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The analysis of the nitrogen (N) isotopic composition of organic matter bound to fossil biomineral structures (BB‐δ15N) using the oxidation–denitrifier (O–D) method provides a novel tool to study past changes in N cycling processes. Methods: We report a set of methodological improvements to the O–D method, including (a) a method for sealing the reaction vials in which the oxidation of organic N to NO3− takes place, (b) a recipe for bypassing the pH adjustment step before the bacterial conversion of NO3− to N2O, and (c) a method for storing recrystallized dipotassium peroxodisulfate (K2S2O8) under Ar atmosphere. Results: The new sealing method eliminates the occasional contamination and vial breakage that occurred previously while increasing sample throughput. The protocol for bypassing pH adjustment does not affect BB‐δ15N, and it significantly reduces the processing time. Storage of K2S2O8 reagent under Ar atmosphere produces stable oxidation blanks over more than 3.5 years. We report analytical blanks, accuracy, and precision for this methodology from eight users over the course of ~3.5 years of analyses at the Max Planck Institute for Chemistry. Our method produces analytical blanks characterized by low N content (0.30 ± 0.13 nmol N, 1σ, n = 195) and stable δ15N (−2.20 ± 3.13‰, n = 195). The analysis of reference amino acid standards USGS 40 and USGS 65 indicates an overall accuracy of −0.23 ± 0.35‰ (1σ, n = 891). The analysis of in‐house fossil standards gives similar analytical precision (1σ) across a range of BB‐δ15N values and biominerals: zooxanthellate coral standard PO‐1 (6.08 ± 0.21‰, n = 267), azooxanthellate coral standard LO‐1 (10.20 ± 0.28‰, n = 258), foraminifera standard MF‐1 (5.92 ± 0.28‰, n = 243), and tooth enamel AG‐Lox (4.06 ± 0.49‰, n = 78). Conclusions: The methodological improvements significantly increase sample throughput without compromising analytical precision or accuracy down to 1 nmol of N. [ABSTRACT FROM AUTHOR]

Nitrogen isotopes are widely used to study the trophic position of animals in modern food webs; however, their application in the fossil record is severely limited by degradation of organic material during fossilization. In this study, we show that the nitrogen isotope composition of organic matter preserved in mammalian tooth enamel (δ15Nenamel) records diet and trophic position. The δ15Nenamel of modern African mammals shows a 3.7‰ increase between herbivores and carnivores as expected from trophic enrichment, and there is a strong positive correlation between δ15Nenamel and δ15Nbone-collagen values from the same individuals. Additionally, δ15Nenamel values of Late Pleistocene fossil teeth preserve diet and trophic level information, despite complete diagenetic loss of collagen in the same specimens. We demonstrate that δ15Nenamel represents a powerful geochemical proxy for diet that is applicable to fossils and can help delineate major dietary transitions in ancient vertebrate lineages. Enamel-bound nitrogen isotopes in modern and fossil mammal teeth preserve dietary and trophic level information. This novel geochemical archive can be used to reconstruct trophic niche in ancient ecosystems. [ABSTRACT FROM AUTHOR]

Nitrogen isotope ratios (δ15N) are a well-established tool for investigating the dietary and trophic behavior of animals in terrestrial and marine food webs. To date, δ15N values in fossils have primarily been measured in collagen extracted from bone or dentin, which is susceptible to degradation and rarely preserved in deep time (>100,000 years). In contrast, tooth enamel organic matter is protected from diagenetic alteration by the mineral structure of hydroxyapatite and thus is often preserved over geological time. However, due to the low nitrogen content (<0.01 %) of enamel, the measurement of its nitrogen isotopic composition has been prevented by the analytical limitations of traditional methods. Here, we present a novel application of the oxidation-denitrification method that allows measurement of δ15N values in tooth enamel (δ15N enamel). This method involves the oxidation of nitrogen in enamel-bound organic matter to nitrate followed by bacterial conversion of nitrate to N 2 O, and requires ≥100 times less nitrogen than traditional approaches. To demonstrate that δ15N enamel values record diet and trophic behavior, we conducted a controlled feeding experiment with rats and guinea pigs (n = 37). We determined that nitrogen concentration in tooth enamel (x̄ = 5.0 ± 1.0 nmol/mg) is sufficient for δ15N enamel analyses with ≥5 mg untreated enamel powder. The nitrogen isotope composition of enamel reflects diet with an enrichment (Δ15N enamel-diet) of ca. 2–4‰. δ15N enamel values differ significantly between dietary groups and clearly record a shift from pre-experimental to experimental diet. The small sample size required (≤5 mg) by this method permits analyses of sample size-limited, diagenetically robust tooth enamel, and, as such it represents a promising new dietary proxy for reconstructing food webs and investigating the trophic ecology of extant and extinct taxa. • Nitrogen isotope analysis of ≤5 mg enamel with 'oxidation-denitrifier' method. • Nitrogen isotopes in tooth enamel record dietary and trophic behavior. • Tooth enamel is enriched in 15N relative to diet by ca. 2 to 4 ‰. • Plant- and meat-based diets are clearly distinguishable in enamel δ15N. • Nitrogen isotopes can also be measured in fossil tooth enamel. [ABSTRACT FROM AUTHOR]