The development of tools and techniques used in paleoenvironmental studies is important for the advancement of geoarchaeological research. Geoarchaeological studies of ancient harbours involve the integration of paleoenvironmental data with archaeological findings, allowing for a more comprehensive understanding of site development and regional maritime trade. This dissertation highlights and/or explores novel applications of microfossils and micro-X-ray fluorescence (μXRF) core scanning data in paleoenvironmental reconstructions within three ancient harbour studies. Foraminifera, diatom, pollen, μXRF, carbon stable isotope, and radiocarbon dating results from underwater cave sediments on the eastern coast of the Yucatán Peninsula indicate that the region’s wetlands developed between 1157 BCE and 312 CE. Continued sea-level rise after ~312 CE allowed canoe access through a channel from the Caribbean Sea to Muyil, a Classic Maya maritime port site. Development of the wetlands may have implications for increased maritime trade on the Yucatán’s eastern coast during the Postclassic (925–1550 CE). This study demonstrates the importance of karst cave systems for obtaining paleoenvironmental records and provides a first assessment of the diatom community that has been transported into the sampled cave system. The novel application of epiphytic foraminifera as biostratigraphic indicators was investigated at Caesarea, Israel. Benthic foraminifera assemblages indicated that the emplacement of hard harbour structures along the sandy coast between 21–10 BCE altered the environmental conditions and impacted the nearby ecological communities. Epiphytic foraminifera, in particular Pararotalia calcariformata, were found to be useful indicators for the timing of harbour construction at Caesarea. μXRF and magnetic susceptibility results also supported the presence of increased harbour material (i.e., kurkar and volcanic ash) in offshore sediments over time. This study demonstrates that epiphytic foraminifera can be cost-effective biostratigraphic indicators in geoarchaeological studies. Results also confirm that P. calcariformata is endemic to Israel and is not a recent arrival following the opening of the Suez Canal. Foraminifera, testate amoebae, μXRF, and radiocarbon dating results from sediment cores collected from two lagoons, Khor Al Balid and Khor Rori, on the southern coast of Oman indicate that the lagoons closed off from the sea between the 12th and 15th centuries CE. Prior to lagoon formation, these two sites formed natural harbours that were the locations of major maritime trade ports. Progressive siltation and sand accumulation along the coastline after the 15th century likely impacted shipping activities, contributing to abandonment of the city near Khor Al Balid. Evidence of a marine overwash event across most cores from both sites dating to the 18th–19th century CE suggests that an extreme wave event (e.g., tsunami or large tropical cyclone) may have also contributed to the decline of the city. The results of this study provide an important paleoenvironmental context for previous archaeological findings. / Dissertation / Doctor of Philosophy (PhD)

This essay focuses on developing non-destructive methods to investigate the relationship between elemental distribution and bone porosity in archaeological bone. The skeletal material, which was analyzed, came from the archaeological sites of Skara (county of Västergötland), Varnhem (county of Västergötland), Sigtuna (county of Uppland) and Kopparsvik (county of Gotland). The essay is based on the results of a previous project, Osteoporosis och osteoarthritis, då och nu (Sten 2012). That project aimed at establishing whether medieval people, buried on the above mentioned archaeological sites, were suffersing from osteoporosis and/or osteoarthritis. This knowledge might help the medical research of today to solve the problem of possibly preventing those bone diseases. The method used was DXA-scanning, which was developed for examine osteoporosis in bone from living people. The result showed that the skeletons from the Skara site had an increased bone mineral density (BMD) compared to the skeletons from the other three sites. This essay investigates why these skeletal remains have a higher BMD and how this affects the results of methods like DXA. In this bachelor project various X-ray instruments were used to analyze the BMD of the skeletal remains. The X-ray pictures were then modified to exhibit high and low density areas in the bone. The elemental distribution of the surface area of the neck of the femur was examined with a μXRF-spectrometer. As a complement to the μXRF-spectrometer a SEM (scanning electron microscope) was used to analyze the elemental distribution of a cross section of the femur neck. Soil samples were collected from Skara and Varnhem and analyzed by using μXRF-spectrometry to find out if there was a correlation between the elemental content of the bone and surrounding soil. The skeletal remains from Skara exhibited increased values of iron and manganese combined with higher bone density. The soil from Skara showed a high level of particularly iron. This could be the reason for the increased BMD of the individuals from Skara when using the DXA-analysis. It is likely that, in each archaeological site, iron and manganese ions have diffused from both ground water and soil into the bones and thus increased the BMD. This is especially notified of the skeletal remains of Skara.

L'objectif de cette étude est d'étudier le potentiel d'Opuntia ficus-indica (Ofi) en tant que biomoniteur dans un environnement contaminé par des dépôts atmosphériques enrichis en métaux lourds. Trois sites d'étude pollués ont été sélectionnés: industriel, minier et routier. Le phénomène de bioaccumulation a été validé en analysant la concentration en métaux lourds par SAA et ICP-MS dans plusieurs raquettes d'âge différent, dans les fruits et les racines. L'identification de l'origine du Pb a été étudiée à partir de la signature isotopique du Pb (TIMS) dans les raquettes, les racines et les sols, avec une caractérisation microscopique (MEB-EDX) des particules déposées à la surface des raquettes. Le taux de bioaccumulation du Pb suite à une exposition racinaire ou foliaire à des particules de fluorapatite synthétisées et enrichies en Pb à permis de préciser les voies de transfert vers la plante. Ainsi, la distribution particulaire ou dispersée du Pb dans les raquettes contaminées a été identifiée au moyen de cartographies MEB-EDX et µXRF. Les résultats obtenus montrent qu'Ofi est un bioaccumulateur du Pb, Cd et Cr. Les signatures isotopiques dans les raquettes révèlent la nature des diverses sources polluantes tout en donnant une indication sur l'évolution de la pollution atmosphérique au cours du temps. Le taux le plus élevé de Pb a été mesuré dans les raquettes contaminées à leur surface par de la fluorapatite. Les analyses microscopiques ont démontré pour les deux approches suivies l'intégration des particules déposées à la surface des raquettes au niveau de la cuticule, des trichomes et des stomates. Cette obsevation est cohérente avec la cartographie obtenue en µXRF. Notre étude démontre le potentiel de cette plante cosmopolite qu'est Ofi en tant que biomoniteur. / This study reports the potential of Opuntia ficus-indica (Ofi) as a biomonitor of atmospheric pollution in three heavily polluted sites, i.e., industrial, mining and road sites. The bioaccumulation phenomenon by Ofi was investigated by studying the heavy metal concentrations in different cladodes of various ages, roots and fruits, using AAS and ICP-MS. To identify the origin of Pb in cladodes, two types of methods were used: TIMS analysis which provided the lead isotopes ratios in cladodes, roots and soil samples, and SEM-EDX analysis at the cladode surface which provided the elemental distribution of Pb and airborne particles and a characterization of their chemical structure at the microscopic scale. A second study was also carried out by testing Pb bioaccumulation after foliar and roots exposure to synthesized fluorapatite particles enriched with Pb. SEM-EDS and µXRF analyses were used to follow the Pb concentration from the contaminated surface to the internal tissues. The results show that Ofi is indeed a bioaccumulator of Pb, Cd and Cr. The Pb isotopic signatures indicate that Ofi is a sensitive species to local aerial pollution and their successive cladodes obviously identify the evolution of air pollution with time. In addition, Ofi cladodes were found more contaminated with lead after direct foliar contamination. SEM-EDS measurements underlined in the two studies the retention of deposited particles on specific zones of the cladode surface (cuticle, trichome and stomata), which were also identified by µXRF cartography. These results represent the first demonstration of the potential of using Ofi, a cosmopolitan plant, as a biomonitor.

La micro-algue verte Chlamydomonas reinhardtii est considérée comme un modèle dans l’étude du stress métallique chez les organismes photosynthétiques. Les mécanismes de tolérance au stress induit par le cadmium ne sont pas encore clairement établis. Afin de déterminer ces mécanismes, la localisation subcellulaire et la spéciation chimique in situ du cadmium ont été déterminées chez trois souches de C. reinhardtii exposées au cadmium en condition mixotrophe (CO2 + Acétate) : (i) une souche de type sauvage (wt), (ii) la souche cell-wall less (cw15) qui est déficiente en paroi cellulaire, (iii) la souche pcs1 qui surexprime la phytochélatine synthase (PCS), enzyme ordinairement cytosolique, directement dans son chloroplaste. Pour ce faire, la toxicité du cadmium a été déterminée en mesurant la croissance ainsi que la teneur en chlorophylle et en amidon des micro-algues. Puis, la localisation du cadmium au niveau subcellulaire a été réalisée par trois techniques complémentaires (fractionnement subcellulaire, µXRF, TEM/X-EDS). La spéciation chimique in situ du cadmium a été effectuée par µXAS et XAS. Enfin, l’imagerie élémentaire et isotopique par nanoSIMS a permis de compléter les distributions élémentaires dans la cellule et de déterminer l’impact du cadmium sur les mécanismes d’assimilation du carbone. (i) Les résultats de ce travail montrent que la souche wt est la plus sensible au cadmium des trois avec une diminution de la croissance et de la teneur en chlorophylle. Lorsqu’elle ne présente pas ces signes de toxicité, le cadmium est séquestré dans l’ensemble de la cellule par des ligands thiolés et de façon mineure par les granules de polyphosphates. Suite à l’exposition à de fortes concentrations en cadmium, le cadmium intracellulaire est lié majoritairement à des ligands carboxylés probablement induits par le stress oxydatif. De plus, la présence du cadmium dans le pyrénoïde bloque l’assimilation du carbone inorganique (CO2), au profit de l’assimilation du carbone organique (acétate), qui est stocké sous forme d’amidon. (ii) La surexpression de la PCS de la souche pcs1 provoque une production d’amidon importante autour du pyrénoïde et protège la chlorophylle du stress lié au cadmium. Bien que la synthèse de phytochélatines soit potentiellement élevée, la moitié du cadmium intracellulaire est séquestrée par les granules de polyphosphates et l’amidon. (iii) La souche cw15 est la plus tolérante des trois souches et n’accumule pas la totalité du cadmium disponible, contrairement aux cellules possédant une paroi cellulaire. Similairement au wt, le cadmium intracellulaire est séquestré majoritairement par des ligands thiolés et de façon mineure par les granules de polyphosphates. L’observation de granules de polyphosphates excrétées par les micro-algues permet l’hypothèse de l’excrétion du cadmium vacuolaire induisant un flux constant de cadmium à travers la cellule. En conclusion, la séquestration du cadmium via des ligands soufre, potentiellement par des polypeptides thiolés, est le mécanisme de tolérance majoritaire mis en place par C. reinhardtii. Néanmoins, la séquestration du cadmium par les granules de polyphosphates semble apporter une plus grande tolérance vis-à-vis du stress lié au cadmium. / The green micro-alga Chlamydomonas reinhardtii is commonly used as a model for the study of the metallic stress in photosynthetic organisms. Tolerance mechanisms against stress induced by cadmium are not well understood. In order to determine these mechanisms, subcellular location and in situ speciation have been determined in three C. reinhardtii strains exposed to cadmium in mixotrophic conditions (CO2 + Acetate) : (i) a wild type strain (wt), (ii) a cell-wall less strain (cw15) which is deficient in cell-wall, (iii) the pcs1 strain which overexpresses the cytosolic enzyme phytochetlatin synthase (PCS) directly in the chloroplast. Cadmium toxicity has been determined by the monitoring of growth and chlorophyll, starch content in micro-algae. Then, cadmium location at subcellular level has been performed using three complementary techniques (subcellular fractionation, µXRF and TEM/X-EDS). In situ cadmium speciation has been studied by µXAS and XAS. Finally, elemental and isotopic imaging by nanoSIMS has allowed to complete elemental distribution in the cells and to determine the impact of cadmium on the assimilation of carbon. (i) The results of this work show that the wt strain is the most sensitive strain to cadmium stress among the three studied strains with a growth and chlorophyll content decrease. When wt cells do not show signs of toxicity, cadmium is mainly sequestered in the whole cell by thiolated ligands and in polyphosphate granules. After an exposure to high concentration of cadmium, intracellular cadmium is mainly bound to carboxylated ligands, probably induced by oxidative stress. Moreover, cadmium located in the pyrenoid blocks inorganic carbon (CO2) assimilation and increases organic carbon (acetate) assimilation which is stored as starch. (ii) The overexpresssion of PCS in the pcs1 strain induces a strong production of starch around the pyrenoid and proctects the chlorophyll against cadmium stress. Although the synthesis of phytocheltins was potentially strong, half of the intracellular cadmium is sequestered in polyphosphate granules and in starch. (iii) Unlike cell-walled cells, the cw15 strain is the most tolerant strain and does not accumulate the totality of available cadmium. Similarly to wt strain, intracellular cadmium is mainly sequestered by thiolated ligands and in polyphosphate granules. The observation of polyphosphate granules excreted by the micro-algae allows the hypothesis of the excretion of vacuolar cadmium, inducing a constant flux of cadmium through the cells. In conclusion, cadmium sequestration by sulfur ligands, potentially by thiolated polypeptides, is the main tolerance mechanism implemented by C. renhardtii. However, cadmium sequestration in polyphosphate granules seems to allow a better tolerance against cadmium stress.