image/svg+xml157 VII/2/2016 InterdIscIplInarIa archaeologIca natural scIences In archaeology homepage: http://www.iansa.eu Variation of Ba/Ca and Sr/Ca Response in Human Hard Tissue from Archaeological Series Anna Pankowska a* , David Milde b , Jana Bohunská b a Department of Anthropology, Faculty of Arts, University of West Bohemia, Sedláčkova 38, Plzeň, 30614, Czech Republic b Department of Analytical Chemistry, Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacký University Olomouc, 17. listopadu 12, 771 46 Olomouc, Czech Republic 1. Introduction The analysis of strontium, barium and calcium in bone (Burton 1996; Burton, Wright 1995) and enamel (Sponheimer et al. 2005; Copeland et al. 2010) has been used to reconstruct diet (Szostek et al. 2009), weaning patterns (Austin et al . 2013; Balter, Simon 2006; Burton, Price 1990), trophic levels in wild animals (Balter 2004), migration (Arnay et al. 2009; Copeland et al. 2008; Lewis et al . 2014) and feeding strategies in herbivores (Sillen 1988).Recently, laser ablation with inductively coupled plasma mass spectrometry (LA ICP-MS) has been used to detect the spatial distribution of Sr, Ca and Ba in separate human skeletal tissues from archaeological samples (Alvira et al. 2010; Austin et al . 2013; Dolphin et al. 2005; Prohaska et al. 2002). Using LA ICP-MS, we can easily observe the diferences between tissues and investigate the causes of these diferences. Diferences between bone and dental tissues (enamel and dentin) may refect three possible factors, other than dietary or geographic diferences. First of all, element ratios in bone, dentin and enamel may refect the specifc responses of each tissue to sampling (ablation). Second, ratios may difer due to the heterogeneity of each element within living tissue caused by an individual’s development, specifc way of metabolism, tissue mineral incorporation, diferences in the elements’ absorption or age-dependent changes (Dolphin et al. 2005) and diseases (Alvira et al . 2011; Gemmel et al. 2002; Malara et al. 2006). Third, each tissue is variably infuenced by diverse post-mortem diagenetic alterations. Bone tissue decomposes more rapidly than enamel, which is known to be less susceptible to diagenesis (Copeland et al. 2008). However, enamel is not altogether resistant to diagenetic processes over longer time scales – similar to that of fossilised remains in paleoanthropological contexts (Sponheimer, Lee-Thorp 2006).It is necessary to consider the reliability of skeletal tissue analysis in the reconstruction of past diets. Are bones less suited for reliable isotope testing than dental tissue, and how are the diferences between them to be interpreted? Volume VII ● Issue 2/2016 ● Pages 157–167 *Corresponding author. E-mail: pankowsk@ksa.zcu.cz ARTiCle inFO Article history: Received: 2 nd May 2016Accepted: 20 th December 2016 Key words: bariumcalciumstrontiumhuman skeletal tissueLA ICP-MS ICP-oa-TOF-MS ABSTRACT This study aims to assess how strontium, barium and calcium ( 138 Ba/ 44 Ca and 88 Sr/ 44 Ca) are incorporated into human hard tissue (enamel, dentin and bone). For this purpose we used laser ablation with inductively-coupled plasma mass spectrometry (LA ICP-MS). Human hard tissue from an archaeological series was analysed to determine isotope signals to investigate possible diferences between enamel, dentine and bone. Signifcant variance in the ratios was identifed by tissue type. The manner in which the type of hard tissue infuences 138 Ba/ 44 Ca and 88 Sr/ 44 Ca ratios is discussed. Possible reasons for distinct isotopic responses are individual ways of metabolism, tissue mineral incorporation, and individual diferences in the elements’ absorption. The validity of dietary and migration studies, based on barium and strontium concentrations, are reconsidered. More than just for dietary and migration pattern reconstruction, this method serves as a chemometric tool for human skeletal remains’ discrimination. Using a discriminant function, we found substantial diferentiation in the hard tissues of investigated individuals.
image/svg+xmlIANSA 2016 ● VII/2 ● 157–167Anna Pankowska, David Milde, Jana Bohunská: Variation of Ba/Ca and Sr/Ca Response in Human Hard Tissue from Archaeological Series 158 Diferences between dental and bone tissue tests are expected. But how do we resolve the distinct isotopic signals between enamel and dentin? The diferent properties of each tissue type, the hardness and irregularity of their surfaces, combined with a lack of solid standard reference materials, can cause the diferent response of isotopic signals and misinterpretation of the date. The use of LA ICP-MS may provide a solution. This method enables the investigation of solid samples using a laser to ablate the isotopes into an inductively coupled plasma (ICP) source for the mass spectrometer and measure the signals of isotopes in their spatial distribution from various parts of the sample.In this brief study we present our pilot research regarding the diferences in isotopic responses of skeletal tissue to various external factors. The present study investigated 88 Sr/ 44 Ca and 138 Ba/ 44 Ca in three hard tissues (enamel, dentin and bone). We expect diferences to be present between dental tissue and bone. Dentin formation follows enamel, therefore they should refect similar 88 Sr/ 44 Ca and 138 Ba/ 44 Ca ratios, in spite of the dentin being partially remodelled during one’s later life, contrary to bone. If this is not the case, then the variances relate to factors that are not related to diet or geography. What could cause such diferences? How reliable then are studies based on the analysis of a single tissue type. 1.1 Utility of Sr/Ca and Ba/Ca ratios in palaeodietary studies The ratio of strontium (Sr) or barium (Ba) to calcium (Ca) fxed into human bone and teeth hydroxyapatite is used to understand the dietary trophic level of past human populations (Burton, Price 2002). Higher concentrations of Sr and Ba, and thus higher Ba/Ca and Sr/Ca ratios, observed in the skeletal tissue are the remarkable indicator of a plant-based diet (Burton 1996). On the other hand, a marine and meat-based diet decreases the value of both these ratios and also the Ba/Sr ratio (Burton, Price 1990). Strontium can be used in this way for diet reconstruction because of its bio-purifcation when rising up through the trophic levels and is thus useful for estimating the proportion of meat and plant food in a diet through a comparison of human and animal skeletal tissue from the same site. The bones and teeth of humans who consume more plants should contain more strontium than those of meat-consumers or omnivores. Barium, similarly to strontium, refects the trophic level. The amount of barium refects certain aspects of a diet such as the consumption of marine sources and meat. Large diferences in barium, but not in strontium, have been evidenced between marine and terrestrial archaeological bone (Burton 2007). The reason for this is the solubility of strontium. Strontium sulphate is soluble in environments rich in sulphate ions, i.e. in the ocean. Barium is not soluble, and is removed from the environment as insoluble barite (BaSO 4 ). Therefore the Ba/Ca ratio is lower in a marine diet (Burton, 2007). Carnivores and consumers of a marine diet have generally lower Ba/Ca ratios than herbivores. Calcium is a major component of skeletal tissue and its levels in humans are strongly regulated, with bone as the main mineral storage tissue. Animals and humans favourably intake calcium, therefore they have lower Sr/Ca and Ba/Ca ratios than the food they eat. However, this efect appears only in single component diets, multi-component diets do not show such simple correspondence. Even food with small amounts of calcium may decrease the Sr/Ca ratio because calcium is preferentially absorbed. Yet absorption depends on the type of food and individual calcium metabolism (Reynard et al. 2011). In plants, calcium is necessary for growth and development. Some plants contain high levels of calcium (seeds, nuts, etc. ), other plants such as grain ( e.g. wheat) contain very low portions of calcium (Pharswan, Farswan 2011). Measurements of calcium content in microanalyses of skeletal tissue are also mainly done with the purpose of assessing the quality of archaeological material in terms of its diagenetic resistance (Allmäe et al . 2012). Diagenetic changes like calcium carbonate precipitation or decalcifcation should also be capable of reconstruction using the Ca/P ratio. 1.2 Limitation of Sr/Ca and Ba/Ca in palaeodietary studies The simple linearity between diet and alkaline elements incorporated in skeletal tissue is highly biased by: a) diagenetic contamination; b) local geographic environment; c) isotopic heterogeneity of a plant-based diet; d) individual variability; and c) a mixed diet (Burton, Price 2002). Diagenetic contamination is caused by many agents, e.g. chemical, mechanical, biological and physical factors (Burton, Price 2002) and the process of diagenesis consists of several mechanisms: dissolution, precipitation, absorption and recrystallization. Elevated levels of Fe, Mn, Si, Al and Ba in fossil teeth indicate the formation of secondary minerals (Kohn et al. 1999; Patterson et al . 1991). Most common is the substitution of elements in the hydroxyapatite. Calcium may be substituted by Sr, Ba, Mg, Na, U and Pb; these substitutions producing changes in the content and structure of the hydroxyapatite. Diagenetic changes in fossil samples can be determined using various methods such as FTIR, NMR and LA ICP-MS (Kang et al. 2004; Prohaska et al. 2002).Barium, strontium and calcium are variably distributed in the subsoil and are geologically-specifc (Burton et al . 2003). Their content in human tissue can refect where the individual lived more than what he or she ate. Burton et al. (2003) recorded high variability of strontium and barium in soil samples in USA. Deviations among geographically diferent regions were signifcantly greater than local variations in skeletons. These variations can afect dietary comparisons among various sites (Burton et al. 2003). Other studies have used the Ba/Sr ratio in teeth for the reconstruction of migration rather than for dietary investigation (Arnay et al. 2009; Brügmann et al. 2012).Even though particular plants may be consumed by both animals and humans, they are absorbed diferently, and will leave diferent trace mineral content in the skeletal and dental tissues of diferent species. Burton and Price (2002)
image/svg+xmlIANSA 2016 ● VII/2 ● 157–167Anna Pankowska, David Milde, Jana Bohunská: Variation of Ba/Ca and Sr/Ca Response in Human Hard Tissue from Archaeological Series 159 present the variability of the dietary Sr/Ca ratio among corn, nuts, potatoes and leaf vegetables (Burton, Price 2002, 165, Figure 8.2). They stress that diferences in Sr/Ca ratios do not show dietary plant/meat proportions, but rather the degree of browsing and grazing. Therefore low-calcium plants like some grains can display the same Sr/Ca ratio as meat or marine food (Ezzo et al. 1995).Strontium, barium and other trace elements’ incorporation into skeletal tissue depends on dietary as well as geological processes (Darrah 2009). Other diferences can be associated with individual metabolic function and incorporation such as inhalation and chemical exposure. Due to the occurrence of individual variation in trace element incorporation in human skeletal tissue, studies like Perrone et al. (2014) and Gonzalez-Rodriguez, Fowler (2013) used trace elements for species and individual classifcation and calculation of minimum number of individuals. These studies have great potential for forensic purposes.The dietary Sr/Ca ratio for mixed diets is not linear; the proportion of plant/meat diet composition is not binary, but rather continuous and highly individually variable. Some plants have more than ten times as much calcium and strontium as meat (Burton, Price 2002) and they have a greater efect on the element composition of connective tissue than meat does. Meat is noticeably up to 90% of its representation in the diet (Burton 2007, 447, Figure 14.2). Only a pure plant or a pure meat diet is visible in the Sr/Ca ratios in human tissue.Comprehension of the variation of Sr, Ba and Ca in human connective tissue and its causes is essential for our investigation. What the skeletal tissues can truly refect is why do they difer from each other and which tissue is the most reliable indicator of diet. 1.3 Subsistence strategy of Early Bronze Age population at Chrášťany site The osteological sample originated from the Early Bronze Age site of Chrášťany. Chrášťany is situated on a small hill (203 m a.s.l.) in the Mojena River valley (194 m a.s.l.) in central Moravia, the eastern part of the Czech Republic (Figure 1). The local subsoil consists of Quaternary loess and loessic loam. The site was settled continually from the 2 nd half of the 3 rd millennium BC (Corded Ware Culture) till the early Middle Ages. Most fnds from the site date from the Early Bronze Age (EBA) from 2200–1500 BC, a period characterized by oak and hornbeam woodlands, and hard- wood foodplains of oak, ash and elm (Kočárová, Kočár 2010).Groundwater at the site is neutral to slightly alkaline (pH 7.0 to 8.3) with a higher than the local average level of calcium, sodium and potassium cations. The area has high levels of strontium (0.24 to 1.22 mg·l –1 ) that probably originate in the