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*Received 8 August 2008; accepted 11 August 2008

University of Oxford, 2008



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Blackwell Publishing Ltd Oxford, UK ARCH Archaeometry 0003-813X 1475-4754 University of Oxford, 2008 XXX

ORIGINAL ARTICLE

Organic residue analysis in archaeology R. 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 the
nature and origins of organic remains that cannot be characterized using traditional
techniques of archaeological investigation (because they are either amorphous or invisible).
The eld is founded upon the principle that the biomolecular, or biochemical, components
of organic materials associated with human activity survive in a wide variety of locations
and deposits at archaeological sites. The archaeological information contained in organic
residues is represented by the biomolecular components of the natural products that
contribute to the formation of a given residue. By applying appropriate separation
(chromatographic) and identication (mass spectrometric) techniques, the preserved, and
altered, biomolecular components of such residues can be revealed. Once identied, the

Archaeological Biomarker Concept

can be applied, wherein the structure and even isotopic
composition(s) of a given biomolecule or suite of biomolecules (the chemical ngerprint)
can be related to the compositions of organisms exploited by humans in the past. As the
organic residue eld emerges from its pre-paradigmatic phase, and the organic residue
revolution gathers pace, the way is open for challenging many long-held archaeological
hypotheses 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 increasing
numbers of archaeologists beginning to consider organic residue analyses amongst the many
scientic tools available to them. A major inuence in the development of the eld was the
emergence of a new generation of analytical chemical methodologies in the middle of the 20th
century that enabled complex environmental materials to be studied in increasingly ne detail.
Spectroscopic methods, such as infrared (IR), Raman and nuclear magnetic resonance (NMR)
spectroscopies, provide insights into bulk compositions that have proved useful in ngerprinting
the sources of certain classes of organic residue; for example, ambers and resins and their
derivatives (Beck

et al

. 1965; Lambert

et al

. 1985). However, the chemical complexity, deriving
from the many constituents (endogenous or exogenous) that comprise residues, constrains the
usefulness of such methodologies beyond broad descriptions of chemical properties; for
example, compound classes present, which are often difcult to relate to specic source
materials, 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
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University of Oxford, 2008,

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50

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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 the
molecular components of complex biological and environmental materials to be separated and
characterized in detail, making the origins of multiple constituents discernable.
The modern era of the organic residue analysis of archaeological materials was heralded by
the paper of Thornton

et al

. (1970), who investigated the origin and identity of the enigmatic
bog butters by GC. This paper was the rst of a number that emerged over the next 25 years
that provided the empirical and experimental framework for our current understanding of the
range of artefacts and deposits likely to contain organic residues preserving archaeological
biomarkers (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; Proefke
and 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). Signicantly,

Archaeometry

was the
venue for the publication of a number of these key papers.
This paper is not intended to be an exhaustive review of the eld of organic residue analysis.
Rather, the aim is to provide a critical appraisal of the current state of knowledge of the eld,
which will consider (i) the

Archaeological Biomarker Concept

, (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 the
future holds.
This paper does not contain specic details of analytical methodologies; nor does it provide
in-depth discussions of the operation of instrumentation, as such matters have been covered
exhaustively elsewhere, although it will discuss the formulation of analytical protocols in the
context 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 or
immunological studies will not be covered, since these are distinctly separate subjects based
on biochemical rather than chemical methods, although many of the matters discussed are
relevant to those areas. Radiocarbon and organic residue analysis are complementary elds,
since, as Bronk Ramsey (2008) points out, the majority of the materials submitted for radio-
carbon analysis are organic residues, puried to the compound or molecular level; thus
chemical purity or structure can be used as quality control criteria for radiocarbon dating.
A fundamental difference between the two elds is that organic residue analyses are generally
performed on components puried from amorphous or invisible organic residues. However,
the two elds become very closely aligned when radiocarbon analyses are performed on
biomolecular components puried from amorphous remains; this combination represents a
potentially 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: rst, that in establishing a new
paradigm, the preferred way for this to be tested is via rigorous scrutiny through the peer
review system; and, second, that a signicant non-peer-reviewed literature appeared early in
the history of the eld (up to

c

. 1990), which initially misled the archaeological community
as to the analytical chemical rigour required for meaningful results to be obtained from
organic residues.
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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 initiatives
occurring at that time. The issue collected together a number of papers encompassing
new approaches to the biomolecular analysis of organic remains at archaeological sites. My
contribution was a paper entitled Biomolecular archaeology and lipids (Evershed 1993).
The motivation for the paper was threefold. First, the eld of organic residue analysis was still
embryonic, although the handful of accounts of molecular-level analyses of organic residue that
existed indicated great promise. The paper drew the available material together and discussed
the opportunities for the development of the eld, attempting to emphasize the potential of low
molecular weight compounds, such as lipids, within the broader scope of the eld of ancient
biomolecule 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 major
impacts on the development of the eldfor example, compound-specic stable isotope
analysisand I felt it important to highlight these. Third, it was clear that a framework
was required to provide continuity between the fragmentary early research and that to be
undertaken in the years ahead. The latter was achieved by dening the concept of

Archaeological
Biomarkers

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

et al

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

Archaeological Biomarker
Concept

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

Archaeological Biomarker
Concept

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, sequences
of DNA ampliable by PCRor building blocks; for example, amino acids or peptides in the
case of ancient proteins.

Analytical prerequisites

The application of the

Archaeological Biomarker Concept

brings with it critical consequences
for the way in which organic residue analyses have to be approached. Most notably, it implicitly
requires 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 a
result of mixing biological materials in, for example, food preparationand then complexity
is 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
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University of Oxford, 2008,

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

Tracing origins from structures

The

Archaeological Biomarker Concept

operating in its simplest mode relies upon matching
the structures or distributions, chemical ngerprints, to the compounds and mixtures known
to exist in extant organisms likely to have been exploited in the past (Evershed 1993). Some-
times, the structure of a single component is sufcient to dene the origin of a constituent of
an organic residue (Fig. 1). For example, the di- and triterpenoid components of modern plant
resins have been widely studied and offer a robust means of assigning their presence as
components of organic residues, sometimes to the botanical species or more commonly to the
genus 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 is
beeswax, which is readily recognized due to the characteristic mixture of aliphatic components
that 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 upon
the specic 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 specic 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.
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wherein consideration of the nature of other constituents of the residue may lead to the
hypothesis 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 prole
shows a textbook

n

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

n

-alkane complement, rather than selected components, means that the null hypothesis must
be acceptedthese compounds do not unambiguously derive from beeswax. The recent report
of 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 rm
conclusions as to the true nature of organic residues can only be reached once more robust
supporting 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 is
as follows: Is the presence of a constituent of a residue based on an observed biomarker
consistent with the archaeology and palaeoecology of the settlement, region and/or period
from which the nd derived? In practice, consideration of the latter actually serves to simplify
interpretations and increase the diagnostic potential of seemingly rather undiagnostic biomarkers,
since alternative sources of biomarkers can legitimately be disregarded if they make no sense
archaeologically or palaeoecologically.

Synergy in structural and isotopic composition

Animal fats and plant oils offer greater challenges because the major components, polyunsaturated
fatty acids in particular, rarely if ever survive (although, as discussed below, their prior presence
can 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 shown
that there is considerable scope for the identication of such materials if the stable isotopic
compositions of the common

n

-alkanoic acid biomarkers are determined. For example, the


13

C
values of the

n

-hexadecanoic and

n

-octadecanoic acids that survive in degraded animal fats can
be used to differentiate their sources: ruminant and non-ruminant fats can be separated and ruminant
carcass fats distinguished from dairy fats, due to metabolic differences between the different animals
and carbon sources utilized in biosynthesis of different fat types (Fig. 2; see also Dudd and Evershed
1998; 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 compositions
alone. Thus, by combining molecular structures in tandem with their stable isotope compositions,
synergy is achieved within the archaeological biomarker approach. Such synergy confers on
even the most mundane of structures a high level of biomarker specicity.
The specicity of this approach is enhanced still further by considering the most likely sources
for 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 only
species that need to be considered in interpretations, as statistically these are the most legitimate
sources likely to leave signicant residues. For example, in our studies of animal product processing
in 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 the
range of species will be different, thereby guiding interpretations accordingly.
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University of Oxford, 2008,

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Source information locked into altered structures

One of the fundamental aspects of the biomarker concept is that of the source being recorded
by 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 that
on observing a given biomarker structure, the presence of a particular constituent could be
conrmed. However, in many cases altered rather than native structures are observed. The
ability to recognize an original constituent, or source, of an organic residue based upon altered
structures surviving in the residues is a powerful aspect of the biomarker approach, which
requires knowledge of the chemical and biochemical mechanisms and pathways likely to be
involved. An example of this phenomenon mentioned above is the recognition that the n-alkanoic
acids that occur very widely in residues derive mainly from triacylglycerols that originally
comprised the parent fats and oils, the former being released by hydrolysis (Fig. 2).
Another example is the recognition of structurally altered components that have obvious
coherency with the carbon skeletons in their precursors, but where signicant structural trans-
formations have occurred. An example of this is seen in the array of diterpenoid structures
observed in heated coniferous resins (Evershed et al. 1985; Robinson et al. 1987). The carbon
skeletons of the observed products leave little doubt as to the generic origin of residues con-
taining such components (Fig. 3). Signicantly, the structural alterations bring with them
additional information concerning the life history of the residue. The high energies required to
produce aromatized and defunctionalized products offer a new level of diagnosis, namely of
the technology of the production process, as well as source information. Analogous examples
of this phenomenon are seen in other heat-treated resins (Fig. 3; see also Hayek et al. 1990; Stern
et al. 2003; Regert et al. 2003a,b; Regert 2004).
Figure 2 Simple saturated C
16:0
and C
18:0
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
13
C 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
condence 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
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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 in
higher plant waxes. The ketones are formed via a free radical-induced dehydration and
decarboxylation, which occurs when acyl lipids are heated in excess of 300C (Fig. 4; see also
Evershed et al. 1995b; Raven et al. 1997). Likewise, -(o-alkylphenyl)alkanoic acids with
1622 carbon atoms (Fig. 5; see also Hansel et al. 2004) are produced when unsaturated fatty
acids are heated to over 270C (Evershed et al. 2008a). Since polyunsaturated fatty acids
rarely survive in appreciable concentrations in organic residues in pottery vessels, these -
(o-alkylphenyl)alkanoic acids, which are more stable compounds, offer a novel means of
detecting the processing of commodities containing unsaturated fatty acids.
A nal example is the observation of dihydroxy fatty acids in archaeological pottery from
monounsaturated fatty acids, wherein the position of the hydroxyl groups effectively added to
the carbon atoms bearing the original double-bond record precisely the original position of
unsaturation, which in combination with the carbon number of the fatty acid can be related
back 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
University of Oxford, 2008, Archaeometry 50, 6 (2008) 895924
In summary, application of the Archaeological Biomarker Concept requires not only
knowledge of the biochemical compositions of the organic commodities exploited by humans
in the past, but also an appreciation of how these materials can be altered by processing and/or
burial. Many of the mechanisms and pathways of molecular structural change resulting from
degradation 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 at
archaeological 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.
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considerable timescale). However, the survival of organic residues is not assured, nor is it
ubiquitousfor reasons that are not yet fully understoodbut it will certainly reect cultural
as well as taphonomic differences in the life history of an organic residue through the
processes of formation, deposition/discard and burial to ultimate recovery. Thus, when
embarking on organic residue analyses of a new class of artefact and/or in a new geographical
region, the presence of organic residues should not be assumed. This conclusion is based
upon the results of organic residue analyses performed on several thousand individual artefacts
and ecofacts recovered from many parts of the world, including those from archaeological
sites located in disparate climatic, ecological and geological zones. However, the occurrence
of organic residues is sufciently widespread that they can be considered as viable sources of
information 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 lls (not to be confused with intrusive post-
burial lls). Instances of the contents surviving in situ are rare, such that few examples exist of
chemical 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. External
sooting and internal carbonized residues are probably the organic residues most familiar to
pottery analysts. The former are presumed to be residues of cooking failures, although visible
residues 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 sediments
Vessel lls* 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 bitumens
Soft tissues*** Natural resins*****
Mummies**** Plant gums**
Fossil resins***
Plant remains Petroleum bitumens***
Waterlogged remains***
Desiccated remains** Miscellaneous
Carbonized(?)* Glass containers**
Metal containers**
Dyes and pigments Stone objects**
Textiles**** Hoards; e.g., bog butter**
Art****
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University of Oxford, 2008, Archaeometry 50, 6 (2008) 895924
2005a), while post-ring treatments are also seen, particularly as copious linings associated
with transport vessels (Beck et al. 1989; Mills and White 1989). Visible residues have been
chemically investigated (e.g., Oudemans and Boon 1991; Oudemans et al. 2007) and are
widely used in dating programmes. A concern when working with such residues is the potential
for them to become contaminated by post-burial or post-excavation contamination due to
their exposed nature. Residues occurring on the exteriors of vessels correspond to either sooting
derived from fuel used in heating on a re, 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 the
organic 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. This
nal category of residue is by far the most common in pottery and probably the most widely
occurring residue type. Analyses performed to date suggest that absorbed organic residues
survive in >80% of domestic cooking pottery assemblages worldwide. Such residues arise
through the processing of plant and animal products, with heat and/or mechanical action
mobilizing fats, waxes and other biochemical components to aid transfer in liquid or solution
form into the fabric of the vessel wall (Charters et al. 1993a; Stern et al. 2000). Laboratory
experiments (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 studies
of ethnographic vessels (Evershed 2008) have contributed greatly to understanding how these
residues arise and their compositional alteration during vessel use and decay.
Chemical investigations of organic residues in archaeological pottery have revealed a wide
range of compound types, which have led to the identication of residues of an impressive
range of commodities from several continents. Commodities recognized include vegetable oils
(Condamin et al. 1976; Kimpe et al. 2002; Copley et al. 2005a), terrestrial animal fatsthat
is, adipose and milk fats (Dudd and Evershed 1998; Mukherjee et al. 2007)marine animal
fats (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 Evershed
2004a,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 appears
to present a signicant challenge, despite the fact that such residues have been reported on a number
of 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 specicity required
to make unambiguous identications of trace constituents of chemically complex residues.
The approach of Guasch-Jan et al. (2004), using high-performance liquid chromatography
MSMS, offers the sensitivity and selectivity essential to provide secure identications based
on 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 nd at archaeological
sites. 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, with
the collagen fraction routinely isolated for stable isotope and/or radiocarbon analysis. As discussed
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above, these types of analysis are viewed separately from organic residue analyses, since the
bulk determination of radiocarbon or stable isotope composition are the primary goals (Vogel
and van der Merwe 1977; Bronk Ramsey 2008), rather than utilizing their structural features
per se to achieve more fundamental links to human activity or taphonomic processes of relevance
to archaeological reconstruction. Where studies of collagen do fall within the paradigm of
organic residue analysis is when individual amino acids are isolated and their stable isotope
signals used to explore aspects of human dietary behaviour intractable to analysis of whole
collagen (Fogel and Tuross 2003; Corr et al. 2005, 2008a). Perhaps more formally recognized
as organic residues are the lipids preserved in skeletal remains, which interestingly are
generally discarded in collagen analyses (Evershed et al. 1995a). These lipid residues provide
an alternative source of stable isotopic information for palaeodietary reconstruction, due their
differing 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, but
where they do survive they attract a high level of interest. As with skeletal remains, the major
surviving biochemical class is protein, which is a common target for radiocarbon and/or stable
isotope analysis. Lipids are also commonly preserved in waterlogged (Evershed and Connolly
1988), 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 balms
applied to many mummies in antiquity to preserve the corpses. The amorphous nature of these
balms requires the use of organic residue analysis techniques to characterize their components
(Rullktter and Nissenbaum 1988; Proefke and Rinehart 1992; Proefke et al. 1992; Mejanelle
et 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 into
why the ancient embalmers chose specic components for their preservative properties or
symbolic signicance (Buckley and Evershed 2001). The chemical analysis of mummies from
other than Egypt is a surprisingly under-investigated area.
Techniques developed for the investigation of organic residues in archaeology have the capacity
to contribute to both palaeo- and contemporary forensic investigations. Recent work with the
Metropolitan Police secured a murder conviction through the complementary use of the DNA
biomarker and compound-specic carbon isotope approaches to provide evidence for the prior
presence 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 concentration
in 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 reference
fats, including those from cadavers comprising the Body Farm experiment in the USA. The
results are consistent with a common origin for the soil, adipocere and victims tissue components.
Resins, fossil resins and bitumens
Humans would have exploited these substances very widely in the past, and analyses of the
remains 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 geographical
provenience. The following major areas are considered:
(i) Natural resins and their derivatives comprise one of the most mature areas of organic
residue analysis. Such materials occur widely either as amorphous deposits or in association with
906 R. P. Evershed
University of Oxford, 2008, Archaeometry 50, 6 (2008) 895924
various classes of archaeological objects, notably as components of the organic balms of
Egyptian mummies and associated objects; for example, cofn varnishes/decorations, internal
coatings and decorations of pottery vessels, as components of glues and hafting materials, and
so on. The natural product chemistry of the di- and triterpenoid constituents of many plant
resins has been widely investigated, providing an invaluable resource for the recognition of
the 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 major
challenges in this area is the recognition of thermally altered forms of natural resins (Evershed
et 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 in
their production.
(ii) Fossil resins attracted attention early in the history of organic residues analysis (Beck
et al. 1965; Lambert et al. 1985), due to the use of amber and copal to fashion ancient art objects
and jewellery (Mukherjee et al. 2008b and references therein). These polymeric materials are
related to the plant resins discussed above in terms of their generic biological origin, but are
considered separately due to their geological ages. While amber is frequently referred to as a
nd in excavations, it is surprising how few rigorous chemical identications exist in the
peer-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
13
C values for the C
16:0
and C
18:0
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 condence ellipses (1), corresponding to cow and sheep
body and butter fat, and further body fats from chickens, geese and pigs (n = 50).
Organic residue analysis in archaeology 907
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(iii) Petroleum bitumens have been widely studied in nds from the Near East due to the
existence of natural petroleum seeps in the region, which would have provided ancient
peoples with the opportunities of exploiting these resources for a wide variety of uses,
including in mummication, 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 of
the bitumen component of these materials has been the use of molecular parameters to
assign 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 favourable
circumstances, organic compounds have been shown to survive in soils and sediments (Bull
et al. 1999b and references therein; Fiore et al. in press). Biomarker analyses have revealed a
number of classes of compound that can be exploited to provide insights into the inputs to
soils and sediments:
(i) Middens and disused watercourses can be identied by detecting 5-stanols and secondary
bile acids (Fig. 1). These biomarkers are present in the faeces of many animals and have been
shown to survive on archaeological timescales (Knights et al. 1983; Pepe et al. 1989; Pepe and
Dizabo 1990; Bethell et al. 1994; Bull et al. 2003). The characteristic compositions exhibited
by different groups of animals allow their faecal contributions to different archaeological
features associated with waste disposal to be assessed.
(ii) The recognition by early farmers that improved crop yields could be achieved by the use
of fertilizers would have been a major step forward in the development of agriculture. While
organic compounds are potentially degradable in the soil, under favourable circumstance
molecular and stable isotope signals can be sufciently long-lived to provide assessments of
manuring 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 burials
provide signicant opportunities for exploiting their constituent organic residues as archives of
biomolecular and stable isotope information for use in reconstructing burial practices and
ritual 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 sites
mainly in the form of seeds and woody tissues. Such remains have been studied to answer a
range of questions relating to plant domestication via PCR-based methods. However, beyond
ancient 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-preserved
biomolecules. Their study has provided many new insights into the factors controlling the
decay and preservation of biomolecules (ODonoghue et al. 1994, 1996a,b; Evershed et al. 1997b;
van Bergen et al. 1997a). The rst direct chemical evidence of DNA in the archaeological
record was derived from such remains (ODonaghue et al. 1994, 1996a,b).
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(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 success
has been achieved in recovering other classes of compound that can be directly related to their
original biomolecule complement.
The biomolecular analyses of plant remains have an important role to play in providing
insights 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 pigments
does not t into the strict denition of organic residues, since they are clearly not invisible.
However, analysis of dyes relies upon analogous biomarker approaches to organic residue
analysis, with their identications relying upon matching the structures of dye components to
the compositions of natural plant and animal pigments. This is a well-developed research eld,
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 the
spectral 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 this
area, analogous to GC/MS, with the potential for on-line separation and UV/visible spectro-
scopic analysis (van Bommel 2005). The eld has been the subject of a number of authoritative
reviews and books, and thus will not be discussed here. Archaeological investigations in this
area 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 dye
installations date back to the third millennium bc. A strong aspect of research in this area has
been 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 highly
developed 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, in
cave or wall paintingsopportunities exist for applying analogous approaches, but a major
challenge lies in resolving contributions from binder components and the organic constituents
of the substrate, which may incorporate signicant microbial or algal contaminant contributions,
highly altered by exposure over the millennia. A recent investigation of hematite pigment
residues from Terra del Fuego showed the presence of fatty acid distributions consistent with
the presence of fat or oils, potentially representing remnants of binders used to apply the
inorganic pigment (Fiore et al. in press).
Miscellaneous organic remains
The archaeological record is a source of continual surprises in terms of the unusual organic
remains recovered. For instance, the identities of bog butter, hoards of animals fat (Thornton
et al. 1970), intentionally deposited in peat bogs, presumably for the purpose of preservation,
have only recently been conrmed (Berstan et al. 2004). These bog butter hoards, found in
Organic residue analysis in archaeology 909
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amounts up to about 50 kg, constitute the largest deposits of organic residues known anywhere
in the archaeological record.
Other notable nds include a Roman tin container with its tightly tting lid and cosmetic
contents in place, which provided a unique opportunity to undertake a total analysis of an
organic residue for the rst time; revealing a mixture of animal fat and puried starch as the
base for the white tin pigment cassiterite (SnO
2
). The cosmetic cream was probably a skin
whitener (Evershed et al. 2004). Roman glass containers have recently been shown to contain
organic 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 at
Qatna, Syria, provided an unusual example of biomarker studies, prompting further morpho-
logical investigations of sediments from archaeological deposits. The biomarkers analyses
revealed purple-coloured solvent extracts containing distributions of indigoid and indirubinoid
components (Fig. 8), which conrmed the presence of the famous Royal Purple dye (James
et al. in press). The dye was presumed to derive from degraded textiles associated with
corpses. This was subsequently conrmed through microscopic examinations of the sediments
from the tomb, which revealed millimetre-sized fragments of fossilized textile (gypsum replicas;
conrmed by X-ray crystallography) displaying the purple dye. The morphologies of the textile
fragments recorded the weave of the textile and pattern of the applied dye. The presence
of 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 systematic
research is rather sparse. Our current understanding of the controls on organic residue preservation
is 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 recalcitrance
conferred by their molecular structures which determine their physico-chemical properties.
Protection of biomarkers within mineral or organic phases
Mineral matrices, such as the inorganic apatite phase of bone and the fabric of pottery, offer
environments in which organic molecules are partially protected from microbiological degradation.
Figure 8 The chemical structures of biomarkers for the shellsh purple dyes.
910 R. P. Evershed
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It is presumed that organic molecules are: (i) contained within pore spaces inaccessible to
exocellular enzymes produced by degrading microbes; or (ii) protected through adsorption on
surfacesfor 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 for
microorganisms. Likewise, protection appears also to be conferred by entrapment within
organic 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), soft
tissue remains (Evershed and Connolly 1988) and so on. Encapsulation of organic residues
within 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 organic
residue preservation. The presence of naturally preservative substancesfor example, antioxidants
or 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 will
account for differential preservation. The susceptibility of the major classes of biomolecules to
structural modication and degradation in the environment generally follows the order: lipids
< carbohydrates lignin < protein < nucleotides. The order can vary somewhat according to
environmental factors and the history of an artefact. However, it is fundamentally determined
by 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, N
and P. One of the primary reasons why lipids survive at archaeological sites is because they are
hydrophobic, which means that they are not readily leached from the site of original deposition
by percolating groundwater, nor made available to microbes as substrates by mobilization
through dissolution in interstitial waters. However, it is vital to appreciate that even within
a 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 animal
fats co-deposited on to replica ceramic, markedly different patterns of decay were revealed,
with the long-chain epicuticular wax components (C
29
n-alkane, 2-alkanol and ketone)
being signicantly better preserved than the fatty acyl lipids (Evershed 2008). This latter obser-
vation has signicant consequences for the assessment of quantitative contributions to organic
residues based on the distributions of components surviving in extracts of artefacts which will
complicate quantitative assessments (Olsson and Isaksson 2008).
The recent discovery described above of Royal Purple dye components surviving chemically
intact, while the textile has been converted to a mineral replica, reects the chemical stability
of 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 inuence organic residue preservation, including
temperature, light exposure, degree of waterlogging, redox conditions and so on (Eglinton and
Logan 1991). Desiccation is highly favourable, as microbial growth cannot occur without
water; however, extensive abiological chemical oxidation of residues will still occur (Glaar
et al. 1989, 1990). Spectacular organic residue preservation by desiccation is seen at desert
Organic residue analysis in archaeology 911
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sites such as Qasr Ibrim, Egypt, where the survival of organic remains, including vast numbers of
seeds and other plant remains, textiles, pottery residues, resins, leather items and so on, is quite
extraordinary, 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). Articial desiccation of Egyptian mummies, together with
the use of balms comprising substances inhibitory to microbial growth, accounts for their
survival 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 such
deposits; 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 acyl
and other aliphatic lipids dosed into replica ceramics have allowed degradation under oxic and
anoxic conditions to be investigated (Evershed 2008): the results showed unequivocally that
acyl 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 oxic
conditions, providing important information concerning the optimal environments for recovery
of 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 of
microbes will inevitably be slowed or halted. While extremes of waterlogging and desiccation
are unquestionably conducive to the survival of organic residues, alternating wetting and drying
in climate zones where seasons of high rainfall are followed by hot dry periods appears to be
detrimental 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 is
predicated on the question (hypothesis) to be answered. The basic philosophy of the analytical
programme will be no different to any other eld, although it is true that organic residue
analysis in archaeology brings with it some considerable challenges on account of the fragmentary
nature of the material record and the unpredictable way in which interesting nds present
themselves. All archaeological science research is interdisciplinary by nature, and thus can
only be effectively performed through collaborations between scientists and archaeologists.
The unifying feature of the most prominent laboratories currently undertaking organic
residues analyses is that the scientists are archaeologically literate. However, the optimal
combination is for such scientists to join forces with scientically empathetic archaeologists.
This combination will ensure a conjoining of minds such that meaningful archaeological
questions will be posed, with realistic chances of achieving success, as optimal analytical
protocols will be devised.
Expert analysts undertake their work systematically, probing aspects of the composition of
materials using often well-established methods, chosen based on well-dened questions. They
fully appreciate why they use particular techniques and the level of interpretation that can be
attached to a particular determination, provided that the context is dened. When questions
are correctly framed, there will probably be surprisingly few choices in the way the analyses
can be undertaken. The nature of the question will dene: (i) the choice of analytical method;
912 R. P. Evershed
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(ii) the sampling protocolin other words, the type, size and number of samples that need to
be taken, including modern reference materials; (iii) the nature of any sample pre-treatment
steps, cleaning, grinding and so on; (iv) whether extractions or separations are required; (v)
the nature of the nal measurement; (vi) the use of standards to validate the methods; and
(vii) the assessment of the nal resultsthat is, the application of statistical methods to
determine whether the results obtained are signicant in the light of the original problem
dened. Organic residue analyses performed with the appropriate degree of rigour are essentially
forensic analyses; thus objective interpretations must consider all lines of archaeological
informationbiomarker, artefactual, geographical and so onin accepting or rejecting the
clearly dened hypotheses.
It cannot be overly stressed how fundamental correctly framing the research question is in
complex analytical work of this nature. The importance of this is emphasized by the enigmatic
paper of Barnard et al. (2007), in which sherds dosed with an organic residue known only to
the 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 that
without proper archaeological contextual information (geographical origin, faunal and palaeo-
botanical information, and human activities deduced from other lines of archaeological
enquiry) and a clearly dened research question, analysts were powerless to devise the most
effective analytical protocol, since they were effectively hindered from (i) being able to choose
the most appropriate analytical technique(s) to tackle the dened question(s), and (ii) selecting
the most appropriate reference materials relevant to the hypothetical ecosystem or settlement
economy from which the organic residue derived. The latter information is absolutely fundamental
to the success of organic residue analysis programmes. Not surprisingly, none of the laboratories
conrmed the presence of camels milk, although to their credit one came close! However, if
the analysts had been told the region of the world from which the residue might theoretically
have originated and been offered some background archaeological informationfor example,
compositions of faunal assemblagesthen the chances of making a correct identication
would have been greatly enhanced.
The most common type of organic residue analysis performed to date has focused on
individual or relatively small collections of nds of amorphous residues. The goal is usually to
determine the nature, origins, technological histories and so on by determining their chemical
compositions. Such analyses are non-trivial because fully characterizing all the original con-
stituents of complex degraded organic residues is an exceedingly complex and analytically
demanding task. Such analyses must bring to bear a range of different analytical techniques to
ensure that all components of residues, organic and inorganic, soluble and insoluble, and so
on are determined (Regert et al. 2003b; Evershed et al. 2004; Ribechini et al. 2008a,b; Stern
et 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 with
the organic constituents of the residues. In all cases, the archaeological biomarker approach
was employed to determine the origins of the organic constituents of the residues. GC/MS was
the universal technique of choice, operated either in full scan or selected ion-monitoring
mode, to capture major and trace constituents. PyrolysisGC/MS (Buckley et al. 1999) direct
exposureMS (Modugno et al. 2006) and electrosprayMSMS (Garnier et al. 2002) techniques
provide access to involatile components potentially intractable to GC/MS. As discussed above,
stable isotope ratio MS, particularly in compound-specic mode (Copley et al. 2003), provides
additional diagnostic information.
Organic residue analysis in archaeology 913
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Interestingly, the majority of organic residue analyses reported to date are qualitative studies; in
other 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 the
various constituents to the residues. Our investigation of a Roman cosmetic from London is, as
far as I am aware, the only example of a total qualitative and quantitative analysis of an
organic residue (Evershed et al. 2004). Although we were unusually fortunate to be presented
with the opportunity to study this remarkable nd, other such opportunities will arise from the
archaeological record and we must be ready to tackle them from this perspective. Gaining
insights into the precise ways in which our ancestors were formulating such commodities is
vital to improving our understanding of their state of knowledge of the properties of raw
materials that they were handling, and of decisions that they would have to have made in
producing commodities with the properties that they required.
Quantication of the biomarker content of artefacts is performed through the addition of
internal 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 planning
a number of aspects of laboratory analyses. For example, the addition of n-tetratricontane
to lipid extractions of pottery allows the concentrations of lipid in the ceramic fabric to be
determined, which has provided important insights into the ways in which lipids are accumulated
in pottery vessels during use (Charters et al. 1993a, 1997; Evershed 2008) and has resulted
in 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 comparisons
to be made, providing vitally important information for use in interpreting the results of
analysis and planning future research. For example, surface nds rarely produce signicant
residues, presumably due to the impacts of weathering, and are thus a high-risk class of artefact
for organic residue analysis.
Another level of quantitative information that can be sought through organic residue
analysis is the intensity of a particular activity within a settlement or region. Using pottery as
an example, we have recently probed the quantitative relationship between the use of Grooved
Ware in relation to other prehistoric British pottery traditions and a range of other archaeological
questions through organic residue analysis. Figure 9 shows clearly that Grooved Ware use is
more strongly associated with pig product processing than any other pottery. This assessment
was achieved by using the compound-specic carbon isotope-based mixing curves shown in
Figure 2. The incidences of pig fat residues (Fig. 9) were shown to correlate with pig skeletal
abundances, thereby establishing an important link between commodity source and processing
(Mukherjee et al. 2007, 2008a). Similar correlations have also been made between milk use
from organic residues in pottery and the abundance of cattle, based on faunal counts, as the
major 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 different
category of analysis, namely that where a particular biomarker proxy, or a group of biomarker
proxies, is brought to bear on a signicant archaeological problem. This category of investigation
is where the interplay between the scientist and archaeologist, or possibly several scientists
and many archaeologists, becomes essential for three important reasons: (i) such studies are
demanding of signicant resource, human and infrastructural, and we must be sure we are
asking the right questions; (ii) it is highly likely such a programme will require assembling
large numbers of artefacts, and this cannot be contemplated without archaeologists and
scientists working together at the planning, iterative and interpretative phases of projects; and
914 R. P. Evershed
University of Oxford, 2008, Archaeometry 50, 6 (2008) 895924
(iii) an assembly of multi-proxy information, derived through several different scientic
approaches, 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 the
early evidence of milk use related to cattle herding in South-East Europe and the Near East
provides 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, already
discussed above (Dudd and Evershed 1998; Copley et al. 2003). The proxy was used to address
an aspect of the late Andrew Sherratts Secondary Products Revolution hypothesis, namely
that of the timing and region of emergence of milk use; a number of other sub-hypotheses
were 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
13
C values of the methyl esters of C
16:0
and C
18:0
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).
Organic residue analysis in archaeology 915
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the pottery used in its processing meant that securing zooarchaeological and organic residue
evidence in tandem was a major feature of the research plan. A signicant aspect of the project
design was the level of sampling required, demanded by the geographical and chronological
range considered, which in turn dictated the resources required to effectively complete the
sampling and analytical phases of the project. We learned a number of lessons from this
project, particularly the importance of maintaining a clear focus on the project goal, and the
need to allow for a degree of exibility in the research design to accommodate unexpected
results; for example, we were forced to investigate three times the number of potsherds, due
to the lower than anticipated organic residue recovery rates from a signicant proportion of the
sites. 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 early
pastoralists. This evidence for extensive milk processing in pottery vessels shows that lactose
intolerance presented no signicant barrier to dairy product consumption, provided that milk
was processed (Fig. 10; see also Evershed et al. 2008b).
THE FUTURE
The eld of organic residue analysis archaeology has witnessed spectacular developments in
recent years, and there is now a well-developed understanding of where residues survive at
archaeological sites and an appreciation of the major classes of biomarker likely to be encountered.
There are, however, signicant opportunities for expanding the range of known biomarkers in
the years ahead. This will occur as organic residue analysis is applied in previously largely
uninvestigated regions of the world, such as Asia, Africa and South America. Organic residues
studies 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 regions
where organic residue preservation is likely to be optimal. Thus, sites located in regions of
constant environmental conditions, especially when combined with low mean annual temperatures
and waterlogging, provide environments conducive to preservation, and thus might be preferred
targets. Likewise, regions of extremely low rainfall, allowing preservation by desiccation, offer
prospects for enhanced preservation and thus increased rates of organic residue recovery. At
the site or artefact level, the indications are that organic residue preservation is enhanced by
encapsulation within organic or mineral aggregates or pores, making pottery, skeletal remains,
resinous or bituminous deposits, organic-rich sediments, soft tissues and plant remains the most
important sources of organic residues. However, we must be careful not to neglect other classes
of artefact; for example, stone artefacts, such as lithics, have yielded generally disappointing results
from organic residues, except in cases where surface deposits are evident. The fundamental
problem is the lack of protection offered to organic residues by non-porous minerals. Preliminary
work in our laboratory suggests that certain porous stone objects may preserve interpretable
organic residues and may be worthy of further study, especially where collections of such
artefacts are sufcient to make systematic study worthwhile. In this respect, querns, grinding
vessels 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 biomarkers
will 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 molecular
weight 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
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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
13
C
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
13
C
values (=
13
C
18:0

13
C
16:0
) 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
signicant (t-test; P < 0.0005). Pig fats have positive
13
C values that do not exhibit signicant variance and the differences in the mean values are also highly
signicant (ANOVA; P < 0.0005 between all three commodity groups; Bonferroni adjustment applied).
13
C = [(
13
C/
12
C)
sample
/(
13
C/
12
C)
standard
] 1, expressed in per
millilitre. All
13
C values are relative to the Vienna PeeDee Belemnite (VPDB) international standard.
Organic residue analysis in archaeology 917
University of Oxford, 2008, Archaeometry 50, 6 (2008) 895924
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-
specic 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 in
stable isotope composition recording climatic/seasonal phenomena relating to the behaviours
of ancient humans and their exploitation of hunted and management of domesticated animals;
(iv) incorporating biomarker analyses into radiocarbon analysis programmesthe combination
of compound-specic radiocarbon analysis and biomarker analysis has the potential to emerge
as a powerful new combination (Berstan et al. 2008); (v) greater integration of analytical
methodologies, to provide a quantitative as well as a quantitative perspective on organic residue
composition; (vi) increasing experimental work to enhance our understanding of the processes
of formation and preservation of organic residues; and (vii) increasing the integration of
organic residue information with other lines of archaeological evidence.
In summary, it is now accepted that molecular approaches alone offer the rigour necessary
for conrming the origins of the constituents of complex organic residues. The information
provided through such analyses ranges from the uses of specic artefacts to provenances of
materials, ancient technologies and wider economic activities. Thus, we are now at the stage
where in order to provide archaeologically meaningful interpretations we must begin to forge
stronger connections between the residues that we observe and the cultural and social attributes
of the individuals and communities that produced them. To achieve this, more emphasis will
have to be placed on establishing the importance of a given residue, or suite of residues, as a
representation of a given activity within a site or region, and the only way of achieving this
will be through the study of statistically meaningfulthat is, largernumbers of systematically
selected artefacts. Accumulating larger data sets brings many other advantages; for example,
opportunities are already emerging for mining existing databases to explore trends in organic
residue preservation with time, environment and ndspot characteristics, which will help to
provide improved predictive frameworks for organic residue preservation/recovery, with obvious
advantages for planning future research programmes.
ACKNOWLEDGEMENTS
This paper was prepared during a sabbatical I spent at the Stanford Archaeology Center. In
presenting it, I would like to express my sincerest thanks to Professor Ian Hodder, for the
invitation to be a guest of the Center. My stay at Stanford University was memorable for so many
reasons, 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, Lynn
Meskell. Melissa Chateld, Cheryl Makarewicz and all the postgraduate students of the Center:
I very much enjoyed sharing with them the innermost secrets of organic residue analysis! Over
the years, the organic residue analysis eld has beneted greatly from the support of a number of
individuals and bodies who recognized its potential; for this, I should like to acknowledge
the Science Based Archaeology Committee of the former UK Science and Engineering
Research Council (SERC), particularly Mark Pollard and Sebastian Payne, for their efforts in
promoting the eld; and the Natural Environment Research Council (NERC) for continued
support, but especially for the Ancient Biomolecules Initiative, coordinated by Geoffrey Eglinton
and Martin Jones, which provided an especially fertile environment for the development of the
eld. The Wellcome Trusts Bioarchaeology Programme provided further opportunities for
918 R. P. Evershed
University of Oxford, 2008, Archaeometry 50, 6 (2008) 895924
developing research in this area. On a personal level, our own research at Bristol would never
have ourished 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 many
postdoctoral researchers, Ph.D. students and visitors to the Bristol laboratory, whose titanic
efforts, creativity and good humour have been vital in maintaining the vigour of our work.
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