Evershed - Biomarker Revolution

*Received 8 August 2008; accepted 11 August 2008 University of Oxford, 2008

Archaeometry

50

, 6 (2008) 895924 doi: 10.1111/j.1475-4754.2008.00446.x

Blackwell Publishing LtdOxford, UKARCHArchaeometry0003-813X1475-4754 University of Oxford, 2008XXX

ORIGINAL ARTICLE

Organic residue analysis in archaeologyR. P. Evershed

*Received 8 August 2008; accepted 11 August 2008

ORGANIC RESIDUE ANALYSIS IN ARCHAEOLOGY: THE ARCHAEOLOGICAL BIOMARKER REVOLUTION*

R. P. EVERSHED

Organic Geochemistry Unit, Bristol Biogeochemistry Research Centre, School of Chemistry, Cantocks Close, Bristol BS8 1TS, UK

Organic residue analysis utilizes analytical organic chemical techniques to identify thenature and origins of organic remains that cannot be characterized using traditionaltechniques of archaeological investigation (because they are either amorphous or invisible).The field is founded upon the principle that the biomolecular, or biochemical, componentsof organic materials associated with human activity survive in a wide variety of locationsand deposits at archaeological sites. The archaeological information contained in organicresidues is represented by the biomolecular components of the natural products thatcontribute to the formation of a given residue. By applying appropriate separation(chromatographic) and identification (mass spectrometric) techniques, the preserved, andaltered, biomolecular components of such residues can be revealed. Once identified, the

Archaeological Biomarker Concept

can be applied, wherein the structure and even isotopiccomposition(s) of a given biomolecule or suite of biomolecules (the chemical fingerprint)can be related to the compositions of organisms exploited by humans in the past. As theorganic residue field emerges from its pre-paradigmatic phase, and the organic residuerevolution gathers pace, the way is open for challenging many long-held archaeologicalhypotheses and offering new perspectives on the study of human activity in the past.

KEYWORDS:

ARCHAEOLOGY, BIOMARKERS, ORGANIC RESIDUES, CHROMATOGRAPHY, MASS SPECTROMETRY, BIOMOLECUES, STABLE ISOTOPES, CHEMICAL

FINGERPRINTS

INTRODUCTION

Organic Residue Analysis

is now an established discipline in archaeology, with increasingnumbers of archaeologists beginning to consider organic residue analyses amongst the manyscientific tools available to them. A major influence in the development of the field was theemergence of a new generation of analytical chemical methodologies in the middle of the 20thcentury that enabled complex environmental materials to be studied in increasingly fine detail.Spectroscopic methods, such as infrared (IR), Raman and nuclear magnetic resonance (NMR)spectroscopies, provide insights into bulk compositions that have proved useful in fingerprintingthe sources of certain classes of organic residue; for example, ambers and resins and theirderivatives (Beck

et al

. 1965; Lambert

et al

. 1985). However, the chemical complexity, derivingfrom the many constituents (endogenous or exogenous) that comprise residues, constrains theusefulness of such methodologies beyond broad descriptions of chemical properties; forexample, compound classes present, which are often difficult to relate to specific sourcematerials, especially where mixtures of natural products may be in evidence (Sherriff

et al

. 1995;Edwards

et al

. 2004). The emergence of chromatographic methods able to achieve molecular-level

896

R. P. Evershed

University of Oxford, 2008,

Archaeometry

50

, 6 (2008) 895924

resolution and recognition during the 1950s and 1960s, particularly the linking of gas chroma-tography (GC) to mass spectrometry (MS)in other words, GC/MSpaved the way for themolecular components of complex biological and environmental materials to be separated andcharacterized in detail, making the origins of multiple constituents discernable.

The modern era of the organic residue analysis of archaeological materials was heralded bythe paper of Thornton

et al

. (1970), who investigated the origin and identity of the enigmaticbog butters by GC. This paper was the first of a number that emerged over the next 25 yearsthat provided the empirical and experimental framework for our current understanding of therange of artefacts and deposits likely to contain organic residues preserving archaeologicalbiomarkers (Condamin

et al

. 1975; Kuksis

et al

. 1978; Lin

et al

. 1978; Passi

et al

. 1981;Knights

et al

. 1983; Rottlnder and Schlichtherle 1983; Morgan

et al

. 1984; Evershed

et al.

1985, 1990, 1991, 1992, 1994, 1995a,b; Patrick

et al

. 1985; Evershed and Connolly 1987,1994; Robinson

et al

. 1987; Rullktter and Nissenbaum 1988; Connan and Dessort 1989;Glaar

et al.

1989, 1990; Hurst

et al

. 1989; Pepe

et al

. 1989; Pepe and Dizabo 1990;Rottlander 1990; Oudemans and Boon 1991; Connan

et al

. 1992; Nissenbaum 1992; Proefkeand Rinehart 1992; Proefke

et al.

1992; Charters

et al.

1993a,b, 1995; Evans and Heron 1993;Bethell

et al.

1994; Heron

et al

. 1994; Boda

et al

. 1995). Significantly,

Archaeometry

was thevenue for the publication of a number of these key papers.

This paper is not intended to be an exhaustive review of the field of organic residue analysis.Rather, the aim is to provide a critical appraisal of the current state of knowledge of the field,which will consider (i) the


, (ii) the occurrence of bio-markers in organic residues in the record, (iii) the survival of biomarkers in organic residues,(iv) the integration of organic residue analysis into research programmes, and (v) what thefuture holds.

This paper does not contain specific details of analytical methodologies; nor does it providein-depth discussions of the operation of instrumentation, as such matters have been coveredexhaustively elsewhere, although it will discuss the formulation of analytical protocols in thecontext of why some approaches may be preferred over others to solve particular archaeo-logical questions. Ancient DNA analysis by PCR and related molecular biological methods orimmunological studies will not be covered, since these are distinctly separate subjects basedon biochemical rather than chemical methods, although many of the matters discussed arerelevant to those areas. Radiocarbon and organic residue analysis are complementary fields,since, as Bronk Ramsey (2008) points out, the majority of the materials submitted for radio-carbon analysis are organic residues, purified to the compound or molecular level; thuschemical purity or structure can be used as quality control criteria for radiocarbon dating.A fundamental difference between the two fields is that organic residue analyses are generallyperformed on components purified from amorphous or invisible organic residues. However,the two fields become very closely aligned when radiocarbon analyses are performed onbiomolecular components purified from amorphous remains; this combination represents apotentially powerful new addition to the organic residues analysts tool kit (Berstan

et al

. 2008).Readers will note that, with only one or two exceptions, the literature cited is drawn from

peer-reviewed journals. The reason for this is twofold: first, that in establishing a newparadigm, the preferred way for this to be tested is via rigorous scrutiny through the peerreview system; and, second, that a significant non-peer-reviewed literature appeared early inthe history of the field (up to

c

. 1990), which initially misled the archaeological communityas to the analytical chemical rigour required for meaningful results to be obtained fromorganic residues.

Organic residue analysis in archaeology

897


Archaeometry

50

, 6 (2008) 895924

THE ARCHAEOLOGICAL BIOMARKER CONCEPT

In 1993, an issue of

World Archaeology

(vol. 25, no. 1) appeared, edited by Kenneth Thomas,that evolved out of a range of conference sessions, discussion meetings and initiativesoccurring at that time. The issue collected together a number of papers encompassingnew approaches to the biomolecular analysis of organic remains at archaeological sites. Mycontribution was a paper entitled Biomolecular archaeology and lipids (Evershed 1993).The motivation for the paper was threefold. First, the field of organic residue analysis was stillembryonic, although the handful of accounts of molecular-level analyses of organic residue thatexisted indicated great promise. The paper drew the available material together and discussedthe opportunities for the development of the field, attempting to emphasize the potential of lowmolecular weight compounds, such as lipids, within the broader scope of the field of ancientbiomolecule research, which at that time was highly focused on ancient DNA analysis. Second,a number of technological developments were on the horizon, which were set to have majorimpacts on the development of the fieldfor example, compound-specific stable isotopeanalysisand I felt it important to highlight these. Third, it was clear that a frameworkwas required to provide continuity between the fragmentary early research and that to beundertaken in the years ahead. The latter was achieved by defining the concept of

ArchaeologicalBiomarkers

as those substances occurring in organic residues that provide informationrelating to human activity in the past. The use of biomarkers in archaeology has many reso-nances with their use in other fields, particularly organic geochemistry, where enduring carbonskeletons reflect the organisms that existed in palaeoenvironments on geological timescales.The application of organic geochemical methods to palaeoenvironmental change in theQuaternary, particularly in the Holocene, is an area of potential coalescence of the organicgeochemical and archaeological biomarker approaches (McClymont

et al

. 2008), while the useof biomarkers to study petroleum bitumen in archaeological contexts is an obvious exampleof convergence of two approaches (see below): beyond this, the

Archaeological BiomarkerConcept

is distinctive, since the focus is specifically on human activity. While my originalpaper (Evershed 1993) focused on lipids (the organic solvent soluble components of livingorganisms; i.e., the fats, waxes and resins of the natural world), the

Archaeological BiomarkerConcept

can be applied to any class of biomolecules; that is, ancient DNA, proteins,carbohydrates, pigments and so on. In the cases of ancient DNA, proteins and carbohydrates,the entire structure of the original biopolymer will rarely, if ever, survive intact, so the struc-turally diagnostic biomarker entity will take the form of fragmentsfor example, sequencesof DNA amplifiable by PCRor building blocks; for example, amino acids or peptides in thecase of ancient proteins.

Analytical prerequisites

The application of the


brings with it critical consequencesfor the way in which organic residue analyses have to be approached. Most notably, it implicitlyrequires that the analytical techniques employed be able to achieve molecular-level resolution.Since all the organic materials encountered at archaeological sites are of biological origin,they will be mixtures. This complexity is increased through human activitiesnamely, as aresult of mixing biological materials in, for example, food preparationand then complexityis increased still further as a result of compositional alteration due to decay during burial.Such complexity means that for the identities of molecular structures to be determined in such

898

R. P. Evershed


Archaeometry

50

, 6 (2008) 895924

mixtures, the use of chromatographic and/or mass spectrometric methods is imperative: this iswhy the emergence of organic residue analysis as a discipline has been so strongly tied toadvances in instrumental methods of this genre.

Tracing origins from structures

The


operating in its simplest mode relies upon matchingthe structures or distributions, chemical fingerprints, to the compounds and mixtures knownto exist in extant organisms likely to have been exploited in the past (Evershed 1993). Some-times, the structure of a single component is sufficient to define the origin of a constituent ofan organic residue (Fig. 1). For example, the di- and triterpenoid components of modern plantresins have been widely studied and offer a robust means of assigning their presence ascomponents of organic residues, sometimes to the botanical species or more commonly to thegenus level (Hayek

et al

. 1990; Charters

et al

. 1993b; Evans and Heron 1993; Evershed

et al

.

1997c; Grunberg 2002; Urem-Kotsou

et al

. 2002; Regert

et al

. 2003a). A further example isbeeswax, which is readily recognized due to the characteristic mixture of aliphatic componentsthat it contains (Heron

et al

. 1994; Charters

et al.

1995; Evershed

et al.

1997b, 2003; Regert

et al

.

2005). Certain other classes of plant lipid can also be assigned to their sources based uponthe specific hydrocarbons, ketone, alcohols and/or fatty acids that they contain (Evershed

et al.

1991; Charters

et al.

1995; Copley

et al.

2001a,b; Reber

et al.

2004).The assignment of a specific source or constituent of a residue based on the presence of a

particular biomarker component or mixture of components demands a high degree of rigour,

Figure 1 Archaeological biomarkers observed in their native state in organic residues.

Organic residue analysis in archaeology

899


Archaeometry

50

, 6 (2008) 895924

wherein consideration of the nature of other constituents of the residue may lead to thehypothesis of a putative source being rejected. An example of this is seen in a recent report(McGovern

et al

. 2004) of the presence of beeswax in an organic residue, based on the pres-ence of C

23

, C

25

, C

27

, C

29

, C

31

and C

33

n

-alkanes. However, the gas chromatographic profileshows a textbook

n

-alkane distribution characteristic of petroleum, wherein both odd and evencarbon number homologues are present at similar abundance: thus consideration of the entire

n

-alkane complement, rather than selected components, means that the null hypothesis mustbe acceptedthese compounds do not unambiguously derive from beeswax. The recent reportof Namdar

et al.

(in press) based an assignment of beeswax on similar

n

-alkane distributions,asserting that they can derive from heated-treated beeswax. Where such ambiguities exist firmconclusions as to the true nature of organic residues can only be reached once more robustsupporting evidence has been obtained, e.g. the presence of hydroxypalmitate wax esters,otherwise the question of origin of such distributions must remain open.

An essential deductive aspect of organic residue analysis is the archaeological and palaeo-ecological context for biomarker-based interpretations. The question that has to be asked isas follows: Is the presence of a constituent of a residue based on an observed biomarkerconsistent with the archaeology and palaeoecology of the settlement, region and/or periodfrom which the find derived? In practice, consideration of the latter actually serves to simplifyinterpretations and increase the diagnostic potential of seemingly rather undiagnostic biomarkers,since alternative sources of biomarkers can legitimately be disregarded if they make no sensearchaeologically or palaeoecologically.

Synergy in structural and isotopic composition

Animal fats and plant oils offer greater challenges because the major components, polyunsaturatedfatty acids in particular, rarely if ever survive (although, as discussed below, their prior presencecan be recorded in the altered components), leaving mainly rather undiagnostic

n

-alkanoic acids(derived mainly through hydrolysis of triacylglycerols; Fig. 2). However, it has been shownthat there is considerable scope for the identification of such materials if the stable isotopiccompositions of the common

n

-alkanoic acid biomarkers are determined. For example, the

13

Cvalues of the

n

-hexadecanoic and

n

-octadecanoic acids that survive in degraded animal fats canbe used to differentiate their sources: ruminant and non-ruminant fats can be separated and ruminantcarcass fats distinguished from dairy fats, due to metabolic differences between the different animalsand carbon sources utilized in biosynthesis of different fat types (Fig. 2; see also Dudd and Evershed1998; Mottram

et al.

1999; Copley

et al.

2003, 2005be; Craig

et al.

2005; Mukherjee

et al.

2007;Evershed

et al.

2008b). Such separations can rarely be achieved based on fatty acid compositionsalone. Thus, by combining molecular structures in tandem with their stable isotope compositions,synergy is achieved within the archaeological biomarker approach. Such synergy confers oneven the most mundane of structures a high level of biomarker specificity.

The specificity of this approach is enhanced still further by considering the most likely sourcesfor these fatty acid components, on the basis of the available palaeoenvironmental information.For instance, in studies of animal fats in cooking pottery, the major domesticates are the onlyspecies that need to be considered in interpretations, as statistically these are the most legitimatesources likely to leave significant residues. For example, in our studies of animal product processingin prehistoric Britain, only cattle, sheep/goat and pigs need to be considered as realistic options(Copley

et al

. 2003, 2005e; Mukherjee

et al.

2007, 2008a): in other regions or periods therange of species will be different, thereby guiding interpretations accordingly.

900

R. P. Evershed


Archaeometry

50

, 6 (2008) 895924

Source information locked into altered structures

One of the fundamental aspects of the biomarker concept is that of the source being recordedby the different arrangements of carbon atoms (i.e., the different carbon skeletons; cf., Fig. 1).The more unique the structures are to a given source organism, the greater is the certainty thaton observing a given biomarker structure, the presence of a particular constituent could beconfirmed. However, in many cases altered rather than native structures are observed. Theability to recognize an original constituent, or source, of an organic residue based upon alteredstructures surviving in the residues is a powerful aspect of the biomarker approach, whichrequires knowledge of the chemical and biochemical mechanisms and pathways likely to beinvolved. An example of this phenomenon mentioned above is the recognition that the n-alkanoicacids that occur very widely in residues derive mainly from triacylglycerols that originallycomprised the parent fats and oils, the former being released by hydrolysis (Fig. 2).

Another example is the recognition of structurally altered components that have obviouscoherency with the carbon skeletons in their precursors, but where significant structural trans-formations have occurred. An example of this is seen in the array of diterpenoid structuresobserved in heated coniferous resins (Evershed et al. 1985; Robinson et al. 1987). The carbonskeletons of the observed products leave little doubt as to the generic origin of residues con-taining such components (Fig. 3). Significantly, the structural alterations bring with themadditional information concerning the life history of the residue. The high energies required toproduce aromatized and defunctionalized products offer a new level of diagnosis, namely ofthe technology of the production process, as well as source information. Analogous examplesof this phenomenon are seen in other heat-treated resins (Fig. 3; see also Hayek et al. 1990; Sternet al. 2003; Regert et al. 2003a,b; Regert 2004).

Figure 2 Simple saturated C16:0 and C18:0 fatty acids generated via hydrolysis of triacylglycerols (LHS) during processing and/or burial of fats (and oils), which on their own have limited diagnostic value as biomarkers. However, the plot (RHS) of the 13C values for these fatty acids shows how the fats of the major Old World domesticated animals can be separated due to differences in the their metabolic and biosynthetic origins. The ellipses are confidence ranges (P = 0.684) and the theoretical mixing ranges. Such plots provide the basis for determining the origins of animal fat residues (adapted from Mukherjee et al. 2005).

Organic residue analysis in archaeology 901

University of Oxford, 2008, Archaeometry 50, 6 (2008) 895924

Further examples of this phenomenon are seen in fats and oils, where more stable structur-ally altered products are formed through human activities, commonly as a result of heating.Examples include the formation of long-chain ketones analogous to components seen inhigher plant waxes. The ketones are formed via a free radical-induced dehydration anddecarboxylation, which occurs when acyl lipids are heated in excess of 300C (Fig. 4; see alsoEvershed et al. 1995b; Raven et al. 1997). Likewise, -(o-alkylphenyl)alkanoic acids with1622 carbon atoms (Fig. 5; see also Hansel et al. 2004) are produced when unsaturated fattyacids are heated to over 270C (Evershed et al. 2008a). Since polyunsaturated fatty acidsrarely survive in appreciable concentrations in organic residues in pottery vessels, these -(o-alkylphenyl)alkanoic acids, which are more stable compounds, offer a novel means ofdetecting the processing of commodities containing unsaturated fatty acids.

A final example is the observation of dihydroxy fatty acids in archaeological pottery frommonounsaturated fatty acids, wherein the position of the hydroxyl groups effectively added tothe carbon atoms bearing the original double-bond record precisely the original position ofunsaturation, which in combination with the carbon number of the fatty acid can be relatedback to the parent plant or animal source exploited in the past (Fig. 6; see also Regert et al. 1998;Colombini et al. 2005; Copley et al. 2005a; Romanus et al. 2007; Hansel and Evershed submitted).

Figure 3 Di- and triterpenoid biomarkers produced by heat treatment of natural products.

Figure 4 Lipid biomarkers produced by heat treatment of saturated fatty acyl lipids.

902 R. P. Evershed


In summary, application of the Archaeological Biomarker Concept requires not onlyknowledge of the biochemical compositions of the organic commodities exploited by humansin the past, but also an appreciation of how these materials can be altered by processing and/orburial. Many of the mechanisms and pathways of molecular structural change resulting fromdegradation and decay are predictable and thus can enhance the interpretative framework.

THE OCCURRENCE OF ORGANIC RESIDUES

Table 1 summarizes the various locations and deposits in which organic residues survive atarchaeological sites. Clearly, organic residues have the capacity to survive widely (and over

Figure 5 Highly unstable unsaturated fatty acids preserved by cyclization and aromatization.

Figure 6 The double-bond position deduced from structures of vicinal diols.



considerable timescale). However, the survival of organic residues is not assured, nor is itubiquitousfor reasons that are not yet fully understoodbut it will certainly reflect culturalas well as taphonomic differences in the life history of an organic residue through theprocesses of formation, deposition/discard and burial to ultimate recovery. Thus, whenembarking on organic residue analyses of a new class of artefact and/or in a new geographicalregion, the presence of organic residues should not be assumed. This conclusion is basedupon the results of organic residue analyses performed on several thousand individual artefactsand ecofacts recovered from many parts of the world, including those from archaeologicalsites located in disparate climatic, ecological and geological zones. However, the occurrenceof organic residues is sufficiently widespread that they can be considered as viable sources ofinformation in many research and post-excavation programmes.

The following section offers further elaboration of the summary given in Table 1.

Pottery and other containers

Organic residues occur widely in association with this major class of artefact, offering a remark-able sink of information relating to vessel use, site and regional economies and technologies.Organic residues survive in three principal forms in archaeological pottery, namely as follows:(i) Actual contents preserved in situ as vessel fills (not to be confused with intrusive post-burial fills). Instances of the contents surviving in situ are rare, such that few examples exist ofchemical investigations of these, a notable example being the contents of Canopic jars (Charri-Duhaut et al. 2007).(ii) Surface residues appearing as visible residues on the interior or exterior of vessels. Externalsooting and internal carbonized residues are probably the organic residues most familiar topottery analysts. The former are presumed to be residues of cooking failures, although visibleresidues in lamps will represent the remains of the fuels and wicks burned in them (Copley et al.

Table 1 Sources of organic residues in the archaeological record. The number of asterisks are scores (***** = high to * = low) relating to the importance of the source, both in terms of its demonstrable capacity to yield

archaeologically relevant information and its occurrence in the record (geographical spread and rates of recovery)

Pottery Soils and sedimentsVessel fills* Midden and other organic wastes***Surface or visible residues**** Agricultural soils*** Absorbed residues***** Habitation deposits**

Ritual deposits**Human and animal remains

Skeletal remains**** Resins and bitumensSoft tissues*** Natural resins*****Mummies**** Plant gums**

Fossil resins***Plant remains Petroleum bitumens***

Waterlogged remains*** Desiccated remains** MiscellaneousCarbonized(?)* Glass containers**

Metal containers**Dyes and pigments Stone objects**

Textiles**** Hoards; e.g., bog butter**Art****

904 R. P. Evershed


2005a), while post-firing treatments are also seen, particularly as copious linings associatedwith transport vessels (Beck et al. 1989; Mills and White 1989). Visible residues have beenchemically investigated (e.g., Oudemans and Boon 1991; Oudemans et al. 2007) and arewidely used in dating programmes. A concern when working with such residues is the potentialfor them to become contaminated by post-burial or post-excavation contamination due totheir exposed nature. Residues occurring on the exteriors of vessels correspond to either sootingderived from fuel used in heating on a fire, applied decorations (Urem-Kotsou et al. 2002;Connan et al. 2004) or, rarely, adhesive used to repair vessels in antiquity (Charters et al.1993b). An under-appreciated source of external surface residues is from carbonization of theorganic components of the contents diffusing through the vessel wall, which burn on the exterior.(iii) As absorbed residues preserved within the vessel wall, invisible to the naked eye. Thisfinal category of residue is by far the most common in pottery and probably the most widelyoccurring residue type. Analyses performed to date suggest that absorbed organic residuessurvive in >80% of domestic cooking pottery assemblages worldwide. Such residues arisethrough the processing of plant and animal products, with heat and/or mechanical actionmobilizing fats, waxes and other biochemical components to aid transfer in liquid or solutionform into the fabric of the vessel wall (Charters et al. 1993a; Stern et al. 2000). Laboratoryexperiments (Rottlnder and Schlichtherle 1983; Patrick et al. 1985; Charters et al. 1997;Raven et al. 1997; Dudd and Evershed 1998; Dudd et al. 1999; Evershed et al. 2008a) and studiesof ethnographic vessels (Evershed 2008) have contributed greatly to understanding how theseresidues arise and their compositional alteration during vessel use and decay.

Chemical investigations of organic residues in archaeological pottery have revealed a widerange of compound types, which have led to the identification of residues of an impressiverange of commodities from several continents. Commodities recognized include vegetable oils(Condamin et al. 1976; Kimpe et al. 2002; Copley et al. 2005a), terrestrial animal fatsthatis, adipose and milk fats (Dudd and Evershed 1998; Mukherjee et al. 2007)marine animalfats (Patrick et al. 1985; Copley et al. 2004; Hansel et al. 2004; Craig et al. 2007), resins(Beck et al. 1989; Mills and White 1989; Stern et al. 2003), birch bark tar (Charters et al.1993b; Urem-Kotsou et al. 2002), plant waxes (Evershed et al. 1991; Reber and Evershed2004a,b; Reber et al. 2004), beeswax (Heron et al. 1984; Charters et al. 1995; Evershed et al.1997a, 2003), palm kernel oil (Copley et al. 2001a,b), petroleum bitumen (Connan et al.2004), cocoa (Hurst et al. 1989, 2002) and so on.

Interestingly, the detection of wine residues and those of other alcoholic beverages appearsto present a significant challenge, despite the fact that such residues have been reported on a numberof occasions (e.g., McGovern et al. 1996, 2004). The recent work of Stern et al. (2008) empha-sizes the importance of employing techniques that possess the molecular specificity requiredto make unambiguous identifications of trace constituents of chemically complex residues.The approach of Guasch-Jan et al. (2004), using high-performance liquid chromatographyMSMS, offers the sensitivity and selectivity essential to provide secure identifications basedon the detection of tartaric and syringic acid biomarkers (Guasch-Jan et al. 2006a,b).

Human and animal remains

Human and animal remains are amongst the most widely surviving class of find at archaeologicalsites. The following three categories of remains are considered in organic residue analysis:(i) Skeletal remains are without question the most widely occurring of this class of remains, withthe collagen fraction routinely isolated for stable isotope and/or radiocarbon analysis. As discussed



above, these types of analysis are viewed separately from organic residue analyses, since thebulk determination of radiocarbon or stable isotope composition are the primary goals (Vogeland van der Merwe 1977; Bronk Ramsey 2008), rather than utilizing their structural featuresper se to achieve more fundamental links to human activity or taphonomic processes of relevanceto archaeological reconstruction. Where studies of collagen do fall within the paradigm oforganic residue analysis is when individual amino acids are isolated and their stable isotopesignals used to explore aspects of human dietary behaviour intractable to analysis of wholecollagen (Fogel and Tuross 2003; Corr et al. 2005, 2008a). Perhaps more formally recognizedas organic residues are the lipids preserved in skeletal remains, which interestingly aregenerally discarded in collagen analyses (Evershed et al. 1995a). These lipid residues providean alternative source of stable isotopic information for palaeodietary reconstruction, due theirdiffering turnover rates and differences in biochemical origin (Stott and Evershed 1995a;Howland et al. 2003; Jim et al. 2004).(ii) Soft tissues survive much less frequently at archaeological sites than skeletal remains, butwhere they do survive they attract a high level of interest. As with skeletal remains, the majorsurviving biochemical class is protein, which is a common target for radiocarbon and/or stableisotope analysis. Lipids are also commonly preserved in waterlogged (Evershed and Connolly1988), desiccated (Glaar et al. 1989) and frozen (Mayer et al. 1990; Corr et al. 2008b)environments. Biomarker analyses have revealed components of bacteria involved in degradation.(iii) Egyptian mummies provide a separate category on account of the copious organic balmsapplied to many mummies in antiquity to preserve the corpses. The amorphous nature of thesebalms requires the use of organic residue analysis techniques to characterize their components(Rullktter and Nissenbaum 1988; Proefke and Rinehart 1992; Proefke et al. 1992; Mejanelleet al. 1997; Koller et al. 1998; Buckley et al. 1999, 2004; Colombini et al. 2000; Maurer et al.2002). The range of natural products used to prepare the organic balms can offer insights intowhy the ancient embalmers chose specific components for their preservative properties orsymbolic significance (Buckley and Evershed 2001). The chemical analysis of mummies fromother than Egypt is a surprisingly under-investigated area.

Techniques developed for the investigation of organic residues in archaeology have the capacityto contribute to both palaeo- and contemporary forensic investigations. Recent work with theMetropolitan Police secured a murder conviction through the complementary use of the DNAbiomarker and compound-specific carbon isotope approaches to provide evidence for the priorpresence of a cadaver in a shallow grave, based on the detection of adipocere (Bull et al. in revision).Fatty acids characteristic of adipocere were several orders of magnitude higher in concentrationin the soils of the grave compared to controls, while Figure 7 shows the stable carbon isotope com-positions of the soil fatty acids, an adipocere particle, subsequently recovered tissues and referencefats, including those from cadavers comprising the Body Farm experiment in the USA. Theresults are consistent with a common origin for the soil, adipocere and victims tissue components.

Resins, fossil resins and bitumensHumans would have exploited these substances very widely in the past, and analyses of theremains of resins, tars and pitches and bitumens can answer questions relating to their botan-ical natural origins, in addition to their modes of acquisition, preparation and geographicalprovenience. The following major areas are considered:(i) Natural resins and their derivatives comprise one of the most mature areas of organicresidue analysis. Such materials occur widely either as amorphous deposits or in association with

906 R. P. Evershed


various classes of archaeological objects, notably as components of the organic balms ofEgyptian mummies and associated objects; for example, coffin varnishes/decorations, internalcoatings and decorations of pottery vessels, as components of glues and hafting materials, andso on. The natural product chemistry of the di- and triterpenoid constituents of many plantresins has been widely investigated, providing an invaluable resource for the recognition ofthe botanical sources of archaeological resins (Mills and White 1994; Evershed et al. 1997c;van Bergen et al. 1997b; Serpico and White 2000a,b). As discussed above, one of the majorchallenges in this area is the recognition of thermally altered forms of natural resins (Evershedet al. 1985; Hayek et al. 1990; Dudd and Evershed 1999; Regert et al. 2003a; Stern et al. 2003;Regert 2004), which raises opportunities to determine the ancient technologies involved intheir production.(ii) Fossil resins attracted attention early in the history of organic residues analysis (Becket al. 1965; Lambert et al. 1985), due to the use of amber and copal to fashion ancient art objectsand jewellery (Mukherjee et al. 2008b and references therein). These polymeric materials arerelated to the plant resins discussed above in terms of their generic biological origin, but areconsidered separately due to their geological ages. While amber is frequently referred to as afind in excavations, it is surprising how few rigorous chemical identifications exist in thepeer-reviewed literature, especially since their determination is non-trivial (Beck et al. 1965;Mills et al. 1984; Anderson and Winans 1991; Mukherjee et al. 2008b).

Figure 7 13C values for the C16:0 and C18:0 fatty acids extracted from grave soils [soil IJH3-8 and IJH8-picking (adipocere)], adipose fat from the suspected victim (Vict 1-3) and reference human adipose from North America (8 Hum samples). Further reference fats are plotted as confidence ellipses (1), corresponding to cow and sheep body and butter fat, and further body fats from chickens, geese and pigs (n = 50).



(iii) Petroleum bitumens have been widely studied in finds from the Near East due to theexistence of natural petroleum seeps in the region, which would have provided ancientpeoples with the opportunities of exploiting these resources for a wide variety of uses,including in mummification, art, as components of building materials and in hafting(Rullktter and Nissenbaum 1988; Connan and Dessort 1989, 1991; Connan et al. 1992, 2004;Nissenbaum 1992; Boda et al. 1996; Connan 1999). The major focus of investigations ofthe bitumen component of these materials has been the use of molecular parameters toassign the origins of the various bitumen sources (Harrell and Lewan 2002; Barakat et al.2005).

Soils and sediments

This is a particularly challenging area of organic residue analysis. However, under favourablecircumstances, organic compounds have been shown to survive in soils and sediments (Bullet al. 1999b and references therein; Fiore et al. in press). Biomarker analyses have revealed anumber of classes of compound that can be exploited to provide insights into the inputs tosoils and sediments:(i) Middens and disused watercourses can be identified by detecting 5-stanols and secondarybile acids (Fig. 1). These biomarkers are present in the faeces of many animals and have beenshown to survive on archaeological timescales (Knights et al. 1983; Pepe et al. 1989; Pepe andDizabo 1990; Bethell et al. 1994; Bull et al. 2003). The characteristic compositions exhibitedby different groups of animals allow their faecal contributions to different archaeologicalfeatures associated with waste disposal to be assessed.(ii) The recognition by early farmers that improved crop yields could be achieved by the useof fertilizers would have been a major step forward in the development of agriculture. Whileorganic compounds are potentially degradable in the soil, under favourable circumstancemolecular and stable isotope signals can be sufficiently long-lived to provide assessments ofmanuring practices and the classes of crop grown (Bull et al. 1999a, 2001; Simpson et al.1999a; Jacob et al. 2008).(iii) The anthropogenic sediments formed pre- and post-use in unrobbed tombs and burialsprovide significant opportunities for exploiting their constituent organic residues as archives ofbiomolecular and stable isotope information for use in reconstructing burial practices andritual activities (James et al. in press). Approaches akin to those used in the forensic investiga-tion described above offer considerable scope in archaeological contexts.

Plant remains

Plant remains of interest to organic residue analysis are preserved at archaeological sitesmainly in the form of seeds and woody tissues. Such remains have been studied to answer arange of questions relating to plant domestication via PCR-based methods. However, beyondancient DNA, under favourable conditions such remains can preserve a wide variety of bio-molecules, in a range of states of preservation, as follows:(i) Desiccated remains only survive in exceptionally arid regions and may contain well-preservedbiomolecules. Their study has provided many new insights into the factors controlling thedecay and preservation of biomolecules (ODonoghue et al. 1994, 1996a,b; Evershed et al. 1997b;van Bergen et al. 1997a). The first direct chemical evidence of DNA in the archaeologicalrecord was derived from such remains (ODonaghue et al. 1994, 1996a,b).

908 R. P. Evershed


(ii) Plant remains from waterlogged deposits. As with (i) above, chemical analyses of bio-molecules from such deposits have provided insights into decay and preservation processes,and are particularly noteworthy in providing insights into the early stages of fossilization(McCobb et al. 2001, 2003).(iii) Carbonized forms. While DNA has been recovered from this class of remains, less successhas been achieved in recovering other classes of compound that can be directly related to theiroriginal biomolecule complement.

The biomolecular analyses of plant remains have an important role to play in providinginsights into the preservational biases that exist in the palaeobotanical record.

Dyes and pigments in art and textiles

Occurring in association with textiles and art objects, the study of ancient dyes and pigmentsdoes not fit into the strict definition of organic residues, since they are clearly not invisible.However, analysis of dyes relies upon analogous biomarker approaches to organic residueanalysis, with their identifications relying upon matching the structures of dye components tothe compositions of natural plant and animal pigments. This is a well-developed research field,in which the archaeological and art/historical periods offer a continuum in practices, under-pinned by an abundant and robust literature (Ferreira et al. 2004; Cardon 2007). Due to thespectral characteristics displayed by the major chromophores contained within the dye com-ponents, the combination of HPLC with photodiode array detection is a powerful tool in thisarea, analogous to GC/MS, with the potential for on-line separation and UV/visible spectro-scopic analysis (van Bommel 2005). The field has been the subject of a number of authoritativereviews and books, and thus will not be discussed here. Archaeological investigations in thisarea are rather rare, due to the poor preservation potential of dyed artefacts; for example,textiles. Chemical investigations are supported by textual evidence and remains of ancient dyeinstallations date back to the third millennium bc. A strong aspect of research in this area hasbeen the study of the vatting technologies used in ancient dying industries, with some remark-able levels of detail emerging (Koren 1995).

Organic residue and pigment analysis overlap most strongly in this area in the characteriza-tion of organic binders used to apply the pigments. The study of these materials is a highlydeveloped area in the study of works of art, providing insights into their production and under-pinning restoration programmes (Mills and White 1984). In archaeologyfor example, incave or wall paintingsopportunities exist for applying analogous approaches, but a majorchallenge lies in resolving contributions from binder components and the organic constituentsof the substrate, which may incorporate significant microbial or algal contaminant contributions,highly altered by exposure over the millennia. A recent investigation of hematite pigmentresidues from Terra del Fuego showed the presence of fatty acid distributions consistent withthe presence of fat or oils, potentially representing remnants of binders used to apply theinorganic pigment (Fiore et al. in press).

Miscellaneous organic remains

The archaeological record is a source of continual surprises in terms of the unusual organicremains recovered. For instance, the identities of bog butter, hoards of animals fat (Thorntonet al. 1970), intentionally deposited in peat bogs, presumably for the purpose of preservation,have only recently been confirmed (Berstan et al. 2004). These bog butter hoards, found in



amounts up to about 50 kg, constitute the largest deposits of organic residues known anywherein the archaeological record.

Other notable finds include a Roman tin container with its tightly fitting lid and cosmeticcontents in place, which provided a unique opportunity to undertake a total analysis of anorganic residue for the first time; revealing a mixture of animal fat and purified starch as thebase for the white tin pigment cassiterite (SnO2). The cosmetic cream was probably a skinwhitener (Evershed et al. 2004). Roman glass containers have recently been shown to containorganic residues comprising fats and waxes, which point to them being the bases for cosmetics,most probably perfumes (Ribechini et al. 2008a,b).

Recent investigations of anthropogenic sediments from the Bronze Age Royal Tomb atQatna, Syria, provided an unusual example of biomarker studies, prompting further morpho-logical investigations of sediments from archaeological deposits. The biomarkers analysesrevealed purple-coloured solvent extracts containing distributions of indigoid and indirubinoidcomponents (Fig. 8), which confirmed the presence of the famous Royal Purple dye (Jameset al. in press). The dye was presumed to derive from degraded textiles associated withcorpses. This was subsequently confirmed through microscopic examinations of the sedimentsfrom the tomb, which revealed millimetre-sized fragments of fossilized textile (gypsum replicas;confirmed by X-ray crystallography) displaying the purple dye. The morphologies of the textilefragments recorded the weave of the textile and pattern of the applied dye. The presenceof the purple dye is indicative of wool being used to fabricate the textile.

THE SURVIVAL OF ORGANIC RESIDUES

The question of why organic residues survive in certain deposits is an area in which systematicresearch is rather sparse. Our current understanding of the controls on organic residue preservationis based almost entirely on empirical observations. All organic compounds are potentially degradable;hence for residues to survive they must be protected in some way, or possess inherent recalcitranceconferred by their molecular structures which determine their physico-chemical properties.

Protection of biomarkers within mineral or organic phasesMineral matrices, such as the inorganic apatite phase of bone and the fabric of pottery, offerenvironments in which organic molecules are partially protected from microbiological degradation.

Figure 8 The chemical structures of biomarkers for the shellfish purple dyes.

910 R. P. Evershed


It is presumed that organic molecules are: (i) contained within pore spaces inaccessible toexocellular enzymes produced by degrading microbes; or (ii) protected through adsorption onsurfacesfor example, polar lipids require the use of a strong base for extraction (Regert et al.1998) and it is assumed that such substances would be largely unavailable as substrates formicroorganisms. Likewise, protection appears also to be conferred by entrapment withinorganic matrices, such as carbonized organic residues on pottery (Patrick et al. 1985; Oude-mans and Boon 1991) and aggregates of organic matter; for example, resin/bitumen deposits(Rullktter and Nissenbaum 1988; Charters et al. 1993b), hoards (Berstan et al. 2004), softtissue remains (Evershed and Connolly 1988) and so on. Encapsulation of organic residueswithin botanical remains (desiccated but not carbonized: ODonaghue et al. 1994, 1996a,b;van Bergen et al. 1997a) and coprolites (Lin et al. 1978) offer further media for organicresidue preservation. The presence of naturally preservative substancesfor example, antioxidantsor enzyme inhibitorsmay also aid preservation of organic residues.

Structural controls on biomarker preservation

There is no doubt that structural differences between different classes of biomarker willaccount for differential preservation. The susceptibility of the major classes of biomolecules tostructural modification and degradation in the environment generally follows the order: lipids< carbohydrates lignin < protein < nucleotides. The order can vary somewhat according toenvironmental factors and the history of an artefact. However, it is fundamentally determinedby the nature of bonds in the different classes of biomolecule. More polar substances will gen-erally be more susceptible to decay, especially where essential elements are present; that is, Nand P. One of the primary reasons why lipids survive at archaeological sites is because they arehydrophobic, which means that they are not readily leached from the site of original depositionby percolating groundwater, nor made available to microbes as substrates by mobilizationthrough dissolution in interstitial waters. However, it is vital to appreciate that even withina class of compounds, substantial differences may exist in the degradation trajectories of differ-ent sub-classes. For example, in the laboratory decay of plant epicuticular waxes and animalfats co-deposited on to replica ceramic, markedly different patterns of decay were revealed,with the long-chain epicuticular wax components (C29 n-alkane, 2-alkanol and ketone)being significantly better preserved than the fatty acyl lipids (Evershed 2008). This latter obser-vation has significant consequences for the assessment of quantitative contributions to organicresidues based on the distributions of components surviving in extracts of artefacts which willcomplicate quantitative assessments (Olsson and Isaksson 2008).

The recent discovery described above of Royal Purple dye components surviving chemicallyintact, while the textile has been converted to a mineral replica, reflects the chemical stabilityof indigoid and indirubinoid structures compared to lability of the protein-based textile (Fig. 8;see also James et al. in press).

Environmental controls

A number of other environmental factors influence organic residue preservation, includingtemperature, light exposure, degree of waterlogging, redox conditions and so on (Eglinton andLogan 1991). Desiccation is highly favourable, as microbial growth cannot occur withoutwater; however, extensive abiological chemical oxidation of residues will still occur (Glaaret al. 1989, 1990). Spectacular organic residue preservation by desiccation is seen at desert



sites such as Qasr Ibrim, Egypt, where the survival of organic remains, including vast numbers ofseeds and other plant remains, textiles, pottery residues, resins, leather items and so on, is quiteextraordinary, presenting numerous opportunities for organic residue analysis (ODonaghue et al.1994, 1996a,b; Evershed et al. 1997b,c; van Bergen et al. 1997a,b; Regert et al. 1998;Copley et al. 2001a,b, 2005a). Artificial desiccation of Egyptian mummies, together withthe use of balms comprising substances inhibitory to microbial growth, accounts for theirsurvival over the millennia, often in a high state of preservation (Buckley and Evershed 2001;Buckley et al. 2004). At the other extreme, burial of artefacts in waterlogged deposits,especially under anoxic conditions, is also favourable for the survival of organic residues:indeed, many of the most remarkable examples of organic preservation come from suchdeposits; for example, bog bodies (Evershed and Connolly 1988, 1994; Evershed 1990, 1992)and bog butters (Berstan et al. 2004). Laboratory experiments involving a range of fatty acyland other aliphatic lipids dosed into replica ceramics have allowed degradation under oxic andanoxic conditions to be investigated (Evershed 2008): the results showed unequivocally thatacyl lipid degradation (animal fat and olive oil) is greatly retarded under anoxic conditions.Indeed, little or no lipid remained after only a few weeks of microbial degradation under oxicconditions, providing important information concerning the optimal environments for recoveryof organic residue-containing sherds for analysis.

Nutrient limitation, especially of N and P, may also serve to limit the progress of degradation:once the concentrations of these, and other, essential elements become limited, the activities ofmicrobes will inevitably be slowed or halted. While extremes of waterlogging and desiccationare unquestionably conducive to the survival of organic residues, alternating wetting and dryingin climate zones where seasons of high rainfall are followed by hot dry periods appears to bedetrimental to residue survival.

INTEGRATING ORGANIC RESIDUE ANALYSIS INTO ARCHAEOLOGICAL RESEARCH PROGRAMMES

As with any area where chemical analysis is involved, the design of the analytical protocol ispredicated on the question (hypothesis) to be answered. The basic philosophy of the analyticalprogramme will be no different to any other field, although it is true that organic residueanalysis in archaeology brings with it some considerable challenges on account of the fragmentarynature of the material record and the unpredictable way in which interesting finds presentthemselves. All archaeological science research is interdisciplinary by nature, and thus canonly be effectively performed through collaborations between scientists and archaeologists.The unifying feature of the most prominent laboratories currently undertaking organicresidues analyses is that the scientists are archaeologically literate. However, the optimalcombination is for such scientists to join forces with scientifically empathetic archaeologists.This combination will ensure a conjoining of minds such that meaningful archaeologicalquestions will be posed, with realistic chances of achieving success, as optimal analyticalprotocols will be devised.

Expert analysts undertake their work systematically, probing aspects of the composition ofmaterials using often well-established methods, chosen based on well-defined questions. Theyfully appreciate why they use particular techniques and the level of interpretation that can beattached to a particular determination, provided that the context is defined. When questionsare correctly framed, there will probably be surprisingly few choices in the way the analysescan be undertaken. The nature of the question will define: (i) the choice of analytical method;

912 R. P. Evershed


(ii) the sampling protocolin other words, the type, size and number of samples that need tobe taken, including modern reference materials; (iii) the nature of any sample pre-treatmentsteps, cleaning, grinding and so on; (iv) whether extractions or separations are required; (v)the nature of the final measurement; (vi) the use of standards to validate the methods; and(vii) the assessment of the final resultsthat is, the application of statistical methods todetermine whether the results obtained are significant in the light of the original problemdefined. Organic residue analyses performed with the appropriate degree of rigour are essentiallyforensic analyses; thus objective interpretations must consider all lines of archaeologicalinformationbiomarker, artefactual, geographical and so onin accepting or rejecting theclearly defined hypotheses.

It cannot be overly stressed how fundamental correctly framing the research question is incomplex analytical work of this nature. The importance of this is emphasized by the enigmaticpaper of Barnard et al. (2007), in which sherds dosed with an organic residue known only tothe lead author were then circulated to a number of participating laboratories for analysis,using a range of techniques of varying suitability for the task in hand. The results showed thatwithout proper archaeological contextual information (geographical origin, faunal and palaeo-botanical information, and human activities deduced from other lines of archaeologicalenquiry) and a clearly defined research question, analysts were powerless to devise the mosteffective analytical protocol, since they were effectively hindered from (i) being able to choosethe most appropriate analytical technique(s) to tackle the defined question(s), and (ii) selectingthe most appropriate reference materials relevant to the hypothetical ecosystem or settlementeconomy from which the organic residue derived. The latter information is absolutely fundamentalto the success of organic residue analysis programmes. Not surprisingly, none of the laboratoriesconfirmed the presence of camels milk, although to their credit one came close! However, ifthe analysts had been told the region of the world from which the residue might theoreticallyhave originated and been offered some background archaeological informationfor example,compositions of faunal assemblagesthen the chances of making a correct identificationwould have been greatly enhanced.

The most common type of organic residue analysis performed to date has focused onindividual or relatively small collections of finds of amorphous residues. The goal is usually todetermine the nature, origins, technological histories and so on by determining their chemicalcompositions. Such analyses are non-trivial because fully characterizing all the original con-stituents of complex degraded organic residues is an exceedingly complex and analyticallydemanding task. Such analyses must bring to bear a range of different analytical techniques toensure that all components of residues, organic and inorganic, soluble and insoluble, and soon are determined (Regert et al. 2003b; Evershed et al. 2004; Ribechini et al. 2008a,b; Sternet al. 2008). The latter studies employed various combinations of bulk methodsthat is,FTIR, SEM and CHN/gravimetric analysisto provide overviews of the elemental composi-tions of both organic and inorganic components, and major functional groups associated withthe organic constituents of the residues. In all cases, the archaeological biomarker approachwas employed to determine the origins of the organic constituents of the residues. GC/MS wasthe universal technique of choice, operated either in full scan or selected ion-monitoringmode, to capture major and trace constituents. PyrolysisGC/MS (Buckley et al. 1999) directexposureMS (Modugno et al. 2006) and electrosprayMSMS (Garnier et al. 2002) techniquesprovide access to involatile components potentially intractable to GC/MS. As discussed above,stable isotope ratio MS, particularly in compound-specific mode (Copley et al. 2003), providesadditional diagnostic information.



Interestingly, the majority of organic residue analyses reported to date are qualitative studies; inother words, the questions asked generally relate to the identity of the natural product(s)present, with little or no information being sought on the quantitative contributions of thevarious constituents to the residues. Our investigation of a Roman cosmetic from London is, asfar as I am aware, the only example of a total qualitative and quantitative analysis of anorganic residue (Evershed et al. 2004). Although we were unusually fortunate to be presentedwith the opportunity to study this remarkable find, other such opportunities will arise from thearchaeological record and we must be ready to tackle them from this perspective. Gaininginsights into the precise ways in which our ancestors were formulating such commodities isvital to improving our understanding of their state of knowledge of the properties of rawmaterials that they were handling, and of decisions that they would have to have made inproducing commodities with the properties that they required.

Quantification of the biomarker content of artefacts is performed through the addition ofinternal standards and potentially leads to information (i) concerning the use-life of an artefact,(ii) relating to the taphonomic or diagenetic history of the artefact, and (iii) that is vital in planninga number of aspects of laboratory analyses. For example, the addition of n-tetratricontaneto lipid extractions of pottery allows the concentrations of lipid in the ceramic fabric to bedetermined, which has provided important insights into the ways in which lipids are accumulatedin pottery vessels during use (Charters et al. 1993a, 1997; Evershed 2008) and has resultedin a complete reassessment of the sampling strategies for organic residue analyses of pottery.Quantitative information of this nature also allows intra- and inter-site and regional comparisonsto be made, providing vitally important information for use in interpreting the results ofanalysis and planning future research. For example, surface finds rarely produce significantresidues, presumably due to the impacts of weathering, and are thus a high-risk class of artefactfor organic residue analysis.

Another level of quantitative information that can be sought through organic residueanalysis is the intensity of a particular activity within a settlement or region. Using pottery asan example, we have recently probed the quantitative relationship between the use of GroovedWare in relation to other prehistoric British pottery traditions and a range of other archaeologicalquestions through organic residue analysis. Figure 9 shows clearly that Grooved Ware use ismore strongly associated with pig product processing than any other pottery. This assessmentwas achieved by using the compound-specific carbon isotope-based mixing curves shown inFigure 2. The incidences of pig fat residues (Fig. 9) were shown to correlate with pig skeletalabundances, thereby establishing an important link between commodity source and processing(Mukherjee et al. 2007, 2008a). Similar correlations have also been made between milk usefrom organic residues in pottery and the abundance of cattle, based on faunal counts, as themajor producing species in Britain (Copley et al. 2005e), South-East Europe and the Near East(Evershed et al. 2008b).

Beyond studies of individual or small collections of organic residues is a quite differentcategory of analysis, namely that where a particular biomarker proxy, or a group of biomarkerproxies, is brought to bear on a significant archaeological problem. This category of investigationis where the interplay between the scientist and archaeologist, or possibly several scientistsand many archaeologists, becomes essential for three important reasons: (i) such studies aredemanding of significant resource, human and infrastructural, and we must be sure we areasking the right questions; (ii) it is highly likely such a programme will require assemblinglarge numbers of artefacts, and this cannot be contemplated without archaeologists andscientists working together at the planning, iterative and interpretative phases of projects; and

914 R. P. Evershed


(iii) an assembly of multi-proxy information, derived through several different scientificapproaches, may be required to provide robust and cohesive interpretations.

Although many opportunities can be envisaged for undertaking such investigations, surpris-ingly few published examples currently exist. Our recently published work concerning theearly evidence of milk use related to cattle herding in South-East Europe and the Near Eastprovides such an example. The work was based on the availability of the robust fatty acid-based stable carbon isotope proxy for dairy fat detection in archaeological pottery, alreadydiscussed above (Dudd and Evershed 1998; Copley et al. 2003). The proxy was used to addressan aspect of the late Andrew Sherratts Secondary Products Revolution hypothesis, namelythat of the timing and region of emergence of milk use; a number of other sub-hypotheseswere also addressed. The inextricable connection between the animals producing the milk and

Figure 9 Scatter plots showing the greater incidence of pig fat in Grooved Ware vessels compared to other prehistoric British pottery, based on the 13C values of the methyl esters of C16:0 and C18:0 fatty acids determined from extracts of all (a) Neolithic (not including Grooved Ware), (b) Grooved Ware, (c) Bronze Age and (d) Iron Age vessels (Copley et al. 2003, 2005be; Mukherjee et al. 2008).



the pottery used in its processing meant that securing zooarchaeological and organic residueevidence in tandem was a major feature of the research plan. A significant aspect of the projectdesign was the level of sampling required, demanded by the geographical and chronologicalrange considered, which in turn dictated the resources required to effectively complete thesampling and analytical phases of the project. We learned a number of lessons from thisproject, particularly the importance of maintaining a clear focus on the project goal, and theneed to allow for a degree of flexibility in the research design to accommodate unexpectedresults; for example, we were forced to investigate three times the number of potsherds, dueto the lower than anticipated organic residue recovery rates from a significant proportion of thesites. Ultimately, the project was successfully completed, with the results having major impli-cations for our understanding of the production and consumption of this key staple by earlypastoralists. This evidence for extensive milk processing in pottery vessels shows that lactoseintolerance presented no significant barrier to dairy product consumption, provided that milkwas processed (Fig. 10; see also Evershed et al. 2008b).

THE FUTURE

The field of organic residue analysis archaeology has witnessed spectacular developments inrecent years, and there is now a well-developed understanding of where residues survive atarchaeological sites and an appreciation of the major classes of biomarker likely to be encountered.There are, however, significant opportunities for expanding the range of known biomarkers inthe years ahead. This will occur as organic residue analysis is applied in previously largelyuninvestigated regions of the world, such as Asia, Africa and South America. Organic residuesstudies are most rewarding when they are targeted at the most important archaeological questions;however, an important factor when deciding where to focus effort is to consider those regionswhere organic residue preservation is likely to be optimal. Thus, sites located in regions ofconstant environmental conditions, especially when combined with low mean annual temperaturesand waterlogging, provide environments conducive to preservation, and thus might be preferredtargets. Likewise, regions of extremely low rainfall, allowing preservation by desiccation, offerprospects for enhanced preservation and thus increased rates of organic residue recovery. Atthe site or artefact level, the indications are that organic residue preservation is enhanced byencapsulation within organic or mineral aggregates or pores, making pottery, skeletal remains,resinous or bituminous deposits, organic-rich sediments, soft tissues and plant remains the mostimportant sources of organic residues. However, we must be careful not to neglect other classesof artefact; for example, stone artefacts, such as lithics, have yielded generally disappointing resultsfrom organic residues, except in cases where surface deposits are evident. The fundamentalproblem is the lack of protection offered to organic residues by non-porous minerals. Preliminarywork in our laboratory suggests that certain porous stone objects may preserve interpretableorganic residues and may be worthy of further study, especially where collections of suchartefacts are sufficient to make systematic study worthwhile. In this respect, querns, grindingvessels and other stone containers may be worthy of further consideration.

Considerable potential exists for increasing the range of natural products that can be recog-nized in the archaeological record via biomarker analyses. The search for new biomarkerswill be enhanced by the application of new analytical approaches, which offer prospects for:(i) extending the range of biomarkers through the detection of high-polarity or high molecularweight substances by application of soft ionization methods (ODonoghue et al. 1996a;Mirabaud et al. 2007; Solazzo et al. 2008); (ii) enhancing the sensitivity and selectivity of

916R. P

. Evershed

U

niversity of O

xford, 2008, Archaeometry

50, 6 (2008) 895924

Figure 10 A map showing the locations of sites providing pottery for organic residue analysis for Early Neolithic milk fats (LHS) and (RHS) plots of the 13C values for archaeological animal fat residues in pottery, showing the most intensive milk use in northwestern Anatolia, dating back to the seventh millennium BC. The pottery was from (a) northwestern Anatolia, (b) central Anatolia, (c) South-East Europe/northern Greece, and (d) eastern Anatolia and the Levant. The 13C values (= 13C18:0 13C16:0) for the ruminant dairy fats are more depleted than for the ruminant adipose fats; the difference in the means is ~2.8, which is highly significant (t-test; P < 0.0005). Pig fats have positive 13C values that do not exhibit significant variance and the differences in the mean values are also highly significant (ANOVA; P < 0.0005 between all three commodity groups; Bonferroni adjustment applied). 13C = [(13C/12C)sample/(13C/12C)standard] 1, expressed in per millilitre. All 13C values are relative to the Vienna PeeDee Belemnite (VPDB) international standard.



biomarker detection by the use of selected ion- or selected reaction-monitoring techniques(ODonoghue et al. 1994; Guash-Jan et al. 2006a,b); and (iii) increasing the range of compound-specific stable isotope investigations of biomarkers to include nitrogen (Simpson et al. 1999b),deuterium and oxygen although archaeological applications of the latter are currently lacking,and such analyses will allow links to be forged between biomarker structures and variations instable isotope composition recording climatic/seasonal phenomena relating to the behavioursof ancient humans and their exploitation of hunted and management of domesticated animals;(iv) incorporating biomarker analyses into radiocarbon analysis programmesthe combinationof compound-specific radiocarbon analysis and biomarker analysis has the potential to emergeas a powerful new combination (Berstan et al. 2008); (v) greater integration of analyticalmethodologies, to provide a quantitative as well as a quantitative perspective on organic residuecomposition; (vi) increasing experimental work to enhance our understanding of the processesof formation and preservation of organic residues; and (vii) increasing the integration oforganic residue information with other lines of archaeological evidence.

In summary, it is now accepted that molecular approaches alone offer the rigour necessaryfor confirming the origins of the constituents of complex organic residues. The informationprovided through such analyses ranges from the uses of specific artefacts to provenances ofmaterials, ancient technologies and wider economic activities. Thus, we are now at the stagewhere in order to provide archaeologically meaningful interpretations we must begin to forgestronger connections between the residues that we observe and the cultural and social attributesof the individuals and communities that produced them. To achieve this, more emphasis willhave to be placed on establishing the importance of a given residue, or suite of residues, as arepresentation of a given activity within a site or region, and the only way of achieving thiswill be through the study of statistically meaningfulthat is, largernumbers of systematicallyselected artefacts. Accumulating larger data sets brings many other advantages; for example,opportunities are already emerging for mining existing databases to explore trends in organicresidue preservation with time, environment and findspot characteristics, which will help toprovide improved predictive frameworks for organic residue preservation/recovery, with obviousadvantages for planning future research programmes.

ACKNOWLEDGEMENTS

This paper was prepared during a sabbatical I spent at the Stanford Archaeology Center. Inpresenting it, I would like to express my sincerest thanks to Professor Ian Hodder, for theinvitation to be a guest of the Center. My stay at Stanford University was memorable for so manyreasons, but especially for the many insights I gained from the time I spent with Neil Brodie,Douglass Bailey, Bjrnar Olsen, Michael Shanks, Kostas Kotsakis, Ian Robertson, LynnMeskell. Melissa Chatfield, Cheryl Makarewicz and all the postgraduate students of the Center:I very much enjoyed sharing with them the innermost secrets of organic residue analysis! Overthe years, the organic residue analysis field has benefited greatly from the support of a number ofindividuals and bodies who recognized its potential; for this, I should like to acknowledgethe Science Based Archaeology Committee of the former UK Science and EngineeringResearch Council (SERC), particularly Mark Pollard and Sebastian Payne, for their efforts inpromoting the field; and the Natural Environment Research Council (NERC) for continuedsupport, but especially for the Ancient Biomolecules Initiative, coordinated by Geoffrey Eglintonand Martin Jones, which provided an especially fertile environment for the development of thefield. The Wellcome Trusts Bioarchaeology Programme provided further opportunities for

918 R. P. Evershed


developing research in this area. On a personal level, our own research at Bristol would neverhave flourished without the sustained support from the SERC, the NERC, The Wellcome Trust,The Leverhulme Trust, English Heritage and The Royal Society. I am also indebted to the manypostdoctoral researchers, Ph.D. students and visitors to the Bristol laboratory, whose titanicefforts, creativity and good humour have been vital in maintaining the vigour of our work.

REFERENCES

Anderson, K. B., and Winans, R. E., 1991, Nature and fate of natural resins in the geosphere 1. Evaluation of pyrolysis-gas chromatography mass-spectrometry for the analysis of natural resins and resinites, Analytical Chemistry, 63,29018.

Barakat, A. O., Mostafa, A., Qian, Y., Kim, M., and Kennicutt, M. C. II, 2005, Organic geochemistry indicates GebelEl Zeit, Gulf of Suez, is a source of bitumen used in some Egyptian mummies, Geoarchaeology, 20, 21128.

Barnard, H., Ambrose, S. H., Beehr, D. E., Forster, M. D., Lanehart, R. E., Malainey, M. E., Parr, R. E., Rider, M.,Solazzo, C., and Yohe, R. M. II, 2007, Mixed results of seven methods for organic residue analysis applied to onevessel with the residue of a known foodstuff, Journal of Archaeological Science, 34, 2837.

Beck, C., Wilbur, E., Meret, S., Kossove, D., and Kermani, K., 1965, The infrared spectra of amber and the identifi-cation of Baltic amber, Archaeometry, 8, 96109.

Beck, C. W., Smart, C. J., and Ossen, D. J., 1989, Residues and linings in ancient Mediterranean transport amphoras,ACS Symposium Series, 220, 36980.

Berstan, R., Dudd, S. N., Copley, M. S., Morgan, E. D., Quye, A., and Evershed, R. P., 2004, Characterisation of bogbutter using a combination of molecular and isotopic techniques, Analyst, 129, 2705.

Berstan, R., Stott, A. W., Minnitt, S., Bronk Ramsey, C., Hedges, R. E. M., and Evershed, R. P., 2008, Direct datingof pottery from its organic residues: new precision using compound-specific carbon isotopes, Antiquity, 82,70213.

Bethell, P. H., Ottaway, J., Campbell, G., Goad, L. J., and Evershed, R. P., 1994, The study of molecular markers ofhuman activity: the use of coprostanol in the soil as an indicator of human faecal material, Journal of Archaeo-logical Science, 21, 61932.

Boda, E., Connan, J., Dessort, D., Muhesen, S., Mercier, N., Valladas, H., and Tisnerart, N., 1996, Bitumen as a haft-ing material on Middle Palaeolithic artifacts, Nature, 380, 3368.

Bronk Ramsey, C., 2008, Radiocarbon dating: revolutions in understanding, Archaeometry, 50, 24975.Buckley, S. A., and Evershed, R. P., 2001, Organic chemistry of embalming agents in Pharaonic and Graeco-Roman

mummies, Nature, 413, 83741.Buckley, S. A., Clark, K. A., and Evershed, R. P., 2004, Complex organic chemical balms in Pharaonic animal mum-

mies, Nature, 413, 83741.Buckley, S. A., Stott, A. W., and Evershed, R. P., 1999, Studies of organic residues from ancient Egyptian mummies

using high temperature gas chromatography mass spectrometry and sequential thermal desorption gas chromato-graphy mass spectrometry and pyrolysis gas chromatography mass spectrometry, Analyst, 124, 44352.

Bull, I. D., Betancourt, P. P., and Evershed, R. P., 2001, An organic geochemical investigation of the practice of man-uring at a Minoan site on Pseira Island, Crete, Geoarchaeology, 16, 22342.

Bull, I. D., Berstan, R., Vass, A., and Evershed, R. P., in revision, Identification of a disinterred grave by molecularand stable isotope analysis. Science & Justice.

Bull, I. D., Elhmmali, M. M., Roberts, D. J., and Evershed, R. P., 2003, The application of steroidal biomarkers to trackthe abandonment of a Roman wastewater course at the Agora (Athens, Greece), Archaeometry, 45, 14961.

Bull, I. D., Simpson, I. A., Dockrill, S. J., and Evershed, R. P., 1999a, Organic geochemical evidence for the originof ancient anthropogenic soil deposits at Toft Ness, Sanday, Orkney, Organic Geochemistry, 30, 53556.

Bull, I. D., Simpson, I. A., van Bergen, P. F., and Evershed, R. P., 1999b, Muck n molecules: organic geochemicalmethods for detecting ancient manuring, Antiquity, 73, 8696.

Cardon, D., 2007, Natural dyes: sources, tradition, technology and science, Archetype, London.Charri-Duhaut, A., Connan, J., Rouquette, N., Adam, P., Barbotin, C., de Rozires, M.-F., Tchapla, A., and Albrecht,

P., 2007, The canopic jars of Rameses II: real use revealed by molecular study of organic residues, Journal ofArchaeological Science, 34, 95767.

Charters, S., Evershed, R. P., Blinkhorn, P. W., and Denham, V., 1995, Evidence for the mixing of fat and wax inarchaeological ceramics, Archaeometry, 37, 13128.



Charters, S., Evershed, R. P., Goad, L. J., Blinkhorn, P. W., and Denham, V., 1993a, Quantification and distribution oflipid in archaeological ceramics: implications for sampling potsherds for organic residue analysis, Archaeometry,35, 21123.

Charters, S., Evershed, R. P., Goad, L. J., Heron, C., and Blinkhorn, P. W., 1993b, Identification of an adhesive usedto repair a Roman jar, Archaeometry, 35, 91101.

Charters, S., Evershed, R. P., Quye, A., Blinkhorn, P. W., and Denham, V., 1997, Simulation experiments for deter-mining the use of ancient pottery vessels: the behavior of epicuticular leaf wax during boiling of a leafy vegetable,Journal of Archaeological Science, 24, 17.

Colombini, M. P., Giachi, G., Modugno, F., and Riberchini, E., 2005, Characterisation of organic residues from pot-tery vessels of the Roman age from Antinoe (Egypt), Microchemical Journal, 79, 8390.

Colombini, M. P., Modugno, F., Silvano, F., and Onor, M., 2000, Characterization of the balm of an Egyptian mummyfrom the 7th century bc, Studies in Conservation, 45, 1929.

Condamin, J., Formenti, F., Metais, M. O., Michel, M., and Blond, P., 1976, The application of gas chromatographyto the tracing of oil in ancient amphorae, Archaeometry, 18, 195201.

Connan, J., 1999, Use and trade of bitumen in antiquity and prehistory: molecular archaeology reveals secrets of pastcivilizations, Philosophical Transactions of the Royal Society of London, Series BBiological Sciences, 354, 3350.

Connan, J., and Dessort, D., 1989, Du bitume de la Mer Morte dans les baumes dune momie gyptienne: identicationpat critres molculaires, Comptes Rendus de lAcadmie des Sciences (Serie II), 309, 166572.

Connan, J., and Dessort, D., 1991, Du bitumen dans les baumes des momies egpytiennes (1295 av JC300 ap JC):determination de son origine et evaluation de sa quantit, Comptes Rendus de lAcadmie des Sciences (Serie II),312, 144552.

Connan, J., Nissenbaum, A., and Dessort, D., 1992, Molecular archaeology: export of Dead Sea asphalts to Canaanand Egypt in the ChalcolithicEarly Bronze Age (4th3rd millennium bc), Geochimica et Cosmochimica Acta,56, 274359.

Connan, J., Nieuwenhuyse, O. P., van As, A., and Jacobs, L., 2004, Bitumen in early ceramic art: bitumen-paintedceramics from Late Neolithic Tell Sabi Abyad (Syria), Archaeometry, 46, 11524.

Copley, M. S., Hansel, F. A., Sadr, K., and Evershed, R. P., 2004, Organic residue evidence for the processing ofmarine animal products in pottery vessels from the pre-Colonial archaeological site of Kasteelberg D East, SouthAfrica, South African Journal of Science, 100, 27983.

Copley, M. S., Bland, M. A., Rose, P., Horton, M., and Evershed, R. P., 2005a, Gas chromatographic, mass spectro-metric and stable carbon isotopic investigations of organic residues of plant oils and animal fats employed asilluminants in archaeological lamps from Egypt, Analyst, 130, 86071.

Copley, M. S., Berstan, R., Dudd, S. N., Straker, V., Payne, S., and Evershed, R. P., 2005b, Dairying in antiquity: Ievidence from absorbed lipid residues dating to the British Iron Age, Journal of Archaeological Science, 32, 485503.

Copley, M. S., Berstan, R., Dudd, S. N., Straker, V., Payne, S., and Evershed, R. P., 2005c, Dairying in antiquity: IIevidence from absorbed lipid residues dating to the British Bronze Age, Journal of Archaeological Science, 32,50521.

Copley, M. S., Berstan, R., Dudd, S. N., Straker, V., Payne, S., and Evershed, R. P., 2005d, Dairying in antiquity:IIIevidence from absorbed lipid residues dating to the British Neolithic, Journal of Archaeological Science, 32,52346.

Copley, M. S., Rose, P. J., Clapham, A., Edwards, D. N., Horton, M. C., and Evershed, R. P., 2001a, Processing palmfruits in the Nile Valleybiomolecular evidence from Qasr Ibrim, Antiquity, 75, 53842.

Copley, M. S., Rose, P. J., Clapham, A., Horton, M. C., Edwards, D. N., and Evershed, R. P., 2001b, Detection ofpalm fruit lipids in archaeological pottery from Qasr Ibrim, Egyptian Nubia, Proceedings of the Royal Society,Series B, 268, 5937.

Copley, M. S., Berstan, R., Dudd, S. N., Aillaud, S., Mukherjee, A. J., Straker, V., Payne, S., and Evershed, R. P.,2005e, Processing of milk products in pottery vessels through British prehistory, Antiquity, 79, 895908.

Copley, M. S., Berstan, R., Dudd, S. N., Docherty, G., Mukherjee, A. J., Straker, V., Payne, S., and Evershed, R. P.,2003, Direct chemical evidence for widespread dairying in prehistoric Britain, Proceedings of the National Acad-emy of Sciences of the USA, 100, 15249.

Corr, L. T., Sealy, J. C., Horton, M. C., and Evershed, R. P., 2005, A novel marine dietary indicator utilisingcompound-specific bone collagen amino acid 13C values of ancient humans, Journal of Archaeological Science, 32,32130.

Corr, T. T., Richards, M. P., Grier, C., Mackie, A., Beattie, O., and Evershed, R. P., 2008a, Probing dietary change ofthe Kwday Dn Tsnchi individual, an ancient glacier body from British Columbia: II. Deconvoluting whole skin

920 R. P. Evershed


and bone collagen 13C values via carbon isotope analysis of individual amino acids, Journal of ArchaeologicalScience, in press.

Corr, L. T., Richards, M. P., Jim, S., Ambrose, S. H., Mackie, A., Beattie, O., and Evershed, R. P., 2008b, Probing dietarychange of the Kwday Dn Tsnchi individual, an ancient glacier body from British Columbia: I. Complementaryuse of marine lipid biomarker and carbon isotope signatures as novel indicators of a marine diet, Journal ofArchaeological Science, 35, 210210.

Craig, O. E., Chapman, J., Heron, C., Willis, L. H., Bartosiewicz, L., Taylor, G., Whittle, A., and Collins, M., 2005,Did the first farmers of central and eastern Europe produce dairy foods? Antiquity, 79, 88294.

Craig, O. E., Forster, M., Andersen, S. H., Koch, E., Crombe, P., Milner, N. J., Stern, B., Bailey, G. N., and Heron,C. P., 2007, Molecular and isotopic demonstration of the processing of aquatic products in northern Europeanprehistoric pottery, Archaeometry, 49, 13552.

Dudd, S. N., and Evershed, R. P., 1998, Direct demonstration of milk as an element of archaeological economies,Science, 282, 147881.

Dudd, S. N., and Evershed, R. P., 1999, Unusual triterpenoid fatty acyl ester components of archaeological birch barktars, Tetrahedron Letters, 40, 35962.

Edwards, H. G. M., Sibley, M. G., Derham, B., and Heron, C. P., 2004, Raman spectroscopy of archaeologicalsamples from the BarberSurgeons medicine chest on the Mary Rose, Journal of Raman Spectroscopy, 35, 74653.

Eglinton, G., and Logan, G. A., 1991, Molecular preservation, Philosophical Transactions of the Royal Society ofLondon, Series B, 333, 31528.

Evans, K., and Heron, C., 1993, Glue, disinfectant and chewing gum: natural products chemistry in archaeology,Chemistry and Industry, 12, 4469.

Evershed, R. P., 1990, Preliminary report of the analysis of lipids from samples of skin from seven Dutch bog bodies,Archaeometry, 32, 13953.

Evershed, R. P., 1992, Chemical composition of bog body adipocere, Archaeometry, 34, 25365.Evershed, R. P., 1993, Biomolecular archaeology and lipids, World Archaeology, 25, 7493.Evershed, R. P., 2008, Experimental approaches to the interpretation of absorbed organic residues in archaeological

ceramics, World Archaeology, 40, 2647.Evershed, R. P., and Connolly, R. C., 1988, Lipid preservation in Lindow Man, Naturwissenschaften, 75, 1435.Evershed, R. P., and Connolly, R.C., 1994, Post-mortem transformations of sterols in bog body tissues, Journal of

Archaeological Science, 21, 57783.Evershed, R. P., Heron, C., and Goad, L. J., 1990, Analysis of organic residues of archaeological origin by high

temperature gas chromatography/mass spectrometry, Analyst, 115, 133942.Evershed, R. P., Heron, C., and Goad, L. J., 1991, Epicuticular wax components preserved in potsherds as chemical

indicators of leafy vegetables in ancient diets, Antiquity, 65, 5404.Evershed, R. P., Jerman, K., and Eglinton, G., 1985, Pine wood origin for pitch from the Mary Rose, Nature, 314,

5285.Evershed, R. P., Anderson-Stojanovic, V. R., Dudd, S. N., and Gebhard, E. R., 2003, New chemical evidence for the

use of combed ware pottery vessels as beehives in ancient Greece, Journal of Archaeological Science, 31, 112.Evershed, R. P., Heron, C., Charters, S., and Goad, L. J., 1992, The survival of food residues: new methods of

analysis, interpretation and application, Proceedings of the British Academy, 77, 187208.Evershed, R. P., Vaughan, S. J., Dudd, S. N., and Soles, J. S., 1997a, Fuel for thought? Beeswax in lamps and conical

cups from Late Minoan Crete, Antiquity, 71, 97985.Evershed, R. P., Arnot, K. I., Collister, J., Eglinton, G., and Charters, S., 1994, Application of isotope ratio monitor-

ing gas chromatography/mass spectrometry to the analysis of organic residues

Documents

Evershed - Biomarker Revolution