Possible diagenetic alterations.

Bio-apatite is extraordinarily chemically reactive because of the living human organism's requirement for the quick regulation of critical elements in the blood [59]. Its good reactivity originates from its high porosity, and the amorphous nature of bio-apatite causes a faster turnover compared to the organic compartment of the bone during life. However, its reactivity does not cease immediately after death, thus making it suspicious for postmortem contaminations. These features of the bone make it susceptible to two main manners of postmortem contamination. Firstly, its high porosity fosters physical infiltration by the soil itself and physical salt deposit from soil solutions. Secondly, the amorphous nature of bio-apatite compared to high crystalline hydroxyl-apatite increases its solubility in wet environments. Additionally, various studies have shown that for all interesting elements contamination can occur in both directions either through enrichment from soil or from depletion to the soil [80]. These processes are unfortunately not unidirectional, furthermore dissolution, permineralization, groundwater replacement, and ion exchange can occur at the same site depending on (ground-)water availability. For this reason, straightforward evaluations of the contamination cannot be made [116]. In general, it seems that virtually all bones buried in soil are affected by some contamination, and so far it does not seem possible to reliably quantify these contaminations [80]. Nevertheless, studies on bone contaminations in various environments prompted tests to find out if massive contamination by the soil has occurred or not [59], [80]. Therefore, possible alteration of Sr/Ca values due to diagenetic changes must be considered in every trace element study.

All investigated human bones and soils were excavated at the foot of the same hill (Panyirdağ) and can be dated within a range of 300 years. We can assume a similar climatic and topographic environment resulting in comparable impact of temperature, rainfall, and ground water conditions for all of the specimens.

All bone samples appeared similar macroscopically with a good status of preservation. Only minor differences of the superficial coloring of the bones were recognized. The same was evident for the hardness of the bones, which could be observed during the homogenization of the bone samples in an agate mortar. On average, about a 15±3% loss of weight during 500°C ashing in the course of sample preparation was detected. This indicates that organic material, believed to be protective for the bone mineral fraction [59], [80], was still present in all of the bone samples.

No quartz residues were found in any of the digestions of bone samples, and measurable concentrations of La in the drill waste bone samples were absent, although La was available (approx. 50 µg/g) in all four investigated soils (Table 2). Presence of quartz and La that are not present in the living bone [117] would be possible predictors for diagenesis in bone due to infiltration of the surrounding soil [59].

However, the prediction of mineral quality is not as simple and straightforward as it is for soil infiltration. Some authors believe that the integrity of the remaining bio-apatite can be checked with the ratio of Ca/P that was found to be about 2.12–2.35 for fresh and archaeological, unaltered bone [102], [104], [118]. Archaeological bones with Ca/P ratios within this range should indicate that no major degradation due to octocalcium phosphate, brushite, whitlockite or significant contaminations with calcium carbonate have occurred. Therefore, a good state of preservation [59] can be assumed. In our study, all investigated bones had Ca/P ratios ranging from 2.16 to 2.35 and therefore meet up to this criterion.

The investigation of the surrounding soil offers more potential to predict significant diagenetic alterations to the Sr/Ca-ratios of the bone. The macroscopic characteristics of all four investigated soils were similarly of dry grey nature. The pH values fell into a range between pH 7.14 and pH 7.72 for all four soils. For soils with pH above 7.0, early trace elements studies postulated that they are less prone to diagenetic alterations of bone than acidic soils (pH<7.0). Nevertheless, [80] found on bones from alkaline soil that they may act as Sr sponges and are able to accumulate Sr to values even higher than 1000 µg/g (mean = 879 µg/g; max = 1488 µg/g), although the values for Sr in the surrounding soil were lower. Such extraordinarily high Sr values were not found in or study. Overall we found a mean Sr concentration of 373.9 µg/g, with individual values ranging from 195.6 µg/g to 724.9 µg/g.

The significantly lower Ca values in the DAM93 soil and therefore the much higher Sr/Ca-ratios in this soil raises concerns whether these conditions caused diagenetic alterations of the bones at this location. But, there are still significant differences in the Sr/Ca-ratios between gladiators (1.26±0.33 µg/mg) and non-gladiators (0.63±0.08 µg/mg) from the DAM93 site buried close to each other. Further, our leaching experiments with bi-distilled H 2 O (simulating rain water) for the different soils have shown that although there were great variations in the Sr/Ca-ratios within the different bones and within the different soils, the H 2 O eluents of all four soils turned out to have similar Sr/Ca-ratios, ranging from 2.84 to 3.36 µg/mg (Table 2). [119] studied the ability of bovine bone meal to act as decontamination sorbent for Sr90 contaminated soils. The study showed that untreated as well as heated (400°C) bone meal is indeed able to absorb small amounts (about 4 µg/g) of Sr when soaked in aqueous solution with Sr concentrations of as high as 526 µg/L. This happens for a wide range of pH (4 to 11) with more or less the same absorption rate. Additionally, a competition between Ca- and Sr-ions for absorption can be observed when an exceeding concentration of Ca is added to the solutions. Ca ions are clearly preferred to bind at the bio-apatite because of their higher binding stability. These findings indicate for our study that absorption of Sr in the buried bones might have been negligible, because Ca was available in 300 times excess in all four soil eluents.

In theory, sufficiently preserved collagen should protect the metastable calcium phosphate which causes the amorphous nature of the bio-apatite. If the organic (collagen) is lost through burning, the crystallinity of the apatite increases and therefore the solubility of the apatite decreases. This makes the bone less prone to contaminations through ion exchanges [59]. In our study, we found that the cremated samples from the Pithoi showed Sr/Ca-ratios of 0.58 µg/mg respectively 0.61 µg/mg comparable to the uncremated bones. This can be taken as a sign that only minor changes to the original Sr/Ca-ratios have occurred, especially if taking into account that the initially intact and sealed ceramic vessels of the Pithoi prevented the cremated bones from direct groundwater contact at least for a certain time.

Finally, and most importantly, the average Sr/Ca-ratio (0.63±0.08 µg/mg) of the two individuals buried in directly adjoining soil (EPH-DAM 106/93 FEM and EPH-DAM 191/93 rHUM) and the female skeleton buried within the gladiator cemetery (EPH-DAM 72/93 rFEM-1) did not differ significantly from the non-gladiator individuals: 0.68±0.09 µg/mg (asymptotic p = 0.392). Additionally, the Sr values of both gladiators (459.9±125.0 µg/g) and non-gladiators (244.8±32.0 µg/g) are within the physiological ranges reported for modern humans. [120] found a mean of 234 µg/g Sr in 227 humans from around the world ranging from values below 100 µg/g to values as high as 772 µg/g. A later study by [121] from worldwide sampling of 190 individuals revealed Sr values ranging from 100 to 430 µg/g.

Because of the experience many colleagues made during their trace element studies, we assume that diagenetic alterations of the bones must have occurred to some extent. However, we believe that in our study these alterations were not substantial enough to change the Sr signals of the bones which therefore originate from real physiological differences these individuals experienced during their life time.