Multianalytical characterization of pigments from funerary artefacts belongs to the Chupicuaro Culture (Western Mexico): Oldest Maya blue and cinnabar identified in Pre-Columbian Mesoamerica

Microchemical Journal 150 (2019) 104101

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Multianalytical characterization of pigments from funerary artefacts belongs to the Chupicuaro Culture (Western Mexico): Oldest Maya blue and cinnabar identified in Pre-Columbian Mesoamerica

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María Luisa Vázquez de Ágredos-Pascuala, , Clodoaldo Roldán-Garcíab, Sonia Murcia-Mascarósb, David Juanes Barberc, María Gertrudis Jaén Sánchezc, Brigitte Faugèred, Véronique Darrasd ⁎

a

Departamento de Historia del Arte, Universidad de Valencia, Avda. Blasco Ibáñez, 28, 46010 Valencia, Spain Instituto de Ciencia de los Materiales, Universidad de Valencia (ICMUV), C/ Catedrático José Beltrán, 2, 46980 Paterna, Valencia, Spain Instituto Valenciano de Conservación, Restauración e Investigación (IVCR+i), C/ Genero Lahuerta, 25-3°, 46010, Valencia, Spain d Centre National de la Recherche Scientifique (UMR 8096, Archéologie des Amériques), University Paris 1 Panthéon-Sorbonne, 21 Allée de l'Université, 92023 Nanterre, Paris, France b c

ARTICLE INFO

ABSTRACT

Keywords: Maya blue Cinnabar Pre-Columbian Mesoamerica Raman EDXRF SEM-EDX LC-MS/MS-TOF/MS

The colours used in Pre-Hispanic Mesoamerica to decorate walls, codices or artefacts have been the subject of numerous studies, with particular attention to Maya blue, red and white pigments. However, most of these studies have been focused on emblematic cultures of the Classic period (ca. 300–1000 CE), such as Teotihuacan and Maya cultures. This work proposes a new chronology of the preparation and use of these pigments, particularly Maya blue, by analysing samples of the Pre-Classic period (ca. 1800 BCE–300 CE). The samples belong to ceremonial artefacts decorated with blue, red and white pigments, in a funerary context from the Chupicuaro culture, which was developed between 600 and 100 BCE in Western Mexico. The analytical results obtained in this research by spectroscopic and chromatographic techniques (EDXRF, SEM-EDX, Raman and LC-MS/MS-TOF/ MS) confirm the presence of indigo (Indigofera suffruticosa L., Indigofera mucrolata or Indigofera jamaicensis, among other local species), iron oxide (α-Fe2O3), cinnabar (HgS) and calcium carbonate (CaCO3) as compounds of these colours. Our findings support the first evidence of the use of the indigo to elaborate a Maya blue pigment outside the Maya region at least four centuries before it was recognized in this region. Moreover, we found an ancient use of cinnabar mixed together with iron-based red pigments to cover bodies in the burial rites in PreColumbian societies, showing a connection between these red pigments and the funerary world in ancient America. These results have a great impact on the history of colour in ancient Mesoamerica from the economic, social, cultural and historical point of view. This study implies a geographical and chronological leap of high impact on the archaeology and history of ancient Mesoamerica.

1. Introduction In ancient cultures, colour was an indicator of technological development and specialisation. The physicochemical studies carried out previously to determine the composition of pigments used in the PreColombian cultures of Mesoamerica, have given an important knowledge about their use over various artistic supports such as architectural [1–5], corporal [6–9] and codices materials [10–13]. This knowledge on the composition of pigments has contributed to complement the economic, social and cultural history of colour in ancient Mesoamerica [14,15]. Among the colours of Mesoamerican art, Maya blue is undoubtedly the most studied to date. The first study of the blue pigments present in

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pre-Columbian cultures corresponds to the mural of the Temple of the Warriors at Chichen Itza [16]. This pigment was named Maya blue several years later [17,18]. Chemistry defined this colour as a pigment that was elaborated with two raw materials: a clay (the inert matrix) and indigo (the organic component) [19,20]. Since then, many physicochemical studies have been carried out and we have learned more about the structure, composition and degradation processes of Maya blue [21–33]. From the middle of the 20th century to date, the three main objectives of the Maya blue physicochemical studies have been: 1) to understand the Maya blue making process; 2) to understand how the indigo dye, which is responsible for this colour, was fixed and stabilized into the palygorskite matrix; 3) to study the aspects of the making

Corresponding author at: Departamento de Historia del Arte, Universidad de Valencia, Avda. Blasco Ibáñez, 28, 46010, Valencia, Spain E-mail addresses: [email protected], [email protected] (M.L.V. de Ágredos-Pascual).

https://doi.org/10.1016/j.microc.2019.104101 Received 31 May 2019; Received in revised form 14 July 2019; Accepted 14 July 2019 Available online 22 July 2019 0026-265X/ Published by Elsevier B.V.

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Fig. 1. Location of the valley of Acambaro (Guanajuato), seat of the Chupicuaro Culture (base map: G. Pereira; map: V. Darras).

in Mesoamerica to paint floor and walls of the tombs, to cover the deceased corpses and to paint and fill the funerary offerings [6]. These rituals were common in many cultures of Antiquity and were not circumscribed exclusively to Mesoamerica [36–39]. The importance of cinnabar in the funeral rites of ancient America extends from Pre-Hispanic Andean cultures to Mesoamerican ones. In this sense, the identification of pigment in Pre-classical funerary contexts, such as the Chupicuaro described here, is also of great socio-cultural and historical interest, because the processing of this pigment required very advanced skills and technical knowledge, which evokes the great development of these ancient societies in Pre-Columbian America. Our work offers new findings on these both important colour topics: the possible origin of the precursor technology of Maya blue in ancient Mesoamerica and the ritual use of cinnabar in Pre-Hispanic funerary contexts. The study is focused on the characterization of pigments present in funerary artefacts from La Tronera site (Chupicuaro culture) by a multi-analytical approach. Scanning electron microscopy with microanalysis (SEM-EDX) and energy dispersive X-ray fluorescence spectroscopy (EDXRF) provides information about the morphology and elemental composition of these pigments, whereas Raman spectroscopy and liquid chromatography-time of flight mass spectrometry (LC-MS/MSTOF/MS) were used to identify the presence of organic colorants in the pigments. The most significant results are discussed, especially in the case of the blue and red colouring materials for its relevance in the Preclassic times, and suggest the early use of Maya blue in this site at least four centuries before the Late Pre-Classic period (c. 300 BCE–300 CE).

process that influencing the characteristic hue of Maya blue. Related to the first two objectives, in the early 1960s Shepard introduced the idea of Maya Blue as the result of an addition reaction between the indigo and a clay [18] and Gettens further established the properties of the pigment following systematic acid attack tests [19]. On the other hand, Van Olphen prepared colouring materials with similar proprieties to that of Maya blue by crushing and heating indigo with palygorskite and sepiolite [21]. This subject was confirmed by several authors from similar experiments and proposed a thermal treatment in which the indigo and palygorskite mixture was heated below 200 °C [22–29]. With regard to the third objective, several studies carried out during the last two decades agree with the hypothesis that the blue coloration is mainly due to bathochromic shift of the indigo absorption bands as a result of the association of the dye with the inorganic support [24–29], and that the hue of Maya blue is connected with dehydroindigo which could be formed by aerobic oxidation of the indigo in the palygorskite matrix [30–33]. It has been very recently that the oldest samples of Maya blue were identified as belonging to the Classic Period of Mayan culture (ca. 300–1000 CE). However, in 2011 the first Maya blue of the Pre-Classic period was identified in the Pre-Columbian city of Calakmul (Mexico), dated by archaeological works around 150 CE, i.e. to the Late PreClassic Period [34]. However, in this work we show that Maya blue technology could be dated back to at least 250 BCE and introduces a new hypothesis, that this technology could have been developed by the Pre-Hispanic Chupicuaro culture, one of the Pre-Classic emblematic cultures of Western Mexico [35]. The archaeological findings and physicochemical results presented in this study could be a turning point in the history of this colour because the use of this technical procedure would go ahead 400 years, and the first manufacture of this colour outside the Mayan civilization would also be relocated. As regard to red pigments, cinnabar was the most important pigment in funerary contexts of Pre-Columbian America and, alone or combined with other iron-based red pigments, the most frequently used

2. Material and methods 2.1. The archaeological context and samples We present the analysis of pigment samples from a burial excavated in 2001 in the JR 24 site La Tronera, located in the east side of the 2

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Acambaro Valley (West Mexico), settling of the Chupicuaro culture in the middle valley of the Lerma River (Fig. 1). The Acambaro Valley had numerous sources of raw materials that were widely used in craft activities. The white (carbonates) and red (hematite) pigments came from hydrothermal sources, widespread in the area. On the contrary, the cinnabar was found in the surrounding hills (Sierra de los Agustinos). While those pigments were widely used in pottery, there were also used in funerary practices [40]. It is nonetheless important to outline that no blue mineral was available locally. The analysis of the chrono-stratigraphic sequence of La Tronera site, supported by the archaeological analyses and 25 radiocarbon dates [41], revealed various stages of occupation over a wide temporal period, from 600 BCE to 400 CE, i.e. from Middle Pre-Classic to Early Classic [42,43]. The samples discussed in this paper come from a funerary context characterized by nine primary burials. Among them, burials 2 and 9 in the area of La Tronera site, that correspond to a levels with presence of ritual hearths (tlecuiles). Burial 2 was a niche-shaped grave with an opening covered by a vertical flagstone, and burial 9 was a simple pit grave. The burial 2 contained human remains and abundant and diversified furnishing that was placed after laying down the body [43]. The furnishing was comprised of ten ceramic vessels, three small balls of white clay sediment resembling chalk, and the rest of a painted jicara (Fig. S1). The jícara, made from the fruit of the jícaro tree, presents remains of white, red and blue pigments. The optical microscopy of the decoration areas on the jícara showed the presence of a whitish layer underneath the pigment layers and directly applied on the vegetal body. The human remains were identified as a child aged between 4 and 6 and had a death mask painted in white and bordered in red strokes on his face and cranium (Fig. S2), as well as a necklace with seashell beads [40]. The quantity and the wealth of objects, as well as the corporal funerary treatment and various health biochemical indicators suggest that the child probably had a particular social status during her life. On the other hand, radiocarbon dates were obtained from a bone sample of the child that was prepared and then analysed at the Centre de Datation par le Radiocarbone of the Université de Lyon 1. The results estimated an age 14C BP of 2295 + 30 which, after calibration to 2 sigmas, allowed positioning the event in the interval from 406 to 231 BCE (n° of sample: Lyon-13535) [41]. The burial 9 contained two individuals. The bone remains of burial 9a correspond to a female adult individual about 35 to 40 years old. The furnishings consisted in two ceramic vessels and a metate (grinding stone). At the feet of burial 9a, bone remains of a very young child (burial 9b) in an extended dorsal position were found accompanied by two ceramic vessels and a chalk ball. Remains of red and white pigments were recovered in the sediment over the child long bones. Other graves discovered in the same stratigraphic context were dated directly by AMS with results also comprised between 400 and 200 BCE. Three pigment samples collected from the painted jícara (Ch-2a, white; Ch-2b: red; and Ch-3: blue) and two samples from the dead mask (Ch-6a: white; and Ch-6b: red) found in burial 2, and one pigment sample from a child long bone (sample Ch-9: red) of the remains found in burial 9b, were analysed for this study (Table 1, supplementary material). For the analyses, due to the high value of these cultural artefacts, small fragments (< 5 mm2) of the pigmented layers were taken from the surface with sterile scalpels and stored in Eppendorf vials to avoid any kind of contamination before the physicochemical analysis had been performed.

2.3. Analytical methods 2.3.1. Energy dispersive X-ray fluorescence spectroscopy (EDXRF) Qualitative analyses were carried out on pigmented and non-pigmented (back side) areas of the samples in order to compare both EDXRF spectra to identify the elements that are present in the coloured zones. The EDXRF spectrometer is equipped with a thermoelectrically cooled Si-PIN detector with 165 eV energy resolution and a low power X-ray transmission tube (silver anode) operating with an excitation potential of 30 kV and working current of 0.004 mA. An aluminium pinhole collimates the X-ray beam on a 2 mm diameter area of the sample surface. Radiation source and detector were fitted on a mechanical device with excitation-detection geometry of 45° and about 2 cm sample-detector distance. EDXRF spectra were processed with the PyMCA code and were normalized to the total count during an acquisition time of 200 s in order to diminish geometrical effects and fluctuations of the tube intensity. Light elements with Z < 14 were not detected. 2.3.2. Scanning electron microscopy (SEM-EDX) The samples were attached on C-tapes and have been analysed using a Hitachi Variable Pressure Scanning Electron Microscope (VP-SEM) model S-3400N, equipped with a Bruker dispersed X-ray energy spectrometer model XFlash® with a silicon drift droplet detector (SDD), with Dura-Beryllium window (8 μm) and energy resolution of 125 eV @ 5.9 keV. The analyses were performed using 20 kV, a working distance of 10 mm, and pressure inside the chamber of 60 Pa. In addition, backscattered electron images were registered for the documentation of the samples. 2.3.3. Raman spectroscopy Unaltered samples of the pigments were analysed by Raman spectroscopy, in backscattering geometry at room temperature, using a HORIBA Jobin Yvon iHR320 spectrometer equipped with a Peltiercooled CCD detector and 785 and 532 nm doubled YAG laser as excitation. A 50× magnification LWD objective was used to focus the laser on the sample and collect the scattered light. Raman spectra were acquired with a laser power between 10 and 30 mW, a grating of 1200 g/mm and hole of 100 mm, an integration time of 120 s and up to 5 spectral accumulations with a resolution of 1.5 cm−1. 2.3.4. Liquid chromatography-mass spectrometry (LC-MS/MS)-time of flight mass spectrometry (TOF-MS) Samples of the indigo reference material and the blue pigment from the jicara, scraped off from the substrate using a scalpel under an Olympus optical microscope, were partially dissolved on acetonitrile (ACN) 2% and trifluoroacetic acid (TFA) 0.1%. Dissolutions of 2 μL of the indigo reference material and 6 μL of the blue archaeological sample that was pre-concentrated on a rotatory evaporator, were analysed and then used for liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) analysis. Sample dissolutions were analysed using an Eksigent NanoLC-Ultra 1D Plus HPLC system that adds an isocratic loading pump for sample enrichment experiments in which sample is loaded onto an Eksigent trap column (NanoLC Column, 3 μ, C18-CL, 350 μm × 0.5 mm) prior to analysis and desalted with 0.1% TFA (trifluoroacetic acid) for 5 min at 3 μL/min. The peptides were then loaded onto a Nikkyo analytical column (LC Column, 3 μ, C18-CL, 75 μm × 12 cm) containing 5% acetonitrile and 0.1% formic acid (FA). Elution was carried out with a linear gradient of two solvents (solvent A: H2O-0.1% formic acid; solvent B: acetonitrile-0.1% formic acid) from 5% to 95% B in A for 15 min and isocratic at 95% B for 5 min. The flow rate of the mobile phase was of 300 nL/min. The temperature was maintained at 20 °C. LC-MS/MS analyses were performed by means of a Triple TOF 5600 ABSCIEX mass spectrometer with ESI ionization source and hybrid triple quadrupole time-of-fly analyser to determine the m/z ratio via a

2.2. Reference material The chromophor of the blue pigment from Chupicuaro was identified by comparison with the reference material Indigofera tinctoria, natural organic powder pigment from Bangladesh (India), supplied by Kremer Pigmente (Germany). Hematite and cinnabar reference powder pigments also supplied by Kremer Pigmente have been used to identify the red pigments of the samples by Raman spectroscopy. 3

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Fig. S3 shows the analysis of the blue side and back side of the sample Ch-3. It also reports the analysis of a sample of sacalum, a local palygorskite that comes from the geographic area of La Tronera site. The composition of the blue side of the sample is similar to that of the palygorskite but also contains Ca and K. The composition of the back side (support) also has Na, P, S, Cl and Ti, which could be due to the contamination by contact with remains inside the burial. Fig. S3 also shows the image of a detail of the morphology of the sample Ch-3 in which the typical structure of the palygorskite is observed. In conclusion, it can be saying that the inorganic part of the Ch-3 sample is a lamellar clay compatible with palygorskite contaminated by the presence of excavation remains or by other clays. Subsequently, the sample Ch-3 was analysed by Raman spectroscopy in order to identify the blue pigment. For this purpose, we compared the Raman spectra of the blue pigment present in sample Ch3 with the reference material Indigofera tinctoria. The spectral region between 800 and 1600 cm−1 was selected because it was the only one in common for all the spectra. The characteristic Raman bands of indigo at 1570 cm−1 (δNeH, δCeH, δCeC ring), 1459 cm−1 (δCeH, δNeH, δCeC ring,), 1363 cm−1 (δNeN, δCeH) and 1248 cm−1 (δCeH, δC]C, δNeH) [44–47] were clearly identify in the reference sample. Nevertheless, it could be observed the significant decrease in the intensity and resolution of the Ch-3 sample bands. The Raman spectra from the blue sample show a very high fluorescence background and the quality of the spectrum is very poor. However, it is possible to eliminate the background and perform a smoothing to observe the presence of some characteristic bands of the indigo molecule (Fig. 2). Maya blue is recognized as a hybrid organic-inorganic material; in particular, an organic dye, as indigo, and an inorganic layered clay, as palygorskite. It is known that the stability of the pigment is related with the interaction between dye and clay, that is between C]O and NeH units from the indigo and SieO, Al or Mg ions from the clay. This pigment could have been prepared using different recipes and local materials. For that, the presence of different chemical forms of indigo on the natural indigo source as well as, the use of impure natural clay makes the interaction very complex and difficult to assign [44,47]. In addition, the complex structure of the sample obtained from the jícara, where the blue pigment was strongly adhered to the vegetal substratum, the presence of several compounds, the degradation of the sample as well as the small amount of sample used in the analysis, made very difficult the unambiguous identification of the blue pigment by Raman spectroscopy. Due to the importance of the possible presence of indigo in these ancient samples, we decided to use also the LC-MS/MS-TOF-MS analysis (in a qualitative mode) to ensure the presence of this pigment [48–50]. After the sample preparation and extraction process detailed in Section 2, reference indigo and sample Ch-3 were analysed by LCMS/MS-TOF-MS and being possible the detection of indigo on basis of their TOF-MS spectra and fragmentation profiles. Fig. S4 shows the representative TOF/MS spectra of the reference material and the sample Ch-3 with a marked signal at m/z 263.08 [M + H]+, characteristic of the atomic mass of the indigo dyestuff. On the other hand, Fig. 3 shows the MS/MS spectra after fragmentation where we can see, in the reference sample and in the sample Ch-3, the representative ion of indigoids at m/z 263.08 [M + H] + and the fragmentation profiles at m/z 206.08, 219.09 and 235.08. These m/z fragments correspond to the loss of CO, NH2 and CO + HCO, respectively [49,50]. Therefore, the reference material and the sample Ch-3 yield similar fragmentation profiles in ESI(+) ion mode, confirming the presence of indigoids in the archaeological sample Ch-3. These results allow us to confirm the use of Maya blue in the decoration of the Chupicuaro jícara. SEM/EDX spectra of the sample Ch-2a (white layer of the jícara) show high peaks of calcium and silicon and lower peaks of sodium, magnesium, aluminium, potassium and iron. The unappreciable presence of sulphur suggests the use of a calcium carbonate white pigment. Raman analyses confirm the presence of CaCO3 in all the white samples

Fig. 2. Raman spectra of the blue indigo reference material and b) blue pigment from the jícara.

time measurement. Analysts were ionized applying 2.8 kV to the spray emitter and the analyses were carried out in a data-dependent mode. MS1 scans were acquired from 100 to 750 m/z for 500 ms. Quadrupole resolution was set to ‘UNIT’ for MS2 analysis, acquired from 100 to 1500 m/z for 125 ms in ‘high sensitivity’ mode. Up to five ions were selected for fragmentation after each survey scan. The switch criteria used were charge (1+ to 2+), minimum intensity and 50 counts per second. Dynamic exclusion was set to 15 s and dynamic collision energy was applied. 3. Results 3.1. Identification of the blue, white and red decorations of the jícara SEM/EDX, Raman and LC-MS/MS-TOF/MS analysis were carried out on a sample of the blue pigment added to the body of the jícara (referenced as Ch-3; see Table 1 in supplementary material). SEM/EDX analysis was applied to identify the elemental composition of the blue pigment and to visualize the grain morphology. The microsample was fixed on the SEM support using a double-sided carbon tape, placing the blue pigment of the sample on the upper part. The images obtained in the electron backscattering mode show an irregular surface with a huge quantity of small particles with different sizes and shapes attached on it. The SEM/EDX spectra show the presence of Na, Mg, Al, Si, P, K, Ca and Fe (Fig. S3). These elements could be related with aluminosilicates, sulphates, carbonates and iron oxides that are characteristics of raw materials but that are not related with key elements for the identification of blue inorganic pigments. Therefore, it is possible to postulate an organic composition of the blue pigment based on light elements. Moreover, elongate particles with fibrous shape, which are present in the sample, show the presence of Mg, Al, Si, K, Ca and Fe. The detection of Mg, Al and Si is compatible with the presence of palygorskite [Si8(Mg2Al2)O20(OH)2(OH2)4·4H2O], a particular phyllosilicate clay mineral that is the substratum of the organic dyestuff indigo (C16H10N2O2) in the Maya blue manufacture. Separately, these compounds have no particular properties but after mixing and thermal treatment a bright blue pigment, chemically and thermally stable and resistant to acids, alkalis and solvents, can be obtained. 4

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Fig. 3. Fragmentation profiles in ES (+) ion mode of the blue pigment from the Chupicuaro sample Ch-3 (blue decoration of the jícara) and the blue indigo reference material.

by the detection of bands at 154, 281 and 1085 cm−1. The two lower wavenumbers bands for calcite arise from the external vibrations of the δ(CO3)2-group that involve the rotatory and translatory oscillations of those groups. The intense sharp Raman band at 1085 cm−1 is assigned to the δ(CO3)2-symmetric stretching mode. The rest of elements can be related with the presence in the ground layers of aluminosilicates and iron oxides. The EDXRF analysis shows again the presence of calcium as key element of the white pigment and iron peaks linked with the materials of the background. In the red areas of the jícara (sample Ch-2b), a mercury-based pigment was found through SEM/EDX and EDXRF analysis. The BSE image and the microanalysis show a support with the mainly presence of iron, calcium, sulphur, silicon and aluminium where several groups of brighter particles have intense peaks of mercury. In addition, EDXRF analysis identifies also the presence of iron, mercury and sulphur (Fig. S5). The comparison between EDXRF spectra from the red pigment and support reveals the presence of mercury only in the red pigment. Iron and calcium are also present in the pigment with most intense peaks than in the support, and suggest a mixture of red pigments in the decoration: red ochre (probably hematite, α-Fe2O3) and cinnabar (HgS). The Raman analysis of the red sample Ch-2b confirms the presence of cinnabar and hematite (Fig. S6, spectra “a” and “d”, respectively).

The Raman spectra of cinnabar is characterized by three bands of the space D3h at 254 cm−1 (active A1 mode), 282 cm−1 (active E mode) and 344 cm−1 (active E mode), while the Raman spectra of hematite shows its characteristic bands at 227 cm−1 (A1g mode), 293 cm−1 (Eg mode) and 412 cm−1 (Eg mode). All these bands are caused by vibrations of tension of the FeeO bonds of the tetrahedral units FeO4 that make up the structure of the hematite. However, the presence of cinnabar in the jícara is much higher in all points than that of hematite, which is only found in very limited areas. The results obtained indicate that the white and red colours preserved on the body of the jícara are inorganic pigments, local origin probably, and its use in the decoration of Mesoamerica's Pre-Hispanic pottery dates back at least to the beginning of Late Pre-Classic times [51–53]. 3.2. The pigments linked to human remains White and red pigments have been found in human remains of the burial 2 (child mask), and burial 9 (sediments next to long bones). The mask that covered the child's face was decorated with white and red pigments (samples Ch-6a and Ch-6b) while the pigment of burial 9 is red (sample Ch-9). 5

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The white pigment of the mask has been identified by SEM/EDX and Raman as a mixture of calcium carbonate and clays. In this sense, it is worth remembering that the calcite was used in medicine and cosmetic science for its anti-microbial properties since early Mesopotamia, where this material was mixed with clays to make facial masks with soft texture and luminous optical properties [54–55]. As regards the red decoration of the mask, the pigment was identified by SEM/EDX as an iron oxide-based pigment with very small cinnabar particles dispersed on an iron oxide matrix. The map distribution of elements obtained clearly showed the cinnabar small (< 10 μm) particles dispersed in some isolated areas of the sample surface (Fig. S7). However, EDXRF analyses only detect the presence of iron oxides (Fig. S8), identified as hematite by Raman (Fig. S6e). The intensity of iron peaks detected by EDXRF on red areas of the mask is greater than those detected in the support, and no other element ascribable to inorganic red pigments was identified. This is undoubtedly due to the small dimensions of the cinnabar particles and their dispersion on the sample. The use of a mercury-based compound, when is applied to the whole corpse, should have slowed down the biological decomposition process mitigating the stench of corruption during funerary ceremonies of rulers or people of a high social category, as it probably happens with the child of this burial of Chupicuaro. On the other hand, the red pigment found in sediments next to the long bones of a child in burial 9 of La Tronera site, was also identified as cinnabar by Raman (Fig. S6b). This could be related to the use of this pigment in a ritual or funerary context in which the red colour plays a symbolism related with life and death in culture, the ancient Mesoamerican cultures.

which revealed the simultaneous presence of mercury and sulphur in red areas of the jícara and in the sediments found next to the long bones of burial 9. These analyses were complemented with the detection of the main Raman bands of hematite and cinnabar. For the white colour, the different spectra show a composition based on calcium carbonate and clays. The same elemental composition has been found in the white coloration of the jícara and the child mask. The red and white pigments identified in the funerary artefacts from the Chupicuaro culture are local raw materials easy to obtain and use. In addition, these are suitable pigments for the ceramic technology and the high temperatures of the ovens in which these pieces were made. It is interesting that here the painter use cinnabar to decor the jícara, which was also reserved to be applied to cover the corpses [6]. Iron oxides, cinnabar and calcite were particularly prominent pigments in elite funerary practices that have been identified in the mask that hid the child's face. These compounds have antibacterial properties of great importance in tombs and burials. Its use on the face of the dead child raises some questions, but specially one: why were they used specifically on the face? In accordance with the writing sources and iconography, the vital importance of the head within the context of the human body was common among all the inhabitants of Pre-Hispanic Mesoamerica, and the pigments that decorate the child's mask buried in the grave of JR 24 site had a symbolism and a cultural and religious meaning [56–58]. 5. Conclusions The multi-analytical study of the pigment decoration of the offerings and human remains found in the burials 2 and 9 of La Tronera site (Chupicuaro culture, Mexico) was successful to identify the use of calcite for the white pigments, cinnabar and iron oxides for the red pigments and Maya blue for the blue pigments. The results presented in this paper bring forward by at least 400 years the origin of the Maya blue and place it in a new non-Mayan culture. Moreover, these results are consistent with the technological advances that had been achieved by the cultures of Western Mexico (including the Chupicuaro culture) by the Late Pre-Classic Period. In that sense, our analytical findings open the door to the study of colouring materials, ritual-mortuary practices, and religious beliefs in one of the most ancient cultures of Pre-Columbian Mesoamerica. At the expense of further studies, this work provides a basis for establishing the ancient use of iron-based red pigments and cinnabar in funerary contexts, a novel information about the precursor technology of Mesoamerican Maya blue and a new chronology for Maya blue much earlier than considered so far. The existence of a new geographical origin for this pigment suggests successive innovations in the production technology from the Pre-Classic times to the Post-Classic period and re-signifies the organic-inorganic chromatic synthesis by locating its origin in an older temporality and in a non-Mayan Pre-Hispanic culture. Considering the historical point of view, our study implies a geographical and chronological leap of high impact on the archaeology and history of ancient Mesoamerica and proposes that this colour technology was subject to ad hoc reinterpretations, especially as regards the choice of the inorganic matrix to which the indigoid dye was to be anchored. These aspects are very interesting in order to looking for links between production centres, materials sources, and commercial routes for Maya blue and other historical pigments in PreColumbian Mesoamerica.

4. Discussion The manufactured Maya blue pigments include different shades of blue from light blues to bluish-greys as the existing in the facade of the tomb of King Yuknom Yichak Kak of Calakmul [34] and in Maya settlements of the Late Pre-Classic period that have not yet been analysed. These different shades of blue are extremely interesting for the study of colour technology in ancient Mesoamerica because they demonstrate the period during which the production of this pigment was “under construction”, which may in turn explains why it did not reach its best expression. In this work, we have identified by means of LC_MS/MS-TOF-MS and Raman spectroscopy the presence of indigo in the blue pigment of a jícara from an archaeological level of the JR 24 site “La Tronera” (Chupicuaro Culture) dated between 400 and 200 BCE. Indigo is an organic dye used by Mesoamerica artisans to prepare Maya blue [16–33] and our work presents analytical data of the oldest Maya Blue samples known today. This finding brings forward the date of this technical procedure by at least 400 years with respect to the Maya blue identified in Calakmul and dated around 150 CE [35]. The Chupicuaro Culture is one of the ancient ceramist cultures from Western Mexico where the artisans experimented combining indigo and fibrous clay silicates (specifically palygorskite) to obtain a new, and more stable, pigment. This suggests that in Western Mexico the technology and know-how needed to manufacture this pigment had already been mastered by the end of the Middle Pre-Classic Period. The sociocultural and economic implications of this finding are clear and very relevant to the technical and cultural history of colour in ancient Mesoamerica. Concerning the red colour, two different inorganic pigments were detected: cinnabar, in the red coloration of the jícara and in the sediments next to the long bones found in burial 9, and red iron oxide in the red coloration of the child mask found in burial 2. The iron oxide-based red pigment of the mask was mixed with small quantities of cinnabar. The EDXRF and SEM-EDX analyses showed the presence of Fe and typical elements as Si, K and Ca associated to clay minerals, which indicates the use of ochre in red areas of the jícara and the child mask. The identification of cinnabar was made by SEM-EDX and EDXRF

Acknowledgments The Chupicuaro Project was directed by Véronique Darras and Brigitte Faugère, and was funded by the Commission des fouilles of the French Ministry for Europ and Foreign Affairs (MAEE, Paris) and the Archéologie des Amériques Laboratory (UMR 8096_CNRS/University Paris 1 Panthéon-Sorbonne). The analysis has been carried out in the 6

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proteomics facility of SCSIE University of Valencia that belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001 of the PE I+D +i 2013-2016, funded by ISCIII and FEDER. Finally, this research and the results outlined would not have been possible without the support of the Ministry of Economy and Competitiveness (Spain), through the funding of coordinated research programmes, reference numbers ARTE Y ARQUITECTURA MAYA. NUEVAS TECNOLOGIAS PARA SU ESTUDIO Y CONSERVACION (BIA2014-53887-C2-2-P).

645–646. [22] Kleber, L. Masschelein-Kleiner, J. Thissen, Étude et identification du bleu maya, Stud. Conserv. 12(2) (1967) 41–56. [23] J. Yacamán, L. Rendón, J. Arenas, M.C. Serra Puché, Maya Blue paint: an ancient nanostructured material, Science 273 (1996) 223–224. [24] G. Chiari, R. Giustetto, G. Ricchiardi, Crystal structure refinements of palygorskite and Maya Blue from molecular modelling and powder synchrotron diffraction, Eur. J. Mineral. 15 (2003) 21–33. [25] E. Fois, A. Gamba, A. Tilocca, On the unusual stability of Maya Blue paint: molecular dynamics simulations, Microporous Mesoporous Mater. 57 (2003) 263–272. [26] B. Hubbard, W. Kuang, A. Moser, G.A. Facey, C. Detellier, Structural study of Maya Blue: textural, thermal and solid-state multinuclear magnetic resonance characterization of the palygroskite-indigo and sepiolite-indigo adducts, Clay Miner. 51 (2003) 318–326. [27] P. Gómez-Romero, C. Sanchez, Hybrid materials. Functional properties. From Maya Blue to 21st century materials, New J. Chem. 29 (2005) 57–58. [28] L.A. Polette-Niewold, F.S. Manciu, B. Torres, M. Alvarado Jr., R.R. Chianelli, Organic/inorganic complex pigments: ancient colors Maya Blue, J. Inorg. Biochem. 101 (2007) 958–1973. [29] D.E. Arnold, Maya Blue and palygorskite: a second possible pre-Columbian source, Anc. Mesoam. 16 (2005) 51–62. [30] A. Doménech Carbó, M.T. Doménech Carbó, M.L. Vázquez de Ágredos Pascual, Dehydroindigo: a new place into the Maya Blue puzzle from the voltammetry of microparticles approach, J. Phys. Chem. B 110 (2006) 6027–6039. [31] F.S. Manciu, L. Reza, L.A. Polette-Niewold, B. Torres, R.R. Chianelli, Raman and infrared studies of synthetic Maya pigments as a function of heating time and dye concentration, J. Raman Spectrosc. 38 (2007) 1193–1198. [32] A. Doménech Carbó, M.T. Doménech Carbó, M.L. Vázquez de Ágredos Pascual, Chemometric study of Maya Blue from the voltammetry of microparticles approach, Anal. Chem. 79 (2007) 2812–2821. [33] M.T. Doménech Carbó, A. Doménech Carbó, C. Vidal Lorenzo, M.L. Vázquez de Ágredos Pascual, L. Osete Cortina, F.M. Valle Algarra, Discovery of indigoid-containing clay pellets from La Blanca: significance with regard to the preparation and use of Maya Blue, J. Archaeol. Sci. 41 (2014) 147–155. [34] M.L. Vázquez de Ágredos Pascual, M.T. Doménech Carbó, A. Doménech Carbó, Characterization of Maya Blue pigment in pre-classic and classic monumental architecture of the ancient pre-Columbian City of Calakmul (Campeche, Mexico), J. Cult. Herit. 12 (2011) 140–148. [35] M. Noé Porter, Excavations at Chupícuaro, Guanajuato, Mexico, Transactions of the American Philosophical Society 46, Philadelphia, 1956. [36] B.T. Arriaza, Beyond Death: The Chinchorro Mummies of Ancient Chile, Smithsonian Institute Press, Washington D.C., 1989. [37] T. Dillehay (Ed.), Tombs for the Living: Andean Mortuary Practice. A Symposium at Dumbarton Oaks, Dumbarton Oaks, Washington D.C., 1995. [38] D. Kennedy, X-ray diffraction studies on ancient hairs, Cosmetics & Toiletries 96 (1981) 121–122. [39] M.S. Walton, K. Trentelman, Romano-Egyptian red lead pigment: a subsidiary commodity of Spanish silver mining and refinement, Archaeometry 51 (2009) 845–860. [40] B. Faugère, V. Darras, Proyecto Dinámicas Culturales en el Bajío, Guanajuato. La cultura Chupicuaro, CEMCA, Mexico City, 2001 Unpublished Report submitted to the Instituto Nacional de Antropología e Historia. [41] CDRC, Résultat d'analyse par le Radiocarbone. Mesure par accélérateur de l'échantillon Chupicuaro-JR24-SEP2 (Lyon-13533), Centre de Datation par le Radiocarbone, université de Lyon1, Lyon, 2016 Unpublished report. [42] V. Darras, B. Faugère, Cronología de la cultura Chupícuaro. Estudio del sitio La Tronera, Puruaguïta, Guanajuato, in: E. Williams, P. Weigand, L. Lopez Mestas, D.C. Grove (Eds.), El Antiguo Occidente de México. Nuevas perspectivas sobre el pasado prehispánico, El Colegio de Michoacán, Zamora, 2005, pp. 255–282. [43] B. Faugère, V. Darras, Shaft tombs in Chupicuaro's funerary practices: Architectural and ritual contents, in C. S. Beekman, R.B. Pickering (Eds.), Shaft Tombs and Figures in West Mexican Society: A Reassessment, Gilcrease Ancient America Series, Thomas Gilcrease Institute of American History and Art, Tulsa, 2005, pp 39 – 54. [44] a A. Doménech Carbó, M.T. Doménech Carbó, H. Edwards, On the interpretation of the Raman spectra of Maya Blue: a review on the literature data, J. Raman Spectrosc. 42 (2011) 86–96; b A. Doménech Carbó, S. Holmwood, F. di Turo, N. Montoya, F. Valle, H. Edwards, M.T. Doménech Carbó, Composition and color of Maya Blue: reexamination of literature data based on the dehydroindigo model, J. Phys. Chem. C 123 (2019) 770–782. [45] H.G. Wiedemann, K. Werner Brzezinka, K. Witke, I. Lamprecht, Thermal and Raman-spectroscopic analysis of Maya Blue carrying artefacts, especially fragment IV of the Codex Huamantla, Thermochim. Acta 456 (2007) 56–63. [46] M. Sánchez del Río, M. Picquart, E. Haro-Poniatowski, E. van Elslande, V.H. Uc, On the Raman spectrum of Maya blue, J. Raman Spectrosc. 37 (2006) 1046–1053. [47] P. Vandenabeele, S. Bodé, A. Alonso, L. Moens, Raman spectroscopic analysis of the Maya wall paintings in Ek´ Balam, Mexico, Spectrochim. Acta A 61 (2005) 2349–2356. [48] P. Zou, H.L. Koh, Determination of indican, isatin, indirubin and indigotin in Isatis indigotica by liquid chromatography/electrospray ionization tandem mass spectrometry, Rapid Commun. Mass Spectrom. 21 (2007) 1239–1246. [49] E. Rosenberg, Characterisation of historical organic dye stuffs by liquid chromatography-mass spectrometry, Anal. Bioanal. Chem. 391 (2008) 33–57. [50] E. Sanz, Á. Arteaga, M.A. García, C. Cámara, C. Dietz, Chromatographic analysis of indigo from Maya Blue by LC-DAD-QTOF, J. Archaeol. Sci. 39 (2012) 3516–3523. [51] F.A. Peterson, Pottery figurines of Chupicuaro, Mexico, Central States

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.microc.2019.104101. References [1] M.L. Vázquez de Ágredos Pascual, C. Vidal Lorenzo, G. Muñoz Cosme, Archaeometrical studies of classic Mayan mural painting at Peten: La Blanca and Chilonche, in: J.L. Ruvalcaba, J. Reyes Trujeque, A. Velázquez Castro, M. Espinosa Pesqueira (Eds.), Materials Research Society Proceedings, Cambridge University Press, Cambridge, 2014, pp. 45–62. [2] M.L. Vázquez de Ágredos Pascual, C. Vidal Lorenzo, G. Muñoz Cosme, The role of the new technology in the study of Maya mural painting: over a century of progress, in: C. Vidal, G. Muñoz (Eds.), Artistic Expressions in Maya Architecture. Analysis and Documentation Techniques, British Archaeological Reports, Oxford, 2014, pp. 165–178. [3] M. Ortega-Avilés, J.A. Ascencio, C.M. San Germán, M.E. Fernández, L. López Luján, J. Yacamán, Analysis of prehispanic pigments from “Templo Mayor” of Mexico City, J. Mater. Sci. 36 (2001) 751–756. [4] M. Ortega-Avilés, C.M. San-Germán, D. Mendoza-Anaya, D. Morales, J. Yacamán, Characterization of mural paintings from Cacaxtla, J. Mater. Sci. 36 (2001) 2227–2236. [5] D. Magaloni Kerpel, The hidden aesthetic of red in the painted tombs of Oaxaca, Res. Anthropol. Aesthet. 57/58 (2010) 55–74. [6] P. Quintana Owen, V. Tiesler, M. Conde, R. Trejo-Tzab, C. Bolio, J.J. Alvarado Gil, D. Aguilar, Spectrochemical characterization of red pigments used in classic period Maya funerary practices, Archaeometry 57 (6) (2015) 1045–1059. [7] M.L. Vázquez de Ágredos Pascual, C. Vidal Lorenzo, P. Horcajada Campos, Las fragancias rituales del Preclásico en Tak'alik Ab'aj, in: C. Schieber de Lavarreda, M. Orrego Corzo (Eds.), The Dimensions of Rituality 2000 Years Ago and Today, Ministerio de Cultura y Deportes de Guatemala, Guatemala, 2016, pp. 211–232. [8] M.T. Doménech Carbó, M.L. Vázquez de Ágredos Pascual, L. Osete-Cortina, A. Doménech Carbó, N. Guasch-Ferré, L.R. Manzanilla, C. Vidal Lorenzo, Characterization of pre-Hispanic cosmetics found in a burial of the Ancient City of Teotihuacan (Mexico), J. Archaeol. Sci. 39 (2012) 1043–1069. [9] M.L. Vázquez de Ágredos Pascual, L.R. Manzanilla Naim, Corporate paint and ancient pharmaceutical mixtures from Teotihuacan: the Teopancazco neighborhood center, Int. J. Pharm. 1 (1) (2016) 11–21. [10] D. Buti, D. Domenici, C. Miliani, C. García Sáiz, T. Gómez Espinoza, F. Jímenez Villalba, A. Verde Casanova, A. Sabía de la Mata, A. Romani, F. Presciutti, B. Doherty, B.G. Brunetti, A. Sgamellotti, Non-invasive investigation of a preHispanic Maya screenfold book: the Madrid Codex, J. Archaeol. Sci. 42 (2014) 166–178. [11] D. Domenici, D. Buti, C. Miliani, B. Brunetti, A. Sgamellotti, The colours of indigenous memory: non-invasive analyses of pre-Hispanic Mesoamerican codices, in: A. Sgamellotti, B. Brunetti, C. Miliani (Eds.), Science and Art: The Painting Surface, Royal Society of Chemistry, London, 2014, pp. 94–119. [12] S. Zetina, T. Falcón, E. Arroyo, J.L. Ruvalcaba, The encoded language of herbs: material insights into the De la Cruz-Badiano codex, in: G. Wolf, J. Connors, L.A. Waldman (Eds.), Colors Between Two Worlds. The Florentine Codex of Bernardino de Sahagún, Max-Planck-Institute, Villa I Tatti, The Harvard University Center for Italian Renaissance Studies, Florence, 2014, pp. 220–255. [13] S. Zetina, J.L. Ruvalcaba, T. Falcón, J. Alatorre Arenas, S. Yanagisawa, M.I. Álvarez Icaza Longoria, E. Hernández, Material study of the codex Colombino, in: A. Sgamellotti, B. Brunetti, C. Miliani (Eds.), Science and Art: The Painting Surface, Royal Society of Chemistry, London, 2014, pp. 120–146. [14] M. Pastoureau, Les couleurs aussi ont une histoire, L'Histoire 92 (1986) 46–54. [15] J. Gage, Color and Culture, Thames and Hudson, London, 2006. [16] R.E. Merwin, Chemical analysis of pigments, in: E.H. Morris, J. Charlot, A.A. Morris (Eds.), The Temple of the Warriors at Chichén Itzá, Yucatán, Carnegie Institution of Washington, Washington D.C, 1931, pp. 355–356. [17] R.J. Gettens, Identification of pigments of mural paintings from Bonampak, Chiapas, México, in: K. Ruppert, J.E.S. Thompson, T. Proskouriakoff (Eds.), Bonampak, Chiapas, México, Carnegie Institution of Washington, Washington D.C, 1955, pp. 501–502. [18] A. Shepard, Maya Blue: alternative hypotheses, Am. Antiq. 27 (4) (1962) 565–566. [19] R.J. Gettens, Maya Blue: an unsolved problem in ancient pigments, Am. Antiq. 27 (4) (1962) 557–564. [20] E.R. Littmann, Maya Blue, Further perspectives and the possible use of indigo as the colorant, Am. Antiq. 42 (2) (1982) 334–339. [21] Van Olphen, Maya blue: a clay-organic pigment? Science 154 (3749) (1966)

7

Microchemical Journal 150 (2019) 104101

M.L.V. de Ágredos-Pascual, et al. Archaeological Journal 1 (1) (1954) 33–40. [52] J.L. Ruvalcaba-Sil, M.A. Ontalba Salamanca, L. Manzanilla, J. Miranda, J. Cañetas Ortega, C. López, Characterization of pre-Hispanic pottery from Teotihuacan, Mexico, by a combined PIXE-RBS and XRD analysis, Nucl. Instrum. Methods Phys. Res., Sect. B 150 (4) (1990) 591–596. [53] D. Reents-Budet Dorie (Ed.), Painting the Maya Universe, Duke University Press, Durham, 1991. [54] G. Rytwo, Clay minerals as an ancient nanotechnology: historical uses of clay organic interactions, and future possible perspectives, MACLA 9 (2008) 15–17. [55] G. Whitney, Role of water in the smectite-to-illite reaction, Clay Clay Miner. 38 (4) (1990) 343–350.

[56] M.L. Vázquez de Ágredos Pascual, C. Vidal Lorenzo, Patricia Horcajada, Face painting among the classic Maya elite. An iconographic study, in: V. Tiesler, M.C. Lozada (Eds.), Social Skins of the Head. Body Beliefs and Ritual in Ancient Mesoamerica and the Andes, 2018, pp. 93–107. [57] V. Tiesler, M.C. Lozada, Introducing the social skins of the head in ancient Mesoamerica and the Andes, in: V. Tiesler, M.C. Lozada (Eds.), Social Skins of the Head. Body Beliefs and Ritual in Ancient Mesoamerica and the Andes, 2018, p. 1. [58] M.L. Vázquez de Ágredos Pascual, Painting the skin in ancient Mesoamerica, in: E. Dupey, M.L. Vázquez de Ágredos (Eds.), Painting the Skin: Pigments on Bodies and Codices in Pre-Columbian Mesoamerica, 2018, pp. 11–23.

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