Stable isotope analysis is essential to reconstruct past diets, mobility, and ecological relationships in archaeological populations. Both bulk (whole sample) and compound-specific (individual biochemical compounds) stable isotope analyses can provide nuanced insights into dietary habits and health status, particularly when applied to human and faunal remains. Archaeologists play a critical role in selecting appropriate samples, documenting context, and interpreting the resulting data within broader archaeological questions. 

Bulk and Compound-Specific Isotope Analysis (CSIA)

Sample selection:  

For carbon and nitrogen bulk isotope analysis, collagen sampled from bone or dentine elements is used. For compound-specific analysis (CSIA), individual amino acids (e.g., glutamic acid and phenylalanine) are isolated from collagen or dentine. The following aspects should be considered during selection: 

  • Preservation: to check this aspect, the collagen yield and C: N atomic ratio for bulk collagen are evaluated following standard quality assurance procedures (DeNiro, 1985; Ambrose, 1990, 1993; van Klinken, 1999; Guiry & Szpak, 2020); additionally, FTIR or other tests can evaluate enamel and dentine diagenesis. 
  • Age and context: Ensure the sample’s archaeological context aligns with your research question. Check whether the age categories are equally represented in the population you are sampling. 
  • Tissue type: The decision of the skeletal element to be sampled should be based on temporal resolution—e.g., ribs reflect short-term diet, femurs long-term, dentine or enamel incremental sections reveal early-life data. 
  • Contamination: Avoid samples with evidence of soil infiltration, adhesives, or conservation treatments. Moreover, avoid samples showing signs of skeletal pathology (unless this is the focus of the study).  

Sample Preparation 

Bulk Analysis of collagen (bone/dentine) (for the complete SIA sampling protocol, see here):

After proper photographic documentation, the sampling of the skeletal or dental elements is performed through drilling or breaking the bone tissue in fragments, which are weighed and placed into a clean plastic zip-lock bag pre-labelled with sample ID and date. Carbon and nitrogen SIA request between 60 and 100 mg of tissue.  

Collagen extraction involves the following steps: 

  • Demineralisation in HCl – removal of the mineral component of the skeletal tissue 
  • Gelatinisation - the collagen is broken down by hydrolysis, making it soluble. 
  • Purification - the large, unwanted particulate matter is removed from the dissolved collagen prior to ultrafiltration (if this is performed). The samples are poured into the syringes and filtered to get rid of the insoluble residues. 
  • Freeze-drying. 
  • Sample weighing into tin capsules (ca. 1 mg of collagen). 

Compound-Specific Stable Isotope Analysis (CSIA): 

After collagen extraction is performed as above, the protein material is processed as follows: 

  • Hydrolysis of collagen to release amino acids 
  • Derivatisation of target amino acids (e.g., using N-acetyl methyl ester) 
  • Purification via gas chromatography 
  • Analysis via GC-C-IRMS (Gas Chromatography–Combustion–Isotope Ratio Mass Spectrometry)  

Data Acquisition

After sample processing, the results will be in this form: 

  • δ¹³C and δ¹⁵N values (collagen) 
  • %C, %N, and C:N ratio (quality control) 
  • Compound-specific (CSIA): 
  • δ¹⁵N values of individual amino acids (glutamic acid, phenylalanine, etc.) 
  • Quality indicators: chromatogram clarity, derivatisation control. 

Interpretation of CSIA data often includes calculating the Trophic Position (TP) using established equations (e.g., Chikaraishi et al. 2009), which helps distinguish dietary protein sources across trophic levels. 

Data Interpretation and Visualisation 

The results of bulk compound-specific SIA are performed in the context of ecological baselines and dietary models. When possible: 

  • Compare with local faunal isotopic baselines 
  • Use biplots of δ¹³C vs. δ¹⁵N (bulk) or TP (Trophic Position) vs. δ¹³C (CSIA) 
  • Consider factors such as breastfeeding/weaning signals, marine vs. terrestrial food sources, and freshwater signature values. 

Data Processing and Analysis 

Software such as R (packages like mixSIAR, IsotopeR) can be used for mixing models and visualisation of stable isotope data, as well as specific statistical software such as IsoReader, FRUITS,  and Lyticos. 

Limitations and Data Quality Concerns 

Key challenges in stable isotope analysis include: 

  • Diagenesis: Chemical alteration of samples occurring post-mortem can compromise collagen and tissue integrity. 
  • Environmental baseline variation: Local ecology and foodweb structure should be available and understood in order to interpret the isotopic data. 
  • Trophic enrichment factors (TEFs): These vary across species and tissues and may affect dietary reconstructions. 
  • Sample preservation: Especially important for CSIA, which requires higher-quality collagen than bulk. 
  • Analytical error: In compound-specific work, poor chromatographic separation or co-elution can sometimes lead to unreliable δ¹⁵N values. 

 

References

Bulk SIA

  • Ambrose, S.H. (1990). Preparation and characterisation of bone and tooth collagen for isotopic analysis. Journal of Archaeological Science 17: 431–451.
  • Schoeninger, M.J., & Moore, K. (1992). Bone stable isotope studies in archaeology. Journal of World Prehistory 6: 247–296. 

Compound-Specific Isotope Analysis: 

  • Chikaraishi, Y. et al. (2009). Determination of aquatic food-web structure based on compound-specific nitrogen isotopic composition of amino acids. Limnology and Oceanography: Methods, 7: 740–750. 
  • O'Connell, T.C. (2017). ‘Trophic’ and ‘source’ amino acids in trophic estimation: a likely metabolic explanation. Oecologia, 184(2): 317–326. 

Method Comparisons and Quality Assurance: 

  • DeNiro MJ. Postmortem preservation and alteration of in vivo bone‐collagen isotope ratios in relation to paleodietary reconstruction. Nature. 1985; 317: 806–809. https://doi.org/10.1038/317806a0. 

ZooMS Workflow 

Lab Work and Data Analysis 

Output and Data Archival 

  • Deposition of final datasets and metadata in ARHUT, the datasets can be entered into ARHUT through the user interface one-by-one or imported using an import tool. 


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