence of diagenetic processes, since the Mn and Fe ions were incorporated in the bone apatite structure by ion ex-
Principal component
Eigenvalue
% of variance
Cumulative%
1 2 3 4
3.935 2.098 1.629 1.381
32.790 17.485 13.582 11.508
32.790 50.275 63.858 75.367
Table 4 Matrix of variable loadings. Highest loading absolute values are signed in bold face. Elements
PC1
Mg Na Mn Al K Fe Ca P Sr Zn Br Cl
PC2
0.801 0.794 0.786 0.763 0.709 0.644 0.5 0.18 0.466 0.153 0.119 0.096
PC3
0.088 0.411 0.415 0.585 0.07 0.014 0.272 0.886 0.574 0.043 0.456 0.067
PC4
0.34 0.065 0.06 0.042 0.277 0.076 0.461 0.112 0.379 0.798 0.49 0.421
0.35 0.284 0.278 0.045 0.514 0.189 0.163 0.109 0.359 0.06 0.184 0.77
changes with the Ca ions during the post-mortem period in the soil [4–6]. It may probably be confirmed by the elevated levels of Fe and Mn in soil samples (Table 1) as compared with those in vertebrae. The heightened levels of Al in the bone samples may possibly be the result of surface adsorption on the bone mineral because gross concentrations of this element were found in the soils of both sites. Elevated levels of Mg were also found in the soil samples (Table 1), but the mean levels of mobile Mg (determined by FAAS) in the aquatic phase of soil extracts (TVN – 33.6 ± 3.85 lg/g; NAN – 12.43 ± 3.19 lg/g were significantly lower than those in the bones (TVN – K–S: D = 1, p = 0.004; NAN – K–S: D = 1, p = 0.002). The pH
Table 2 The correlation coefficient matrix (according to Pearson) of the elements based on bone samples derived from the Tiszavasvári-Nagy Gyepáros (a) and Nagycserkesz-Nádasibokor (b) sites. Significant values which match in both sites are signed in bold face. Na
a b
Mg
Al
P
Cl
K
Ca
Mn
Fe
Zn
Br
(a) Na Mg Al P Cl K Ca Mn Fe Zn Br Sr
0.995b 0.968b 0.748b 0.16 0.647a 0.464 0.567a 0.705b 0.028 0.301 0.028
0.967b 0.758b 0.199 0.662a 0.478 0.538 0.695b 0.022 0.352 0.034
0.729b 0.189 0.55 0.429 0.507 0.616a 0.112 0.358 0.055
0.007 0.793b 0.902b 0.596a 0.886b 0.168 0.383 0.039
0.065 0.036 0.067 0.132 0.053 0.229 0.096
0.772b 0.321 0.885b 0.188 0.213 0.107
0.521 0.869b 0 0.196 0.012
0.695b 0.039 0.116 0.521
0.133 0.142 0.102
0.67a 0.421
0.467
(b) Na Mg Al P Cl K Ca Mn Fe Zn Br Sr
0.987b 0.085 0.334 0.276 0.205 0.29 0.343 0.376 0.023 0.471a 0.14
0.044 0.356 0.312 0.21 0.304 0.305 0.40 0.064 0.472a 0.103
0.201 0.308 0.617b 0.174 0.298 0.747b 0.018 0.105 0.085
0.073 0.008 0.928b 0.405 0.527a 0.161 0.165 0.06
0.032 0.021 0.018 0.211 0.109 0.382 0.385
0.049 0.335 0.647b 0.011 0.248 0.338
0.261 0.53b 0.412 0.212 0.088
0.438a 0.344 0.311 0.154
0.121 0.318 0.098
0.042 0.051
0.232
Correlation is significant at the 0.05 level (2-tailed). Correlation is significant at the 0.01 level (2-tailed).
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Fig. 2. Box plots of principal component values (PC1, PC2, PC3 and PC4) of the two past populations derived from the principal component analysis. Tiszavasvári NagyGyepáros (TVN) site and Nagycserkesz-Nádasibokor (NAN) site are compared with Kolmogorov–Smirnov (K–S) and Mann–Whitney (M–W) tests based on the first four principal components. (a) PC1 – K–S: D = 0.297, p = 0.397, M–W: U = 106, p = 0.213; (b) PC2 – K–S: D = 0.279, p = 0.474, M–W: U = 105, p = 0.2; (c) PC3 – K–S: D = 0.164, p = 0.966, M–W: U = 128, p = 0.620; (d) PC4 –K–S: D = 0.402, p = 0.105, M–W: U = 104, p = 0.188.
Fig. 3. 2D scatter plots of the individual values of the four principal components (PC1, PC2, PC3, and PC4).
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values of the soil samples coming from the burial environment were alkaline in both sites: 9.12–9.53 in TVN and 8.14–8.9 in NAN which are typical of the calcareous sandy soils. Comparing with data of modern bones, the Sr represents elevated levels which may be in accordance with past dietary habits (presumably consumption of plant foods). Knowing that Sr is also able to substitute Ca in the hydroxiapatite lattice, in case of Sr contamination strong negative correlation can be expected. Here, however, the former hypothesis cannot be supported since the Ca–Sr correlation coefficients are close to zero in both sites and the means of Sr concentrations of soil samples fall short of the mean values of bones (Tables 1 and 2). Although the soil Sr content was significantly higher in TVN than that in NAN (K–S: D = 1, p = 0.033), this element does not show significant differences in bones between the two sites (K–S: D = 0.283, p = 0.458). The correlation coefficient matrix represents quite good accordance between samples of the two populations regarding pairs of certain elements but do not in case of other elements. Matching values of significant correlation coefficients are marked by bold face in Table 2. Despite the fact that there are more significant correlation pairs in TVN than those in NAN, the main tendencies are similar in both cases. The minor dissimilarities might have been derived from population differences or from the random effects of the different sample size. In order to compare the two groups of samples (cemetery) on the basis of their elemental status, principal component analysis (without Kaiser normalisation) were carried out on the entire data set. Four principal components (with an eigenvalue higher than 1) could be extracted (Table 3). These principal components explain 75.4% of total variance. The results represented that P, Mg, Na, Fe, Ca, Mn and Br have similar loading on the first principal component (PC1). The second principal component (PC2) mainly correlates with Al and K as well as Mg, Na, Fe and Cl to a lesser degree. Sr and Cl have higher (and Zn smaller) loadings on the third principal component (PC3) and only one element, the Zn, can be classified to the fourth principal component (PC4) (Table 4). By fitting Kolmogorv–Smirnov and Mann–Whitney tests for PC1, PC2, PC3 and PC4, we found that there are no significant differences between the elemental statuses of bones belonging to the two contemporary populations (Fig. 2). This suggests that similar burial environment conditions (Table 1) have existed and similar changes have taken place during the post mortem period over time. However, looking at the scatter plots of the principal components, it is conspicuous that the factor values of NAN have higher heterogeneity than those of TVN which may derive from a minor population differences (Fig. 3). The results suggest that the elemental status of buried bones might be typical of a given ancient living ethnic group of humans and a certain archaeological period. Between the two populations, the K, Fe and Sr show minor (not significant) differences.
Secondly, the correlation coefficient matrix provided important information about the inter-elemental relationships: negative correlations were found between Fe–Ca and Mn–Ca in both populations that could probably be accounted for by the above mentioned ion exchange processes since the Mn and Fe might have incorporated in the bone apatite structure replacing Ca ions during the post-mortem period. The strong positive correlation between Mn and Fe in the bone samples can be explained by the similar ion radius of the Mn(II) as well as Fe(II) and Fe(III) ions which resulted in parallel mobility of these elements towards the bone tissue in accordance with their concentrations. The elevated levels of Al in the osseous material could be related to the attributes of Al ions which have gross affinity to surface adsorption on bone tissue in the soil. These observations can serve as evidence for diagenetic processes. Thirdly, the mean Zn concentrations denoted slightly lower levels than those of the reference data (Table 1) but fell within the typical range of diets high in foods of vegetable origin and low in meats. This element likely accumulated during the past life. Contrarily, the Sr indicated elevated levels in both sites which might probably also be connected with heightened consumption of plant foods (see the values listed in the Introduction chapter). The modern nutrition is usually more diverse and in most cases omnivorous, therefore the Zn and Sr concentrations in bone samples derived from healthy humans represent mainly moderate, intermediate levels. Depending on the main diet component the levels of these two elements can shift into opposites directions of each other. Lastly, comparing the two populations, it could be said that population of Nagycserkesz-Nádasibokor denoted higher heterogeneity in elemental values but there were no significant differences between the two populations regarding their bone elemental contents on the basis of statistical tests. In our opinion, a given archaeological period can presumably be characterised by typical levels of elements detected in human bones in similar environmental circumstances. On the basis of the elemental concentrations of bone samples, population (ethnic) differences will probably be detected between these series and samples derived from other contemporary cemeteries in the 10th century. Further examinations will be focused on analysing other type of bones within these series by different analytical methods and will be aimed at drawing conclusions regarding biological attributes such as age, sex and diet of these populations. Acknowledgements The authors thank all the colleagues who have helped the completion of this work. The anonymous reviewer also deserves thanks for his/her helpful comments and suggestions on an earlier version of the manuscript.
4. Conclusions References Taking it all round, by elemental XRF analysis of 10th century human bone samples from two excavation sites (Northeastern Hungary), at this stage of research, the following conclusions can be drawn: Firstly, seeing the elemental values as compared with those known from the literature, the data showed Na, Mg and Cl leaching which were probably due to the prevalent environmental circumstances over time. Despite the high concentrations of Mg in the soil samples (Table 1) the leaching seems to be feasible due to the significant rainfalls over time. Additionally, the mobile Mg, which was available for diffuse moving towards the bones in the aquatic soil extracts, showed significantly lower levels than the Mg in the solid soil and bone samples. The great part of soil Mg is appears to be bound in the soils in both sites.
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