Chromatography and compound specific isotope analysis of lipid remains in ancient pottery tell a lot about ancient food habits and culinary practices. In the last two decades, this technique has successfully been employed to unravel ancient food practices of several archaeological sites in the world. Researchers have applied this technique recently to the potteries collected from multiple archaeological sites of Indus Valley Civilisation. The key scientific finding was dominance of non-ruminant fats in the cooking vessels implying non-ruminant animals (such as horse, pigs, poultry, fowl, rabbit, etc) were cooked in the vessels over a long period. This contradicts the long held view (based on faunal evidence) that ruminant animals (such as cattle, buffalo, deer,etc) were consumed as food by Indus Valley people.
Archaeological excavations of important sites in the past century provided lot of information about the culture and practices of ancient people. However, understanding diet and subsistence practices prevalent in ancient prehistoric societies with no written records used to be an uphill task because not much of what constituted ‘food’ were left due to almost complete natural degradation of food and biomolecules. In the last two decades, the standard chemical techniques of chromatography and compound specific analysis of the ratio of stable isotopes of carbon have made inroads in archaeological studies enabling researchers to pinpoint sources of lipids. As a result, it has become possible to investigate diet and subsistence practices using molecular and isotopic analyses of absorbed food residues based on the δ13C and Δ13C values.
Plants are the primary producers of food. Most plants use C3 photosynthesis to fix carbon, hence are called C3 plants. Wheat, barley, rice, oats, rye, cowpea, cassava, soybean etc are the main C3 plants. They form the staple food of mankind. C4 plants (such as corn, sugarcane, millet, and sorghum) on the other hand, use C4 photosynthesis for carbon fixation.
Carbon has two stable isotopes, C-12 and C-13 (the third isotope C-14, is unstable hence radioactive and is used for dating organic archaeological finds). Of the two stable isotopes, the lighter C-12 is preferentially taken up in photosynthesis. Photosynthesis is not universal; it favours fixation of C-12. Further, C3 plants take up lighter C-12 isotope more than C4 plants do. Both C3 and C4 plants discriminate against heavier C-13 isotope but C4 plants do not discriminate as heavily as C3 plants. Put conversely, in photosynthesis, both C3 and C4 plants favour C-12 isotope over C-13 but C3 plants favours C-12 more than C4 plants. This results in differences in ratio of stable isotopes of carbon in C3 and C4 plants and in animals that feed on C3 and C4 plants. An animal fed on C3 plants will have more of lighter isotopes than an animal fed on C4 plants meaning a lipid molecule with lighter isotope ratio is more likely to have originated from an animal fed on C3 plants. This is the conceptual basis of compound specific isotope analysis of lipid (or any other biomolecule for that matter) that helps in identifying sources of lipid residues in the pottery. In a nutshell, C3 and C4 plants have different carbon isotopic ratios. The δ13C value for C3 plants is lighter between −30 and −23‰ while for C4 plants this value is between −14 and −12‰.
After extraction of lipid residues from the samples of potteries, the first key step is to separate different lipid constituents using the technique of Gas chromatography-mass spectrometry (GC-MS). This gives a lipid chromatogram of the sample. Lipids degrade over time so what we usually find in ancient samples are fatty acids (FA), especially palmitic acid (C16) and stearic acid (C18). Thus, this chemical analysis technique helps in identification of fatty acids in the sample but it does not give information about origin of fatty acids. It needs to be further ascertained whether a specific fatty acid identified in the ancient cooking vessel originated from dairy or animal meat or plant. The fatty acid residue in the potteries depend on what was cooked in the vessel in the ancient times.
C3 and C4 plants have different ratios of stable isotopes of carbon due to preferential uptake of lighter C12 isotope during photosynthesis. Similarly, animals fed on C3 and C4 plants have different ratios, for example, domesticated cattle (ruminant animals such as cow and buffalo) fed on C4 food (such as millet) will have different isotope ratio than the smaller domesticated animals like goat, sheep and pig that usually graze and thrive on C3 plants. Further, dairy products and meat derived from ruminant cattle have different isotope ratios due to differences in synthesis of fats in their mammary gland and adipose tissue. Ascertaining origin of a specific fatty acid identified earlier is done by way of analysis of ratios of stable isotopes of carbon. The technique of Gas chromatography-combustion-isotopic ratio mass spectrometry (GC-C-IRMS) is used to analyse isotope ratios of the identified fatty acids.
Importance of ratio analysis of stable carbon isotopes in lipid residues in archaeological studies of prehistoric sites was demonstrated in 1999 when the study of archaeological site in Welsh Borderlands, U.K., could make a clear distinction between fats from non-ruminant (e.g., porcine) and ruminant (e.g., ovine or bovine) origins1. This approach could provide conclusive proof of first dairying in green Saharan Africa in the fifth millennium BC. North Africa was green with vegetation then and prehistoric Saharan African people had adopted dairying practices. This was concluded on the basis of δ13C and Δ13C values of the major alkanoic acids of milk fat identified in potteries2. Similar analyses provided earliest direct proof of dairy processing and consumption by pastoral neolithic societies in eastern Africa3 and in early Iron Age, north China4.
In South Asia, evidence of domestication dates back to the 7th millennium BC. By 4th millennium BC, domesticated animals like cattle, buffalo, goat, sheep etc were present across various Indus Valley sites. There were suggestions of utilisation of these animals in food for dairy and meat but no conclusive scientific evidence to support the view. Stable isotope analysis of lipid residue extracted from ceramic shreds collected from Indus Valley settlements provide the earliest direct evidence of dairy processing in South Asia5. In another recent, more elaborate, systematic study of lipid residues from pot fragments collected from multiple Indus Valley sites, researchers tried to establish type of foodstuffs used in the vessels. Isotope analysis confirmed use of animal fats in vessels. Key scientific finding was dominance of non-ruminant fats in the cooking vessels6 implying non-ruminant animals (such as horse, pigs, poultry, fowl, rabbit, etc) were cooked in the vessels over a long period and consumed as food. This contradicts a long held view (based on faunal evidence) that ruminant animals (such as cattle bovine, buffalo, deer, goats etc) were consumed as food by Indus Valley people.
Unavailability of local modern reference fats and possibility of mixing of plants and animal products are limitations of this study. To overcome possible effects resulting from the mixing of plant and animal products, and for a holistic view, starch grain analysis was incorporated into lipid residue analyses. This supported cooking of plants, cereals, pulses etc in the vessel. This helps overcome some of limitations7.
***
References:
- Dudd SN et al 1999. Evidence for Varying Patterns of Exploitation of Animal Products in Different Prehistoric Pottery Traditions Based on Lipids Preserved in Surface and Absorbed Residues. Journal of Archaeological Science. Volume 26, Issue 12, December 1999, Pages 1473-1482. DOI: https://doi.org/10.1006/jasc.1998.0434
- Dunne, J., Evershed, R., Salque, M. et al. First dairying in green Saharan Africa in the fifth millennium BC. Nature 486, 390–394 (2012). DOI: https://doi.org/10.1038/nature11186
- Grillo KM et al 2020. Molecular and isotopic evidence for milk, meat, and plants in prehistoric eastern African herder food systems. PNAS. 117 (18) 9793-9799. Published April 13, 2020. DOI: https://doi.org/10.1073/pnas.1920309117
- Han B., et al 2021. Lipid residue analysis of ceramic vessels from the Liujiawa site of the RuiState (early Iron Age, north China). Journal Of Quaternary Science (2022)37(1) 114–122. DOI: https://doi.org/10.1002/jqs.3377
- Chakraborty, K.S., Slater, G.F., Miller, H.ML. et al. Compound specific isotope analysis of lipid residues provides the earliest direct evidence of dairy product processing in South Asia. Sci Rep 10, 16095 (2020). https://doi.org/10.1038/s41598-020-72963-y
- Suryanarayan A., et al 2021. Lipid residues in pottery from the Indus Civilisation in northwest India. Journal of Archaeological Science. Volume 125, 2021,105291. DOI:https://doi.org/10.1016/j.jas.2020.105291
- García-Granero Juan José, et al 2022. Integrating Lipid and Starch Grain Analyses from Pottery Vessels to Explore Prehistoric Foodways in Northern Gujarat, India. Frontiers in Ecology and Evolution, 16 March 2022. Sec. Paleontology . DOI: https://doi.org/10.3389/fevo.2022.840199
Bibliography
- Irto A., et al 2022. Lipids in Archaeological Pottery: A Review on Their Sampling and Extraction Techniques. Molecules 2022, 27(11), 3451; DOI: https://doi.org/10.3390/molecules27113451
- Suryanarayan, A. 2020. What’s cooking in the Indus Civilisation? Investigating Indus food through ceramic lipid residue analysis (Doctoral thesis). University of Cambridge. DOI: https://doi.org/10.17863/CAM.50249
- Suryanarayan, A. 2021. Lecture – Lipid Residues in Pottery from the Indus Civilization. Available at https://www.youtube.com/watch?v=otgXY5_1zVo
***
