Methodological Considerations for the Use of Acid-Based Pre-Treatment Protocols for Carbon and Oxygen Analysis of Tooth Enamel

Rationale

Chemical pre-treatment is a common methodological step aimed to remove exogenous materials introduced to archaeological tooth enamel in the burial environment through diagenetic processes. However, some of these methods, involving the use of oxidising reagents such as NaClO, H₂O₂, as well as weak acids like CH₃COOH, have been shown to alter the chemical composition and stable isotope values of enamel. Here, we aim to re-examine the effects of commonly used pre-treatment protocols on bioapatite δ¹³C and δ¹⁸O values, and investigate the relationship between diagenetic alteration and measured isotope values, as indicated by pre-screening using attenuated total reflectance-Fourier transform infrared (ATR–FTIR) spectroscopy.

Methods

Modern and archaeological samples were subjected to 10 commonly used pre-treatment protocols that apply NaClO, H₂O₂ and/or CH₃COOH to tooth enamel powders at treatment lengths. Preservation status and diagenetic alteration prior to and after treatment were investigated using ATR–FTIR. δ¹³C and δ¹⁸O values were measured before and after treatment to determine if different wet chemistry protocols induced isotopic shifts.

Results

The results show that all pre-treatment protocols imparted shifts in δ¹³C and δ¹⁸O values of up to ± 1.5‰ in both archaeological and modern samples. Most treated samples display increased crystallinity, likely indicating sample recrystallisation. We suggest that these changes indicate the removal of contamination and diagenetic alteration, and also the dissolution and restructuring of enamel carbonate leading to changes in the in vivo isotope signal.

Conclusions

We discourage the use of H₂O₂ and NaClO to remove organic matter from samples as it incurs unwanted changes to the enamel structure and carbon and oxygen isotope ratios. We also recommend the use of only short-duration acetic acid treatment protocols to avoid recrystallisation caused by prolonged acid exposure and concomitant unwanted change to in vivo isotope values.