Organic Residue Analysis

Organic residue analysis of Saxo-Norman pottery from Glebe Field, Siston: Report

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Julie Dunne, Toby Gillard and Richard P. Evershed

Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock’s Close, Bristol, BS8 1TS, UK

November 7th 2022

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1.      Introduction

Lipids, the organic solvent soluble components of living organisms, i.e. the fats, waxes and resins of the natural world, are the most frequently recovered compounds from archaeological contexts. They are resistant to decay and are likely to endure at their site of deposition, often for thousands of years, because of their inherent hydrophobicity, making them excellent candidates for use as biomarkers in archaeological research (Evershed 1993).

Pottery has become one of the most extensively studied materials for organic residue analysis (Mukherjee et al. 2005) as ceramics, once made, are virtually indestructible and thus are one of the most, if not the most, common artefacts recovered from archaeological sites from the Neolithic period onwards (Tite 2008). Survival of these residues occurs in three ways; rarely, actual contents are preserved in situ (e.g. Charrié-Duhaut et al. 2007) or, more commonly, as surface residues (Evershed 2008). The last, most frequent occurrence is that of absorbed residues preserved within the vessel wall, which have been found to survive in >80% of domestic cooking pottery assemblages worldwide (Evershed 2008).

The application of modern analytical techniques enables the identification and characterisation of these sometimes highly degraded remnants of natural commodities used in antiquity (Evershed 2008). Often, data obtained from the organic residue analysis of pottery or other organic material provides the only evidence for the processing of animal commodities, aquatic products or plant oils and waxes, particularly at sites exhibiting a paucity of environmental evidence. To date, the use of chemical analyses in the reconstruction of vessel use at sites worldwide has enabled the identification of terrestrial animal fats (Evershed et al. 1997a; Mottram et al. 1999), marine animal fats (Copley et al. 2004; Craig et al. 2007), plant waxes (Evershed et al. 1991), beeswax (Evershed et al. 1997b) and birch bark tar (Charters et al. 1993; Urem-Kotsou et al. 2002). This has increased our understanding of ancient diet and foodways and has provided insights into herding strategies and early agricultural practices. Organic residue analysis has also considerably enhanced our understanding of the technologies involved in the production, repair and use of ancient ceramics.

Preserved animal fats are by far the most commonly observed constituents of lipid residues recovered from archaeological ceramics. This demonstrates their considerable significance to past cultures, not just for their nutritional value but also for diverse uses such as binding media, illuminants, sealers, lubricants, varnish, adhesives and ritual, medical and cosmetic purposes (Mills and White 1977; Evershed et al. 1997a).

Today, the high sensitivities of instrumental methods such as gas chromatography and mass spectrometry allow very small amounts of compounds to be detected and identified. Furthermore, higher sensitivity can be achieved using selected ion monitoring (SIM) methods for the detection of specific marine biomarkers (Evershed et al. 2008; Cramp and Evershed 2013). The advent of gas chromatography-combustion-isotope ratio mass spectrometry in the 1990s introduced the possibility of accessing stable isotope information from individual biomarker structures, opening a range of new avenues for the application of organic residue analysis in archaeology (Evershed et al. 1994; 1997a).

This stable carbon isotope approach, using GC-C-IRMS, is employed to determine the δ13C values of the principal fatty acids (C16 and C18), ubiquitous in archaeological ceramics. Differences occur in the δ13C values of these major fatty acids due to the differential routing of dietary carbon and fatty acids during the synthesis of adipose and dairy fats in ruminant animals, thus allowing ruminant milk fatty acids to be distinguished from carcass fats by calculating Δ13C values (δ13C18:0 - δ13C16:0) and plotting that against the δ13C value of the C16:0 fatty acid. Previous research has shown that by plotting ∆13C values, variations in C3 versus C4 plant consumption are removed, thereby emphasizing biosynthetic and metabolic characteristics of the fat source (Dudd and Evershed 1998; Copley et al. 2003).

1.      Aims and objectives

The objective of this investigation was to determine whether absorbed organic residues were preserved in potsherds excavated from Glebe Field, Siston.

2.      Materials and analytical methods

Lipid analysis and interpretations were performed using established protocols described in detail in earlier publications (Correa-Ascencio and Evershed 2014). Briefly, ~2 g of potsherd were sampled and surfaces cleaned with a modelling drill to remove exogenous lipids. The cleaned sherd powder was crushed in a solvent-washed mortar and pestle and weighed into a furnaced culture tube (I). An internal standard was added (20 µg n-tetratriacontane; Sigma Aldrich Company Ltd) together with 5 ml of H2SO4/MeOH 2 - 4% (δ13C measured) and the culture tubes were placed on a heating block for 1 h at 70 °C, mixing every 10 min. Once cooled, the methanolic acid was transferred to test tubes and centrifuged at 2500 rpm for 10 min. The supernatant was then decanted into another furnaced culture tube (II) and 2 mL of DCM extracted double distilled water was added. In order to recover any lipids not fully solubilised by the methanol solution, 2 x 3 mL of n-hexane was added to the extracted potsherds contained in the original culture tubes, mixed well and transferred to culture tube II. The extraction was transferred to a clean, furnaced 3.5 mL vial and blown down to dryness. Following this, 2 x 2 mL n-hexane was added directly to the H2SO4/ MeOH solution in culture tube II and whirlimixed to extract the remaining residues, then transferred to the 3.5 mL vials and blown down until a full vial of n-hexane remained. Aliquots of the TLE’s were derivatised using 20 µl BSTFA, excess BSTFA was removed under nitrogen and the derivatised TLE was dissolved in hexane prior to GC, GC-MS and GC-C-IRMS. Firstly, the samples underwent high-temperature gas chromatography using a gas chromatograph (GC) fitted with a high temperature non-polar column (DB1-HT; 100% dimethylpolysiloxane, 15 m x 0·32 mm i.d., 0.1 μm film thickness). The carrier gas was helium and the temperature programme comprised a 50°C isothermal followed by an increase to 350°C at a rate of 10°C min−1 followed by a 10 min isothermal. A procedural blank (no sample) was prepared and analysed alongside every batch of samples. Further compound identification was accomplished using gas chromatography-mass spectrometry (GC-MS). FAMEs were then introduced by autosampler onto a GC-MS fitted with a non-polar column (100% dimethyl polysiloxane stationary phase; 60 m x 0.25 mm i.d., 0·1 μm film thickness). The instrument was a ThermoFinnigan single quadrupole TraceMS run in EI mode (electron energy 70 eV, scan time of 0·6 s). Samples were run in full scan mode (m/z 50–650) and the temperature programme comprised an isothermal hold at 50°C for 2 min, ramping to 300°C at 10° min-1, followed by an isothermal hold at 300°C (15 min). The instrument was a ThermoFinnigan single quadrupole TraceMS run in EI mode (electron energy 70 eV, scan time of 0·6 s). Samples were run in full scan mode (m/z 50–650) and the temperature programme comprised an isothermal hold at 50°C for 2 min, ramping to 300°C at 10° min-1, followed by an isothermal hold at 300°C (15 min). Data acquisition and processing were carried out using the HP Chemstation software (Rev. C.01.07 (27), Agilent Technologies) and Xcalibur software (version 3.0). Peaks were identified on the basis of their mass spectra and gas chromatography (GC) retention times, by comparison with the NIST mass spectral library (version 2.0).

1.      Results

Lipid analysis and interpretations were performed using established protocols described in detail in earlier publications (e.g. Dudd and Evershed 1998; Correa-Ascencio and Evershed 2014).  Thirty sherds were analysed with the lipid recovery rate being 73%, with 22 vessels yielding interpretable lipid profiles.

The mean lipid concentration from the sherds (Table 1) was 1.3 mg g-1, with a maximum lipid concentration of 6.1 mg g-1 (GLB21). A number of the potsherds contained high concentrations of lipids (e.g. GLB03, 3.4 mg g-1, GLB06, 2.5 mg g-1,  GLB12, 3.5 mg g-1 and GLB25, 5.8 mg g-1,), demonstrating excellent preservation. This likely indicates that these Glebe Field vessels were subjected to sustained use in the processing of high lipid-yielding commodities. The lipid profiles (Figure 1a and b) comprised the free fatty acids, palmitic (C16) and stearic (C18), typical of a degraded animal fat (Evershed et al. 1997a; Berstan et al. 2008).

Extracts from a number of sherds (GLB03, GLB04 and GLB21) include a series of long-chain fatty acids (in low abundance), containing C20 to C26 carbon atoms. It is thought these LCFAs likely originate directly from animal fats, incorporated via routing from the ruminant animal's plant diet (Halmemies-Beauchet-Filleau et al. 2013; 2014).

k

        k

Figure 1. Partial gas chromatograms of acid-extracted FAMEs from Glebe Field Saxo-Norman pottery extracts of GLB04 (ruminant dairy) and GLB25 (ruminant adipose), circles, n-alkanoic acids (fatty acids, FA); IS, internal standard, C34 n-tetratriacontane.

GC-C-IRMS analyses were carried out on the sherds (n=22; Table 1) to determine the δ13C values of the major fatty acids, C16:0 and C18:0, and ascertain the source of the lipids extracted, through the use of the Δ13C proxy. The δ13C values of the C16:0 and C18:0 fatty acids from the lipid profiles are plotted onto a scatter plot along with the reference animal fat ellipses (Figure 2a). It has been established that when an extract from a vessel plots directly within an ellipse, for example, ruminant dairy, ruminant adipose or non-ruminant adipose, then it can attributed to that particular source. If it plots just outside the ellipse then it can be described as predominantly of that particular origin. However, it should be noted that extracts commonly plot between reference animal fat ellipses and along the theoretical mixing curves, suggesting either the mixing of animal fats contemporaneously or during the lifetime of use of the vessel (Mukherjee 2004; Mukherjee et al. 2005).

In this instance, one of the lipid residues plots within the dairy reference ellipse (GLB14, Figure 2a), suggesting this vessel was solely used to process dairy products, with a further two (GLB04 and GLB09, Figure 2a) plotting quite close to the ellipse. Nine vessels plot within the ruminant adipose ellipse (GLB02, GLB05, GLB07, GLB11, GLB13, GLB16, GLB17, GLB18 and GLB26, Figure 2a) with a further five plotting just outside the ellipse (GLB06, GLB12, GLB15, GLB21 and GLB25, Figure 2a) suggesting these vessels were solely/mostly used to process ruminant carcass products. A further five vessels (GLB01, GLB03, GLB10, GLB23 and GLB29, Figure 2a) plot between the ruminant and non-ruminant ellipses, suggesting some mixing of these animal products, whether contemporaneously or during the lifetime use of the vessel.

Ruminant dairy fats are differentiated from ruminant adipose fats when they display Δ13C values of less than -3.1 ‰, known as the universal proxy (Dunne et al. 2012; Salque 2012). Lipid residues from 3 of the 22 (14%) lipid-yielding vessels plot within the ruminant dairy region (GLB04, GLB09 and GLB14 with Δ13C values of -3.4, -4.8 and -4.6 ‰, respectively, Figure 2b and Table 1) confirming that these vessels were used to process secondary products, such as milk, butter and cheese. However, it should be noted that vessel GLB04 plots at the extent of the dairy range, suggesting some mixing with ruminant carcass products in this vessel.  Seventeen vessels (77%, GLB02, GLB03, GLB05, GLB06, GLB07, GLB10, GLB11, GLB12, GLB13, GLB15, GLB16, GLB17, GLB18, GLB21, GLB23, GLB25 and GLB26 with Δ13C values of -1.6, -1.6, -2.0, -1.4, -1.9, -1.5, -1.5, -1.2, -2.0, -1.3, -1.8, -2.0, -2.5, -1.5, -2.3, -1.5 and -1.7 ‰, respectively, Figure 2b and Table 1) plot within the ruminant adipose region. One vessel (5%, GLB01 with a Δ13C value of 1.2 ‰) plots within the non-ruminant region and one vessel plots between the ruminant and non-ruminant regions (GLB29, with a Δ13C value of -0.5 ‰, Figure 2b and Table 1) suggesting mixing of these animal products, whether contemporaneously or during the lifetime use of the pot.

Figure 2. Graphs showing: a. δ13C values for the C16:0 and C18:0 fatty acids for archaeological fats extracted from the Glebe Field Saxo-Norman ceramics. The three fields correspond to the P = 0.684 confidence ellipses for animals raised on a strict C3 diet in Britain (Copley et al. 2003). Each data point represents an individual vessel. b shows the Δ13C (δ13C18:0 – δ13C16:0) values from the same potsherds. The ranges shown here represent the mean ± 1 s.d. of the Δ13C values for a global database comprising modern reference animal fats from Africa (Dunne et al. 2012), UK (animals raised on a pure C3 diet) (Dudd and Evershed, 1998), Kazakhstan (Outram et al. 2009), Switzerland (Spangenberg et al. 2006) and the Near East (Gregg et al. 2009), published elsewhere.

1.      Discussion and conclusion

The objective of this investigation was to determine whether organic residues were preserved in Saxo-Norman vessels excavated from Glebe Field, Siston. The results, determined from GC, GC-MS and GC-C-IRMS analyses, demonstrate that the majority of vessels (77%) were used to process ruminant carcass products (i.e. from cattle, sheep and goat). Information from any faunal remains excavated would help establish which animals may have been exploited at Siston. A further 14% of vessels were used to process dairy products such as butter and cheese, sometimes referred to as ‘white meats’ of the poor and known to have been one of the mainstays of the medieval peasant diet (Black 2003; Adamson 2004). One vessel was used to process solely pig products and another, mixtures of ruminant and non-ruminant carcass products. This confirms the importance of ruminant carcass products, likely used to prepare the stews or potages known to be the mainstay of the medieval diet. These results are somewhat comparable to those obtained from ceramics at medieval West Cotton (c. 950 to 1450 AD) where the majority of the assemblage was used to process ruminant carcass products (57%), a further 27% of vessels were used to process dairy products and 10% to process pig products (Dunne et al. 2019). 

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