Name: sampling and pretreatment of tooth enamel carbonate for stable carbon and oxygen isotope analysis
Uploaded: 2018-08-15T14:30:00
Description: Watch this Scientific Journal Video about Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis at JoVE.com

We would like to thank the Max Planck Society for funding this research as well as the recent setting up of a Stable Isotope laboratory at the Department Archaeology, Max Planck Institute for the Science of Human History.

The following protocol follows the guidelines of the Light Isotope Mass Spectrometry Laboratory at the Max Planck Institute for the Science of Human History. Appropriate ethics permissions from national and international committees should be sought for analyses involving endangered modern or historical faunal specimens, and for the use of archaeological and faunal material of interest to contemporary stakeholders. In this paper, the samples used were archaeological and fossil specimens. No living humans were used in this...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isotopic+generation:+four+decades+of+stable+isotope+analysis+in+African+archaeology.">An isotopic generation: four decades of stable isotope analysis in African archaeology.</a> Azania: Archaeological Research in Africa. 51 (1), 88-114 (2016).</li><li>Ventresca Miller, A. R., Makarewicz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopic+approaches+to+pastoralism+in+prehistory:+Diet,+mobility,+and+isotopic+reference+sets.">Isotopic approaches to pastoralism in prehistory: Diet, mobility, and isotopic reference sets.</a> Isotopic Investigations of Pastoralism in Prehistory. , 1-14 (2018).</li><li>Hollund, H. I., Ariese, F., Fernandes, R., Jans, M. M. 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The challenges of successful sampling (bulk and incremental) of dentition relies on the access to knowledge regarding drilling techniques and sample preparation, alongside the investment in relatively inexpensive equipment. These challenges are easily surmountable when clear instructions are available concerning&#160;sampling and pretreatment approaches. In this article, we hope to have disseminated these in a clear, concise fashion for researchers new to these methods. Scholars applying these methods for the first time ...

Stable carbon and oxygen isotope analyses of tooth enamel carbonate has been used to study past human dietary intake, weaning, and mobility, as well as faunal reliance on vegetation, the movement of animals, and livestock foddering. These applications have been comprehensively discussed and reviewed for a variety of environmental conditions indicating the effects of local aridity, temperature, water sources, and vegetation compositions1,2,3,4,5,6. The diversity of potential applications in archaeology and paleontology, as well as the good preservation of tooth enamel carbonate, has made it an attractive material for stable isotope work3. Methods of sampling, pretreatment, and diagenesis screening are briefly described in a number of previous publications1,7. However, thorough verbal and visual demonstrations remain largely unavailable, particularly to people outside of archaeological science laboratories and among laboratory groups with limited funding where the interest in the use of this technique is increasing5.
Tooth enamel is primarily made up of hydroxyapatite (bioapatite) crystallites8 larger than those in bone, making it more resistant to post-mortem diagenetic ionic substitutions and contamination3. Modern studies have demonstrated that stable carbon isotope (&#948;13C) measurements of faunal tooth enamel reliably record animal diet and behavior9,10. The stable oxygen isotope (&#948;18O) value of tooth enamel is determined by the oxygen isotopic composition of ingested water, which includes water in plant and animal foods, drinking water, respiration, as well as various environmental impacts on the water which can lead to further isotopic fractionation (e.g., aridity, temperature, altitude, rainfall amount, continental location)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 years12. 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 period13. Chronologically-ordered dietary change within a given tooth is observable for humans and other animals1,14, 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 observed15,16, 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 contexts17,18,19,20. However, detailed protocols and recording standards remain relatively inaccessible to many people outside the fields of geochemistry or material science.
Reaction times and the chemicals employed by researchers in the pretreatment of tooth enamel also vary considerably in the literature, often with limited consideration as to what this variability may do to stable carbon and oxygen isotope values of the sample21,22. Here, we report an approach that uses dilute acetic acid (0.1 M) for the pretreatment of enamel powder samples. However, given that the differences in isotopic measurements resulting from pretreatment are relatively minor for tooth enamel, it is best for the researchers to follow the protocols for datasets with which they wish to compare their data to11. Furthermore, where small sequential samples are taken, particularly on Holocene samples, no pretreatment may be chosen (following pilot diagenetic tests) to avoid sample wastage.
Although the methods we report here are by no means new, to our knowledge, this is the first time that a thorough written and visual documentation of bulk and sequential sampling, pretreatment choices, and diagenetic check methods (in the form of FTIR) for tooth enamel have been made widely available to a varied academic audience. While we hope our efforts will make this approach more easily accessible to a wider number of individuals and laboratories, researchers who want to apply and publish this technique must be aware of minimum reporting standards, diagenetic considerations, and presentation requirements overviewed elsewhere20, as well as potential interpretive complexities that will be unique to their study region, taxa analyzed, and time period5.

<table>
	<tbody>
		<tr>
			<td>Dremel Micro</td>
			<td>Dremel</td>
			<td></td>
			<td>https://www.dremel.com/en_US/products/-/show-product/tools/8050-micro</td>
		</tr>
		<tr>
			<td>Diamond-tipped drill bit</td>
			<td>Dremel</td>
			<td></td>
			<td>https://www.dremel.com/en_US/products/-/show-product/accessories/7122-diamond-wheel-point</td>
		</tr>
		<tr>
			<td>1.5 mL micro-centrifuge tube</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>https://www.sigmaaldrich.com/catalog/product/sigma/t2422?lang=de&#38;region=DE&#38;gclid=EAIaIQobChMI7pHRpauW2QIV77ftCh1p1wjhEAAYASAAEgKzkvD_BwE</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td></td>
			<td></td>
			<td>Linear Formula:&#160;CH3OH</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td></td>
			<td></td>
			<td>Linear Formula:&#160;CH3CO2H</td>
		</tr>
		<tr>
			<td>Dremel rig set-up (workstation)</td>
			<td>Dremel</td>
			<td></td>
			<td>https://www.dremel.com/en_US/products/-/show-product/tools/220-01-workstation</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>http://www.thermofisher.com/order/catalog/product/75002401</td>
		</tr>
		<tr>
			<td>Mini-centrifuge</td>
			<td>Sprout</td>
			<td></td>
			<td>http://www.heathrowscientific.com/sprout-mini-centrifuge-4</td>
		</tr>
		<tr>
			<td>Freeze drier</td>
			<td>Zirbus Technology</td>
			<td></td>
			<td>http://www.zirbus.com</td>
		</tr>
	</tbody>
</table>

sampling and pretreatment of tooth enamel carbonate for stable carbon and oxygen isotope analysis

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.

Using the sampling procedure presented above, incremental enamel bioapatite samples were prepared. The analysis of bioapatite in enamel depends on the accuracy of sampling, whether bulk or incremental. In this case, we have chosen to present the results of archaeological samples (two sheep) from different climatic zones. Incremental samples were analyzed from sheep second molars and labelled starting at the ERJ (Figure 4). Incremental sample locations were nu...

Watch this Scientific Journal Video about Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis at JoVE.com

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

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

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