An Integrated Multianalytical Approach to the Reconstruction of Daily Activities at the Bronze Age Settlement in Peñalosa (Jaén, Spain)

Microchemical Journal 122 (2015) 127–136

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Microchemical Journal

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An integrated multianalytical approach to the reconstruction of daily activities at the Bronze Age settlement in Peñalosa (Jaén, Spain)

E. Manzano a,⁎,A.Garcíab, E. Alarcón b,S.Cantareroa,F.Contrerasb,J.L.Vílcheza 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 article info abstract

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 first Available online 3 May 2015 findings on domestic activities four millennia ago at the Peñalosa settlement in Jaén, Spain. The bands observed in FTIR and ATR–FTIR spectra of the archaeological residues showed a peak assignment that confirmed the silicate Keywords: GC–MS components of the clay as raw material and the presence of organic matter. The amorphous organic materials – – – fl GC–C–IRMS were analyzed by GC MS, HRMS and GC C IRMS, and the mineral residues by non-invasive portable X-ray uo- HRMS rescence (pXRF). We identified α,ω-dicarboxylic and ω-(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 profile, fatty acid ratios and the δ13Cvaluesof Peñalosa 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 fish- 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 first 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 difficult 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, specific methodologies dues and every remain artifact and ecofact in the archaeological record for extraction, purification and further analysis of the total lipid extract of a site is not only a reflection 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 chromatography–mass spectrom- remains we know where (space) and in what (ceramic vessels) people etry (GC–MS) [5–8], high-performance liquid chromatography–mass stored, cooked and ate food four thousand years ago, but answering spectrometry (HPLC–MS) [9,10] and pyrolysis–gas chromatography– questions as to what they cooked or what they ate remains difficult. mass spectrometry (Py–GC/MS) [11]. More recently the determination In order to find out which foods were cooked or consumed in an an- of the ratios of the stable isotopes of carbon (12Cand13C) and nitrogen cient potsherd, it is necessary to analyze the organic residues absorbed (14N and 15N) using gas chromatography–combustion–isotope ratio by the pores in the pottery or those adhering to its inner surface. In addi- mass spectrometry (GC–C–IRMS) [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 whichwasthecomparisonof“chemical fingerprints” 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: [email protected] (E. Manzano). materials.Theratiosofsomecommonfattyacids,thepresenceofspecific

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) 127–136 archaeological biomarkers and the stable carbon isotope values of specif- 4000 cm−1, with a resolution of 2 cm−1 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 five ce- – – ramic vessels recovered from the Peñalosa site (Baños de la Encina, 2.2.2. Gas chromatography mass spectrometry (GC MS) Jaén, 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 μ culture that flourished 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 scientific community, as exemplified 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 fi [2]. In fact, in research into the Bronze Age in Southeast Spain, only commencing the analysis. In order to achieve the most ef cient chro- fi two publications have analyzed the organic remains found in ceramic matographic separation, ve different temperature gradients were fl fl fl vessels and both of these were from funerary finds [18,19].Thenovelas- assayed. As regards gas ow, several ows were tested with a ow of −1 pect of our research is that it is the first study in which domestic ce- 1mLmin 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 μ 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, ATR–FTIR, HRMS, GC/MS and tained with the 3 EV gain factor. Finally, we optimized the m/z range, – GC–C–IRMS) with two ambitious goals: (1) to identify the compounds selecting the 50 520 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 Peñalosa Wiley mass spectra database. cooked and ate. The results obtained using this combination of analytical techniques showed unambiguous identifications of constituents of 2.2.3. Gas chromatography–combustion–isotope ratio mass spectrometry chemically complex residues and offer a rigorous scientific insight into (GC–C–IRMS) everyday activities in this part of southeastern Spain four millennia ago. The GC–C–IRMS equipment was based on a Thermo Trace GC ultra and Thermo Delta V Advantage as an IRMS detector (Thermo Fisher 2. Experimental Scientific, Waltham, MA). Conflo III was selected as an interface and reactor temperature (Cu–Ni–Pt) 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 μm) 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 min−1 for 20 min. Carbon isotope ene (Analytical Grade) and Meth-Prep II (m-trifluoromethylphenyl 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 δ13C (%) = vent and the reagent for the derivatization process prior to gas chroma- [(R − R )/R ], where R is 13C/12Cinpermil. tography analysis. Both were purchased from Sigma-Aldrich (St. Louis, sample standard standard 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, phy–mass spectrometry, focusing specifically on the identification 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 β 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: −1 dard solutions of compounds (1000 mg mL ) 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 flow, 40 L h−1; desolvation gas flow, Working standard mixtures were prepared by diluting the individual 400 L h−1;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 flow. 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 fluorescence (pXRF) 2.2.1. Attenuated total reflectance–Fourier transform infrared spectroscopy Portable X-ray fluorescence analyzer, with a 40 kV X-ray tube with (ATR–FT-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 fixed 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) 127–136 129

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 50–520 uma Gas ﬂow 1.0 mL min−1 (helium) Voltage 2235 V Mode Splitless Quadrupole temperature 150 °C Column HP-5 Mode Scan Temperature gradient 12 °C min−1 until 250 °C Scan velocity 2.12 scans/s 20 °C min−1 until 290 °C Injection volume 1.5 μL Gain factor 3 EV

2.3. Archaeological samples. Criteria for selecting the artefacts highest part of the settlement. Archaeological research has deﬁned House 25 as a large social space, measuring approximately 20 m2.Itis 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 Peñalosa site. This domestic E–W orientation, as do most of the documented houses in Peñalosa. space is located on the western side of the oriental acropolis of the set- We studied ﬁve 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 five ceramic vessels studied (25567, 25617, 25638, 25705 and 25789). 130 E. Manzano et al. / Microchemical Journal 122 (2015) 127–136 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 floor 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) 0.3–1 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)mixturewas 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 50–60 °C; 6) fi prehistory of southern Spain, since it would be the rst Argaric goblet 500 μL of Toluene and 37.5 μL 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) finally, the derivatized samples were injected into the chromatograph (1 μL). 2.4. Sampling and sample handling The procedure for analyzing the proteins by HRMS was based on Chertov et al. [20].Thefinal extracts were diluted in a mixture of meth- fl In-situ measurements were carried out using a portable X-ray uo- anol with 0.1% of formic acid and were directly injected (100 μL) 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 fine powder were collected for FTIR, ATR–FTIR, GC–MS, HRMS and GC–C–IRMS 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 fi - 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 pieces—25567, 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 materials, a combination of analytical approaches (GC–MS, HRMS and GC– C–IRMS) was used to ensure comprehensive analysis. Since few studies The samples analyzed using the attenuated total reflectance (ATR) have focused on HRMS, in this paper we support the lipid residues – sampling accessory was taken finely ground and did not require prepa- obtained from GC MS 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- fl sion mode, the samples were mixed with KBr (sample:KBr, 1:100) and invasive X-ray uorescence (pXRF). pressed into discs. For GC–MS a small amount of fine 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 ATR–FTIR 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 GC–C–IRMS 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 3700–3600 cm−1 (O–Hstretchingbands gloves and a mask when handling the vessels and the samples. at 3694 and more intense at 3619 cm−1), 1100–1000 cm−1 (asymmet- ric Si–O–Si stretching bands) and 910–830 cm−1 (Si–O stretching 2.5. Sample treatment bands) [22]. This band contains contributions from various silicate minerals typically found in clay. Quartz is identified by a characteristic dou- 2.5.1. Extraction and derivatization blet around 779 and 797 cm−1 and the Si–O stretch around 1000 cm−1. The samples were examined for lipids and proteins. Organic resi- The weak broad band around 3440 cm−1 and less intensive absorption dues were extracted from the pottery using the procedure first present- at 1635 cm−1 in all FTIR spectra can be attributed to the O–H stretching ed by Evershed et al. [5], which we optimized for these purposes, mode and H–O–H bending mode respectively from water from crystal- 2− assaying various solvents, mixed in different proportions, namely meth- lization and/or hydroxyl groups from the clay. A CO3 band is observed anol, dichloromethane, chloroform and toluene. The best results were at 1420–1440 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 δ(N–H) and supported by a mild band (N–H) 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 filtered through 0.22 μm Organic materials (wax traces) could be inferred from the features nylon Millipore filters before injection into the high resolution mass around 2900 cm−1 characteristic of C–Hstretchingin25789, 25638, spectrometer (100 μL). 25705 and 25791,afinding later confirmed by GC–MS. E. Manzano et al. / Microchemical Journal 122 (2015) 127–136 131

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

3.2. GC–MS and GC–C–IRMS analysis compounds, most of which are fatty acid methyl esters (FAMEs) which result from the transesterification of the acyl glycerides and the The gas chromatograms obtained from the organic residues extract- esterification of fatty acids. Table 2 reports the peak assignment of the ed from the ceramic vessels show the presence of different classes of compounds identified in all samples (assignment percentage of over

Table 2

Peak assignment to compounds identiﬁed (% assignment greater than 90%), retention time (tR)andm/z selected.

#tR m/z Compound identiﬁed 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) 127–136

90%), the retention time (tR)andm/z selected. The compounds identi- [23]. A lipid profile exhibiting a C16:0/C18:0 ratio of 1.75 was interpreted fied in each of the 8 samples are specified 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 GC–MS analysis, 25638). compounds characteristic of ruminants [25]. The consumption of red meat by the Peñalosa people has been reported in previous paleopatho- 3.2.1. Ceramic goblet and the associated soil residue inside the goblet logicalstudiesofhumanbones[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 profile 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 finding 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 identified by GC–MS 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 confirms 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) 127–136 133

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 fish residue [27]. that it once contained animal fat, an assumption confirmed by archaeo- There is little evidence for fish consumption at Peñalosa. Nevertheless, logical data from the Peñalosa area [2]. the chemical analysis of bone samples from an adult woman found in Significant amounts of odd number fatty acids (C15:0 and C17:0)could this settlement (Log Ba/Sr = −1.55) confirmed repeated ingestion of indicate bacterial action and suggest that ruminant animals were prob- fish 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 difficult 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 fits the ruminant fat profile. The presence of bovine and ovine an- attribution of fish residues in goblet 25567 still remains ambiguous imals was previously demonstrated by experimental studies on animal but GC–MS analyses provide an indicator to justify further study. and human bones found at the Peñalosa 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, flax 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 Peñalosa site reinforces the evidence for the repeated heating of cooking vessels or oxidative and microbial pro- vegetable oils [28].Theidentification 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 identified as ‘green’ plants and next to saturated fatty acids, may be justified 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 Peñalosa 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 archaeological 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 identified from GC–MS 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 (C ,C , acids [32–34], while citric acid, a natural preservative used in 12:0 14:0 C ,C ,C ,C ) originating from both animal and plant lipids. winemaking, and the aconitic acids derivatives (tributyl aconitate) 16:0 18:0 20:0 30:0 The significant content of C and C fatty acids and the saturated hy- identified in the residue, are a strong indication of wine or other grape 20 30 drocarbon (pentacosane, C H )identified in the residues suggests derivatives. The unsuccessful search for other potential wine bio- 25 52 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 Peñalosa settlement, next to the river. Although the identification In addition, the biplot of fatty acids C /C against the C /C of the aforementioned grape markers suggests the presence of wine or 12:0 14:0 16:0 18:0 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 identification by IR spectrometry [18] and the discovery of carbonized grape seeds at Castellón Alto [35], Fuente Amarga and Fuente Álamo 3.2.4. Ceramic bowl and associated soil residue inside the bowl (25705 and [36] and Peñalosa [28] all point in this direction. This paper represents 25706 samples) a step forward in that for the first 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 identified (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 fires 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 fire [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 identified in the residue support the idea of a vegetable oil. vant finding 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 identified, 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 significant information and there was no evidence ganisms. Thus, the dehydroabietic acid identified 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 profile 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 identification 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) 127–136

Table 3 Table 5 13 −1 Mean δ C and standard deviation (SD) values of C16:0 to C18:0 fatty acids to 25706 and Quantitative results (μgg ) 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 δ13C value SD δ13C 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 identified by GC–combustion–iso- Rb 25567 92.770 6.320 tope ratio MS (GC–C–IRMS) (Table 3). δ13Cdata(defined 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 Δ13Cvalue 25638 2221.400 57.725 fi δ13 δ13 δ13 (de ned as C18:0- C16:0) plotted against C16:0 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 13 13 25638 155.770 25.450 obtained δ C(−31.06%0; −31.06%0)andΔ C (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 Δ13C value obtained for this residue plotted 25638 18,747.440 355.460 13 Mn 25567 335.445 83.400 against δ C16:0 falls between the reference area for porcine adipose 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 13 the GC–MS and archaeological data [2]. In sample 25706,theδ C values Ti 25567 3332.600 231.105 25638 3092.840 241.310 obtained for C16:0 and C18:0 fatty acid (−28.3 ± 2.0) were consistent with animals that subsisted mainly on C3 plants, according to data pre- Ca 25567 14,447.430 452.870 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 sufﬁcient precaution during excavation and subsequent (Table 2) shed little light on the diet of the prehistoric people from handling and washing. Peñalosa. Nevertheless, the chromatogram for the associated soil (sample 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 related to animal fat. Some quinone derivatives, Gibberellin A3, and oleic Although there is extensive bibliography about lipids retained in and adipic acids identified 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 Δ13C value obtained from sidual peptides have already been added together to lipid analyses and 13 the residue plotted against δ C16:0 falls between the reference area the benefit 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 identified 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 Peñalosa [2]. According to the results shown in Table 4, four peptide sequences AscanbeseeninTable 2, the analysis of residues from ceramic bowl have been undoubtedly identified 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 significant 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) 127–136 135

Fig. 4. Isotopic peak profile based on organic residues (peptides) extracted from bowl sample 25638. same house in Peñalosa, 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 Peñalosa 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 finding 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 identified in the potteries production along with wheat and olive oil. between 13,000 and 28,000 μgg−1 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 2200–3400 μgg−1 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 Andalucía (FQM 338) and 320 μgg−1. and the R & D Project HAR2011-30131-CO2-01 funded by the Spanish Ministry of Economy and Competitiveness. A. García acknowledges sup- 4. Conclusions port from a FPU 13/02389 scholarship from the Spanish Ministry of Ed- ucation. ERDF Funds and Junta de Andalucía'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. 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