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Microchemical Journal 122 (2015) 127136

Contents lists available at ScienceDirect

Microchemical Journal

journal homepage: www.elsevier.com/locate/microc

An integrated multianalytical approach to the reconstruction of daily


activities at the Bronze Age settlement in Pealosa (Jan, Spain)
E. Manzano a,, A. Garca b, E. Alarcn b, S. Cantarero a, F. Contreras b, J.L. Vlchez a
a
Department of Analytical Chemistry, University of Granada, E-18071 Granada, Spain
b
Department of Prehistory and Archaeology, University of Granada, E-18071 Granada, Spain

a r t i c l e i n f o a b s t r a c t

Article history: This paper seeks to reconstruct everyday human activities in a Bronze Age Argaric site in southeastern Spain. We
Received 4 April 2015 used an integrated multi-analytical approach to study residues extracted from 5 ceramic pieces and 3 soil sam-
Accepted 26 April 2015 ples. The results of these analyses were complemented with archaeological interpretations to produce the rst
Available online 3 May 2015
ndings on domestic activities four millennia ago at the Pealosa settlement in Jan, Spain. The bands observed
in FTIR and ATRFTIR spectra of the archaeological residues showed a peak assignment that conrmed the silicate
Keywords:
GCMS
components of the clay as raw material and the presence of organic matter. The amorphous organic materials
GCCIRMS were analyzed by GCMS, HRMS and GCCIRMS, and the mineral residues by non-invasive portable X-ray uo-
HRMS rescence (pXRF). We identied ,-dicarboxylic and -(o-alkylphenyl) alkanoic acids produced by oxidative
Organic residue degradation of unsaturated fatty acids, as well as monocarboxylic acids, n-alkanes, diterpenoid acids, cholesterol,
Bronze Age dyes and chemical grape markers. Assignments were based on lipid prole, fatty acid ratios and the 13C values of
Pealosa site C16:0 and C18:0. The results are consistent with the presence of tissues from ruminant (ovine or bovine) and non-
ruminant (swine and equine) animals, vegetable oils, waxes, conifer resin and grape seeds. The presence of sh-
based fats was suggested in one of the residues. Our evidence also suggests that grape juice or wine may have
been consumed at this Bronze Age site. As far as we know this is the rst investigation of the organic residues
absorbed into an Argaric goblet used in a domestic situation.
2015 Elsevier B.V. All rights reserved.

1. Introduction long time in a ceramic matrix, although this depends on their ability to
withstand biodegradation. The chemical characterization of solid resi-
Food preparation and consumption are essential daily activities for dues is a difcult task due to their complex composition and their de-
the survival of any social group. These timeless practices generate resi- graded state. In order to identify these residues, specic methodologies
dues and every remain artifact and ecofact in the archaeological record for extraction, purication and further analysis of the total lipid extract
of a site is not only a reection of the decisions taken by a group of (TLE) must be developed. The analytical techniques used most common-
humans and the relations between them [1,2] but is also often the prod- ly for this purpose are Fourier transformed infrared spectroscopy (FTIR)
uct of a particular phase in the food production chain. Thanks to these [3], Raman spectroscopy (RS) [4], gas chromatographymass spectrom-
remains we know where (space) and in what (ceramic vessels) people etry (GCMS) [58], high-performance liquid chromatographymass
stored, cooked and ate food four thousand years ago, but answering spectrometry (HPLCMS) [9,10] and pyrolysisgas chromatography
questions as to what they cooked or what they ate remains difcult. mass spectrometry (PyGC/MS) [11]. More recently the determination
In order to nd out which foods were cooked or consumed in an an- of the ratios of the stable isotopes of carbon (12C and 13C) and nitrogen
cient potsherd, it is necessary to analyze the organic residues absorbed (14N and 15N) using gas chromatographycombustionisotope ratio
by the pores in the pottery or those adhering to its inner surface. In addi- mass spectrometry (GCCIRMS) [12] has provided insights into the
tion to foods, these vessels may also have contained beverages, medi- type of food consumed and the source of the original lipids giving rise
cines, dyestuffs, essential oil, wine, etc. For archaeologists, residues to these residues. A better understanding of the analytical data can be ob-
containing lipids are perhaps of most interest, because the altered prod- tained by establishing the relationship between the molecular constitu-
ucts formed as a result of burial or other human activities (for example ents that remain in the organic residues and the source from which
by heating) are relatively stable. They can therefore remain intact for a they originate. To this end, we used various criteria, the simplest of
which was the comparison of chemical ngerprints of molecules that
Corresponding author. Tel.: +34 958 243388; fax: +34 958 243328. have remained in the ceramics for many millennia with the reference
E-mail address: emanzano@ugr.es (E. Manzano). materials. The ratios of some common fatty acids, the presence of specic

http://dx.doi.org/10.1016/j.microc.2015.04.021
0026-265X/ 2015 Elsevier B.V. All rights reserved.
128 E. Manzano et al. / Microchemical Journal 122 (2015) 127136

archaeological biomarkers and the stable carbon isotope values of specif- 4000 cm1, with a resolution of 2 cm1 and 200 scan. The spectrometer
ic lipid components have also proved useful for this characterization [13, has a TGS Detector. The spectrum of pure KBr was used as background.
14].
This paper focuses on the analysis of residues discovered in ve ce-
ramic vessels recovered from the Pealosa site (Baos de la Encina, 2.2.2. Gas chromatographymass spectrometry (GCMS)
Jan, Spain) and the associated soil residues found inside three of The chromatographic system consisted of an Agilent 6890 N series
these vessels. These vessels date from the Bronze Age and in chronolog- (Agilent Technologies, Palo Alto, CA, USA) equipped with an automatic
ical and cultural terms are associated with the Argar people, an ancient injector (model 7683) and automatic sample tray (model 7683). An
culture that ourished in south-east Spain around the period 2250 HP-5 (5% Phenylsiloxane 30 m 0.25 mm, 0.25 m of particle size)
1550 BC [15]. Since the 19th century, this ancient culture has been a was used as a capillary column. In addition, an Agilent 5973N mass
subject of great interest among the scientic community, as exemplied spectrometry detector (Agilent Technologies, Palo Alto, CA, USA) was
by the abundant bibliography [16,17]. The analysis and study of their fu- coupled to the chromatograph. The detector consisted of an ionic im-
nerary rituals have been a recurrent theme, to the extent that it has pact (70 eV) and a quadrupole as an ionization source and analyzer re-
eclipsed other possible lines of investigation such as everyday activities, spectively. Table 1 shows the parameters used for gas chromatography
which in many ways offer a broader view of life in ancient civilizations mass spectrometry analysis. Each parameter was optimized before
[2]. In fact, in research into the Bronze Age in Southeast Spain, only commencing the analysis. In order to achieve the most efcient chro-
two publications have analyzed the organic remains found in ceramic matographic separation, ve different temperature gradients were
vessels and both of these were from funerary nds [18,19]. The novel as- assayed. As regards gas ow, several ows were tested with a ow of
pect of our research is that it is the rst study in which domestic ce- 1 mL min1 producing the best results. Four different injection volumes
ramics, rather than funerary objects, have been analyzed. To this end, were tested and the best chromatogram was obtained using a volume of
we embarked on interdisciplinary research using a combination of cur- 1.5 L. After assaying four voltages, we found that the best result was ob-
rent analytical techniques (pXRF, FTIR, ATRFTIR, HRMS, GC/MS and tained with the 3 EV gain factor. Finally, we optimized the m/z range,
GCCIRMS) with two ambitious goals: (1) to identify the compounds selecting the 50520 uma range as the m/z working range. Peak assign-
present in the organic residues extracted from the vessels, (2) to discuss ments were performed on the basis of the analysis of available standard
the possible biomolecular origin of these organic residues in order to compounds and by comparing their mass spectra to those from the
better understand what the people from the Argaric site in Pealosa Wiley mass spectra database.
cooked and ate. The results obtained using this combination of analyti-
cal techniques showed unambiguous identications of constituents of
2.2.3. Gas chromatographycombustionisotope ratio mass spectrometry
chemically complex residues and offer a rigorous scientic insight into
(GCCIRMS)
everyday activities in this part of southeastern Spain four millennia ago.
The GCCIRMS equipment was based on a Thermo Trace GC ultra
and Thermo Delta V Advantage as an IRMS detector (Thermo Fisher
2. Experimental
Scientic, Waltham, MA). Cono III was selected as an interface and
reactor temperature (CuNiPt) was set at 940 C. The mass spectrom-
2.1. Chemicals and reagents
eter source pressure was 1.9 10 06 mBar. HP-1 (30 m 0.25 mm
ID 0.25 m) was used as a GC column for assessing chromatographic
Dichloromethane and methanol (Analytical Grade) purchased
conditions. The carrier gas was helium with the GC oven programmed
from Fluka (St. Louis, MO, USA) were used as extraction solvents. Tolu-
from 60 (hold 1 min) to 320 C at 6 C min1 for 20 min. Carbon isotope
ene (Analytical Grade) and Meth-Prep II (m-triuoromethylphenyl
ratios are reported in the standard delta notation relative to the Pee Dee
trimethylammonium hydroxide) were respectively selected as the sol-
Belemnite (PDB) standard. The results were expressed as 13C (%) =
vent and the reagent for the derivatization process prior to gas chroma-
[(Rsample Rstandard) / Rstandard], where R is 13C/12C in per mil.
tography analysis. Both were purchased from Sigma-Aldrich (St. Louis,
MO, USA). In order to prevent degradation and obtain high levels of re-
producibility, Meth-Prep II was stored at 4 C in the freezer. Further- 2.2.4. High resolution mass spectrometry (HRMS)
more, LC-MS grade Formic Acid and Methanol (Sigma-Aldrich) were HRMS analysis was performed to identify other residues apart from
used in High Resolution Mass Spectrometry assays. fatty acids and lipids, which cannot be detected by gas chromatogra-
Analytical grade standards of fatty acids (C10, C12, C13, C14, C15, C16, phymass spectrometry, focusing specically on the identication of
C16:1, C18, C18:1, C18:2, C19, C20, C22, C26, C30) and Cinnamic acid, Azelaic peptides and proteins.
acid, Suberic acid, Adipic acid, Tartaric acid, Syringic acid, Cholesterol An LCT Premier (Waters, Manchester, UK) spectrometer was used
and -Sitosterol were purchased from Sigma-Aldrich (St. Louis, MO, for the HRMS analysis and was operated with electrospray ionization
USA). Fatty acid C13 was used as an Internal Standard. Individual stan- (ESI) in positive mode. Mass spectrometer parameters were as follows:
dard solutions of compounds (1000 mg mL1) were prepared in meth- capillary voltage, 2.60 kV; source temperature, 100 C; desolvation tem-
anol and stored at 20 C. These solutions were prepared fresh monthly. perature, 400 C; cone gas ow, 40 L h1; desolvation gas ow,
Working standard mixtures were prepared by diluting the individual 400 L h1; nitrogen (99.995%) was used as cone and desolvation gas
stock solution in methanol. They were stored at 4 C and prepared and cone gas ow. It is important to mention that before injection the
fresh weekly. All solutions were stored in dark glass bottles to prevent samples were diluted with a mixture of methanol (0.1% acid formic)
photodegradation. to ionize the compounds, so enabling them to be detected by HRMS.

2.2. Instrumentation
2.2.5. Portable X-ray uorescence (pXRF)
2.2.1. Attenuated total reectanceFourier transform infrared spectroscopy Portable X-ray uorescence analyzer, with a 40 kV X-ray tube with
(ATRFT-IR) and Fourier transform infrared spectroscopy (FT-IR) Ag anode target excitation source, and a silicon PIN-diode with a Peltier
The FT-IR spectra were collected using a JASCO Spectrometer 6200, cooled detector. The analyzer was initially calibrated using the silver
working in transmission mode and diamond micro-ATR accessory. The and tungsten shielding on the inside of the shutter, and the source
instrument was connected to a Pentium 200 and the instrument soft- count time for analysis was xed in 90s.
ware was SPECTRA MANAGER v2. The FT-IR spectra were registered Direct measurements can be performed with portable instruments
from 400 cm 1 to 4000 cm 1 and the ATR spectra from 600 to that do not affect the integrity of the sample.
E. Manzano et al. / Microchemical Journal 122 (2015) 127136 129

Table 1
Gas chromatography-mass spectrometry parameters.

Gas chromatography Mass spectrometry

Injector temperature 250 C Interface temperature 230 C


Initial column temperature 70 C (2 minute hold) m/z range 50520 uma
Gas ow 1.0 mL min1 (helium) Voltage 2235 V
Mode Splitless Quadrupole temperature 150 C
Column HP-5 Mode Scan
Temperature gradient 12 C min1 until 250 C Scan velocity 2.12 scans/s
20 C min1 until 290 C
Injection volume 1.5 L Gain factor 3 EV

2.3. Archaeological samples. Criteria for selecting the artefacts highest part of the settlement. Archaeological research has dened
House 25 as a large social space, measuring approximately 20 m2. It is
Archaeological samples were taken from ceramics found in situ at completely open and has no subdivisions. Its rectangular layout has an
the occupation level of House 25 of the Pealosa site. This domestic EW orientation, as do most of the documented houses in Pealosa.
space is located on the western side of the oriental acropolis of the set- We studied ve ceramic vessels (Fig. 1) and the associated soil resi-
tlement, near a further three domestic and productive spaces. Together dues preserved inside them. These vessels were selected in accordance
they present a complex constructive and urban system, located in the with a series of archaeological criteria, including their good state of

Fig. 1. The ve ceramic vessels studied (25567, 25617, 25638, 25705 and 25789).
130 E. Manzano et al. / Microchemical Journal 122 (2015) 127136

preservation (complete vessels), the context they were found in (all the So as to analyze lipid matter and fatty acids by gas chromatography
ceramics were found in primary position on the paved oor of the coupled mass spectrometry, a derivatization reaction was required prior
room), their possible uses and functions and their position in the food to injection into the chromatograph. Meth-Prep II was selected as the
production chain (relations morphology-use/function). These vessels derivatization reagent because it can derivatize both fatty acids and
included a medium-sized spherical pot with a convex base, three lipid materials simply in a single step without further collateral reac-
bowls (one spherical, one semi-spherical and one with open sides), tions. The derivatization procedure was based on a procedure for char-
and a goblet with a tall, thin stem, the most characteristic and emblem- acterizing drying oil in paintings that we developed and successfully
atic element of the El Argar Culture. All these pieces belong to different tested in previous research [21].
phases in the food preparation process and provide a fascinating insight
into daily life 4000 years ago. 2.5.2. Analytical procedure
The goblet is perhaps the most interesting of all these pieces. Archae- The analytical procedure for GC comprises the following steps:
ologists believe that in the Argaric Culture, these kinds of goblets were 1) 0.31 g of sample were weighed and placed in an ultrasound vessel;
associated with high social status and hierarchical organization, because 2) 15 mL of the dichloromethane and methanol (2:1 v/v) mixture was
of their design and technical complexity. This goblet is especially inter- added; 3) lipids, fatty acids and other compounds were extracted
esting because although similar vessels have been found among Argaric by immersing the vessel in an ultrasonic bath (Mod 5133 JP Selecta,
grave goods, this is almost the only goblet thought to have been used for Barcelona, Spain) for 15 min (twice); 4) the extracts were collected
everyday domestic purposes. Chemical residue analysis on this goblet and centrifuged at 3500 rpm for 5 min; 5) the two extracted liquids
can provide an insight into the daily life of this culture from the recent were collected and dried in a nitrogen atmosphere at 5060 C; 6)
prehistory of southern Spain, since it would be the rst Argaric goblet 500 L of Toluene and 37.5 L of Meth-Prep II were added to carry out
to be analyzed from a domestic context from the recent prehistory of the derivatization reaction in the ultrasonic bath, which took 30 min
southern Spain. at ambient temperature; and 7) nally, the derivatized samples were
injected into the chromatograph (1 L).
2.4. Sampling and sample handling The procedure for analyzing the proteins by HRMS was based on
Chertov et al. [20]. The nal extracts were diluted in a mixture of meth-
In-situ measurements were carried out using a portable X-ray uo- anol with 0.1% of formic acid and were directly injected (100 L) into the
rescence (pXRF) spectrometer for trace elements from samples 25567 high resolution mass spectrometer.
and 25638. XRF analysis does not require sampling.
Small amounts of ne powder were collected for FTIR, ATRFTIR,
GCMS, HRMS and GCCIRMS analysis by: 3. Results and discussion

- scraping the inner surface and the bottom of the 5 pieces of pottery Since the origin of the organic residues was unknown, we began by
(samples 25567, 25617, 25638, 25705 and 25789), analyzing the samples using infrared spectroscopy. The preliminary
- taking the associated soil residues (samples 25568, 25706 and FTIR and FTIR-ATR analysis provided a ngerprint which suggested pos-
25791) found inside 3 of the above pieces25567, 25705 and sible initial hypotheses about the substances present in the residues and
25789, respectively. pottery characterization. Due to the complex nature of the organic ma-
terials, a combination of analytical approaches (GCMS, HRMS and GC
CIRMS) was used to ensure comprehensive analysis. Since few studies
The samples analyzed using the attenuated total reectance (ATR) have focused on HRMS, in this paper we support the lipid residues
sampling accessory was taken nely ground and did not require prepa- obtained from GCMS with residues of peptide chains provided by
ration. For Fourier transform infrared spectroscopy (FTIR), in transmis- HRMS. Finally, the mineral residue was analyzed by portable non-
sion mode, the samples were mixed with KBr (sample:KBr, 1:100) and invasive X-ray uorescence (pXRF).
pressed into discs. For GCMS a small amount of ne powder from each
of the 8 samples was weighed and analyzed (samples 25567 (0.9264 g), 3.1. FTIR and ATR analysis
25617 (0.3675 g), 25638 (0.2771 g), 25705 (0.5235 g), 25789 (0.3678 g),
25568 (0.9772 g), 25706 (0.9961 g) and 25791 (0.9919 g)). Each sample The FTIR and ATRFTIR spectra for the eight residues analyzed are
was crushed and ground in an agate mortar for chromatographic analy- very similar. Fig. 2 shows the FT-IR spectrum for one of the samples
sis. For GCCIRMS similar amounts of samples were processed. This (sample 25789) with the main wavenumbers and their corresponding
technique was only applied to samples 25706 and 25791 due to the un- intensities. All samples analyzed are rich in clay minerals showing char-
availability of samples. To avoid contamination, researchers wore kid acteristic bands in the region 37003600 cm1 (OH stretching bands
gloves and a mask when handling the vessels and the samples. at 3694 and more intense at 3619 cm1), 11001000 cm1 (asymmet-
ric SiOSi stretching bands) and 910830 cm 1 (SiO stretching
2.5. Sample treatment bands) [22]. This band contains contributions from various silicate min-
erals typically found in clay. Quartz is identied by a characteristic dou-
2.5.1. Extraction and derivatization blet around 779 and 797 cm1 and the SiO stretch around 1000 cm1.
The samples were examined for lipids and proteins. Organic resi- The weak broad band around 3440 cm1 and less intensive absorption
dues were extracted from the pottery using the procedure rst present- at 1635 cm1 in all FTIR spectra can be attributed to the OH stretching
ed by Evershed et al. [5], which we optimized for these purposes, mode and HOH bending mode respectively from water from crystal-
assaying various solvents, mixed in different proportions, namely meth- lization and/or hydroxyl groups from the clay. A CO2 3 band is observed
anol, dichloromethane, chloroform and toluene. The best results were at 14201440 cm 1 in the IR spectra of sample 25789 and its inner
achieved with mixtures of methanol:dichloromethane (1:2) and sediment 25791 due to calcite remains probably originated from soil
methanol:chloroform (1:2). Due to the high toxicity of chloroform, depositions. The weak band at 1538 cm 1 in samples 25789 and
the methanol:dichloromethane mixture was selected as our extraction 25791 attributed to (NH) and supported by a mild band (NH) at
solvent. Proteins were extracted using a procedure based on Chertov 3127 cm 1 may be attributed to traces of proteinaceous material.
et al. [20]. The extracts we obtained were ltered through 0.22 m Organic materials (wax traces) could be inferred from the features
nylon Millipore lters before injection into the high resolution mass around 2900 cm 1 characteristic of CH stretching in 25789, 25638,
spectrometer (100 L). 25705 and 25791, a nding later conrmed by GCMS.
E. Manzano et al. / Microchemical Journal 122 (2015) 127136 131

Fig. 2. FT-IR spectrum of organic residues extracted from bowl sample 25789.

3.2. GCMS and GCCIRMS analysis compounds, most of which are fatty acid methyl esters (FAMEs)
which result from the transesterication of the acyl glycerides and the
The gas chromatograms obtained from the organic residues extract- esterication of fatty acids. Table 2 reports the peak assignment of the
ed from the ceramic vessels show the presence of different classes of compounds identied in all samples (assignment percentage of over

Table 2
Peak assignment to compounds identied (% assignment greater than 90%), retention time (tR) and m/z selected.

# tR m/z Compound identied

1 11.229 153.0,184.0, 125.0 Aconitic acid


2 11.418 138.0, 171.0 Octanodioic acid (suberic acid)
3 11.630 143.0, 101.0, 175.0 Trimethyl citrato (citric acid)
4 12.439 143.0, 171.1, 183.1 Dodecanoicacid (lauric acid)
5 12.753 167.1, 182.0 2-Allyl-1-methylnaphthalene
6 13.963 153.1 Methyldihydrojasmonate
7 14.780 143.1, 199.1 Tetradecanoic acid (myristic acid)
8 15.086 115.0, 129.0, 145.1 Hexylcinnamicaldehyde
9 15.864 143.1, 213.1 Pentadecanoic acid
10 16.359 111.1, 239.2 Methyl 1-anthraquinonesulfenate
11 16.665 236.1, 194.1, 111.1, 152.1 9-Hexadecenoic acid (palmitoleic acid)
12 16.940 143.1, 227.2, 270.2 Hexadecanoic acid (palmitic acid)
13 17.003 291.1 7-Ethoxy-2-hydroxy-8-methoxy-3-methyl-5,6-
methylenedioxy-1,4-naphthoquinone
14 17.882 143.1, 241.2, 284.2 Heptadecanoic acid (margaric acid)
15 18.652 264.2, 111.0 9-Octadecenoic acid (oleic acid)
16 18.841 143.1, 298.2, 256.2 Octadecanoic acid (stearic acid)
17 19.132 157.0, 112.0 Tributylaconitate
18 19.352 185.0, 129.0, 259.1 Tributylcitrate
19 19.791 109.1 Retinaldehyde
20 19.886 186.0, 259.1 Tributylacetylcitrate
21 20. 561 178.0, 161.0 2-Ethylhexyl p-methoxycinnamate
22 20.616 326.3, 143.0, 283.2 Eicosanoic acid (arachidic acid)
23 20.781 239.1 Dehydroabietic acid
24 20.844 109.0 Sanguinone a
25 20.938 126.1 9-Octadecenamide
26 21.119 129.0 Hexanodioicacid (adipic acid)
27 22.030 113.1 Pentacosane
28 23.766 382.3 Tetracosanoic acid (lignoceric acid)
29 25.341 410.4 Hexacosanoic acid (cerotic acid)
30 25.832 386.3 Cholestan-6-one
31 27.395 207.0, 105.0, 386.4 Cholesterol
32 27.477 438.4 2,3-Didecyl-1,4-naphthoquinone
33 29.756 207.0 Gibberellin a3
34 30.600 466.5 Triacontanoic acid (melisic acid)

Sample 25567: 1; 3, 4; 5; 6; 8; 11; 12; 15; 16; 17; 18; 20; 21; 22; 25; 26; 28.
Sample 25568: 12.
Sample 25617: 2; 4; 7; 9; 11; 12; 14; 15; 16; 20; 21; 22; 28; 29; 31.
Sample 25638: 2; 4; 7; 12; 14; 15; 16; 22; 23; 27; 34.
Sample 25705: 7; 8; 10; 12; 13; 19; 23; 24.
Sample 25706: 11; 12; 15; 30.
Sample 25789: 7; 15; 16.
Sample 25791: 4; 12; 15; 16; 22; 26; 29; 31; 32; 33.
132 E. Manzano et al. / Microchemical Journal 122 (2015) 127136

90%), the retention time (tR) and m/z selected. The compounds identi- [23]. A lipid prole exhibiting a C16:0/C18:0 ratio of 1.75 was interpreted
ed in each of the 8 samples are specied at the bottom of the as a non-ruminant animal according to the ratio value proposed by
table. Fig. 3 shows a chromatogram for the residues extracted from Romanus et al. [24]. This conclusion is also supported by the absence of
the inner surface of the goblet (sample 25567) and the bowl (sample C15:0, C17:0, C19:0 and branched-chain fatty acids from the GCMS analysis,
25638). compounds characteristic of ruminants [25]. The consumption of red
meat by the Pealosa people has been reported in previous paleopatho-
3.2.1. Ceramic goblet and the associated soil residue inside the goblet logical studies of human bones [2]. Biochemical paleonutritional indica-
(samples 25567 and 25568) tors (Sr/Ca(c) and Zn/Ca ratios) have improved previous knowledge
The general chromatographic pattern of the fatty acid prole in the or- about human diets in the Argaric culture, which seems to have been
ganic residue extracted from goblet 25567 shows that it contained animal mainly based on non-ruminants (swine or equine) and ruminants
fat as the original raw materials (Table 2). This nding is supported by the (ovine or bovine) animals, as well as cereals and legumes. Lignoceric
relatively high proportion of saturated fatty acids (C16:0 and C18:0) com- and arachidic acids identied by GCMS in the scraped sample from gob-
pared to unsaturated fatty acids (C16:1 and C18:1) in the residues analyzed. let 25567 are compounds that are naturally abundant in the surface of
The presence of 9-octadecenamide conrms the presence of animal fat leaves and the skins of berries and fruits, which in this case could be at-
since this compound is considered an important marker of animal lipids tributed to the presence of plant waxes [26].

Fig. 3. Chromatograms for the organic residues extracted from vessels 25567 and 25638.
E. Manzano et al. / Microchemical Journal 122 (2015) 127136 133

The important presence of C16:0, and C16:1 and C18:1 acids together level of stearic acid and the traces of cholesterol support the assumption
with a low C18:0/C16:0 ratio could infer a possible sh residue [27]. that it once contained animal fat, an assumption conrmed by archaeo-
There is little evidence for sh consumption at Pealosa. Nevertheless, logical data from the Pealosa area [2].
the chemical analysis of bone samples from an adult woman found in Signicant amounts of odd number fatty acids (C15:0 and C17:0) could
this settlement (Log Ba/Sr = 1.55) conrmed repeated ingestion of indicate bacterial action and suggest that ruminant animals were prob-
sh proteins (crustaceans, river carbs, molluscs, prawns, etc.) possibly ably cooked in this pot [25]. This hypothesis is supported by the fact that
from the River Rumblar [2]. Fish lipid residues are very difcult to distin- the value for the C15:0 + C17:0/C12:0 + C14:0 + C16:0 + C18:0 ratio is great-
guish from plant lipid residues mainly because the n-alkanoic acid dis- er than 0.04 as proposed by Malainey [38]. The residue from sample
tribution of both is strongly dominated by the C16:0. Consequently the 25617 ts the ruminant fat prole. The presence of bovine and ovine an-
attribution of sh residues in goblet 25567 still remains ambiguous imals was previously demonstrated by experimental studies on animal
but GCMS analyses provide an indicator to justify further study. and human bones found at the Pealosa settlement [2].
The high content of C16:0 in the sample in addition to the relatively Moreover, the presence of unsaturated fatty acids (C16:1 and C18:1),
higher levels of C16:0 compared to C18:0 (C16:0/C18:0 = 1. 75) could sug- dicarboxylic acid (suberic acid) and traces of short chain fatty acids
gest that the goblet once contained vegetable oils. The archaeological (C12:0 and C14:0) in residue indicates that pot 25617 once contained veg-
documentation describing the presence of cereal, ax seeds, lavender etable oils. Eerkens diagram [39] has been used with the necessary cau-
(culinary and aromatic plants such as rosemary, thyme, basil, etc.) and tion because of possible changes in chemical composition arising from
carbonized olive stones at the Pealosa site reinforces the evidence for the repeated heating of cooking vessels or oxidative and microbial pro-
vegetable oils [28]. The identication of adipic acid indicates original un- cesses occurring as a result of use and burial. Thus, in both of the graphs
saturated fatty acids in prehistoric pottery which also supports the use proposed by Eerkens (C12:0/C14:0 against the C16:0/C18:0 ratios and
of vegetable oils [29,30]. The absence of other dicarboxylic acids, such C15:0 + C17:0/C18:0 against C16:1/C18:1 ratios), ceramic pot 25617 tends
as azelaic acid, that usually result from the oxidation of the original un- to fall into elliptical groupings identied as green plants and next to
saturated fatty acids, may be justied by their susceptibility to leaching the Eerkens region for terrestrial mammals. Consequently, both raw
into groundwater, given the proximity of the Rumblar river to the materials proposed (vegetable and animal oil) are consistent with the
Pealosa settlement (in the High Guadalquivir, Spain). These dicarbox- Eerkens results and also with previous archaeological data [2].
ylic acids and other polar compounds are only encountered in archaeo-
logical samples from especially arid environments [31].
3.2.3. Ceramic bowl 25638
It seems likely that goblet 25567 was used for a variety of purposes.
The list of compounds identied from GCMS analysis of scrapings
Chromatographic analysis has detected other compounds containing
from the inside surfaces of bowl 25638 is reported in Table 2. The chro-
phenolic acids often related to wine or grape products, such as cinnamic
matogram shows a large amount of saturated fatty acid (C12:0, C14:0,
acids [3234], while citric acid, a natural preservative used in
C16:0, C18:0, C20:0, C30:0) originating from both animal and plant lipids.
winemaking, and the aconitic acids derivatives (tributyl aconitate)
The signicant content of C20 and C30 fatty acids and the saturated hy-
identied in the residue, are a strong indication of wine or other grape
drocarbon (pentacosane, C25H52) identied in the residues suggests
derivatives. The unsuccessful search for other potential wine bio-
some kind of waxy material in the vessel. The absence of lignoceric
markers such as tartaric acid and other phenolic compounds could be
acid means that beeswax can be ruled out, although vegetable wax
due to their higher water solubility. As discussed above, these com-
may be possible. The high content of oleic acid together with suberic,
pounds may well have leached away, given the damp conditions of
lauric and myristic acids indicate the use of vegetable oil in the bowl.
the Pealosa settlement, next to the river. Although the identication
In addition, the biplot of fatty acids C12:0/C14:0 against the C16:0/C18:0
of the aforementioned grape markers suggests the presence of wine or
falls next to the seeds ellipse in Eerkens graphs [39]. Finally
grapes in 25567 residues, the consumption of grape juice or wine in
anthracological researches suggest that the dehydroabietic acid could
the Bronze Age in the southeast of Spain has not yet been proved by ar-
be attributable to pine resin from pinus carrascus [28].
chaeological research (dates) at Argaric sites, although tartrate band
identication by IR spectrometry [18] and the discovery of carbonized
grape seeds at Castelln Alto [35], Fuente Amarga and Fuente lamo 3.2.4. Ceramic bowl and associated soil residue inside the bowl (25705 and
[36] and Pealosa [28] all point in this direction. This paper represents 25706 samples)
a step forward in that for the rst time an Argaric goblet has been ana- The content of the extract obtained from ceramic bowl 25705 and
lyzed using an integrated analytical approach. The compounds pre- from the soil found inside (25706) is quite poor in organic components
served within ceramic lead us to the conclusion that grape juice/wine as can be seen in Table 2. Two saturated fatty acids were identied (C14:0
was consumed in this area 4000 years ago. and C16:0) together with dehydroabietic acid and cinnamaldehyde,
Polycyclic aromatic hydrocarbons (PAHs) are common in smoke which seem to suggest raw materials of plant origin. The essential oils
condensates from wood res and offer a useful record of past human ac- could be used mixed with polycyclic aromatic hydrocarbon (PAH), as
tivities. Thus the presence of 2-allyl-methyl naphthalene is considered anthraquinone and naphthoquinone derivatives, and Sanguinone A, all
in the literature to be the result of cooking on an open re [37]. The pres- used as dyes for making ointments [40]. In addition, unsaturated fatty
ence of contaminants of pyrolitic origin in the goblet would be a rele- acids identied in the residue support the idea of a vegetable oil.
vant nding because it would indicate possible use of culinary In order to provide a robust framework for interpreting the com-
techniques such as roasting, boiling or cooking. This implies that the pounds we identied, it is essential to make a clear distinction between
people who cooked with it had some degree of technical knowledge. the lipids absorbed into the pottery and the lipids from the associated
Finally, the content of residue 25568 from the inside of bowl 25567 soil residue that resulted from the decay of plants, animals and microor-
did not provide any signicant information and there was no evidence ganisms. Thus, the dehydroabietic acid identied in residue 25705 could
of migration of fatty acids to the soil. be attributed to the leaching of terpenic compounds from pine trees.
Nevertheless, the absence of this marker compound in the associated
3.2.2. Ceramic pot 25617 soil residue found inside the bowl (25706 sample) proves that this
The biomolecular origin of the lipid prole could indicate an animal acid was not a product of a pine environment, as had been hypothe-
lipid. Residues extracted from ceramic pot 25617 (Table 2) are charac- sized, and may instead have been used as an ointment. By contrast,
terized by high amounts of saturated fatty acid (C12:0, C14:0, C16:0, the identication of cholestan-6-one compound and the high content
C18:0, C20:0, C24:0, C26:0). Most prominent among these is lignoceric of C18:0 vs C16:0, provides evidence of animal fat remains in the sur-
acid (C24:0), a characteristic compound of beeswax. The relatively high rounding soil.
134 E. Manzano et al. / Microchemical Journal 122 (2015) 127136

Table 3 Table 5
Mean 13C and standard deviation (SD) values of C16:0 to C18:0 fatty acids to 25706 and Quantitative results (g g1) obtained by non-invasive pXRF analysis from the 25567 and
25791 samples. Triplicate sample analysis. 25638 potsherds. The average concentration for n = 2 measurements is given in the third
column and the standard deviations (SD) in the last column.
C16:0 C18:0
Sample Mean SD
Sample 13C value SD 13C value SD
Sb 25567 81.000 27.920
25706 31.06 0.11 31.06 0.09
25638 87.100 27.425
25791 30.26 0.19 30.64 0.25
Sn 25567 66.845 27.075
25638 86.490 26.720
Sr 25567 114.940 6.205
The origin of this animal fat in sample 25706 (or a possible mixture 25638 79.640 5.295
of fats from different species) was identied by GCcombustioniso- Rb 25567 92.770 6.320
tope ratio MS (GCCIRMS) (Table 3). 13C data (dened as 13C/12C, iso- 25638 124.010 7.260
Pb 25567 26.185 8.445
topic composition of the palmitic and stearic acids ratio) and 13C value 25638 2221.400 57.725
(dened as 13C18:0-13C16:0) plotted against 13C16:0 provide a robust Zn 25567 66.005 17.025
criterion to differentiate between fats from ruminant and non- 25638 51.460 15.895
ruminant animals [41]. The comparison of the experimental values we Cu 25567 56.795 19.430
25638 155.770 25.450
obtained 13C (31.06%0; 31.06%0) and 13C (0) with those for mod-
Ni 25567 bLD 70.635
ern reference fats using data from a diagram by Mottram [30,41,42], 25638 85.135 48.140
allowed us to conclude that the 25706 residue falls within the ruminant Fe 25567 24,145.160 409.930
adipose fats cluster. The 13C value obtained for this residue plotted 25638 18,747.440 355.460
against 13C16:0 falls between the reference area for porcine adipose Mn 25567 335.445 83.400
25638 209.915 74.035
fats (non-ruminant) and the area for ruminant adipose fats [43]. The re-
Cr 25567 121.575 29.950
sults indicate a mixture of fats in the original pottery, a fact supported by 25638 317.665 34.805
the GCMS and archaeological data [2]. In sample 25706, the 13C values Ti 25567 3332.600 231.105
obtained for C16:0 and C18:0 fatty acid ( 28.3 2.0) were consistent 25638 3092.840 241.310
Ca 25567 14,447.430 452.870
with animals that subsisted mainly on C3 plants, according to data pre-
25638 13,063.480 424.105
sented by Bender [44]. K 25567 27,744.530 862.275
25638 26,203.900 821.875

Cd, Ag, Se, As, Hg, Co, V, Sc b LD.


3.2.5. Ceramic bowl and the associated soil residue found inside the bowl
(samples 25789 and 25791)
The residues extracted from ceramic 25789 (C14:0, C18:0 and C18:1) treated with sufcient precaution during excavation and subsequent
(Table 2) shed little light on the diet of the prehistoric people from handling and washing.
Pealosa. Nevertheless, the chromatogram for the associated soil (sam-
ple 25791) is more complex and characterized by certain fatty acids 3.3. HRMS analysis
(C12:0, C16:0, C18:0, C20:0, C26:0) and cholesterol (Table 2) that may be re-
lated to animal fat. Some quinone derivatives, Gibberellin A3, and oleic Although there is extensive bibliography about lipids retained in
and adipic acids identied are typically attributed to leafy greens. Mea- pottery, there are surprisingly few reports on the occurrence of other
surements of the relative abundances of 13C/12C in organic residues types of compounds, e.g. proteins and carbohydrates, the major molec-
from 25791 [30,41] allowed us to conclude that the bowl had once ular constituents of plants and animals. In this study, results for some re-
contained meat from ruminant cattle. The 13C value obtained from sidual peptides have already been added together to lipid analyses and
the residue plotted against 13C16:0 falls between the reference area the benet of additional information has been considered.
for porcine adipose fats (non-ruminant) and ruminant adipose fats, in- The HRMS technique allowed us to detect the monoisotopic molecu-
dicating animals that subsisted mainly on C3 plants [44]. Since interpre- lar weight of substances to four decimal places. Compounds can there-
tation of chromatographic and isotope analyses remains challenging, fore be identied with high accuracy. In this study, the assumed error
the results help us to distinguish between the ruminant and non- was established at 10 ppm. Taking this into account, only the compounds
ruminants fats that the vessels once contained. Both animal species below this cut-off level were considered and presented as results.
are supported by archaeological reports from Pealosa [2]. According to the results shown in Table 4, four peptide sequences
As can be seen in Table 2, the analysis of residues from ceramic bowl have been undoubtedly identied in 4 of the 7 samples analyzed using
25789 and the associated soil residues 25791 produced no signs of pos- high resolution mass spectrometry (HRMS). It is worth to mention that
sible exchange of signicant amounts of organic material between neither peptides nor amino acids were detected in the blank sample. It
them. It is possible that the residues once absorbed into the pottery is therefore assumed that the samples contained animal or plant proteins.
are no longer preserved.
In all the samples, we found evidence for contamination with 3.4. pXRF analysis
phthalates and derivatives. These compounds have been excluded
from Table 2 because they are not intrinsic materials from the residues. The XRF results obtained for each ceramic investigated without sam-
This suggests unfortunately that the material reported here was not pling are summarized in Table 5. Since all the vessels originate from the

Table 4
HRMS analysis.

Sample Molecular mass (high resolution) Deviation (ppm) Molecular formula Peptide sequence

25638 452.1808 1,5 C22H24N6O5 L-Asparticacid, N-(4-(((2,4-diamino-5-ethyl-6-quinazolinyl) methyl) amino)benzoyl)


25617 626.2417 7 C24H38N10O6S2 Cys-cys-his-lys-his
25789 542.2740 0,6 C28H38N4O7 Phe-tyr-leu-thr
25789 709.3184 7,4 C33H43N9O9 Glu-ala-his-try-ala-pro
E. Manzano et al. / Microchemical Journal 122 (2015) 127136 135

Fig. 4. Isotopic peak prole based on organic residues (peptides) extracted from bowl sample 25638.

same house in Pealosa, one would not expect big differences in leaching into groundwater, given the proximity of the Rumblar river
the concentrations of the chemical elements that make them up. It is in- to the Pealosa settlement, the occurrence of the aforementioned com-
teresting to note therefore that Ni is only present in bowl 25638, whose pounds could indicate that this ancient people drank grape juice or
Cr concentration is twice that of goblet 25567. Similarly its Cu concen- wine. The chemical evidence for wine is an important nding when
tration is three times higher than that of the goblet and its Pb concentra- tracing the prehistoric origins of this drink in Spain, which by Roman
tion is eighty times higher. These quantitative data are very interesting times had become one of the three basic pillars of Mediterranean food
for provenance studies. The major elements identied in the potteries production along with wheat and olive oil.
between 13,000 and 28,000 g g1 were Ca, Fe and K. These elements
were associated with the minerals (such as calcite, potassium feldspar,
iron oxides, hydroxides and silicates) typically found in potter's clay. Acknowledgments
Other elements found between 22003400 g g1 were Ti and Pb;
while Sb, Sn, Sr, Rb, Pb, Zn, Cu, Ni, Mn and Cr were found between 50 This research has been supported by Junta de Andaluca (FQM 338)
and 320 g g1. and the R & D Project HAR2011-30131-CO2-01 funded by the Spanish
Ministry of Economy and Competitiveness. A. Garca acknowledges sup-
4. Conclusions port from a FPU 13/02389 scholarship from the Spanish Ministry of Ed-
ucation. ERDF Funds and Junta de Andaluca's support to acquire the FT-
An integrated multi-analytical investigation of organic residues pre- IR spectrophotometer Jasco 6300 is acknowledged. The authors would
like to thank to F. Martin (UGR) for handling the pXRF.
served in Bronze Age vessels from the southeast of Spain has been pro-
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