Matthieu Lebon

Imaging fossil bone alterations at the microscale by SR-FTIR microspectroscopy

Diagenetic alterations modifying fossil bones over geological time can limit their use as archaeological and paleontological proxies. The understanding of fossilization processes and the evaluation of the extent of diagenetic alterations of bones therefore constitute major issues in current research. The complex hierarchical structure of bone tissue and the spatial heterogeneity of the diagenetic alterations induce significant chemical variations in fossil bones at the microscale. We adapted a sample preparation procedure based on PMMA impregnation that allowed us to obtain bone thin sections independent of their level of degradation. As a first step to investigate the variations in bone composition at the histological scale, synchrotron radiation FTIR micro-spectroscopy (FTIRM) was applied on two fossil bone samples displaying distinct preservation states: (a) a bone from Magdalenian layers dated to 15 ka of the Bize-Tournal cave (Aude, France) and (b) another from Song Terus site (Java island, Indonesia, 60 ka). The first one is partially recrystallized but still contains zones with locally preserved collagen and biogenic carbonates, whereas the other consists of a recrystallized mineral fraction without any remaining collagen fraction. These data obtained on the microscale were compared to bulk measurement data and the relationships between different IR diagenetic parameters explored. This analytical approach allowed the characterization of diagenetic alterations such as collagen loss, carbonate uptake and mineral recrystallization in heavily altered fossil bone tissue at the histological scale. Using the presented procedure, the histological bone structures could be studied, even for brittle samples altered by an extensive loss of their collagen matrix during diagenesis.

Microscale imaging of the preservation state of 5,000-year-old archaeological bones by synchrotron infrared microspectroscopy.

AbstractArchaeological bone materials record characteristic markers of life in prehistoric times (dating, climate, environment, diet, human migration) in their isotopic and chemical composition in addition to palaeontological, archaeozoological, anthropological and palaeogenetic information. Thus, the discovery and conservation of archaeological bone materials is of great importance to get access to this information. However, archaeological materials are altered by different postmortem processes and it appears necessary to estimate if the archaeological information is still reliable or if it has been modified during burial. As archaeological bone materials present a high structural hierarchy at the micro- and nanoscale, changes induced by diagenetic phenomena have to be observed at these scales. One method for revealing post mortem changes of the bone structure and composition at the microscale is synchrotron radiation micro-FTIR imaging (SR micro-FTIR). Thus, thin sections of about 5,000-year-old archaeological bones have been analysed in transmission mode at the IRIS beamline (BESSY II, HZB Berlin) to determine markers of the state of bone preservation at the microscale. The archaeological bone material comes from station 19 of the Neolithic site of the Chalain Lake. By using SR micro-FTIR it was possible to image characteristic bone structures, e.g. osteons (the constitutive histological unit of cortical bone), using the absorption band ratios corresponding to different chemical bone constituents (collagen content and quality, phosphate crystallinity, carbonate content). These data allow us to precisely evaluate the state of preservation of a 5,000-year-old bone at the histological level. FigureChemical mapping of a thin section of the archaeological bone AB_CH19nb1 from the Neolithic station 19 at Chalain Lake

First use of portable system coupling X-ray diffraction and X-ray fluorescence for in-situ analysis of prehistoric rock art.

Study of prehistoric art is playing a major role in the knowledge of human evolution. Many scientific methods are involved in this investigation including chemical analysis of pigments present on artefacts or applied to cave walls. In the past decades, the characterization of coloured materials was carried on by taking small samples. This procedure had two main disadvantages: slight but existing damage of the paintings and limitation of the number of samples. Thanks to the advanced development of portable systems, in-situ analysis of pigment in cave can be now undertaken without fear for this fragile Cultural Heritage. For the first time, a portable system combining XRD and XRF was used in an underground and archaeological environment for prehistoric rock art studies. In-situ non-destructive analysis of black prehistoric drawings and determination of their composition and crystalline structure were successfully carried out. Original results on pigments used 13,000 years ago in the cave of Rouffignac (France) were obtained showing the use of two main manganese oxides: pyrolusite and romanechite. The capabilities of the portable XRD-XRF system have been demonstrated for the characterization of pigments as well as for the analysis of rock in a cave environment. This first in-situ experiment combining X-ray diffraction and X-ray fluorescence open up new horizons and can fundamentally change our approach of rock art studies.

Rapid Quantification of Bone Collagen Content by ATR-FTIR Spectroscopy

Expensive and time-consuming preparation procedures for radiocarbon and stable isotope analyses can be conducted on archaeological bone samples even if no collagen is preserved. Such unsuccessful preparation can lead to the partial destruction of valuable archaeological material. Establishing a rapid prescreening method for evaluating the amount of bone collagen while minimizing the impact of sampling constitutes a challenge for the preservation of archaeological collections. This study proposes and discusses a new methodology to detect and quantify collagen content in archaeological bone samples by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. A total of 42 Pleistocene to modern bone samples were selected according to their nitrogen content measured using an elemental analyzer. Comparison of collagen content estimation using ATR-FTIR and mass spectrometry reveals that some of the studied samples are contaminated by a nitrogen source coming from the burial environment. Two different FTIR calibration approaches were tested on the uncontaminated samples: peak-to-peak ratio and multivariate regression (PLS). The two approaches yield similar results with a good correlation of ATR-FTIR analyses and N wt% from 0.7 to 4wt% (R²=0.97–0.99; standard error of estimation ±0.22 to 0.25wt%). While collagen content remains difficult to detect in poorly preserved bones (less than ~3wt%), ATR-FTIR analysis can be a fast alternative for sample screening to optimize the sampling strategy and avoid partial destruction of valuable samples that do not contain enough collagen for further analysis.

Can we Use Calcined Bones for 14C Dating the Paleolithic

This work aims to test the reliability of calcined bones for radiocarbon dating of the Paleolithic. Fifty-five calcined bone samples coming from Aurignacian and Gravettian layers at Abri Pataud (Dordogne, France) were selected based on their macroscopic features. For each sample, the heating state was estimated on the basis of bone crystallinity (splitting factor [SF] using FTIR) and δ 13 C value. Twenty-seven bone samples (3 unburnt and 24 calcined) from 5 different levels were prepared for 14 C dating. The majority (15/24) of the calcined samples had to undergo a sulfix treatment prior to graphitization, probably due to the presence of cyanamide ion in these samples. The comparison between our results and recently published dates on bone collagen for the same levels shows that unburned bone apatite is systematically too young, while a third of the calcined bones fall within or very near the range of expected age. No clear correlation was found between 14 C age offset and δ 13 C value or SF. Most of the sulfixed samples (14/16) yielded ages that were too young, while almost all of the non-sulfixed samples (8/9) gave ages similar or 2 gas from well-calcined Paleolithic bones can provide reliable 14 C ages.

Non-Destructive Portable Analytical Techniques for Carbon In-Situ Screening Before Sampling for Dating Prehistoric Rock Paintings

Direct dating of prehistoric paintings is playing a major role in Paleolithic art studies. Very few figures can be directly dated since the necessary condition is that they contain organic carbon-based material. Thus, it is very important to check the presence of organic carbon-based material in situ before sampling in order to protect the visual integrity of the paintings or drawings. We have tested and compared 3 different portable analytical systems that can be used in cave environments for detecting carbon in prehistoric paintings: (1) a very compact X-ray fluorescence (XRF) system in Villars Cave (Dordogne, France); (2) a portable micro-Raman spectrometer in Rouffignac Cave (Dordogne, France); and (3) an infrared reflectography camera in both caves. These techniques have been chosen for their non-destructiveness: no sample has to be taken from the rock surface and no contact is made between the probes and the paintings or drawings. The analyses have shown that all the animal figures have been drawn with manganese oxides and cannot be directly dated by radiocarbon. However, carbon has been detected in several spots such as black dots and lines and torch marks. 14C results were obtained from 5 torch marks selected in Villars Cave, with ages between 17.1–18.0 ka cal BP. Three methods were used to identify carbon in black pigments or to confirm the presence of torch marks by carbon detection. Thanks to these new analytical developments, it will be now possible to select more accurately the samples to be taken for 14C dating prehistoric paintings and drawings.

Collagen Extraction and Stable Isotope Analysis of Small Vertebrate Bones: A Comparative Approach

Bone remains of small vertebrate fossils provide valuable information for paleoenvironmental and paleoclimatic reconstructions. However, direct radiocarbon dating of small vertebrates remains challenging as the extraction of sufficient good quality collagen is required. The efficiency of eight collagen extraction protocols was tested on seven samples, representative of different ages and burial environments, including both macro and small vertebrate taxa. First, the samples were prescreened using attenuated total reflectance–Fourier transform infrared spectroscopy (ATR-FTIR) to quantify collagen content in archaeological bones, revealing that one should be discarded for 14 C dating. Then, the quantity of protein extracted (yield) and collagen integrity were checked using conventional elemental analysis. The results show that one protocol was not able to accurately extract collagen from the samples. A soft HCl-based protocol seems more appropriate for the pretreatment of archaeological small mammal bones, whereas a harsher protocol might be more efficient to extract a higher amount of collagen from large mammals as well as amphibian bones. The influence of the tested protocols on carbon and nitrogen isotope values was also investigated. The results showed that isotopic variability, when existing, is related to the interindividual differences rather than the different protocols.

Direct Dating and Physico-Chemical Analyses Cast Doubts on the Coexistence of Humans and Dwarf Hippos in Cyprus

In the Mediterranean, the island dwarf megafaunas became extinct around the end of the Pleistocene, during a period of rapid and global climate change. In Cyprus, this coincided with the first human presence on the island, as attested by the rock shelter of Akrotiri-Aetokremnos where an Epipaleolithic anthropogenic layer (stratum 2) was found overlying a massive accumulation of pygmy hippopotamus (Phanourios minor (Desmarest, 1822)) [Boekschoten and Sondaar, 1972] bones (stratum 4). The relationship between the two layers is highly controversial and the role played by humans in hippo extinction remains fiercely debated. Here, we provide new, direct radiocarbon and physico-chemical analyses on calcined bones which elucidates the complex depositional history of the assemblage. Bone turquoise was identified using micro-PIXE analysis and depth-profiling together with Vis spectroscopy, demonstrating that these bones were not freshly burned. Bayesian modeling of the radiocarbon dates indicates that stratum 4 accumulated during the first half of the 13th mill cal BP and that calcination occurred several hundred years later. We conclude that accumulation occurred naturally during the beginning of the Younger Dryas and that Epipalaeolithic visitors subsequently used the bones as fuel, starting from the mid-13th mill cal BP. At that time, dwarf hippos were probably already extinct or at least highly endangered. Our results shed new light on the possible causes of hippo extinction, on the subsequent introduction of the wild boar and on the earliest occupation of the island by humans.