Received: 30 September 2022 Revised: 10 March 2023 Accepted: 13 March 2023

DOI: 10.1002/xrs.3344

RESEARCH ARTICLE

Elemental characterization of a Puma concolor fossil by

non-destructive X-ray techniques

C. M. S. F. F. dos Santos 1 | C. R. Moraes-Neto 2 | O. M. O. Araújo 1 |

1 3 3 2
A. S. Machado | R. S. dos Santos | M. J. Anjos | L. O. Salles |
D. F. Oliveira 1 | R. T. Lopes 1

1
Nuclear Instrumentation Laboratory,
COPPE, Federal University of Rio de
Janeiro, Rio de Janeiro, Brazil
2
Vertebrate Departament (Mastozoology),
National Museum, Federal University of
Rio de Janeiro, Rio de Janeiro, Brazil
3
Physics Institute, State University of Rio
de Janeiro, Rio de Janeiro, Brazil
Correspondence
C. M. S. F. F. dos Santos, Federal
University of Rio de Janeiro, Federal
University of Rio de Janeiro, Rio de
Janeiro, Brazil.
Email: caio.santos@coppe.ufrj.br
Funding information
Conselho Nacional de Desenvolvimento
Científico e Tecnol

ogico; Coordenação de
Aperfeiçoamento de Pessoal de Nível
Superior
Abstract
Over the lifetime of an animal, metabolic processes in the body maintain bone
tissues in a dynamic state of equilibrium. After the death of the organism, bone
tissues undergo diagenesis, an intense transformation that affects its physico-
chemical constitution. Although many recent works have greatly contributed
to the understanding of the processes surrounding diagenesis, there is still
much yet to be understood about this multifaceted phenomena. In this work,
XRF and microCT were used to characterize the specimen elemental configu-
ration and investigate the fossilization process of a recovered Puma concolor
(cranium, mandible and teeth elements), found submerged in a lake in the
Impossível-Ioiô Cave System (Bahia, Brazil). MicroCT enabled the identifica-
tion of the density distribution throughout the specimens, which were used as
reference to XRF analyses. The XRF spectra of the analyzed specimen showed
the presence of 11 elements: P, Cl, Ca, Mn, Fe, Cu, Zn, Br, Sr, Y and
Hg. Yttrium counts proved to be regular throughout the specimen surface and
decreased in the interface between bone surface and interior, evidencing their
correspondence to sample environmental exposure. Furthermore, a correlation
between fossil surface coloration, element configuration and density is pre-
sented. The relatively low presence of Fe and Y in the enamel elicits its ele-
mentary and structural preservation. The combined results contribute to the
understanding of the physicochemical process of paleomastozoological fossili-
zation, further enriching the comprehension of the process of fossilization in a
submerged environment.

KEYWORDS
diagenesis, fossil, microCT, XRF, undestructive analysis

1 | INTRODUCTION minerals for ionic homeostasis.1–3 As a main constituent

of the skeleton, the bone is a highly complex and dynamic
The skeleton provides structural and mechanical support tissue, with hierarchical organization that spans through
to vertebrate animals, contributes to protection and move- several length scales.3–7 Bone is composed of organic
ment and is a reservoir of calcium, phosphorus and other (mostly collagen) and inorganic constituents. The

X-Ray Spectrom. 2023;1–9. wileyonlinelibrary.com/journal/xrs © 2023 John Wiley & Sons Ltd. 1

2 dos SANTOS ET AL.
inorganic part of bone consists mainly of carbonate-
substituted hydroxyapatite [Ca10(PO4)6(OH)2] crystals,
and water.4–7
Over the lifetime of an animal, metabolic processes in
the body maintain bone tissues in a dynamic state of
equilibrium.2,6 After being removed from an organism,
bone tissues undergo diagenesis, a transformation
derived from numerous biological, chemical and physical
processes that occur between bone and environment and
are affected by several parameters (e.g., pH, temperature,
microbiological, soil and water compositions).2,7 During
this process, most organic elements are removed, giving
room to chemical composition changes, as inorganic sub-
stances deposit in the new available voids.2,8 Addition-
ally, the inorganic material is altered through a variety of
chemical processes, depending on environmental condi-
tions, resulting in further elemental constitution
changes.2,8–10 Hydroxyapatite structure can be altered by
a variety of processes, such as calcium substitution by
rare earth elements or trace metals (e.g., Fe, Mn, Zn, Sr,
Mg and others), recrystallization, or replacement of car-
bonate ions in the crystal lattice.11–16
Bone, dentine and enamel share chemical similarities,
but differ significantly in micro- and submicro-
architecture and porosity. Compared to bone, enamel has
larger crystals, lower collagen and porosity.7,8,14,17
Because of these differences in physical properties,
enamel is a thermodynamically more robust tissue and
less susceptible to be transformed during diagenesis com-
pared to bone8,14,17 Therefore, enamel is the mineralized
tissue that better retains initial chemical properties of the
organism, making it a fine candidate in palaeoecological,
palaeoenvironmental, palaeoclimatic and paleodietary
investigations.7,14,17–20
In paleontology and related fields, specimen preserva-
tion is of utmost importance, and the use of techniques
capable of probing without damaging samples is increas-
ingly common. Among the techniques used in paleonto-
logical studies, X-ray fluorescence (XRF) stands out. As a
multielemental nondestructive analytical technique, XRF
has been used to characterize the elemental distribution
of fossils, providing valuable information on the fossiliza-
tion processes by mapping the element configuration
throughout the surface of the studied material. The pres-
ence of each element can be evaluated either quantita-
tively or through qualitative analysis, allowing
researchers to unveil taphonomic and preservation infor-
mation of fossilized materials as well as facilitate fossil
detection.21–23 On the other hand, non-destructive imag-
ing techniques such as X-ray computed microtomography
(microCT) have also been used in combination with XRF
in several studies.24–28
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Without requiring special sample preparation, the
imaging process of a microCT experiment starts with the
acquisition of a series of 2D projection images, acquired
as the sample is rotated around a stationary axis. Mathe-
matical methods are then applied in this series of projec-
tions to digitally reconstruct the sample, resulting in a
tridimensional image where the voxels represent the
attenuation of the material in that point. This attenuation
is primarily caused by X-ray absorption, which is intrinsi-
cally related to material density; hence, the contrast in
the grayscale reconstructed images is given by the differ-
ence in density in the sample. This allows the evaluation
of the relative density of samples and their internal mor-
phology in the micron scale,24–27 which is of particular
importance in the evaluation of diagenesis transforma-
tions since the process significantly alters the physico-
chemical characteristics; and morphological properties of
bone, for instance, the porosity on bone surface (BS) and
the appearance of fractures in the enamel that facilitate
environment material deposition.28
In this work, XRF and microCT were used to charac-
terize the specimen elemental configuration and investi-
gate the fossilization process of a recovered Puma
concolor (cranium, mandible and teeth elements), found
submerged in a lake in the Impossível-Ioiô Cave System
(12
23'13.56''S, 41
32'57.48''W).
2 | MATERIALS AND METHODS
2.1 | Puma concolor skull
The analyzed specimens in this work are the cranium,
mandible and dental elements of a Puma concolor
(Linnaeus, 1771) fossil material, deposited in the Verte-
brate Department, Mammals Sector of the National
Museum of the Federal University of Rio de Janeiro,
Brazil, and registered under the number MN 57461. This
specimen was recovered from a submerged deposit on
the karstic region of Iraquara (Chapada Diamantina,
Bahia, Brazil) at the Impossível-Ioiô Cave System. The
material was collected under submerged conditions by
hand with the use of surgical gloves and plastic zip bags.
Technical procedures for the preparation of the material,
as well as a thorough description of all collected bones of
the specimen, can be found in a previous work [29].
Through the analysis of the preserved parts of this fossil
material, it is possible to ascertain that the specimen was
found in an excellent state of preservation, with little
influence of external factors, given the condition of a sub-
aquatic cave. In this analysis, we selected the cranium,
mandible and dental elements since it is believed that
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dos SANTOS ET AL. 3

these pieces fully represent the condition of fossil preser-
vation of this specimen.
2.2 | MicroCT analysis
MicroCT images were acquired using a phoenix vjtomejx
m system, from General Electric (GE), equipped with a
microfocus X-ray tube and a 2024  2024 pixels digital
detector array. Cranium, mandibles and a canine tooth
were scanned separately, and a corresponding polystyrene
holder was used to better accommodate each sample dur-
ing scans. Operational setup for voltage and current dur-
ing scans were 80 kV and 320 μA, respectively. In order to
pursue the best possible voxel size for each sample while
still scanning the whole sample, the number of projections
and voxel size were different for each scan. The number
of projections were 2100, 1600 and 1200 for the canine
tooth, mandibles and cranium, respectively, with corre-
sponding isotropic voxel sizes of 108, 80 and 31 μm. For
each projection, an average of 3 frames was taken, with
500 ms exposure time per frame. All scans were
performed with full sample rotation (360
). To minimize
the occurrence of beam hardening artifacts due to low-
energy photons, a 0.3 mm copper filter was used. Figure 1
shows the reconstructions of the cranium (Figure 1a–c),
mandible (Figure 1d) and canine tooth (Figure 1e).
After scanning, the images were reconstructed using
datosjx reconstruction software (GE), version 2.5, result-
ing in 16-bit datasets. VGStudio Max was used for visuali-
zation and analysis of the samples. Analyzing the
internal volume of the cranium, it was possible to assess
the preservation state of the fossil.
Avizo software (version 2021) was used to generate a
heatmap image based on the grayscale histogram of the
cranium reconstruction. MicroCT images were then used
to map areas of interest for XRF analyses.
2.3 | XRF analysis
The X-ray fluorescence spectra were acquired from differ-
ent regions of the samples using an ARTAX 200 system
(Bruker), equipped with a Mo target X-ray tube of

F I G U R E 1 The 3D reconstructions of all samples used in this work are shown: (a) cranium surface on a superior view, along with two
cross sections, showing in (b and c) the well preserved state of the bone interior, (d) mandible reconstruction, where the pink arrow
indicates region of interface analyses and (e) the canine tooth, where XRF measurements were performed both in enamel and dentine (blue
and yellow arrows, respectively). The color was added in VGStudio to improve the visualization of details in the images.
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4 dos SANTOS ET AL.

maximum voltage and current of 50 kV and 1 mA,

975 ± 139
739 ± 193
respectively, and a Flash Silicon Drift Detector (SDD),
with an energy resolution of 145 eV for the Mn-Kα

ND
ND
ND
ND
ND
Hg
energy peak. The measurement area is 500 μm2. For
alignment of the incident X-ray beam and detector, the

18,947 ± 1814
16,379 ± 1899
7934 ± 1121

6518 ± 1016
2548 ± 483
system has a laser and CCD camera with resolution of

668 ± 263
500  582 pixels. In this work, the X-ray tube operated at

486 ± 7
40 kV and 500 μA voltage and current, respectively, with

Y
an acquisition time set to 300 s for each point.

2193 ± 252
2172 ± 248
3627 ± 392
1662 ± 403
2918 ± 150
3021 ± 104
2906 ± 133
Using the microCT images as a reference, regions
could be selected by density differences throughout the
cranium and mandible matrix for XRF. Measurements of

Sr
a canine tooth were performed in both dentine and

815 ± 105
enamel regions. The mandible was connected when col-

525 ± 65
469 ± 89

727 ± 79
894 ± 48
lected and was disconnected during curatorial proceed-

ND
ND
Br
ings. To characterize the elemental difference among
different exposure regions, several points were analyzed

1557 ± 122
1289 ± 206
1326 ± 178
1503 ± 133
1505 ± 118
1559 ± 117
1454 ± 152
along the interface between the internal region of the
mandible and the surface of the sample.

Zn
A total of 108 points were measured in the cranium,

Intensities (number of photons detected) of the analyzed regions (mean ± Standard deviation, nMIN = 5).
mandibles and canine tooth regions, with a minimum of

491 ± 44

409 ± 66
426 ± 21
441 ± 60
n = 5 measurements per region. The acquired XRF spec-

ND
ND

ND
tra were analyzed using PyMCA open-source software.30

Cu
The average intensities were obtained through deconvo-

34,885 ± 4448
lution of the spectra, and are reported as the counts of

4582 ± 1148
2426 ± 306

2483 ± 171
2478 ± 184
2340 ± 273
the Kα peak of each element (also Lα for Hg).

551 ± 64
Fe

3 | R E SUL T S
5278 ± 1248

1324 ± 284
498 ± 105
382 ± 31
493 ± 51
493 ± 26
The XRF spectra of the cranium, mandibles and canine
Mn

ND

tooth regions showed the presence of 11 elements: P, Cl,

Ca, Mn, Fe, Cu, Zn, Br, Sr, Y and Hg. Table 1 shows the
169,497 ± 11,810
130,455 ± 20,909
120,707 ± 14,263
184,106 ± 28,209
217,575 ± 20,127

intensities of the analyzed regions. 196,712 ± 12,349

200,233 ± 7733

The visual inspection of the fossil showed significant

differences in the BS coloration. Most of the BS shows a
lighter yellow-like color, whereas a brown to dark-brown
Ca

coloration could be observed in some regions (Dark Bone

Surface—DBS), as shown in the photograph (Figure 2a).
391 ± 36

As described in a previous work,29 the water flux inside

ND
ND
ND
ND

ND
ND
Cl

the cave is slow enough not to cause abrasion to the skel-

eton, so this hypothesis was discarded as a possible expla-
1334 ± 232
1305 ± 170
2305 ± 288
3833 ± 381
1870 ± 73
2356 ± 49
2354 ± 86

nation to the bone coloration. The microCT

reconstruction evidenced the difference in density
P

throughout the sample surface, which can be observed in

Abbreviation: ND, not detected.

Figure 2b, where the original gray scale reconstruction

Dark Bone Surface (DBS)

was used in the 3D rendering of a heatmap image, depict-

ing lower density regions in blue-green and higher den-
Interface-A (Int-A)

Interface-C (Int-C)
Interface-B (Int-B)
Bone Surface (BS)

sity in orange-red. The observed differences in MicroCT

Dentine (D)

correspond to the coloration observed on the sample sur-

Enamel (E)
TABLE 1

face, with dark-brown and light yellow-like regions in

Region

the photograph corresponding to higher and lower den-

sity areas of the BS, respectively.

dos SANTOS ET AL.
XRF measurements were made at different points
both in the BS and DBS throughout the cranium (light
yellow-like and dark-brown regions in Figure 2a, respec-
tively), and the elements detected were P, Ca, Mn, Fe,
Zn, Br, Sr, Y and Hg, as shown in the spectra of
Figure 2c. As XRF analyzes were not performed in vac-
uum, Ar is present in all acquired XRF spectra.
As expected, the bone is rich in hydroxyapatite con-
stituents (P and Ca) and essential trace elements in ani-
mal nutrition (Mn, Fe, Zn, Br and Sr).13,31,32 Strontium is
present in animals in vivo as Ca substitute in the bone
crystal lattice at low levels and may be retained in bones
on Holocene time scales.17 However, strontium incorpo-
ration is also characteristic of diagenetic change.17,28,33–35
Hg, on the other hand, is more commonly related to envi-
ronmental contamination from polluting agents,36–38
which is probably the case of the water where the fossil
was found. As it is not present in the dentine or enamel,
its presence along the BS is not likely due to the animal
diet, as reported in the literature.39 Analysis of BS and
DBS showed that most elements were present in the
same relative amounts (P, Ca, Zn, Sr, Y and Hg). How-
ever, significant differences were identified in the relative
counts of Fe (approx. relative ratio DBS/BS 7.6) and
Mn. In the BS region, the presence of artifacts interfered
F I G U R E 2 Analysis of the
cranium surface density. It is
possible to correlate the (a) fossil
photograph colors with the
(b) colored microCT image,
where blue-green and orange-
red pixels represent lower and
higher density regions in the
material, respectively. (c) XRF
spectra obtained from both
lower and higher density regions
(light and dark areas in (a),
respectively).
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5
with the accurate identification of the Kα line of Mn, thus
the element was not considered in the region since its
intensity peak is below the detection limit. The signifi-
cant higher presence of Fe and Mn, along with the dark
color on DBS, suggests that the staining is caused by iron
and manganese oxides, a common process during
diagenesis,12,15,40,41 as both elements can usually be
found in high concentrations in rivers and groundwater
as hydroxides and oxides.12
The microCT reconstruction of a canine tooth
extracted from the cranium is shown in Figure 3a (axial
view) and 3b (3D model), where it is possible to see the
difference in density between tooth dentine and enamel,
with the latter being the higher density part of the sam-
ples analyzed (brighter pixels), as expected. XRF analyses
were carried out on the loose canine surface, both in the
enamel and dentine (spectra shown in Figure 3c,d,
respectively). The enamel showed significant differences
in the fluorescence spectrum, marked especially by the
presence of Cl, a minor element found naturally in tooth
enamel and dentine.20,42 The presence of only Cl in the
enamel and the low intensity of Y there, indicate that
part of the elemental configuration of the enamel was
preserved, in accordance with previous surveys.7,14,17–
20,43,44
Furthermore, a comparison between the elemental

6 dos SANTOS ET AL.
compositions of dentine and bone showed both materials
share most elements, as shown in Table 1, which is
expected due to the chemical similarities between
them.7,44
The fossil was recovered with its mandible intact
(both parts connected) and was separated into its two
connected parts during curatorial procedures. Where the
parts were connected, the separation resulted in an inter-
face between BS, exposed to the liquid environment, and
the interior of the bone (Figure 4a,b), less subject to
external fluids. The analyzed areas are comprised of com-
pact cortical bones, which are less subject to alteration,
thus making them an adequate region to investigate dia-
genetic changes.44
Measurements were divided into three separate
regions in order to assess the different stages of diagene-
sis in the bone, as the probing area goes from the surface
into its interior. The same elements were detected in all
three regions, most with approximately the same relative
counts (Mn, Fe, Cu, Zn, Br and Sr), though with striking
differences in Y, Ca and P (Figure 4c). As the probing
moves from the BS BS to the inner regions of the bone,
Yttrium counts abruptly fade (Y counts of Int-A, Int-B
and Int-C represent 40%, 16% and 4% of that observed
in BS, respectively), while in contrast Ca and P increases.
P loss during early diagenesis is expected because of the
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F I G U R E 3 Tooth enamel
and dentine analysis: (a) 2D
cross-section of the tooth, where
it is possible to see the enamel
(brighter pixels) and
microcracks in the dentine,
which enable mineral
deposition during fossilization.
(b) 3D model of the tooth, with
a red dashed line highlighting
the position of the cross-section
shown in (a). (c) XRF spectrum
of the enamel, and (d) XRF
spectrum of the dentine.
incorporation of carbonate ions in place of the phosphate
ions in hydroxyapatite.8 Yttrium enrichment is character-
istic of diagenetic processes,28,34,35,45–49 as it can be incor-
porated in the apatite crystal by calcium substitution or
absorption. Deposition of Y in the bone porous surface
media (see red arrows in Figure 4d) can also occur, and is
more likely to happen in Holocene fossil materials, how-
ever the depletion of Ca accompanied by the increase of
Y on the surface points to the substitution of Y for Ca, in
accordance with reports found in the literature.28,50 The
presence of Y has been reported in natural waters,45,46
indicating that its incorporation to the BS was probably
enabled by its presence in the water where the fossil was
found, and the higher counts on the BS indicates the
environmental connection of the measured areas with
the water network.28 Figure 4d shows possible mineral
entries near the measured areas (left) and Figure 4e
shows the corresponding deposited minerals. Bone may
gain low levels of Yttrium throughout its life, which
could explain the relatively same amounts of the element
in both Int-C and the enamel. Furthermore, Sr, which is
also present in animals in vivo as a Ca substitute in the
bone crystal lattice, was found in the same quantities in
all three interface measured regions (Int-A, B and C),
whilst showing a small but notable difference from BS
and DBS, suggesting minor Sr depletion in the BS. The
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dos SANTOS ET AL. 7

F I G U R E 4 Analysis of the interface between bone surface (BS) and interior: (a) Four measured regions are marked as blue, red, green
and black squares, representing BS, Interface a, b and c, respectively, where the latter three represent three different steps towards the bone
interior. (b) Larger view of the mandible, where red dashed square represents the area visualized in (a) and the red dashed line marks the 3D
rendering cross-section viewed in (d) and (e). (c) The four spectra are shown, where the striking difference is in Y and Fe counts, with both
elements being significantly more abundant on the BS. (d) MicroCT renderization of a subvolume near the analyzed area, showing different
porous entries that facilitate mineral deposition (red arrows on the left) and (e) the corresponding minerals deposited with significantly
higher density than the bone matrix and dentine.

preservation of Sr is expected in bones from the Holo- 4 | CONCLUSION

cene, as bones have been reported to retain biogenic Sr in
this time scale. However, to actually ascertain if the
found Sr is biogenic or diagenic, it would require isotope
analyses.17 It is interesting to notice that the relative
counts found in the dentine differs strongly from those in
the BS and are the same, considering the deviation of the
Int-A region, as depicted in Figure 4a. For instance, Cu,
which is an element present in the diet of mammals,19
showed the same relative intensities in the spectra of BS
and all three interface regions (Table 1). This suggests
that although the dentine has suffered diagenetic
changes, it was not as strong as in the bone, in accor-
dance with previous works,7,44 and the elemental config-
uration of dentine is similar to the first shallower inner
layer of bone beneath the surface.
The combined use of microCT and XRF techniques pro-
vided valuable insights to the diagenetic changes the
specimen has sustained and its present elemental config-
uration. The nondestructive analysis of data obtained
through the techniques used in this work contributes to
the understanding of the physicochemical process of
paleomastozoological fossilization in its early stages,
which, within the studies developed in fossil materials,
demonstrates particularities in the process of fossilization
in a submerged environment.
A C KN O WL ED G EME N T S
This study was financed in part by Coordination for the
Improvement of the Higher Education Personnel

8 dos SANTOS ET AL.
(CAPES)—finance Code 001 (Brazil)—(grant
88887.476179/2020-00 to OOA) and the Brazilian Council
of Science and Technological Development—CNPQ. The
authors would like to thank the graphic designer Bianca
Gribel for her kind and detailed work with the final
image compositions.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are avail-
able from the corresponding author upon reasonable
request.
ORCID
C. M. S. F. F. dos Santos https://orcid.org/0000-0001-
7023-8119
C. R. Moraes-Neto https://orcid.org/0000-0002-3470-
3391
O. M. O. Araújo https://orcid.org/0000-0002-6287-0592
A. S. Machado https://orcid.org/0000-0002-7973-9611
R. S. dos Santos https://orcid.org/0000-0001-9443-9062
M. J. Anjos https://orcid.org/0000-0003-4227-2138
L. O. Salles https://orcid.org/0000-0002-5502-4718
D. F. Oliveira https://orcid.org/0000-0003-2038-1480
R. T. Lopes https://orcid.org/0000-0001-7250-824X
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How to cite this article: C. M. S. F. F. dos Santos,
C. R. Moraes-Neto, O. M. O. Araújo,
A. S. Machado, R. S. dos Santos, M. J. Anjos,
L. O. Salles, D. F. Oliveira, R. T. Lopes, X-Ray
Spectrom 2023, 1. https://doi.org/10.1002/xrs.3344