Biomarkers in archaeology

3.1.2.1. Fatty acids

Fatty acids from natural substances generally consist of an even number of carbon at-oms (between 14 and 22) arranged linearly with a group of carboxylic acid at the end of the hydrogen-carbon chain (Christie 1989).

Fatty acids are the most common compounds in archaeological ceramics. Palmitic acid (C16:0) and stearic acid (C18:0) are the most common, followed by lauric acid (C12:0) and

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myristic acid (C14:0). These acids may be the result of the degradation of the triaciglicer-ides found in animal fats, although they may also be the result of microbial endogenous or exogenous postdepositional contamination.

Unsaturated fatty acids are also frequentely detected: mono- and di-unsaturated acids with 18 carbon atoms (C18:1 and C18:2), as well as to a lesser extent C16:1. C16:1 appears in large quantities when it comes from both marine and freshwater aquatic resources (Ail-laud 2001, Malainey et al. 1999b).

Odd-named fatty acids of carbon atoms (C15:0 y C17:0) appear in archaeological vessels as linear or branched isomers. These compounds are present in ruminant fats (Dudd et al.

1999).

The occurrence of long chain fatty acids (from C20:0 to C30:0) can be attributed to inputs of lipids with an aquatic origin (Craig et al. 2011, 2013, Heron et al. 2015) or waxes of higher plant or honey from bees (Charters et al. 1995, Evershed et al. 1997, Regert et al.

2001a). Long chain unsaturated fatty acids, such as C20:1 can also be related to fats of aquatic origin (Craig et al. 2011, 2013, Heron et al. 2015).

3.1.2.2. TriacylglYcerols

Triacylglicerols are the main components of natural fats and oils. They are composed of a glycerol base, which includes each hydroxy group is linked to a fatty acid through an ester connection (Christie 1989). The position of each of the fatty acids that make up the TAG, as well as their nature (chain length, number and position of insaturations) result from the mode of synthesis of TAGs by enzymatic mechanism within living beings and are highly variable according to the natural origin of the fat. In order to identify TAGs and trace the natural origin of a fat, it is necessary to know the composition of the fatty acids, but also their distribution in the glycerin skeleton.

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The distribution and concentration of triglycerides allows us to get closer to the origin of the fat. Thus, classical ruminant fat profiles are distributed between T46 and T54, with T50 and T52 predominant (Dudd et al. 1999, Mukherjee et al. 2007, Regert et al. 1999).

Dairy products are characterised by a wider distribution, between T40 and T54, with spe-cial concentrations of T50 and T52 (Dudd and Evershed 1998, Mirabaud et al. 2007). Fi-nally, non-ruminant fat profiles are distributed between T46 and T54, with T52 being the most predominant (Dudd et al. 1999).

Figure 3.1. Histograms of characteristic triacylglycerols distributions of various reference contemporary animal fats (taken from Mukjerjee et al. 2007).

However, the identification of unsaturated TAGs, often dominated by T54, corresponds to a classical distribution of vegetable oils (Copley et al. 2005e, Drieu 2017).

3.1.2.3. Alkanes

Alkanes are molecular compounds characterized by a saturated hydrogen-carbon chain and the absence of a functional group (Killops and Killops 2009: 30).

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Alkane profiles are often presented in analyses of archaeological ceramic vessels and may have different origins. Odd alkane profiles dominated by C27 are characteristic of beeswax (Charters et al. 1995, Evershed et al. 1997c, Regert et al. 2001a), whereas when the concentration falls on C29 and C31 alkanes, they are interpreted as epicuticular waxes (Eglinton and Hamilton 1967). The thermal or natural degradation of beeswax can vo-latilize the lighter alkanes and present profiles dominated by C29, so its presence must be interpreted with caution (Regert et al. 2001a).

On the contrary, the presence of alkanes with carbon atom pair name chains is related to a petrogenic origin (Killops and Killops 2009).

3.1.2.4. Alcohols

Linear alcohols, sometimes called fatty alcohols, have a hydroxy group at the end of their hydrogen-carbon chain. Like fatty acids, they rarely exist in free form, but rather in com-bination with other molecules.

Long-chain linear alcohols called carbon atom pairs (C20OH-C32OH) often appear in ar-chaeological ceramics, dominated by C26OH or C28OH, which are identified in vegetal waxes (Egliton and Hamilton 1967), or by C30OH and C32OH, characteristic of degraded beewaxes (Regert et al. 2001a, Evershed et al. 1997c, Charters et al. 1995).

3.1.2.5. Wax sters

Wax esters consist of fatty acids (mainly linear and saturated but sometimes branched or unsaturated), bound to long chain linear alcohols by an ester bond.

Wax esters are identified in archaeological materials with a distribution between W40

and W52, which are interpreted as beeswax (Charters et al. 1995, Evershed et al. 1997c, Garnier et al. 2002, Heron et al. 1994, Regert et al. 2001a). The wax esters correspond to acid fragments from palmitic acid, detected by m/z 257 ions (Regert et al. 2001a). The

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presence of wax esters of different acids is interpreted as plant esters (Ribechini et al.

2008).

3.1.2.6. Sterols

Sterols are molecules formed by steranes and functional groups, such as cholesterol, formed by 27 carbon atoms, which is found in much of animal fat (Evershed et al. 1993, Heron and Evershed 1993). Classic plant sterols, called phytosterols, are composed of 29 carbon atoms, such as stigmasterol, ß-sitosterol, campesterol (Evershed 1993).

3.1.2.7. Terpenes

Terpenes are compounds derived from isoprene, a hydrocarbon of 5 carbon atoms. Ter-penes originate from the enzymatic polymerization of two or more isoprene units, so most terpenes have polycyclic structures, which differ from each other not only in func-tional group but also in their basic carbon skeleton.

The diterpenes commonly identified in archaeological samples come from conifer resin, such as pimaric acid, isopimaric acid and abietic acid. The latter is characteristic of pine resins. All of them degrade and oxidize easily, by exposure or thermal alteration (Colom-bini et al. 2005b, Regert and Rolando 2002).

The presence of triterpenes, formed by 30 carbons, is usually found in the form of lupeol, betulin and hopans. These are the evidence of plant exudates, such as birch bark (Heron 1998, Binder et al. 1990, Rageot 2015, Regert 2004).

3.1.2.8. Exogenous lipids

Since many of the compounds identified through organic residue analysis are of a com-mon nature, the presence of lipids that do not come from anthropic activities should be taken into account so as not to bias archaeological information.

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These contaminants can have two sources of origin: anthropic or taphonomic. Contam-inants of anthropogenic origin, i.e. the result of the manipulation of the containers by archaeologists, can have different sources. The presence of squalene and cholesterol, characteristic molecules of fat secreted by human epithelial cells, can be transferred during the manipulation of ceramic vessels. Squalene has a polyunsaturated structure that degrades easily and is not usually preserved in archaeological contexts. In short, when squalene is detected in the analysed sample, it is automatically assumed that the detected cholesterol is not archaeological. Another modern source of anthropic con-taminants are phthalate plasticisers, which are easily introduced by contact with plastic bags (Pollard et al. 2007, Stacey, 2009). To avoid this, the use of nitrile gloves during the manipulation of the vessels, as well as the storage of the ceramics in aluminium foil to avoid the migration of compounds such as phthalates, can optimize the analysis of or-ganic residues.

Taphonomic or environmental contaminants are more difficult to control (Condamin et al. 1976). Although the migration of lipids from the sediment is almost insignificant (Rot-tländer, 1990, Heron et al. 1991), some samples show evenly distributed alkanes of car-bon atoms indicating their petrogenic origin (Steele et al. 2008).

Due to underlying problems with contamination, Evershed (2008) suggested that the minimum amount of total lipid extract (TLE) recovered, which can be used for a reliable identification, should not be less than 5μg of lipid per gram of sherd.

3.2. Applied methods