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Stable isotope analysis of bone and dental collagen is one of the most common methods to investigate the ecology of modern and extinct human and animal populations. However, since bone and dentine are composite materials with both organic and mineral components, the mineral component must be removed prior to analysis. In this study we investigated the timing and efficacy of mineral removal from bone and dentine. We performed a series of time-step experiments that show that mineral removal can be quantified over short periods of time using Fourier Transform Infrared Spectroscopy (FTIR), and collagen alteration can be tracked using a combination of stable isotope analysis and elemental analysis. We tested our methods on three modern materials: mammalian bone, mammalian dentine, and shark dentine. Our results show: 1) mineral removal is a necessary step, as structural carbonate has a strong influence on stable isotope compositions; 2) demineralization using weak acid (0.1M HCL) does not appear to alter the elemental and isotopic compositions of collagen. Our methods can be used as a framework to evaluate the need-for and efficacy of other demineralization methods in use today including EDTA-demineralization and lipid removal. 

Abstract Diet is a crucial trait of an animal’s lifestyle and ecology. The trophic level of an organism indicates its functional position within an ecosystem and holds significance for its ecology and evolution. Here, we demonstrate the use of zinc isotopes (δ 66 Zn) to geochemically assess the trophic level in diverse extant and extinct sharks, including the Neogene megatooth shark ( Otodus megalodon ) and the great white shark ( Carcharodon carcharias ). We reveal that dietary δ 66 Zn signatures are preserved in fossil shark tooth enameloid over deep geologic time and are robust recorders of each species’ trophic level. We observe significant δ 66 Zn differences among the Otodus and Carcharodon populations implying dietary shifts throughout the Neogene in both genera. Notably, Early Pliocene sympatric C. carcharias and O. megalodon appear to have occupied a similar mean trophic level, a finding that may hold clues to the extinction of the gigantic Neogene megatooth shark. 

Nitrogen isotope ratios in fossil teeth place extinct megatooth sharks at the top of the marine food web.