Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Abstract

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings. Video LinkThe video component of this article can be found at https://www.jove.com/video/58002/ 11 . This has made it a popular method for dietary and environmental reconstruction in archeological, paleoecological, and paleontological research.The period of tooth enamel formation is relatively short (years) and differs depending on the tooth being sampled. For humans, first molar enamel mineralizes between birth and 3 years of age, premolars mineralize between 1.5 and 7 years of age, second molars mineralize between 2.5 and 8 years of age, and third molars mineralize during adolescence, between 7 and 16 years 12 . Given that tooth enamel forms incrementally over its period of formation, it can be sampled in bulk along the entire growth axis or sampled sequentially in order to investigate the changes in diet and environment that have occurred during the formation period 13 . Chronologically-ordered dietary change within a given tooth is observable for humans and other animals 1,14 , providing information regarding inter-annual seasonal and dietary variation.While enamel is usually resistant to diagenesis, isotopic modifications resulting from the burial environment are possible and have been observed 15,16 , making experimental checks and pretreatment choices useful. While it is not the only available method, Fourier transform infrared spectroscopy (FTIR), particularly in Attenuated Transmission Mode, has emerged as a quick, inexpensive, and relatively accessible method for assessing taphonomic alteration in tooth enamel, particularly in paleontological contexts 17,18,19,20 . However, detailed protocols and recording standards remain relatively inaccessible to many people outside the fields of geochemistry or material science.

Isotopic evidence for human mobility in late antique Bulla Regia (Tunisia)

“…Enamel carbonate pre-treatment took place at BioArCh at the University of York following the method of Miller et al (2018) . Powdered dental enamel was pre-treated by adding 0.1 ml of 0.1 M of acetic acid per mg of enamel to each sample for 10 min.…”

Osteological, multi-isotope and proteomic analysis of poorly-preserved human remains from a Dutch East India Company burial ground in South Africa

“…As the interest of this research focuses local vs. non-local origins, the first (M1) and second (M2) molars were selected for sampling. During amelogenesis of these teeth, the individual's dietary intake containing strontium, oxygen and carbon isotopes representative of their geographical environment and diet, are incorporated into the enamel’s calcium hydroxyapatite (HAP, chemical formula; Ca 10 (PO 4 ) 6 (OH) 2 ) 64 – 69 . Strontium can be incorporated into HAP through substitution at the calcium (Ca) sites, while oxygen and carbon can be incorporated via structural carbonate (–CO 3 ) substitution at both –PO 4 and –OH sites 64 , 70 .…”

“…Then, approximately 10 mg of enamel powder was removed in a band across the height of the crown (from the cementoenamel junction to the occlusal surface) using a Dremel rotary tool with diamond burr accessories. Initial indicators of enamel preservation were considered at various stages of sampling by assessing hardness of tooth enamel, response of enamel to drilling, and colour of raw enamel powder 69 . As all drilled teeth had well-preserved enamel, and the duration of interment was expected to be no more than 300 years, no further preservation checks (i.e., FTIR or FTIR-ATR) were deemed necessary.…”

Sample Preparation Methods Used at the Australian National University Radiocarbon Facility

“…>0.8% C and unexpected δ 13 C values may indicate severe contamination (Wood et al 2021), and so IRMS is undertaken on the cleaned enamel. FTIR, a technique routinely used to assess preservation in the fields of δ 13 C and δ 18 O (Miller et al 2018), is Sample Preparation at ANU 14 C Facility 581 also undertaken although it is not yet possible to identify which indicies provide the best indication of exogenous carbonate contamination. Calcite has never been observed in the tooth enamel dated, and brushite formed during acidification is rare.…”