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157
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 2016
Accepted: 20
th
December 2016
Key words:
barium
calcium
strontium
human skeletal tissue
LA 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.
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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)
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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
Figure 1.
Chrášťany site, Moravia, Czech Republic. Plan of the site by Martin Paulus (Archeologické centrum Olomouc).
0 40 km
0 200 km
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clayey sediments of the Menilite and Submenilite formations of
the Ždánice Unit of the Carpathian Flysch Belt (Müller 1994).
Agriculture was the dominant subsistence strategy and the
daily activity for most of the EBA population (Barker 1985).
The distribution of grain storage pits at excavated sites is
indicative of substantial agricultural activity. The storage
pits at Chrášťany were concentrated in clusters and used
primarily for cereal (or pulse) crops and other food storage.
Storage pits were dug in the loess, which is well suited for
the dense networks of deep pits. The pits were concentrated
in discrete areas of the settlement, and are thought to have
been a valuable fxed asset of the community.
Macroremains of hulled wheat (einkorn and wild emmer)
have been discovered in these storage pits (Kočárová,
Kočár 2010). Zooarchaeological analyses have identifed
the remains of cattle, pigs, sheep or goats, and some dogs
and horses. Hunted wild game included bear, roe deer,
red deer, hare and pike. The bones of most farm animals,
including dog, had been butchered and most likely modifed
by cooking. Young animals were butchered for meat, whilst
older animals were exploited for milk, labour and breeding
(Holub 2010). Animal bones were frequently used for tools
such as awls and spear points. It is presumed that stored
grain was only used for human consumption. Animals grazed
outside the settlement and were slaughtered before winter so
that they did not require winter feeding.
2. Material and methods
2.1 Osteological sample
The osteological material consists of six burials from fve
settlement features excavated at Chrášťany (Paulus 2011).
Two radiocarbon dates from sites with pit burials range from
3460±25 BP to 3580±25 BP (cal BC: 1804±55 to 1938±30,
Weninger
et al
. 2007). Individuals were selected for analysis
according to their preservation. All individuals were buried
inside the settlement in storage pits scattered across the
Figure 2.
Settlement burials. Drawings of skeletons by Antonie Pešková (Archeologické centrum, Olomouc).
0 1 m
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excavated area (Figure 2). Six human teeth (frst or second
molars) and six human bone samples taken from cortical part
of their femoral midshaft, along with one pig bone (
Sus scrofa
f. domestica
) were chosen for isotopic analysis (Table 1).
Pigs often eat human waste and consume a diet similar to
humans, and are the domesticated animals most likely to
have lived locally (Bentley 2004). The isotopic content of
their hard tissues should correspond to their geological and
temporal context.
2.2 Sample Preparation
Bone and teeth samples were dry-cut (to prevent sorption)
with a 200 mm diamond saw. Bones were cut across, teeth
longitudinally. The samples were stabilized in 25 mm
lenses flled with epoxy glue (Araldite 2020, Huntsman,
Switzerland). The sample and epoxy were left to harden for
two days at a temperature of 55 °C on a heating plate (Stuart,
Germany). After removal from their forms, samples were
Table 1.
Sample under study.
IDBone sampleTooth sampleSexAge (yr)C
14
(calBP)*
107/804femurM1 sin (enamel, dentin)male19–34
196/812femurM2 dx (enamel, dentin)female15–213754±55
230/813femurM2 dx (enamel, dentin)indiferentadult
374/820femurM1 sin (enamel, dentin)female25–45
194/809femurM1 dx (enamel, dentin)female14–18
229/818femurM3 sin (enamel, dentin)female25–343888±30
Sus scrofa f. domestica
femur indiferentadult
* Laboratory code UGAMS# 9503, 9504.
Figure 3.
Example of the ablation line.
Photo by J. Bohunská.
abraded, and washed with denatured alcohol.
Using LA ICP-MS, we analyzed standard reference materials
and the samples of bones and teeth. We determined the part of
the sample to be analyzed using a micro-camera and drew an
ablation line on the sample (see Figure 3). The measuring time of
one ablation line was set at 10 s (in this way, the mean intensity
of isotopes was determined). In the case of measurement of the
correlation between intensity (cps) and measuring time, 35–40
points were selected according to sample type, each measured
for 1 s so that each measurement took 30–45 s and the mean
value of background intensity was discounted from the intensity
values of the isotopes of the sample.
2.3 Elemental Analyses
LA connected to the ICP-MS with Tygon tubing was used
for the elemental analysis of all bone and teeth samples. The
LA Analyte G2 (Photon Machines, USA) equipped with a
HelEx ablation cell uses an argon fuoride excimer laser with
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a wavelength of 193 nm and helium (He) 5.5 is used as an
ablation gas (Siad, Czech Republic). The following parameters
were used: laser fuence of 7.4 J cm
–2
, pulse frequency of 10 Hz,
line of 50 µm spots, scan speed of 30 µm s
–1
, He fow of 0.7 L
min
–1
. Inductively coupled plasma with a time-of-fight mass
spectrometer (ICP-TOF-MS) Optimass 9500 (GBC, Australia)
was used to analyse
43
Ca,
44
Ca,
86
Sr,
87
Sr,
88
Sr,
137
Ba,
138
Ba
isotopes. The instrument settings are summarized in Table 2.
Optimization of the LA ICP-MS was performed using SRM
612 and 610 (NIST, USA). Mass calibration was achieved with
7
Li
+
,
115
In
+
and
238
U
+
in above mentioned reference materials.
2.4 Use of NIST SRM 1486 Bone Meal tablet
We followed Uryu
et al
. (2003) when preparing the NIST
SRM 1486 Bone Meal tablet. Measured values of diferent
isotopes’ intensities in cps units, and correlation of intensities
to measuring time were processed using the linear inversion
method.
Diferent isotopes of reference samples SRM 610 and
SRM 612 were measured with a laser beam 40 μm, 50 μm
and 65 μm thick. The intensity of the laser was 100%
(10.59 J/cm
2
), the frequency of its pulses 10 Hz, speed of
motion of the sample 30 μm/s. The optimal diameter of
the laser beam was 50 μm. Using Sr intensity from SRM
1486 reference material in a linear regression counted from
SRM 610 and SRM 612, the calculated concentration of
Sr was 2365.0 μg/g. The certifed concentration of Sr in
the standard reference material SRM 1486 Bone Meal was
264±7 μg/g (Table 3). The signifcant diference between the
certifed and calculated value was given by a diferent type of
material,
i.e.
the hardness of the glasses SRM 610 and SRM
612, and the prepared tablet composed of a material similar
to bone.
2.5 Statistical Analysis
Initially,
88
Sr/
44
Ca and
138
Ba/
44
Ca ratios were correlated in each
separate tissue in each individual. This is necessary as both
ratios refect the same process. We used the nonparametric
Spearman Correlation on data with non-normal distributions
obtained from bone (Shapiro-Wilk Tests; N=34; p≤0.05) and
dentin (Shapiro-Wilk Tests; N=21; p≤0.05), and Pearson’s
Correlation on data with standard distributions obtained from
enamel (Shapiro-Wilk Tests; N=22; p≥0.05). The second
step used the Kruskal-Wallis test for independent samples.
The null hypothesis was that the distribution of
88
Sr/
44
Ca and
138
Ba/
44
Ca is the same across the three categories of tissue
(bone, dentin, enamel). Linear discriminant analyses (LDA)
were carried out in order to identify the ratio intensities
which best classify individuals. LDA was performed using
the open source PAST software (Hammer
et al.
2001) for
n=25. Univariate statistical analyses were undertaken with
SPSS (version 22.0) software.
Table 2.
Instrumental operating conditions for ICP-MS.
ICP source
RF power (27.12 MHz)
1200 W
Plasma gas fow rate
10 l min
–1
Auxiliary gas fow rate
0.5 l min
–1
Nebuliser gas fow rate
0.7 l min
–1
Mass spectrometer
Skimmer–1250 V
Extraction–1000 V
Z1
–900 V
Y mean–750 V
Y defection
0 V
Z lens mean
–1290 V
Z lens defection
0 V
Lens body–190 V
Blanker150 V
Refectron
650 V
Electron multipier gain2450 V
Table 3.
Standard reference material.
Element
SRM
*
610SRM
*
612SRM
*
1486
mg/kgmg/kg
μg/g
Ca26.6±0.3
1)
Sr515.5±0.578.4±0.2264±7
Ba453.5±3738.6±2.6
Isotope
**
Average (n=4)RSD (%)Average (n=4)RSD (%)Average (7–16 s)RSD (%)
44
Ca 28461.12.124820.88.3968652.44.6
88
Sr138448.54.326754.45.7611055.04.8
137
Ba 24260.73.4 2580.74.9123695.15.2
87
Sr 62041.07.110456.44.3 52499.96.0
86
Sr 22627.12.0 7637.47.2 63520.84.7
138
Ba157092.64.014829.64.7817909.24.5
*
Standard Reference Material (SRM);
1)
in weight %.
**
Average cps of the measured isotopes and their RSD (%) for SRM 610, SRM 612 a SRM 1486 (n = number of measurements).
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3. Results
The correlation of
88
Sr/
44
Ca and
138
Ba/
44
Ca was signifcant
within each separate tissue (Figure 4A). The signifcant
correlation in all tissues shows the close connection of
the
88
Sr/
44
Ca and
138
Ba/
44
Ca ratios. The lowest correlation,
though still signifcant, was found in enamel.
In Table 4, we present additional measurements within
each individual according to the type of tissue. All individuals
were compared with a pig bone (
Sus scrofa f. domestica
) that
originated from the same site and same stratigraphy. Figure
4B shows signifcant diferences in the ratio of
88
Sr/
44
Ca
(N=69; Kruskal-Wallis=35.02, df=2; p<0.05) and
138
Ba/
44
Ca
(N=65; Kruskal-Wallis=28.76, df=2; p<0.05) by tissue. In
Figure 4.
Isotopic correlation by tissue and diferent responses in each tissue. A: Correlation of
88
Sr/
44
Ca and
138
Ba/
44
Ca ratios by tissue; B: Diferent signal
response of
88
Sr/
44
Ca and
138
Ba/
44
Ca ratios by tissue.
Table 4.
Means, standard errors, and sample sizes, by tissue and individual. N = number of measurements in one individual.
BoneDentinEnamel
Individual IDn
138
Ba/
44
Ca
88
Sr/
44
Ca
138
Ba/
44
Ca
88
Sr/
44
Ca
138
Ba/
44
Ca
88
Sr/
44
Ca
MeanSDMeanSDMeanSDMeanSDMeanSDMeanSD
10740.0130.0050.0310.0100.0140.0030.0220.0040.0010.0000.0100.003
19440.0120.0010.0400.0020.0180.0020.0340.0040.0010.0000.0090.000
23040.0120.0010.0350.0010.0010.0010.0110.0010.0050.0000.0140.003
37440.0180.0040.0340.0050.0190.0040.0300.0050.0020.0010.0060.002
22940.0360.0050.0610.0020.0280.0020.0380.0020.0010.0000.0120.001
19650.0180.0030.0330.0050.0010.0010.0060.0010.0030.0010.0100.003
Pig (
Sus scrofa
f. domestica)
4
0.0320.0030.0480.009––––––
––
n = number of measurements in one individual.
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both observations, the lowest value of each ratio was found
in enamel and the highest one in bone. In dentin, there was
the widest range of values caused by the variability among
the individuals. However, the means between dentin and
bone were similar for
138
Ba/
44
Ca. When we combined the
values from enamel and dentin together, there was no efect
on the values of dentin, because of the low signals in enamel.
We used a pairwise comparison as per the Kruskal-Wallis
test. The
138
Ba/
44
Ca distribution was the same across dentin
and bone (Table 5), otherwise all other combinations difered
signifcantly by tissue.
Figure 5 presents the mean intensity ratios of
88
Sr/
44
Ca and
138
Ba/
44
Ca by individuals and tissue. While these results were
difcult to extrapolate due to the small sample size, it seems
that enamel deviated signifcantly from bone and dentin in
both ratios. Values were usually lower in dentin than in bone,
and in enamel than in dentin and bone. Higher intensity
ratios in dentin can be explained by biogenic factors. The
enamel values signifcantly deviated from dentin although
their development was simultaneous. Intensity ratios found
in enamel are similar to the bones of carnivores (for example,
according to Burton
et al
. 1999). Overall,
138
Ba,
88
Sr and
44
Ca
intensities were very low in enamel and, in connection with
the lowest correlation of both ratios in enamel, these values
possibly do not refect the biogenic signal.
However, there were interesting correlations found in the
bone and dentin. For example, the third molar of individual
229, a young adult female, showed higher intensity ratios
of both elements in both dentin and bone. It was expected
that data on diet obtained from both the tooth and bone
would be in accord, as they refected a similar episode in
the individual’s life (
i.e.
the mineralization of the third molar
was variable, but more or less synchronous in a juvenile).
Dentin data were similar for individuals 196 and 230, as
both were derived from the second permanent molar whose
mineralization occurs broadly between 2 and 5 years of age.
In the remaining individuals, the frst molars were analysed.
These precede the second molars mineralize prior to three
years of age. The lower values of
88
Sr/
44
Ca in M1 compared to
M3 are to be expected, since the M1 may have been afected
by lactating and thus had a higher trophic level (Austin
et al.
2013). The lower values in teeth, compared to bone, could be
expected for the same reasons.
Figure 6 shows the clusters of individuals based on
their isotopic signals, which are individually specifc.
All individuals (100%) from the reference groups were
classifed correctly. However, bone and dental tissues
classifed individuals with varying degrees of reliability.
Isotopic signals from bone tissue classifed individuals less
(83.33%) than dental tissue (100%). Using a cross validation
(Jackknife) method, the correct classifcation rate was
66.67% in bone and 95.83% in enamel and dentin.
4. Discussion
The intensities of the isotopes signifcantly difer by tissue
and by individual. They are highest in bone, and lowest in
enamel. There is a similar ratio signal between bone and
dentin, while the enamel is diferent from both. Correlation
Table 5.
Pairwise comparison between samples.
88
Sr/
44
Ca
138
Ba/
44
Ca
Sample vs. SampleTest Statistic*SEp valueTest Statistic*SEp value
Enamel-dentin13.7235.980.02222.8525.920.000
Enamel-bone34.6785.920.00028.6895.580.000
Dentin-bone20.9555.850.0005.8385.800,315
**
*
Kruskal-Wallis test
**
null hypothesis is accepted; the ratio distribution is the same in the samples
Figure 5.
Distribution of
88
Sr/
44
Ca and
138
Ba/
44
Ca ratios according to individuals within each tissue. Scatter plots showing distribution, by individual and by
tissue. Bone is compared against pig (
Sus scrofa f. domestica
). F=female; M=male; i=indiferent individual.
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Anna Pankowska, David Milde, Jana Bohunská: Variation of Ba/Ca and Sr/Ca Response in Human Hard Tissue from Archaeological Series
165
of both ratios (
88
Sr/
44
Ca and
138
Ba/
44
Ca) was signifcant within
each tissue, but less signifcant in enamel. The positive
correlation, however, shows that all tissues followed similar
processes (biogenic and diagenetic) as indicated by both
ratios. Furthermore, the intensities of the isotopes classifed
individuals into clusters and it is apparent that the isotope
signals refect more an individual’s incorporation of minerals
and absorption of elements from their diet rather than their
general dietary pattern. Although the ratios are lower in
dentin, they are similarly distributed in all individuals,
similar to that in bone. In both cases, individual 229 stands
out, with high values of both
88
Sr/
44
Ca and
138
Ba/
44
Ca ratios,
caused by a poorer Ca signal when compared to individuals
374, 194 and 107. All have similar intensities of
138
Ba and
88
Sr. Individuals 230 and 196 are anomalous in their signal
for dentin. Individual 230 shows a decline of
44
Ca and
88
Sr
over time, possibly caused by a variable deposition of the
elements.
44
Ca was low in both individuals (lower than in
individual 229). Individual 230, however, showed high
88
Sr
values. Traces of Ba were comparatively very low in both
individuals. The growth of
88
Sr and decline of
44
Ca may also
refect some contamination. However, when we measured the
elements’ concentration with pXRF in all samples, the Ca/P
ratio was 2.20: meaning the skeletons were not infuenced by
contamination during their deposition.
We suppose that the reason for the variation in isotopic
signals is each individual’s incorporation of minerals into their
hard tissue. Recent studies (Gonzalez-Rodriguez, Fowler
2013; Perrone
et al.
2014) have used X-ray fuorescence
spectrometry (XRF) for discriminating human remains by
analyzing the element concentration in bones. During one
individual’s life the bones are sculpted by process modelling,
which allows the formation of new bone. Most of an adult’s
skeleton is replaced every 10 years or so (Currey 2002).
During remodelling, new minerals are incorporated into the
formation of bone and partially into dentin. However, the
dentin mineral is remodelled to a much lesser extent and
the remodelling is under cellular control (Boskey 2007).
Enamel, by contrast, does not remodel itself and retains the
isotopes of elements incorporated at the time of its formation.
Individual skeletal mineral composition is associated with
uptake from the diet, water, metabolic function, inhalation,
and environment exposure of an individual, as well as
with specifc tissue formation during development
in utero
(Dolphin
et al
. 2005). Darrah (2009) has shown that trace
element incorporation occurs systematically according to
predictable physiochemical parameters. Elements that are
not strongly afected by metabolic activity (
e.g.
Ba and Sr)
may be directly linked to individual dietary inputs. Trace
elements difer in relation to individuality and may thus be
also useful for forensic studies (Perrone
et al.
2014).
In addition, diagenetic processes also afect the element
concentration individually. Barium and strontium are located
in group II of the periodic table along with calcium. These
elements substitute for calcium in hydroxyapatite, the
mineral component of bone. LA ICP-MS is, however, well
suited to detect contamination (Prohaska
et al.
2002). Its
high spatial resolution makes it possible to analyse isotope
ratios in selected regions of the teeth and detect altered zones.
Contamination may come from, for example, a crack in the
tooth or from many other factors (Hedges 2002). With the
ablation, a signifcant drop of intensities in individual 230
was recorded and the value discarded. Contamination can
be ruled out as a cause of the variability as it would have
to infuence all the isotopes in the same way to preserve the
correlations we presently observe. The correlation between
the ratios does not imply just a degree of bio-purifcation but
also the exposure to diagenetic processes. Diagenesis alters
Figure 6.
Linear discriminant analysis (LDA) scatter plot. Scatter plot shows clusters of individuals according to
88
Sr/
44
Ca and
138
Ba/
44
Ca ratios in hard
tissue; n=25 measurements (in six individuals).
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Anna Pankowska, David Milde, Jana Bohunská: Variation of Ba/Ca and Sr/Ca Response in Human Hard Tissue from Archaeological Series
166
bone ratios towards an equilibrium with soil ratios, so there
is no guarantee that the bone Ba/Ca and Sr/Ca ratios remain
correlated (Burton
et al
. 1999).
Another issue is the diferences in mineral absorption by
hard tissue. The intake of elements in enamel is lower than
in dentin and bones (Eggins
et al.
2003). The intake of Ca
increases during mineralization at the expense of Ba and Sr
so that these latter two are less represented in enamel and
dentin (Balter 2004). Though tooth tissue is resistant due to its
high mineralization, and conserves ontogenetic information,
it can show higher chemical variability depending on the
changes over a person’s lifetime. Element distributions
in one tooth may be more variable than between several
teeth of one individual. This is caused by the chronology
of individual development, the degree of mineralization
in diferent periods of a person’s life, their diet, stress and
diseases (Dolphin
et al.
2005). The chemical composition
of dentin is largely infuenced by the life-long deposition
of elements, and related to gender and age (Kumagai
et al
.
2012). As well as diferences in Sr concentrations having
been observed between the sexes (Li
et al
. 2013), positive
correlations have been observed between Sr concentrations
and an individuals’ age (Kumagai
et al.
2012).
Another signifcant factor is the analysis of the tissues, and
the ability to distinguish between natural isotopic composition
and numerical values infuenced by methodology. Laser
ablation ICP-MS measurement is infuenced by unequal
ablation and by the diferential vaporization of isotopes in
plasma. Because of the values of certain isotopes depended
to a certain extent on the time of measurement, the signal
could be variable during measurement, dropping in any
particular moment due to inhomogeneity. However, such
anomalies were deleted. Nonetheless, signals of certain
isotopes fuctuated over time in individuals. In dentin, the
signal remained relatively constant over time, whilst in
enamel it fuctuated more strongly, so that enamel sufered
major deviations in its record. Fluctuations in enamel may
be caused by the inhomogeneous distribution of elements
in the material, and by its hardness infuencing the rate of
vaporization.
5. Conclusion
This study shows that there is high variability in the isotopic
response of skeletal tissue. Great care must be taken when
interpreting isotope measurement results to reconstruct diet
and geographic origins. Many other factors, such as biogenic
processes, diagenetic alteration and ablation have been shown
to bias the data. Our results indicate that isotope signals in
human hard tissue serve rather better as a chemometric tool
than as a tool for dietary pattern reconstruction. Furthermore,
enamel deviates isotopically from bone and dentin. The
reason for this deviation may be the inability of enamel to
be remodelled during an individual’s life: we are thus unable
to detect individual metabolic function, diet pattern, and
mineral incorporation. The enamel also records the episodes
of growth and development of an individual, as these episodes
are characterized by variability in their exposure to chemical
elements. Stress and diseases can leave typical traces in
enamel in the form of defects and undermineralization.
Another reason for this enamel deviation may be due to
its hardness and the “noisy” signal of its isotopes during
ablation. Furthermore, enamel is also not immune to the
diagenetic processes. It can be structurally and isotopically
altered just as much as other tissues.
The analysis of isotope ratios in human skeletal remains
must take into account the complex and unique chemical,
diagenetic, biogenic and ablative properties of diferent
tissue types. Without the careful integration of primary and
secondary corroborating evidence, signifcant unintended
bias in the interpretation of bioarchaeological data will result
from straightforward laser ablation-ICP-MS analysis.
Acknowledgement
The authors gratefully acknowledge the support by the
project POSTOC-16 (University of West Bohemia, Pilsen)
and Ministry of Education Youth and Sports of the Czech
Republic (projet LO 1305). We would like to thank Martin
Paulus and Arkadiusz Tajer from the Archaeological Centre
in Olomouc and Martin Moník for their help.
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