burned by the fire: isotopic effects of experimental …...1.1. enamel composition and previous...

Journal of Archaeological Science: Reports 34 (2020) 102593

2352-409X/© 2020 Elsevier Ltd. All rights reserved.

Burned by the fire: Isotopic effects of experimental combustion of faunal tooth enamel

Joshua R. Robinson a,b,*, John D. Kingston c

a Archaeology Program, Boston University, 675 Commonwealth Avenue, Suite 347, Boston, MA 02215, USA b Geography-Anthropology Program, University of Southern Maine, 300 Bailey Hall, Gorham, ME 04038, USA c Department of Anthropology, University of Michigan, 1085 S. University Avenue, Ann Arbor, MI 48109, USA

A R T I C L E I N F O

Keywords: Stable isotopes Archaeological fires Paleoecology Paleodiets African herbivores

A B S T R A C T

Paleoenvironmental reconstructions of hominin fossil localities and archaeological sites are a critical component of understanding the selective pressures involved in the biological and behavioral evolution of our ancestors. Increasingly, these reconstructions are based on the carbon and oxygen stable isotope (δ13Cenamel and δ18Oenamel) composition of herbivore tooth enamel from these sites. Although tooth enamel is subject to diagenetic processes, it is generally assumed to be a refractory substrate that retains the in vivo isotopic signal during fossilization. However, the effects of burning, for which there is extensive evidence, on the isotopic composition of herbivore tooth enamel is often overlooked. Here, the consequences of burning on δ13Cenamel and δ18Oenamel values of five African herbivore taxa are directly tested in a series of controlled combustion experiments up to 1100 ◦C. Results suggest that δ13Cenamel values are minimally affected by burning up to 700 ◦C for all taxa, and up to 1100 ◦C for all but the thin-enameled Madoqua (dik-dik). δ18Oenamel values, on the other hand, are significantly altered at temperatures beyond 300 ◦C for all taxa, with enamel becoming increasingly and systematically 18O depleted at higher temperatures. This is suggestive of a temperature-dependent biochemical process of exchange with at-mospheric oxygen. These results suggest that while δ13Cenamel values from burned teeth are potentially reliable for typical hearth temperatures, strong caution is needed when interpreting δ18Oenamel values from context with evidence of extensive burning.

1. Introduction

Paleoanthropological and archaeological studies are increasingly reliant on stable isotope analyses of fossil faunal and hominin tooth enamel for reconstructing past environments and dietary paleoecology. Unlike bone, enamel bioapatite has been shown to maintain in vivo biogenic signals into the deep past (Ambrose, 1990; Wang and Cerling, 1994; Lee-Thorp, 2002; Lee-Thorp and Sponheimer, 2003). Teeth, however, are not entirely resistant to diagenetic alteration (Kohn and Cerling, 2002; Kohn et al., 1999; Zazzo, 2014). Kohn et al. (1999) have shown that fossil tooth chemistry is controlled by both diagenetic in-clusion of secondary minerals at ~0.3% and chemical alteration of the bioapatite during fossilization, particularly in the hydroxyl group (OH) component of teeth. An additional source, and a possible catalyst for chemical alteration of enamel bioapatite, is the burning of faunal re-mains evident at many archaeological and paleontological sites (White, 1992; Shahack-Gross et al., 1997; Schiegl and Conard, 2006; Wadley

et al., 2011; Chazan, 2017). Fourier transform infrared spectroscopy (FTIR) of consolidated reddened sediment and the presence of Thermal Curve Fragments (TCF) from knapped material exposed to sources of high heat suggests that early hominins may have used fire as early as 1.5 Ma at Koobi Fora (Hlubik et al., 2017, 2019). In addition to anthropo-genic contexts, the burning of teeth and bone in paleoanthropological settings may also be the result of natural landscape fires.

Archaeological evidence and experimental studies have shown that bone chemistry is altered through cremation, food waste disposal, and cooking processes (Pate and Hutton, 1988; Ambrose, 1990; Lee-Thorp, 2002), with alteration of δ18Obone carbonate at ~250 ◦C (Munro et al., 2008) and ~675 ◦C for δ13Cbone carbonate (Munro et al., 2008; Huls et al., 2010; although see Snoeck et al., 2016). Despite these studies, little attention has focused on if and how the chemistry of tooth enamel, much more widely used as a source for paleodietary and paleoenvir-onmental interpretations, is affected by burning.

* Corresponding author at: Archaeology Program, Boston University, 675 Commonwealth Avenue, Suite 347, Boston, MA 02215, USA. E-mail address: [email protected] (J.R. Robinson).

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Journal of Archaeological Science: Reports

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https://doi.org/10.1016/j.jasrep.2020.102593 Received 20 June 2020; Received in revised form 4 September 2020; Accepted 8 September 2020

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1.1. Enamel composition and previous burning experiments

Tooth enamel is composed of ca. 98% highly substituted bioapatite with a general composition of Ca4(PO4)3(OH) (Driessens and Verbeeck, 1990). Carbon occurs in two components in enamel bioapatite, struc-tural carbonate (CO3) substituting for phosphate (PO4) (B site) and hydroxl (OH) (A site). Oxygen occurs in four sites in the bioapatite structure, as PO4 (as PO4 or HPO4), as CO3, and as OH (Driessens and Verbeeck, 1990). Relative to bone, enamel is less porous, has larger crystals and less organic matter, which are features that tend to limit alteration and exchange. Studies of the response of enamel to thermal stress indicate structural and chemical alteration, especially at temper-atures >600 ◦C (Beach et al., 2008; Karkhanis et al., 2009; Reyes-Gasga and Vargas-Becerril, 2019; Vargas-Becerril et al., 2019; Taylor et al., 2020). Similar to bone, there is a progressive heat related sequence of color change up to 1100 ◦C (yellow to brown to light gray mottled in the case of enamel) (Shipman et al., 1984; Karkhanis et al., 2009), accom-panied by fragmentation and fissuring along planes of weakness, such as the enamel-dentine junctions and crown cusp intersections (Taylor et al., 2020). Crystalline indices (CI) of enamel treated thermally to 1200 ◦C reveal an increase in CI (the volume fraction of the crystalline phase) due to the elimination of organic matter, expulsion of structural water, and an increase in crystal size (Shipman et al., 1984; Vargas- Becerril et al., 2019). Despite these changes in form and texture, SEM studies indicate that the characteristic topography of crystallites in enamel structure are still recognizable at 1100 ◦C (Karkhanis et al., 2009). As an example of the refractory nature of enamel, results from a study of strontium isotope ratios of cremated teeth from a Late Bronze

Age/Early Iron Age cemetery in Hesse, Germany indicate that the bioavailable strontium isotopic composition of enamel is preserved even after exposure to very high temperatures (Taylor et al., 2020).

In this study, we subject faunal tooth enamel to controlled combus-tion experiments in order to determine if δ13Cenamel and/or δ18Oenamel carbonate values are altered in the calcination process. If stable isotope values are altered by burning, understanding the chemical changes and isotopic consequences will allow for more accurate paleoecological in-terpretations at sites associated with extensive burning.

1.2. Burned faunal remains in archaeological contexts

The presence of burned faunal remains has often been noted from archaeological contexts, but only recently have detailed examinations of burned bone been conducted (Cain, 2005; Dibble et al., 2009; Clark and Ligouis, 2010). Burning in anthropogenic contexts is usually attributed to cooking and/or food preparation (Brain and Sillen, 1988; Gowlett and Wrangham, 2013; Wrangham, 2009; 2017), as well as the production of bone fuel (Costamagno et al., 2005; Thery-Parisot et al., 2005). Addi-tionally, Wadley et al. (2011) has suggested that modern humans during the Middle Stone Age at Sibudu in South Africa may have also engaged in regular burning for refuse disposal and removal of insects and other pests from living areas in caves. Burned bones and teeth are abundant in archaeological sites with burning even being a significant factor in whether a bone is identifiable or not due to increased friability and fragmentation (i.e., Stiner et al., 1995; Villa et al., 2004; Morin, 2012). At some sites, such as the Middle Paleolithic site of Lakonis I in Greece (Starkovich et al., 2020) and Sibudu (Clark, 2019) and Blombos Cave

Fig. 1. Example sampling schematic showing how teeth were sub-divided to produce 18 distinct longitudinal samples. Each sample provides a similar, if not the same, average of intra-tooth variation related to any dietary changes during crown formation. This example image depicts eight potential samples, with sample #8 removed and ready for combustion from the lingual aspect of a lower right third molar of an Oryx beisa.

J.R. Robinson and J.D. Kingston


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(Thompson and Henshilwood, 2011) in South Africa, it is common for ~40% or more of the identifiable bone assemblage in certain layers to reflect moderate to severe burning. While these studies do not report the proportions for burned dental remains separately, it is clear that teeth were also anthropogenically burned – either intentionally through cooking or as a byproduct of the disposal of food waste (Cain, 2005; Clark and Ligouis, 2010) – in the Paleolithic.

1.3. Characteristics of archaeological and natural fires

Anthropogenic fires in the paleoanthropological and archaeological records provide glimpses into controlled fire use, typically in the form of hearths. Reconstructing the characteristics of these fires in terms of maximum temperature and length of burning time, however, are diffi-cult processes. Microcontextual analyses can provide some evidence for the nature of anthropogenic fires (Gowlett, 2016; Aldeias, 2017; Gold-berg et al., 2017). Archaeological fires dated to ~1.0 Ma at Wonderwerk Cave in South Africa have been characterized by FTIR, which identifies characteristic peaks in the infrared spectrum caused by recrystallization under high temperatures, on bone and sediment heated to between ~400 ◦C and ~700 ◦C (Berna et al., 2012). At Qesem Cave in Israel, Karkanas et al. (2007) found evidence for heating of bone fragments in the Acheulo-Yabrudian occupation (~400–200 kya) to ~500 ◦C with FTIR. In order to characterize the variables involved in simple archae-ological hearths, Sievers and Wadley (2008) built a series of controlled fires constructed following evidence from both archaeological sites and ethnographic records. Their experimental fires were built with Euca-lyptus wood directly on flat sand without a rock or brick retaining wall. A maximum temperature of 670 ◦C was reached, but cooled in approxi-mately 15–20 min to a range of ~250 ◦C to 350 ◦C. It took six hours for the experimental fires to fall below 100 ◦C.

Further burning experiments (Wadley, 2009; Aldeias et al., 2016) have shown that the effect of fire on artifacts is a result of the temper-ature reached during combustion and the distance of the artifact to the fire. Artifacts placed directly in a fire are subject to substantial thermal

alteration, while those just outside, as little as 15–20 cm from the direct source of the fire, are largely unaltered (Stiner et al., 1995; Sievers and Wadley, 2008; Aldeias et al., 2016). Experimental open flat fires inten-ded to mimic natural landscape, or anthropogenic fires that become uncontrolled, reach maximum temperatures of up to 1000 ◦C and may last for days as long as fuel wood remains available (Stiner et al., 1995; Canti and Linford, 2000). While short-term flares in natural fires may reach higher temperatures (Dennison et al., 2006), these experiments are in general agreement with a measured maximum sustained tem-perature for wildfires of ~900 ◦C (Ice et al., 2004). Maximum temper-atures and length of burning time of both controlled anthropogenic and natural fires are determined by fuel type, with wood and sedges having higher maximum temperatures and longer fire durations than grass- based fires (Aldeias, 2017).

2. Methods

Modern tooth enamel from five African ungulate taxa – Connochaetes taurinus (blue wildebeest), Madoqua kirkii (Kirk’s dik-dik), Giraffa camelopardalis (giraffe), Equus quagga (zebra), and Phacochoerus africa-nus (warthog) – were heated in a Thermolyne Type 2000 Muffle Furnace. These specific taxa provided a range of tooth sizes and enamel thicknesses. Samples were from modern teeth collected from skeletal assemblages by J.D.K. during surface surveys in the Eyasi Plateau, Tanzania in 2005. A single second or third molar for each species was sectioned with a high-speed Brasseler dental drill (Forza L50k) with a diamond-tipped wheel to generate eighteen separate samples per tooth per taxon. Samples were primarily taken from the lingual and buccal aspects, although all aspects of the Madoqua tooth were sampled. Each enamel sample represented the full longitudinal dimension of the tooth, from the cemento-enamel junction to the crown, in an effort to stan-dardize a bulk isotopic value for each species, although it is recognized that the thickness of enamel, and hence the amount of time that is averaged, varies across different aspects (Zazzo et al., 2012; Fig. 1).

Five controlled furnace experiments were conducted at the following temperature settings on the furnace controller: 300 ◦C, 500 ◦C, 700 ◦C, 900 ◦C, and 1100 ◦C. For each of the five temperature conditions three samples of a second or third molar (Supplemental Dataset 1) from each taxon were heated in separate ceramic crucibles. The furnace was interfaced with a K-Type Thermocouple to provide a more accurate temperature reading for each trial. After the furnace reached the desired temperature, determined by a steady thermocouple reading for 15 min, this temperature was maintained for an additional forty-five minutes, for a total of one hour. The furnace’s heating element was then shut off and the furnace allowed to cool naturally to <100 ◦C before removing the samples. Cool down for 300 ◦C, 500 ◦C, and 700 ◦C typically took 6–9 h. At 900 ◦C and 1100◦, samples were left to cool in the furnace overnight for at least 14 h. The furnace door remained closed during the cooling process to maintain gradual cooling. These controlled experi-ments were designed to mimic the heating and cooling characteristics of archaeological fires as determined by the experiments referenced earlier. The experimental temperature range includes, and exceeds, the maximum temperatures recorded in both anthropogenic and wildfires (Ice et al., 2004; Aldeias, 2017), and the experiments maintained the maximum temperature longer than observed in experimental fires (Sievers and Wadley, 2008; Aldeias et al., 2016). A Thermolyne furnace was used instead of an experimental fire to allow for greater control of temperature uniformity and to assure that the tooth enamel was isolated from byproducts of fuel combustion, such as ash and smoke. Three additional samples of each taxon were analyzed isotopically without heating to provide a baseline for assessing any changes in δ13Cenamel and δ18Oenamel.

In both anthropogenic and natural fires, we expect tooth enamel to be buffered from the heat by surrounding cranial bone and flesh. As such, we consider these experiments to mimic aerobic, or directly in the fire, heating conditions, the most extreme encountered in natural or

Table 1 Combustion experiment δ13Cenamel and δ18Oenamel values (‰ VPDB) compared with modern isotope data from eastern Africa and the Laetoli area. Values in parenthesis represent sample sizes.

Taxon East Africaa

Laetoli Areab

Control Values

Control Mean

Connochaetes taurinus

δ13C 1.4 ± 1.3 (65)

0.6 (1) 0.7; 0.5; 0.1 0.4 ± 0.3

δ18O — 1.5 (1) 3.1; 3.2; 3.6 3.3 ± 0.3

Madoqua kirkii

δ13C − 11.9 ±1.7 (40)

− 11.1 (1) − 12.0; − 12.4; − 13.0

− 12.5 ±0.5

δ18O — − 2.2 (1) 2.4; 3.3; 6.0 3.9 ± 1.9

Equus quagga

δ13C 0.0 ± 1.4 (128)

− 1.4; − 1.1 (2)

0.1; − 0.5; − 0.9

− 0.4 ±0.5

δ18O — − 1.7; − 2.0 (2)

2.3; 2.8; 3.1 2.7 ± 0.4

Giraffa camelopardalis

δ13C − 12.0 ±1.6 (58)

− 11.3 ±2.1 (8)

− 10.3; − 10.4; − 10.6

− 10.4 ±0.2

δ18O — 6.1 ± 1.5 (8)

6.1; 8.0; 8.1 7.4 ± 1.1

Phacochoerus africanus

δ13C − 1.0 ±1.3 (61)

− 1.4 ±0.9 (12)

− 0.8; − 0.8; − 1.0

− 0.9 ±0.1

δ18O — 3.0 ± 1.0 (12)

3.6; 4.0; 4.3 4.0 ± 0.4

a δ13Cenamel-equivalent values from Cerling et al., 2015 Supplemental File 1. No δ18Oenamel values published.

b δ13Cenamel and δ18Oenamel values from Kingston, 2011.



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hearth type fires. While human cremation fires in the archaeological record have been measured at temperatures below 900 ◦C, many are estimated to have occurred at temperatures exceeding 1000 ◦C (Piga et al., 2008a; Iriarte et al., 2020). Furthermore, cremation burning times at 900 ◦C or higher are estimated at an hour, with longer burning times at lower temperatures (Piga et al. 2008b). While in many ways our ex-periments simulate cremation fires in terms of high temperatures and extended burning times, we note that we are explicitly trying to dupli-cate archaeological hearths and/or natural fire conditions so our ex-periments may not fully capture the characteristics of a cremation fire.

Following the experimental combustion trials, all samples were crushed using an agate mortar and pestle. Typically, archeological or paleontological samples are pretreated with H2O2 or NaOCl to oxidize any organics that might generate interfering masses during mass

spectrometry and 0.1 M CH3COOH to eliminate any exogenous or diagenetic carbonate. Given the minimal organic matter in enamel and the lack of diagenesis in modern teeth, we avoided these pretreatment processes to minimize the introduction of additional chemical variables into the analyses (Munro et al., 2008). Enamel samples were freeze dried and analyzed in a Kiel III carbonate extraction device interfaced with a Finnigan-MAT 252 isotope ratio mass spectrometer at the Light Stable Isotope Laboratory, Department of Geosciences, University of Florida, Gainesville. Results are reported using the standard ‰ notation where:

δ13C(18O) = (Rsample/Rstandard – 1) × 1000

and R = (13C/12C) or (18O/16O)

Precision was measured via repeated measures of the international

Fig. 2. Relationship between CO2 produced (in mmol/kg h) and combustion temperature (◦C). Control samples plotted at a combustion temperature of 0 ◦C. Blue line and 95% confidence intervals show the linear fit. Open circles represent samples that produced <100 mmol/kg h of CO2.

Table 2 Results of the regression analysis between CO2 yield and temperature, with bold p-values indicating significant relationships. Abbreviations: df, degrees of freedom of the residuals; SEE, standard error of the estimate.

Family Taxon df F-statistic SEE Slope r R2 p-value

Bovidae Connochaetes taurinus 16 37.470 146.721 − 0.578 0.837 0.701 <0.001 Bovidae Madoqua kirkii 16 5.505 225.145 − 0.340 0.506 0.256 0.032 Equidae Equus quagga 16 14.140 170.009 − 0.412 0.685 0.469 0.002 Giraffidae Giraffa camelopardalis 16 8.738 181.947 − 0.346 0.594 0.353 0.009 Suidae Phacochoerus africanus 16 27.504 154.031 − 0.520 0.795 0.632 <0.001



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calcium carbonate standard NBS-19 during sample analysis (n = 20; δ13C: 1.95 ± 0.02‰; δ18O: − 2.21 ± 0.06‰). Replicate measurements of the standards during analysis had errors of ~0.05‰ for carbon and ~0.1‰ for oxygen. δ13Cenamel and δ18Oenamel values are reported rela-tive to VPDB (Vienna Pee Dee Belemnite).

All statistical analyses were conducted in R v3.6.1 (R Core Team). δ13Cenamel, δ18Oenamel, and CO2 produced values for each taxon were regressed against combustion temperature using linear models with the lm() function in R. To visualize patterns, scatter plots were generated of δ13Cenamel, δ18Oenamel, and CO2 produced values with linear fits using geom-smooth() in ggplot2 (Wickham, 2016). Regression analyses were conducted on these data in two ways: 1) only samples which produced >100 mmol/kg h of CO2 are included in the first set of regression ana-lyses as isotope values derived from samples producing less than this amount of CO2 are more variable as determined by the standard devi-ation of replicate analyses of those samples (Supplemental Dataset 1), and 2) regression analyses run on all eighteen data points for each taxon regardless of the amount of CO2 produced. All summary statistics were also completed in R.

3. Results

δ13Cenamel control values for all taxa are consistent with modern isotope data from these taxa in eastern Africa (Table 1; Kingston and Harrison, 2007; Kingston, 2011; Cerling et al., 2015). Few datasets of modern δ18Oenamel values have been published for equatorial Africa, but the control values here are similar to those yielded by modern samples from the Laetoli area (Kingston and Harrison, 2007; Kingston, 2011). Modern herbivore teeth typically incorporate variable degrees of intra- tooth variation in both oxygen and carbon, ranging up to 7-8‰ (Kohn et al., 1999; Lee-Thorp, 2002; Kingston, 2003). These differences pre-sumably reflect seasonally mediated changes in foraging strategy and drinking water during the time in which a tooth is mineralizing (i.e., Janzen et al., 2020). To minimize the confounding effects of this vari-ation on interpreting the dietary signatures of each taxon at different

temperatures based on different samples, each tooth was strategically sampled in a way to include enamel forming at roughly the same interval of time (Fig. 1). However, it was not possible to completely eliminate δ13Cenamel and δ18Oenamel differences between samples as indicated by the isotopic values of three control samples for each of the taxa. Intra- tooth variation in δ13Cenamel values for the three control samples from each taxon range from 0.2 to 1.0‰ (Table 1) and 0.5 to 3.6‰ for δ18Oenamel (Supplemental Dataset 1). These ranges are inherent in the sample sets combusted at variable temperatures and need to be considered when interpreting the effects of burning.

3.1. CO2 yields

Heating tooth enamel to higher temperatures resulted in diminished CO2 yields, as determined by CO2 gas evolved during enamel digestion in phosphoric acid (Fig. 2). This is particularly true for Madoqua and Connochaetes, which have thinner enamel than the non-bovid taxa (Supplemental Dataset 1). For all taxa, the amount of CO2 produced is greatly reduced (~77–86%; Table 2) at the 900 ◦C and 1100 ◦C condi-tions (Fig. 2). Yields below 100 mmol/kg h indicate substantial alter-ation and produced isotopic values that are not considered generally reliable. We include these values in the figures and some of the discus-sion to assess the analytical issues involved in burning enamel at high temperatures. CO2 yield for all five taxa are significantly negatively related to combustion temperature but it should be noted that there might be a non-linear relationship between temperature and CO2 yield (Fig. 2). Based on the linear models, temperature is moderately (Madoqua: R2 = 0.256) to strongly (Connochaetes: R2 = 0.701) predictive of CO2 yield (Table 2), although this relationship does appear to be primarily driven by decreased yields at 900 ◦C and 1100 ◦C.

One measure of the reliability of isotopic values is the standard de-viation of replicates of the same sample. There also appears to be a relationship between CO2 produced and these standard deviations, particularly for samples characterized by <100 mmol/kg h of CO2 produced. Standard deviations of δ13Cenamel and δ18Oenamel values at

Table 3 Results of t-tests on δ13Cenamel and δ18Oenamel standard deviations (Stdev) of replicate samples between those that yielded >100 mmol/kg h of CO2 (top) and those that yielded <100 mmol/kg h of CO2 (bottom).

Taxon n Mean Stdev Range df t-statistic p-value


δ13C 9 0.012 0.010 0.01–0.03

13 4.301 <0.001 6 0.113 0.070 0.04–0.21

δ18O 9 0.022 0.013 0.01–0.04

13 4.533 <0.001 6 0.137 0.075 0.06–0.25

Madoqua kirkii

δ13C 9 0.012 0.004 0.01–0.02

13 6.256 <0.001 6 0.462 0.222 0.28–0.77

δ18O 9 0.019 0.006 0.01–0.03

13 5.797 <0.001 6 0.572 0.287 0.20–0.96

Equus quagga

δ13C 11 0.013 0.008 0.01–0.03

13 3.301 0.006 4 0.243 0.248 0.05–0.59

δ18O 11 0.028 0.022 0.01–0.09

13 2.916 0.012 4 0.378 0.426 0.05–0.29


δ13C 12 0.016 0.012 0.01–0.05

13 6.561 <0.001 3 0.107 0.047 0.07–0.16

δ18O 12 0.048 0.077 0.01–0.29

13 1.955 0.072 3 0.14 0.044 0.09–0.17


δ13C 12 0.013 0.006 0.01–0.02

13 5.940 <0.001 3 0.15 0.09 0.06–0.24

δ18O 12 0.032 0.024 0.01–0.10

13 4.969 <0.001 3 0.11 0.026 0.08–0.13



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900 ◦C and 1100 ◦C for Madoqua and Connochaetes are higher and sta-tistically different than those from between 300 ◦C and 700 ◦C (Table 3; Supplemental Dataset 1). Samples of Giraffa and Phacochoerus at the 900 ◦C condition produce between 200 and 100 mmol/kg h of CO2, but at 1100 ◦C all samples of these two taxa fall below the 100 mmol/kg h threshold. Equus is intermediary with one sample in the 900 ◦C condition below 100 mmol/kg h CO2 produced, but the other two samples be-tween 200 and 100 mmol/kg h. As with the other taxa, all Equus samples in the 1100 ◦C heating condition produced <100 mmol/kg h of CO2. In all of these cases, except for Giraffa δ18Oenamel values, the standard de-viations of samples that produced <100 mmol/kg h of CO2 are statisti-cally significantly larger than the standard deviations of samples that produced >100 mmol/kg h of CO2 (Table 3), indicating greater vari-ability and potentially suspect isotopic values.

3.2. Carbon

Almost all samples that produced >100 mmol/kg h of CO2 fall within 1.0‰ of the respective taxon’s mean control δ13Cenamel value (Table 1, 4; Supplemental Dataset 1). Samples that fall outside this range include three samples of Phacochoerus, five samples of Equus, and two samples of Madoqua. At 1100 ◦C, where no samples of any taxon produced >100 mmol/kg h of CO2, almost all δ13Cenamel values fall within 1.0‰ of the respective control mean. Madoqua samples in the 900 ◦C and 1100 ◦C conditions fall ~4‰ to ~10‰ above the Madoqua control mean and one

sample of Equus at 1100 ◦C is 5.2‰ above the control value. This lack of any clear or consistent relationship between δ13Cenamel and combustion temperature is supported by our regression analyses (Fig. 3). When only those samples that produced >100 mmol/kg h of CO2 are considered, only Equus δ13Cenamel values have a significant relationship with tem-perature. This is a negative relationship with samples at 700 ◦C and 900 ◦C that fall >1.0‰ below the control mean. In this case, temperature is a relatively good predictor of Equus δ13Cenamel values, explaining 44.1% of variation. However, when all samples are included in the regression analysis this relationship is no longer negative or significant. When all samples are included, Madoqua δ13Cenamel values are found to have a significant, positive relationship with temperature where tem-perature explains 32.3% of the variation (Table 5). All other taxa have non-significant relationships, regardless of whether samples that pro-duced <100 mmol/kg h of CO2 are included, with temperature being a poor predictor of δ13Cenamel values (explaining only between 1% and 8% of variation).

3.3. Oxygen

All samples at 300 ◦C have δ18Oenamel values within 3.6‰, the range of natural intra-tooth variation associated with this sampling strategy, of their respective control means (Table 1). No samples of any taxa are within 3.6‰ of their respective control mean, or any single species- specific control value, in the 500 ◦C condition or at any higher tem-perature. At and above 500 ◦C, all samples yield δ18Oenamel values well below the control values (Table 4; Fig. 4). The qualitative examination of the relationship between δ18Oenamel values and temperature is sup-ported by a regression analysis. δ18Oenamel values of all five taxa have strong and significant negative relationships with temperature whether those samples that produced <100 mmol/kg h of CO2 are included or not (Table 6). Temperature is a reliable predictor of δ18Oenamel values, explaining from 77.0% (Madoqua) to 94.4% (Connochaetes) of the variation when all samples are analyzed. Since individual taxa have different control δ13Cenamel and δ18Oenamel values its difficult to inves-tigate whether there is a general relationship between isotopic values and combustion temperature. One possible taxon-free way would be to calculate the offset of the mean values at each experimental temperature for each taxon from the control values (Fig. 5). When this is done, a strong and significant relationship (p > 0.001) between δ18Oenamel offset values and combustion temperature is found where temperature ex-plains 78.7% of the variation in δ18Oenamel offset. The relationship for δ13Cenamel is not significant (p = 0.101; Fig. 5).

4. Discussion

4.1. Effects of burning on enamel isotopic composition

The isotopic composition of tooth enamel appears to be more resis-tant to the effects of burning than that of bone (Munro et al., 2008; Hüls et al., 2010). Munro et al. (2008) found significant changes in structural carbonate yields of bone starting at a temperature of ~325–350 ◦C, with no structural carbonate present in bone burned to ≥725 ◦C. Structural carbonate yields appear to be maintained in enamel up to ~700 ◦C. δ13Cenamel values were preserved in all samples up to ~700 ◦C for thinner-enameled taxa Madoqua and Connochaetes and up to ~900 ◦C for thicker-enameled Giraffa and Phacochoerus. CO2 yields further support the persistence of structural and chemical integrity of carbonate up to ~700 ◦C and beyond with significant changes occurring only once yields fall to less than 20–25% of the quantity originally present (Table 4). While there is a significant negative relationship between Equus δ13Cenamel values and combustion temperature, this remains difficult to interpret because there is very little variation from the control values and this relationship is not significant when all samples are included in the analysis. Furthermore, these slight deviations from the control δ13Cenamel mean are primarily in the direction of lower enamel

Table 4 Summary statistics from combustion experiments, including mean δ13Cenamel, δ18Oenamel, and CO2 yield values per taxon. Each taxon has a sample size of three at each combustion temperature.

Taxon K-typea δ13C δ18O CO2


Control 0.4 ± 0.3 3.3 ± 0.3 588.00 ± 138.17

313 ◦C − 0.2 ± 0.1 1.7 ± 0.6 636.33 ± 57.01 516 ◦C − 0.7 ± 0.1 − 1.9 ± 0.2 658.33 ± 54.52 710 ◦C 0.4 ± 0.6 − 4.3 ± 0.8 361.67 ± 69.01 905 ◦C − 0.7 ± 0.2 − 7.1 ± 2.1 86.00 ± 3.61 1101 ◦C 0.4 ± 0.1 − 10.5 ±

0.9 75.00 ± 1.00

Madoqua kirkii Control ¡12.5 ± 0.5

3.9 ± 1.9 282.67 ± 99.03

313 ◦C − 13.5 ± 0.1 1.8 ± 1.2 565.33 ± 65.06 516 ◦C − 13.5 ± 0.1 − 2.9 ± 0.8 714.00 ± 96.60 710 ◦C − 9.8 ± 7.2 − 6.0 ± 3.3 400.00 ± 73.01 905 ◦C − 5.5 ± 3.5 − 20.4 ±

5.3 69.33 ± 3.51

1101 ◦C − 7.6 ± 4.1 − 16.3 ±4.8

68.00 ± 5.29

Equus quagga Control ¡0.4 ± 0.5 2.7 ± 0.4 403.33 ± 39.37 313 ◦C − 2.0 ± 0.2 1.8 ± 0.6 679.00 ± 90.60 516 ◦C − 1.6 ± 0.1 − 3.0 ± 0.8 572.00 ± 56.70 710 ◦C − 1.7 ± 0.6 − 5.1 ± 0.8 442.67 ± 11.06 905 ◦C − 1.9 ± 0.1 − 7.7 ± 0.8 154.33 ± 98.31 1101 ◦C 1.5 ± 2.8 − 13.4 ±

6.7 73.67 ± 7.51


Control ¡10.4 ± 0.2

7.4 ± 1.1 353.00 ± 147.65

313 ◦C − 11.2 ± 0.1 6.3 ± 0.6 575.33 ± 45.08 516 ◦C − 11.3 ± 0.3 1.9 ± 1.0 639.67 ± 10.79 710 ◦C − 11.2 ± 0.4 − 1.0 ± 1.4 437.67 ± 52.62 905 ◦C − 10.6 ± 0.5 − 1.7 ± 2.4 147.33 ± 17.01 1101 ◦C − 10.6 ± 0.3 − 6.8 ± 2.0 77.33 ± 3.22


Control ¡0.9 ± 0.1 4.0 ± 0.4 549.67 ± 32.59 313 ◦C − 1.9 ± 0.5 3.2 ± 0.8 621.33 ± 15.82 516 ◦C − 1.9 ± 0.7 − 1.1 ± 0.6 692.33 ± 23.35 710 ◦C − 1.1 ± 0.5 − 3.7 ± 0.1 462.33 ± 11.24 905 ◦C − 1.7 ± 0.3 − 5.1 ± 0.5 119.00 ± 5.29 1101 ◦C − 1.1 ± 0.5 − 9.5 ± 2.6 75.33 ± 3.51

Values in italics indicate samples producing < 100 mmol/kg h. Bold indicates control samples that were not heated.

a Thermocouple temperature reading.



7

Fig. 3. Relationship between δ13Cenamel values and combustion temperature (◦C). Control samples plotted at a combustion temperature of 0 ◦C. Red line and 95% confidence interval show linear fit for samples that produced >100 mmol/kg h of CO2. Blue line and 95% confidence intervals show the linear fit for all samples. Open circles represent samples that produced <100 mmol/kg h of CO2. Dotted line is the mean of the control values. R2 and p-values color coded by condition. Bolded and italicized values indicate significant relationships at α < 0.05.

Table 5 Results of the regression analyses between δ13Cenamel and temperature, with bold p-values indicating significant relationships. Top line for each taxon is regression of only those samples that produced >100 mmol/kg h of CO2 and bottom line is all samples. Abbreviations: df, degrees of freedom of the residuals; SEE, standard error of the estimate.

Taxon df F-statistic SEE Slope r R2 p-value

Connochaetes taurinus 10 0.329 0.594 <0.001 0.178 0.032 0.579

16 0.158 0.581 <0.001 0.099 0.010 0.696

Madoqua kirkii 10 0.614 3.518 0.003 0.240 0.058 0.452

16 7.618 3.727 0.007 0.568 0.323 0.014

Equus quagga 12 9.470 0.535 − 0.001 0.664 0.441 0.010

16 1.151 1.626 0.001 0.259 0.067 0.299

Giraffa camelopardalis 13 0.601 0.471 <0.001 0.210 0.044 0.452

16 0.032 0.467 <0.001 0.045 0.002 0.861

Phacochoerus africanus 13 1.135 0.594 − 0.001 0.283 0.080 0.306

16 0.023 0.594 <0.001 0.038 0.001 0.882



8

Fig. 4. Relationship between δ18Oenamel values and combustion temperature (◦C). Control samples plotted at a combustion temperature of 0 ◦C. Red line and 95% confidence interval show linear fit for samples that produced >100 mmol/kg h of CO2. Blue line and 95% confidence intervals show the linear fit for all samples. Open circles represent samples that produced <100 mmol/kg h of CO2. Dotted line is the mean of the control values. R2 and p-values color coded by condition.

Table 6 Results of the regression analysis between δ18Oenamel and temperature, with bold p-values indicating significant relationships. Top line for each taxon is regression of only those samples that produced >100 mmol/kg h of CO2 and bottom line is all samples. Abbreviations: df, degrees of freedom of the residuals; SEE, standard error of the estimate.

Taxon df F-statistic SEE Slope r R2 p-value

Connochaetes taurinus 10 118.408 0.914 − 0.011 0.960 0.922 <0.001

16 269.631 1.222 − 0.013 0.972 0.944 <0.001

Madoqua kirkii 10 39.464 2.078 − 0.014 0.893 0.798 <0.001

16 53.657 4.754 − 0.022 0.878 0.770 <0.001

Equus quagga 12 125.296 1.206 − 0.012 0.955 0.913 <0.001

16 65.383 2.810 − 0.015 0.896 0.803 <0.001

Giraffa camelopardalis 13 62.040 1.793 − 0.012 0.909 0.827 <0.001

16 115.122 1.899 − 0.013 0.937 0.878 <0.001

Phacochoerus africanus 13 144.785 1.119 − 0.011 0.958 0.918 <0.001

16 163.613 1.514 − 0.012 0.954 0.911 <0.001



9

δ13Cenamel values, opposite to the pattern found in the Munro et al. (2008) experiments. Of these samples, only a single sample of Madoqua heated to 700 ◦C indicates 13C enrichment (Fig. 3), similar to what Munro et al. (2008) found for all bone samples combusted at >650 ◦C. The 13C enrichment documented for this sample, as well as the Madoqua samples heated to 900 ◦C and 1100 ◦C (Table 4), likely reflects the fact that 13C is more tightly bonded to the bioapatite structure than 12C. Unlike bone, this kinetic fractionation may not occur in enamel until temperatures reach at least 700 ◦C, and possibly higher depending on the thickness of enamel. Furthermore, in the case of Madoqua, future work should investigate whether differences in thickness and growth rates of specific enamel aspects have a role in some of the variation seen in these experiments (i.e., Zazzo et al., 2012).

δ18Oenamel values also appear to be more stable during heating than δ18Obone values, with control δ18Oenamel values maintained up to ~300 ◦C while δ18Obone values begin to deviate from the control value at ~225 ◦C (Munro et al., 2008). At temperatures greater than ~300 ◦C, however, δ18Oenamel values of all taxa decrease and exhibit a clear negative relationship with combustion temperature. Based on the trends in δ18Oenamel values it appears that temperatures > ~ 300 ◦C may be sufficient for breaking the covalent bonds in the carbonate CO3

2–, allowing it to react with atmospheric H2O (and possibly CO2), resulting in uptake of atmospheric 18O or 16O (e.g., Kohn and Cerling, 2002; Lee- Thorp, 2002). It is also possible that there is exchange between car-bonate and hydroxyl groups would occur as the smaller reservoir size and weaker bonds suggests loss of oxygen here first (Sponheimer and Lee-Thorp, 1999). In the first step during heating, protonation of CO3

2–

forms HCO3– leaving behind OH–. Since OH– is unstable in isolation, it

precipitates protonation of HCO3–, yielding H2CO3. The unstable H2CO3

allows for the parent oxygen in the enamel to trade places with atmo-spheric H2O resulting in a reformed carbonate containing alien oxygen: CO*O2

2–. Loss of structural carbonate during burning may also be asso-ciated with isotopic fractionation whereby CO2 enriched in 16O is preferentially lost resulting in 18O depletion of the surviving carbonate (Sharma and Clayton, 1965; Munro et al., 2008). Such a process explains both why the δ18Oenamel values change, but the δ13Cenamel values remain mostly consistent, and why there appears to be a linear relationship between temperature and δ18Oenamel. This explanation suggests that burning affects structural carbonate in stages, first at the enamel surface and then proceeding inwards towards the dentino-enamel junction. This can be explained by lower temperatures only breaking bonds on the surface of the enamel while higher temperatures lead to ‘more complete’

breakage of bonds throughout the enamel crystal fragment. This pattern may explain why thicker-enameled taxa maintain control isotope values at higher temperatures (Sponheimer and Lee-Thorp, 1999; Kohn and Cerling, 2002; Lee-Thorp, 2002). The strong and significant relation-ships between δ18Oenamel values and combustion temperature (Table 6; Fig. 4), suggests that it may be possible to ‘correct’ for δ18Oenamel values of enamel burned at high temperatures. This would assume that one is able to independently determine the approximate temperature of com-bustion, through FTIR or another method, the taxon-free offset means approach (Fig. 5) offers a potential linear function for determining the offset (y) from the actual δ18Oenamel value:

y = -0.015 T + 1.697

where ‘T’ is temperature in ◦C.

4.2. Implications for dietary and paleoecology studies

While experimental and archaeological evidence suggests that anthropogenic fires can reach ~700 ◦C (Sievers and Wadley, 2008; Karkanas et al., 2007; Berna et al., 2012), this combustion experiment indicates that original, in vivo, δ13Cenamel values are preserved beyond these temperatures. Alteration of δ13Cenamel values start to occur at temperatures over ~700 ◦C and such alterations appear to be minimal (< ±2‰ except for Madoqua samples at 900 ◦C and 1100 ◦C). Even for Equus, where a relationship is indicated, slight shifts within 1.5‰ are unlikely to be relevant to the purposes of reconstructing diets or past environments. δ18Oenamel composition, on the other hand, is clearly affected by burning at temperatures above ~300 ◦C. Shifts in the range of ~4–10‰ would have significant implications for paleo-aridity studies (i.e., Levin et al., 2006; Blumenthal et al., 2017). δ18Oenamel values from contexts where there is evidence for burning should be used cautiously in climate analyses and environmental reconstructions, unless effort is made to ‘correct’ those values.

5. Conclusions and future directions

While it is known that tooth enamel is potentially susceptible to diagenetic alteration (e.g., Kohn et al., 1999; Zazzo, 2014), the degree to which natural or anthropogenic burning may affect the isotopic composition of enamel carbonate has previously been unknown. These experiments indicate that sustained burning at temperatures above ~300 ◦C results in altered δ18Oenamel values. On the other hand,

Fig. 5. Relationship between offset of mean δ13Cenamel and δ18Oenamel values at each temperature for each taxon from control mean values. Blue line and 95% confidence intervals show the linear fit for all data.



10

δ13Cenamel values are maintained to ~700 ◦C for all taxa and up to ~900 ◦C for thicker enameled species. Based on experimental fires intended to simulate archaeological hearths (Sievers and Wadley, 2008) and FTIR measurements from archaeological sites (e.g., Karkanas et al., 2007; Berna et al., 2012), δ13Cenamel values should be considered reliable even when there is evidence of extensive burning. Some caution, how-ever, may be warranted in the interpretation of δ18Oenamel values from similar sites as combustion temperatures have been documented to reach above 300 ◦C. Future research needs to investigate how burning specifically alters the chemistry and microstructure of tooth enamel and if and how these changes are linked to visible changes in texture and color. Developing a list of criteria by which teeth can be assessed as potentially having altered chemistry prior to sampling will act to in-crease the reliability of our stable isotope datasets.

CRediT authorship contribution statement

Joshua R. Robinson: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review & editing, Visualiza-tion. John D. Kingston: Conceptualization, Methodology, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank John Krigbaum and Jason Curtis for supporting mass spectrometry in the Departments of Anthropology and Geosciences and Light Stable Isotope Laboratory at the University of Florida.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.jasrep.2020.102593.

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