An archaeometric approach to biocontamination with manganese pigments in ancient marine hunter-gatherers of the Atacama Desert: health, ideological, and socioecononic considerations

The use of poisonous pigments for mortuary preparation in ancient hunter-gatherers allows us to investigate the relationship between this practice with socio-economic organization, mortuary ideology, and potential impacts on human health from overexposure to these dangerous compounds. About 6000 years ago, the Chinchorro people, maritime hunter-gatherers in the Atacama Desert, northern Chile, began mining manganese to ornament the dead, representing the earliest form of artificial mummification in the world. We present the analysis of the bones from 68 Chinchorro mummies using flame atomic absorption spectrometry, revealing, for the first time, the presence of manganese in their bodies. We excluded the potential diagenesis by comparison of manganese concentration difference between the cemetery soils and outer coating of the mummy and by manganese source identification by SEM/EDX. Approximately 84% (57/68) of individuals have manganese concentrations beyond normal 1 ppm and 20.6% (14/68) were overexposed to levels of higher toxicity (≥ 10 ppm), with no sex differences, suggesting equal participation in manganese handling. We conclude that manganese exploitation and refining for the ritualistic treatment of the dead posed a serious occupational hazard for the living, with evidence that many of the Chinchorro people were likely involved in these activities. This is relevant, considering manganese dust inhalation produces neurological disorders and Parkinson-like symptoms. These findings open new avenues for research into the long history of anthropogenic pollution in the Americas and the importance of mineral gathering and processing as part of a broader subsistence spectrum undertaken by these ancient hunters, gatherers, and fishers.


Introduction
About 7000 years ago, ancient coastal marine hunter-gatherers (MHG) who settled along the Atacama coast of southern Peru and northern Chile-known today as Chinchorrotransformed their dead into effigy-like figures using clay, mineral pigments, reeds, and sticks (Fig. 1A-F) (Arriaza 1995;Marquet et al. 2012;Schiappacasse and Niemeyer 1984;Standen 1997).This is thought to be the first artificial mummification in the world.A millennium later, the Chinchorro became fascinated by the ornamental properties of manganese (Mn) and its intense black color, which they extracted from host rocks and river terraces to use as pigments to paint the bodies of their deceased for the afterlife (Arriaza et al. 2023a,b;Sepúlveda et al. 2013).Manganese, however, is a highly toxic element as will be discussed later 188 Page 2 of 15 (Mena et al. 1967;O'Neal and Zheng 2015;Schüler et al. 1957).
The early peopling of the Arica-Camarones coast and complex mummification developed by the Chinchorro have been associated with the abundance of marine resources and year-round occupation of the littoral region (Arriaza 1995(Arriaza , 2005;;Guillén 1997;Marquet et al. 2012;Montt et al. 2021;Schiappacasse and Niemeyer 1984).In MHG societies, stability in food subsistence is provided by marine and coastal resources, which can result in the formation of early and more permanent forms of territorial occupation than in non-marine hunter-gatherer societies (Arriaza 1995;Claassen 1991;Marquet et al. 2012;Standen 1997;Waselkov 1987).It has been argued that the natural mummification of bodies in this extremely arid environment resulted in intricate burial practices and associated mortuary ideology (Arriaza 2005;Marquet et al. 2012;Montt et al. 2021).This social preoccupation has led to other aspects, unrelated to food acquisition, being central to MHG subsistence activities, such as cultural innovations and experimentation for the treatment of the dead and their final disposal.All of this implies economic aspects and cultural behavior for material acquisition, improvement of pigment quality and preparation, color selection, and decision making.The Chinchorro people are a good example of this social dynamic with its intensive and extensive use of pigments for the complex, intricate, and artistic preparation of the dead.
From a socio-economic point of view, mineral acquisition may have been a collective effort by the Chinchorro, gathered from highly visible and exposed Mn veins using simple tools such as stone hammers, grinding stones, and baskets (Arriaza et al. 2023a;Standen and Arriaza 2016).These variables combined (i.e., intense black color, availability, and easy acquisition) permitted Mn pigment to become a central economic element within their worldview, and the preservation of artificial (anthropogenic) mummification traditions (black and red mummies) for multiple generations.Complex Chinchorro mummies are found in many sites along the Atacama coast (Arriaza 1995;Arriaza et al. 2005Arriaza et al. , 2008;;Arriaza and Standen 2016;Guillén 1997;Montt et al. 2021;Schiappacasse and Niemeyer 1984;Standen 1997), suggesting a common mortuary ideology, longitudinal mobility, social interaction, and transmission of knowledge (e.g., how to prepare the mummies and the type of mineral pigment gathered) with other Chinchorro populations that settled along the Atacama Desert coast.In addition, further specialization of these mortuary practices occurred with some mummification techniques requiring large amounts of refined Mn (e.g., red  (Baeza et al. 2014;García et al. 2004).C Adult female mummy completely painted with Mn.D Child with open mouth and face painted with Mn.E Chinchorro mummy head showing the Mn paste used to secure a long wig.F Chinchorro mummy head with multiple layers of painting and open mouth mummies head treatment, Fig. 1D and E).Thus, it is evident that for the Chinchorro groups, the social practices related to Mn use were of paramount importance.
The Arica-Camarones area was the geographic center for the origin of the Chinchorro artificial mummification practices during the archaic period (Arriaza 1995(Arriaza , 2005) ) (Fig. 1).Here, Mn was intensively used during two main cultural moments.First, during the classic so-called black mummies (ca.6000 to 4750 BP) (Arriaza 1995), where the body was completely covered with a few millimeters of Mn dioxide (MnO 2 ) paint, except for the hair (Figs.1C and 3).The second moment (ca.4750 to 3950 BP), associated with the "red mummies" painted with red ochre (Fe 2 O 3 ) which have a thick Mn-based paste applied only to the face and scalp to secure a long black wig (Arriaza 1995;Arriaza et al. 2008;Arriaza and Standen 2016;Sepúlveda et al. 2013;Standen and Arriaza 2016;Van Hoesen et al. 2019) (Fig. 1D and E).The face of the individual was reconstructed using a black or reddish facial mask (Fig. 1D and  F) implying mortuary art specialization (Arriaza et al. 1988).
We propose that Chinchorro exposure at levels of toxicity may have occurred from direct contact during the process of Mn mining extraction, accumulation of this mineral in their camps, during processing, and application of the Mn coating to the dead (Figs. 1 and 3).Thus, considering the cultural mortuary importance of Mn in Andean antiquity and its neurotoxicity, we investigate: Is there evidence that Mn was inadvertently biologically incorporated into the bodies of the Chinchorro people during their lives?And what are the socio-economic, ideological, and behavioral implications of Mn use in these MHG people?Using a dependable, Fig. 2 Manganese ore mixed with cobbles in the Lluta River.A Strata of Mn near the river mouth at the coast, sample 04 (latitude 18° 24′ 16.374′′ S, longitude 70° 19′ 6.054′′ W).B Localized pocket of Mn embedded within river cobbles in the mid-valley section, about 8 km from the coast, sample 12 (latitude 18° 24′ 25.722′′ S, longitude 70° 15′ 3.656′′ W).Scale: 10 cm, each colored segment.SEM/EDX point analysis reveals up to 62.4% of Mn in A and 66.1% in B (see Supplementary Information, Tables SI1 and SI2) easily accessible, and low-cost instrumental technique (flame atomic absorption spectrometry or FAAS), we explore to what extent the Chinchorro were overexposed to Mn as an occupational hazard of mineral gathering.Using the 1-ppm concentration value as a baseline, we evaluated Mn poisoning through FAAS in 68 rib samples from the Chinchorro people of northern Chile (Supplementary Information, Table SI1) and discuss its biocultural significance.

Materials and methods
The Archaeology Museum of the University of Tarapacá in San Miguel de Azapa (MASMA), Arica, Chile, houses Chinchorro mummies with and without artificial mummification that were excavated during different field seasons and in different states of preservation and completeness.Mummies that were in an exhibit or in impeccable condition could not be sampled.Under Fondecyt Grant 1210036 and with permission from the Arica archaeological museum, we sampled 69 of these individuals for chemical analysis (see Supplementary Information, Table SI1).Sixteen cases are artificially prepared bodies: one black mummy type, four red mummies, and eleven mud-coated mummies, whose treatment is mainly a thin layer of mud applied externally.Fiftythree of the bodies analyzed had natural mummification.

Age and sex estimation of the mummies
The biological sex of most of these individuals is known because their external sex organs remained intact.When soft tissue was not present, sex was estimated using standard protocols for sexual dimorphism in the pelvis and skull (Buikstra and Ubelaker 1994).Thirty-five individuals were estimated as female and 34 were male.In many cases, the breakage of the mummified material allowed for visual observation to assess the biological profiles.Age was estimated by observing the stage of dental eruption and bone growth (e.g., epiphyseal fusion stages), for sub-adults (<18-20 years), and by using standard scoring of the pubic symphysis and the auricular surface for adults (Brooks and Suchey 1990;Buikstra and Ubelaker 1994;Lovejoy et al. 1985).To facilitate analysis, we grouped the individuals into age categories (Buikstra and Ubelaker 1994): two were children (3-12 years), four were adolescents (12-20 years), 37 were young adults (20-35 years), 25 were middle adults (35-50 years), and one was an older adult (50+ years).

Archaeological sites background
The Chinchorro samples came from the Morro 1 (n = 41), Morro 1-5 (n = 1), Morro 1-6 (n = 25), and Camarones 15D (n = 2) funerary sites in northern Chile (Supplementary Information, Table SI1 and Fig. SI1).These archaic cemeteries are located on the slope of the coastal hills (about 30 masl) of Arica, in northern, Chile.The bioarchaeology of these sites has been extensively described in the literature (Allison et al. 1984;Arriaza et al. 2005;Focacci and Chacón 1989;Guillén 1992;Muñoz et al. 1991;Standen 1997;Standen and Arriaza 2016).In general, the bodies were inhumed laying on their back, wrapped in reed mats, and buried at a shallow depth (ca. 1 m).Grave goods are minimal and associated with coastal subsistence (e.g., fishhooks and harpoons), in addition to atlatl, reed baskets, and lithic points.A recent detailed depiction of Chinchorro artifacts can be found in Standen and Arriaza (2016), and many of the Arica-area Chinchorro mummies with artificial mummification are illustrated in a catalogue (Arriaza and Standen 2016).Both cultural remains and mummies are stored at MASMA.

Morro 1 (ca. 5434 ± 59 to 3670 ± 59 BP)
This is a large funerary site (latitude 18° 28′ 53.33′′ S, longitude 70° 19′ 17.47′′ W) located on the northern slope of a large rocky promontory known as Morro de Arica near the coast and was excavated in 1984 (Standen 1997).On this site, 152 individuals were found with various mortuary treatments, including black, red, bandaged, mudded, and natural mummies (Allison et al. 1984;Arriaza et al. 2005;Standen 1997;Standen and Arriaza 2016).This funerary site (latitude 19° 12′ 7.10′′ S, longitude 70° 16′ 2.14′′ W) is located about 100 km from the Morro bluff sites.This site is located at the mouth of the Camarones River area, on the south coastal border, and on a steep slope of the hill flanks.It was excavated in 1991 by Muñoz, Rivera, and collaborators (Muñoz et al. 1991;Rivera and Aufderheide 1995).Twenty-four individuals associated with the late Chinchorro period, some had clay ocher masks, and were exhumed here.

Rib bone samples
Bone samples (approximately 1-2 g) were taken from Chinchorro mummies' ribs housed at the MASMA museum to investigate endogenous Mn levels.Rib fragments of intentionally prepared bodies and natural bodies were included in the sampling and analysis.The rib samples were readily available, and their sampling caused minimal damage to the mummies.Ribs, therefore, allowed for a large sample of individuals to be studied.The bone samples were analyzed at the analytical chemistry laboratory, Department of Chemistry, University of Tarapacá, Arica, Chile.

Lab sample preparation method for bone dissolution and Mn determination
Prior to destructive chemical analyses, the mummy rib fragments were thoroughly cleaned, first using mechanical cleaning with new scalpel blades and then using distilled water to remove any adhered external residues to obtain a bulk sample that approximates the composition of living bone.The cleaned rib samples were then dried at 105°C for 6 h and crushed into bone powder.One gram of the bone powder was put in a mixture of hydrochloric and nitric acid to dissolve the hydroxyapatite matrix and to oxidize remnants of organic materials (Eastman et al. 2013;Rasmussen et al. 2019).The Mn physiologically incorporated into the bone tissue was measured using FAAS, Agilent 240FS Series AA Model 200.Data were collected using the SpectrAA software, version 5.4.Measurement system: PROMT (precision optimized measurement time).A lighter with a 10-cm-long groove was used along with an air-acetylene-oxidizer flame, with three measuring replicates in the standard and sample.The read delay time was 2 s (seconds), and the measuring delay time was 2 s, while the wavelength was 279.5 nm with a slit width of 0.2 nm.The background correction was with a deuterium-D 2 lamp, and the calibration range of the standard was 0.5 to 2.0 μg*mL −1 .The limit of quantification of 0.14 mg*L −1 of this equipment is suitable for the proposed bioarcheological work considering we are measuring significant levels of Mn exposure rather than trace level metals exposure.

Controls for diagenesis
Diagenesis, and subsequent exogenous incorporation of ecotoxic Mn mineral into bone, is reported to be minimal for the Atacama mummy samples due to near-neutral soil pH, oxic conditions, absence of rain, and low humidity (Arriaza et al. 2021;Cahiez et al. 2004;Husson 2013;Kakoulli et al. 2014;Quinn et al. 2004).Nevertheless, we minimized any contamination by cleaning the rib samples of individuals with deionized water thoroughly.Importantly, we also controlled for this by sampling distinct types of mummies (natural and artificially prepared) and by analyzing cemetery soil samples from the burial matrix to check for exogenous incorporation (see Supplementary Information, Table SI3).Of the total rib samples, a random subset of 25 cases were additionally tested for bone surface contamination.These bones were washed and sonicated for 5 min.The sonication energy permitted the extraction of any foreign material attached to the bone tissue, primarily dust particles that adhered to the bone surface.These residues become partly solubilized in the water.The sonication was done by controlling for the volume of water added and the mass of bone used.The total of this extracted solution was stored in 50-mL containers and the Mn concentration was subsequently directly measured for 2 s in all samples using a 0.63-mm (internal diameter) capillary tube and FAAS.

SEM/EDX analysis of possible Mn sources and mummy external black coating
About 1 mg of the black pigment taken from two sediments coming from fluvial gravels along the Lluta River terrace exposed face (Fig. 2) and the external black coating of seven Chinchorro mummies was analyzed using an EVO LS scanning electron microscope (SEM), in environmental mode (8.5 WD and 20 kV) to evaluate Mn quality.The images were taken using a backscattered electron detector at ×100, ×600, and ×2000 magnification.The samples were also analyzed using an Oxford EDX detector, selecting mapping, and point test analysis for Mn.The topographic qualitative information, spectrometry data, and semi-quantitative results (detection sensitivity down to 0.1% by weight) were recorded and interpreted using the INCA software.These analyses were carried out at the bioarchaeology laboratory, Instituto de Alta Investigación, University of Tarapacá, Arica, Chile.

Field method
Using 50 × 50 cm test pits, at the Morro cemetery area, 17 soil samples were collected at different depths, ranging from the surface to about 240 cm depth.Test pits were dug in a Chinchorro cemetery area, including Morro 1 and Morro 1-6 sites, under permit number 4518 of the Chilean National Council of Monuments (Supplementary Information, Fig. SI1).

Soil laboratory analysis
The seventeen archaeological cemetery soil samples were analyzed at the analytical chemistry laboratory, Department of Chemistry, University of Tarapacá, Arica, Chile, using FAAS (Supplementary Information, Table SI3).The Mn extraction was carried out on a mass of 20.00 g of dried soil with an exact volume of 50 mL of distilled water with electrical conductivity ≤ 2.5 μS/cm, in a ratio of 1:2.5 solid phase to liquid phase, using a 100-mL capacity polypropylene cup container tube with hermetic closure.The extraction was carried out in a batch system, shaking for 2 h in an orbital shaker at 180 r.p.m.Subsequently, the liquid phase was separated from the solid phase by filtration through Whatman ashless filter paper.The aqueous extract was transferred to a polypropylene container and identified according to the corresponding soil sample.The resulting solutions were available for direct measurement of the element Mn by FAAS.The Mn values obtained in mg/L were transformed into Mn mg/kg soil (ppm).The FAAS characteristics were similar to those used to measure Mn in the bone matrix, except in the calibration range of 0.5 to 3.0 μg*mL −1 and the characteristic concentration of 0.035 μg*mL −1 .

Results
It should be noted that one female case (red mummy code MO 1 T7 C1) presented an extreme concentration of Mn and was therefore considered an outlier (z score ≥ 3) and was excluded from further statistical analysis (Supplementary Information, Table SI1).Thus, bearing in mind the z scores, the whole dataset analyzed corresponds to 99.7% of the representative sample, reducing the number of acceptable cases to 68.The solution of the dissolved cleaned bone samples presented a 9 ppm mean value and 100% of the cases had evidence of Mn ≥ 0.1 ppm (Table 1 and Supplementary Information, Table SI1).However, approximately 84% (57/68) of individuals have Mn concentrations beyond normal 1 ppm, and 20.6% (14/68) were overexposed to levels of higher toxicity (≥ 10 ppm) (Table 1 and Supplementary Information, Table SI1).The mean of Mn in natural mummification cases was 7.9 ppm while that in the artificial mummification cases was 12.8 ppm (Table 2).The dataset by mummification type is not normally distributed according to Kolmogorov-Smirnov (n > 50) and Shapiro-Wilk tests (n < 50), and the mean differences of endogenous Mn between these types of mummies are not statistically significant (Mann-Whitney U, p = 0.85).
The Mn quantification of the residues from sonication presented a low average Mn concentration of 0.7 ppm (see Table 1 and Supplementary Information, Table SI1).Both datasets (sonication residues and digested bone sample solution) are not normally distributed (Kolmogorov-Smirnov, p < 0.001), but the Wilcoxon tests show that their mean values (0.7 ppm versus 9 ppm) are significantly different (p < 0.001).The same occurs when a pairwise (n = 25) comparison is undertaken between the residues (mean = 0.7 ppm) versus the digested bone (mean = 8.7 ppm).Here, the Wilcoxon test also shows there are significant differences between the digested bone and sonicated residues (p < 0.001) with minimal presence of Mn in the external residues.
Eighty-four percent (57/68) of the Chinchorro mummies with and without artificial mummification treatment have more than 1 ppm of Mn in their bone samples with a mean of 10.6 ppm (Table 3).This subset of data is not normally distributed according to the Shapiro-Wilk test.Yet again, there were no significant differences in bone Mn concentrations by mummification type (natural, n = 43 versus artificial mummification, n = 14) when considering the cases ≥ 1 ppm (Mann-Whitney U, p = 0.55).No sex differences in Mn concentrations were observed within the different mummification types.Regarding age, adolescents had a higher mean (29.1 ppm) than the other age categories (Table 4).But there is a variable mean distribution (≥ 1 ppm) by age category (Fig. 4).However, the presence of Mn according to age using the Shapiro-Wilk test showed that this age data is not normally distributed.A Kruskal-Wallis test found no significant differences between the age categories (p = 0.63).
The water-based soil Mn leaching analyses of the Chinchorro cemetery soil show extremely low concentrations at various depths (mean = 0.1 ppm), indicating little diagenetic contribution of Mn from soil at the burial sites (Supplementary Information, Table SI3).Finally, the SEM/EDX analysis of the external black coating of seven Chinchorro mummies with complex treatment revealed a high concentration of Mn with a range of 41.8 to 91.4% (point analysis, see Supplementary Information, Table SI2) and the two black sediment samples presented in Fig. 2 (Lluta River's terraces exposed face): one near the Lluta River mouth and another about 8 km from the coast showed 62.4% and 66.1% of Mn (point analysis, see Supplementary Information, Table SI2).

Discussion
The Mn quantification of the external residues resulting from sonicating the bone to remove external debris presented a low mean Mn concentration of 0.7 ppm.The data show that the bone surface has an outer layer with minimal contamination and/or that the immersion in water and sonication extracted some of the Mn present in the bone.
In both cases, the amount of Mn present in the residues is statistically lower compared with the 9 ppm (mean) obtained from the cleaned and digested bone samples (Table 1 and Supplementary Information, Table SI1), suggesting that any Mn observed in the residues or environmental contamination does not affect the Mn content in bone.In addition, the water-based Mn leaching analyses in the soil of the Chinchorro cemetery area near the coastal bluff (Morro 1 and Morro 1-6) where the mummies were excavated show extremely low concentrations at various depths (mean = 0.1 ppm) (Supplementary Information, Table SI3).In summary, the Mn in bone is 90 times (0.1/9) higher than in the soils where the mummies were found, indicating little diagenetic contribution of Mn from soil at the burial sites.In addition, these mummies are found in the highly arid coastal Atacama region (Quinn et al. 2004) where microbial activity, which usually favors diagenesis, is minimal.Furthermore, MnO 2 (pyrolusite), one of the compounds found in the coating of the Chinchorro mummies (Sepúlveda 2014(Sepúlveda , 2015)), is insoluble in water (Cahiez et al. 2004); and thus, it is not probable to transfer into inner tissues by percolation through soils.
Given that Atacama soils are oxic and pH ranges between 5.5 and 8.5 (Quinn et al. 2004) and interpreting the Pourbaix diagram (Eh versus pH) of Mn chemical species (Husson 2013) in aqueous media, it is improbable there has been migration of soluble Mn 2+ species into the inner rib bone.Thus, it is unlikely the mummy rib samples have undergone diagenetic changes.High concentrations of Mn in bone were observed in the bone not only of those bodies with artificial mummification, but also of those without mortuary treatment, 12.8 versus 7.9 ppm, respectively (Table 2) with no statistically significant differences.These results show that individuals who have been intentionally mummified could have had the same level of exposure as those naturally mummified, and if the slight differences in Mn amounts found (more concentrated in artificial mummies) were due to post-burial contact, this contamination was not significant.This is relevant because the external painting of the seven black and red mummies coating analyzed with SEM/EDX showed Mn values ranging from 41.8 to 91.4% (Supplementary Information, Table SI2), but the Chinchorro bodies were wrapped meticulously with mats made from reeds (Schoenoplectus californicus and Typha sp.), without direct contact of the bones with the cemetery soil.Additionally, other studies show that the mean range of Mn by weight (w/w) in the paint used to coat the black mummies varies between 24.0 and 35.1%.In contrast, the mean range of Mn in the thick black cap that covers the head of the red mummies (Fig. 1E) ranges from 46.0 to 60.7% (w/w) (Arriaza et al. 2008;Sepúlveda et al. 2013;Van Hoesen et al. 2019).The red mummies (Fig. 1D) have higher concentrations of Mn in circumscribed areas (face and head, Fig. 1E and F), while the black mummies (Fig. 1C) have a greater surface area to cover.Moreover, our SEM/EDX analysis of the two Lluta River possible Mn sources reaches 62.4 to 66.1% purity (see Fig. 2A, B; and Supplementary Information, Table SI2).In summary, our data show that (a) they used processes to purify the Mn to obtain a highgrade product, (b) they exposed themselves to Mn poisoning, (c) the Chinchorro cemetery soils did not contribute to Mn concentration readings of the bones, (d) the high Mn concentration used to coat the mummies did not permeate into the cemetery soil, and (e) the endogenous Mn found in natural and artificially prepared mummies comes from Mn mining and other social activities that involved intensive handling of this mineral.Thus, at different Chinchorro cultural periods, the risk of Mn overexposure to ancient morticians and those who supplied or managed the raw material was likely always present.In brief, our data suggest that the high amount of Mn quantified in bones is not a consequence of post-depositional percolation processes through the bone from Mn used in mummification rites.Rather, Mn overexposure occurred during the individuals' lives, independently of whether they received a complex mummification treatment or not.

Health aspects
Manganese is an essential element for bodily function, with a healthy body requiring only a few milligrams (ATSDR 2012;Lucchini et al. 2015;Mena et al. 1967;O'Neal and Zheng 2015;Ramírez and Azcona-Cruz 2017;Riojas-Rodríguez et al. 2010).Any portion of the ingested Mn not absorbed by the body is excreted through feces (ATSDR 2012; Lucchini et al. 2015;Malecki et al. 1996;Mena et al. 1967;O'Neal and Zheng 2015;Ramírez and Azcona-Cruz 2017;Riojas-Rodríguez et al. 2010).Modern autopsies reveal about 40% of endogenous Mn in the body is present in the bones, making them an excellent biomarker of ancient Mn poisoning (Chen et al. 2018;O'Neal 2015;O'Neal and Zheng 2015).However, experimental models show that rats continuously fed orally with Mn (50 mg Mn/kg/day) do not accumulate Mn as a long-term biomarker (Conley et al. 2022) and high bone Mn levels may be a useful biomarker of recent oral Mn exposure.In addition, Conley et al. (2022) reported on 49 adult individuals (41-95 years) who underwent hip replacement (bone femur sample) and lived near an industrial area of ferromanganese production on the Province of Brescia (Italy).They found bone Mn levels (range, 0.014 to 0.170 μg/g) show no differences between sex and parity history, and that Mn tended to decrease with age.Notwithstanding this work, mining evidence showed that drilling in shafts, and thus direct respiratory exposure to Mn dust, is more harmful than digestive intake, resulting in a higher endogenous Mn concentration (Rodier 1955;Schüler et al. 1957).
Thus, considering the 9 μg/g (ppm) mean Mn concentration obtained in the dissolved bone samples, the low Mn contents obtained from the control samples (residues and burial soils), and high quality of the Mn used to decorate the bodies, Chinchorro Mn overexposure is likely to have occurred.This poisoning was most likely the consequence of Mn mining or other exposure and inhalation of fine Mn dust in the last years of the individuals' life.
In more complex societies, the acquisition and uses of chromophores can expose the population to severe environmental stress and health complications as part of their economic and cultural development (Álvarez-Fernández et al. 2020;Arriaza and Galaz-Mandakovic 2022;Cooke et al. 2009).Thus, the types of mineral pigments used, their toxicity levels, and health consequences may be highly relevant to the study of MHG techno-economic and socio-cultural development.It is likely that the cultural innovation of complex mortuary treatment using Mn processing had a downside for the Chinchorro.Overexposure to Mn dust may cause manganism, characterized by severe motor and neurological problems, affecting posture, walking, speech, and facial expressions (Arriaza and Galaz-Mandakovic 2022;ATSDR 2012;Lucchini et al. 2015;Malecki et al. 1996;Mena et al. 1967;O'Neal and Zheng 2015;Ramírez and Azcona-Cruz 2017;Riojas-Rodríguez et al. 2010).
Chinchorro adolescents appear to have a higher exposure to Mn (with a mean of 29.1 ppm) than other age categories (Table 4).This suggests that adolescents could have higher exposure in recent years or that the older individuals are not as exposed (Fig. 4).Modern miners are poisoned by inhaling Mn dust particles deposited in their upper respiratory tract (Rodier 1955;Schüler et al. 1957).Thus, Mn health hazards will depend on the size of the particles absorbed, entering as either respirable dust or particulate matter (PM 10 and PM 2.5 ), respectively.The direct respiratory exposure via inhalation to Mn dust is more pathogenic than gastrointestinal or oral intake (Rodier 1955;Schüler et al. 1957).As such, Mn toxicity is related to mode of extraction, particle size (smaller more toxic), overexposure time, Mn concentration, and route of exposure (for example, the braunite appears to be more toxic Mn than other forms) (Rodier 1955).In the Chinchorro, cryptomelane appears to be more common (Arriaza et al. 2023a,b;Sepúlveda et al. 2014Sepúlveda et al. , 2015)).Thus, the processes of extraction, purification, grinding, and application of manganese-based black pigments could increasingly expose the population at different ages.Also, it may indicate that older individuals no longer fulfill these tasks in the same capacity, or they eliminated more of the Mn intake.However, the presence of Mn according to age determined no significant differences between the age categories.Also, no sex differences in Mn concentrations were observed within the different mummification types.Thus, exposure to Mn was ubiquitous in the population studied, likely an environmental contaminant due to the heavy use of Mn black pigments.
We know from historical and current clinical literature that continuous mineral mining was potentially harmful to humans.Among the most intrinsically toxic metallic elements are the heavy metals Pb and Hg, and the semimetal As, with mining being one of the main sources of contamination (Ferrer 2003;Londoño-Dranco et al. 2016;López-Bravo et al. 2016).In historic NW Iberian populations, quantification of bones with ICP-MS and MC-ICP-MS for lead and lead isotopes, and for mercury concentrations determined with a Milestone DMA-80 analyzer, shows that Roman populations have greater exposure than post-Roman populations, while the form of contamination was probably an atmospheric source (Álvarez-Fernández et al. 2020).Likewise, no other significant correlations were found in gender, age, etc. (López -Costas et al. 2020).Mercury contamination from mining and the use of cinnabar has a history in China, Japan, Europe, and the Americas; it is detected in populations from the Late Neolithic to Antiquity in Portugal and Spain (Álvarez-Fernández et al. 2020;Emslie et al. 2015Emslie et al. , 2022)).
Today, Mn mining in shafts produces Mn-related mental illnesses.Affected workers develop multiple health 188 Page 10 of 15 problems including a psychiatric syndrome characterized by compulsive behavior, irritability, aggressiveness, emotional lability, and hallucinations (Barceloux 1999;Finkelstein et al. 2008;Lucchini et al. 2015;Mena et al. 1967;Rodier 1955).Manganese overexposure also causes psychomotor problems and a Parkinson-like condition in miners.Involuntary movements, facial spasms, pathological laughter, and dystonia are present among other conditions (ATSDR 2012;Lucchini et al. 2015;O'Neal and Zheng 2015;Rodier 1955;Schüler et al. 1957;Solís-Vivanco et al. 2009).Additionally, a solid testimony of manganeserelated health problem is the dramatic case of miners from Morocco (Rodier 1955) and Andacollo in northern Chile (Schüler et al. 1957) who suffered terrible consequences of Mn overexposure.Thus, the use of minerals in antiquity, and Mn in particular, cannot be detached from their concealed health hazards.Selection and accumulation of minerals, cultural practices of mineral acquisition (enclosed or open cast mining), the potential health hazard, which varies according to the element (mercury > lead > manganese > copper (for example)), the size of the particulate matter, and inhalation during mineral processing need to be considered.The time of exposition, toxicity, forms of transportation, and preparation of the minerals may lead to incorporation into the human system, including by food contamination as reported for other social and mortuary rituals employing cinnabar for example (Ávila et al. 2014).Consequently, continued overexposure to mineral dust or tiny particulate matter can produce acute and chronic mineral poisoning (Arriaza and Galaz-Mandakovic 2022;ATSDR 2012;Lucchini et al. 2015;Malecki et al. 1996;Mena et al. 1967;O'Neal and Zheng 2015;Ramírez and Azcona-Cruz 2017;Riojas-Rodríguez et al. 2010;Tchernitchin et al. 2015).Thus, our data allow us to hypothesize that a segment of the Chinchorro population may have suffered from manganism.

Chinchorro Mn overexposure
A significant segment of the Chinchorro population has evidence for the bioaccumulation of Mn, suggesting people of both sexes and different ages participated in the manipulation and overexposure of Mn.We posit that Mn mining, handling, inhaling particulate matter (Fig. 5), and processing for the purpose of Chinchorro mortuary preparation, or perhaps intentional body painting of the living during final rituals, were significant pathways to overexposure and affected the entire community.Mining of minerals requires stockpiling of the material and in antiquity the Mn collected was likely kept around settlements.Hypothetically, the entire community at different periods (peak and decline of mummification practices) could have participated in grinding and handling of the mineral.This is particularly likely, considering that in the Arica lowlands high-grade Mn ores are abundant along the Lluta River, a few kilometers from the main Chinchorro sites (Fig. 1A and B; Fig. 2A and B).Many of these minerals are extracted from fluvially transported clasts derived from the Lauca and Huaylas Formations (Van Hoesen et al. 2019), about 80-90 km from the coast.As such, high-purity Mn could have been easily collected by the group along the riverbanks with subsequent grinding, sifting, refining, and application to the mummies conducted at nearby camps (Fig. 3).All ages are affected by Mn overexposure, suggesting that all age groups may have had an important role as collectors and/or handlers.Scholars have reported that children are more susceptible to Mn toxicity as they accumulate higher Mn concentration levels and excrete less Mn than adults (Lucchini et al. 2015).Modern children living near stockpiles or mining areas or playing with contaminated soils are at higher risk of exposure (Riojas-Rodríguez et al. 2010).In addition, Mn is incorporated into breast milk (Lucchini et al. 2015), and its concentration in the water varies regionally (Tchernitchin et al. 2015).However, there is no record in this region of modern poisoning due to ingestion of Mn-contaminated water (Tchernitchin et al. 2015).
Chinchorro mining artifacts and grave goods that could help discern who was directly engaged in Mn mining are scarce.However, it is worth noting that several Chinchorro artifacts (e.g., grinding stones, pestle, and containers) show Mn residues, associated with mineral processing.Portable X-ray florescence (XRF) analyses reveal that these artifacts processed minerals that contained 1 to 63.7% (w/w) Mn (Arriaza et al. 2023a).Moreover, the Chinchorro have an abundance of finely ground and high-grade Mn resources available near their settlements that were easy to mine.Thus, in addition to Mn applied to the black and red mummies, the Chinchorro artifacts with high Mn concentration and the abundance of local Mn sources (Arriaza et al. 2023b) Fig. 5 Analysis of archaeological Mn pigments using SEM/EDX.These Mn residues came from the head of a Chinchorro mummy, case PLM8T4, similar to those shown in Fig. 1D and E. The image shows large and small Mn particles.Magnification is ×2000.The white arrow highlights a Mn particle 3.3 μm in diameter, containing 21.7% concentration of Mn Page 11 of 15 188 add further support for potential poisoning hazard for these ancient coastal people.Consequently, it will be unlikely that a population using large amounts of high-grade Mn for long periods would not present evidence of endogenous or premortem Mn.In addition to psychomotor problems, overexposure to Mn can also cause emotional lability (Barceloux 1999;Lucchini et al. 2015), irritability, aggressiveness, pathological laughter, and fixed facial expression referred to as wooden face (ATSDR 2012;Lucchini et al. 2015;O'Neal and Zheng 2015;Solís-Vivanco et al. 2009).Many manganism psychomotor symptoms cannot be assessed in the mummies, but the high concentrations of Mn found on artifacts and now in the bone tissue force us to rethink the risk of ancient mineral mining and Mn overexposure in the Andes and elsewhere.We suspect that the vivid fixed facial expression of the artificially prepared Chinchorro mummies (Fig. 1D and F), with a gaping mouth, may be a representation of the consequence of the physical symptoms of manganism, a proposition that will need further testing.Chinchorro overexposure to Mn was dangerous and significant.This is particularly noteworthy considering these populations exploited and used high-purity Mn for multiple generations and their sites represent a continuous chronological sequence of at least two millennia distributed along the Atacama Desert coast.
Even though we posit that Mn mining was a collective effort, the fact that about 21% of the individuals show endogenous Mn above 10 ppm (mean = 29.01;median = 21.20,DE =16.72, n = 14) hints to labor specialization.A couple of possible scenarios could explain the high values of Mn in their bodies.These individuals may have been leading the exploitation, collection, refinement, and handling of the Mn, or even painting themselves with this pigment.Perhaps adolescents and young adults were involved at this stage.The development of the complex Chinchorro mummies certainly required specialization.Some of the population would have taken an active role during the mummification process exposing themselves to Mn dust during the filling, painting, and/or retouching of the mummies with Mn.For example, some mummies (e.g., case MO1T25C3) have grave goods with evidence of Mn residues in two Choromytilus chorus valves and in an unhafted hand hammer stone (chopper) presumably used for Mn grinding or refining (see Supplementary Information, Table SI4, Arriaza et al. 2023a, Standen andArriaza 2016).Most likely middle-aged and older individuals could have been involved in the complex mummy preparation process.
Considering those individuals with high endogenous Mn values (14/68), males (n = 8) and females (n = 6) may have been equally involved in the many tasks pertaining to Mn acquisition and handling, which indicates an active role by females and likely in an equal basis participation to males in Chinchorro society.Certainly, Chinchorro females were buried with artifacts related to hunting activities and other grave goods that attested to their importance in their community (Standen 1991, Standen andArriaza 2016).

Socio-economic, ideological, and behavioral implications of Mn use in the Chinchorro people
The collection of Mn pigments was an important economic and social activity for the Chinchorro.Notwithstanding the negative health effects of Mn, most likely unknown to the Chinchorro, an ideological mortuary tradition linked to Mn may help explain the endurance of its use.Colors represent relationships and experiences and have social significance (Gibson et al. 2017;Sahlins 1976;Turner 1970).Although we do not know the meaning of the colors for the Chinchorro people, black and red are colors that tend to be universally used (Sahlins 1976;Turner 1970).For example, the former often symbolizes eternity, sadness, or the end of a cycle, and the latter, vitality and life itself (Turner 1970).The use of these colors in Chinchorro children, and adults of both sexes, speaks of its centrality to the group.The Chinchorro searched for and refined the black Mn (and red ocher) most likely as part of their daily subsistence, which has been hypothesized to be linked to notions of their expected eternal life (Arriaza 1995).The use of the Mn pigments in Chinchorro populations for thousands of years meant a deepquested cultural and symbolic ideology.
The economic aspects of Mn acquisition and its artistic palette on the Chinchorro bodies provide a useful lens into cultural evolution of early MHG and their social complexity.Yet, these cultural practices and processes ultimately left a record in the bones of the Chinchorro people.The high concentration of Mn in non-artificially mummified Chinchorro individuals suggests that there are probably other social practices beyond the application on the mummies that need further studies (e.g., mining and processing).Considering that the finished Mn-coated mummies were kept nearby their camps and that there is accumulating evidence that the mummies formed part of the social milieu of the living including practices of visiting and retouching them (Arriaza 1995;Sepúlveda et al. 2015), then hypothetically these effigies of the dead could became a source of Mn contamination, affecting the living.It is likely that touching and repairing the mummies was a lower health risk compared with the direct exposure of inhaling Mn mining dust particles during its sifting and refining.Mn residues around the camp could also present a potential risk, especially to children playing with contaminated soils.
Working with minerals is a dangerous affair for miners and artists, among others.Throughout the centuries, many artists were unknowingly exposed to lead and other heavy metal-based pigments when preparing and mixing their color for their paintings.It has been suggested that well-known 188 Page 12 of 15 artists like Goya suffered from Saturnism or lead poisoning.Saturn was a Roman God, and centuries ago, the common belief was that the Saturn planet irradiated melancholy or that some people were under its influence.Lead poisoning produces many health problems including melancholy, colic, and attacks on the central nervous system.Many artists working with lead saw a significant decline in their health (Cipriani et al. 2018;Montes-Santiago 2013).However, artists continued to work with toxic pigments as they did not know the exact cause of their maladies.An example of Mn poisoning from mining is from Corral Quemado (Chile).Here, they suffered terrible consequences of manganese "madness" and were aware that their work made them sick, including mood changes, and speech, behavioral, and psychomotor problems.However, due to economic reasons, many continued to risk their health and went on working under terrible conditions (Arriaza and Galaz-Mandakovic 2022).We may wonder what the Chinchorro thought of the people affected by Mn poisoning.Perhaps, they may have associated their health problem with evil spirits possession, witchcraft, or some supernatural forces of the environment.Even today, we carry our own cargo of disease nomenclature based on ancient beliefs.In addition to saturnism, for example, the term "influenza" is derived from an epidemic attributed to "influence of the stars in 15 th century Italy," and the term malaria (meaning bad air) is derived from the belief that the inhalation of foul air of marshy areas caused sickness.
For the Chinchorro, it was likely that during the initial stage of Mn use they were unaware of its harmfulness, but perhaps with time they realized its detrimental effects.Despite the endurance of these mortuary practices for millennia, Mn overexposure can hypothetically be linked to their ultimate decline as well, in terms of both mortuary ideology and in their bodies in life through recurrent exposure causing behavioral and neurological problems.It was a serious occupational hazard for these archaic populations, affecting a significant percentage of individuals, putting them at risk of mineral poisoning.Our findings show that the Chinchorro had a broader subsistence spectrum than originally understood, where mining and mineral collecting were paramount to their socio-economic and world view.These findings open new avenues of research to study the long history of mineral use and anthropogenic pollution in the Americas (Cooke et al. 2009) and to reconsider the Chinchorro as having a broader subsistence spectrum of hunters, gatherers, fishers, and mineral collectors.
There is evidence for a long mining tradition for the Chinchorro.Like Mn, the use of hematite (Fe 2 O 3 ) likely also played an important symbolic meaning in the daily life of early maritime communities.Hematite mining activities occurred very early in South America (ca.12,000 BP) as reported for the San Ramon site in Taltal, Chile (Castelleti et al. 2015;Castro et al. 2012;Salazar et al. 2010Salazar et al. , 2011Salazar et al. , 2015;;Vaughn et al. 2005Vaughn et al. , 2007Vaughn et al. , 2013)), and were recurrent in the Chinchorro culture including being used in mortuary treatment and rock art (Arriaza et al. 2005;Ogalde et al. 2015;Sepúlveda et al. 2014).This red mineral pigment was also extensively exploited by the maritime communities in the Pacific coast of Peru, around 3200-3030 BP (Prieto et al. 2016).

Final comments
The archaeological and archaeometric analyses show the Chinchorro used Mn for thousands of years, which they refined to create intense black pigments and was a communal and recurrent effort.Our analysis of the bones from 68 Chinchorro mummies reveals, for the first time, the presence of Mn in their bodies.About one out of five of the Chinchorro had higher Mn poisoning, hinting to specialization or more direct involvement in Mn handling by some individuals.Young individuals, and adult males and females played an active role in the social spheres and chaîne opératoire of Mn production and usages.The continuous exposure of these populations to Mn occurred at different moments and places affecting children, adolescents, and adults.First, overexposure during the mining in Mn-rich areas (e.g., LLuta Valley, see Arriaza et al. 2023b), involving extraction, refinement, and bringing the desired mineral to their camp.Second, contamination during the artistic or pictorial moment comprising the application of Mn to the bodies under preparation.Here, morticians may have been exposed to Mn dust and residues when applying the highly refined black pigment to their mortuary tool kit and to the bodies to produce a final colorful mummy.Third, contamination may have occurred during daily life activities.Manganese residues left around camp, dirty tool kits with Mn, or bodies in the process of preparation, or cracking of artificially prepared bodies that need retouching could all potentially have contaminated their environment.Last, but not least, Mn overexposure during post body preparation ritualistic events.This stage involved mummy revisiting, retouching, feasting with the dead, mummy display, and handling, potentially exposing many individuals to Mn dust.
The Chinchorro contamination was due to Mn dust particles entering the body via the respiratory tract, which is more dangerous than other means of access into the body.The high-grade Mn pursued in their ornamentation of the dead put the Chinchorro unknowingly at continual health risks of this silent anthropogenic contamination.The Chinchorro individuals with higher concentrations of Mn in their bones may have suffered from some degree of manganism in the last years of their life.This needs to be investigated in future studies using brain tissue if available.

Fig. 1
Fig. 1 Main areas of the Chinchorro culture under study and characteristics of the mummies.The circular magenta areas A and B show the main areas with high Mn concentration in northern Chile along the Lluta and Camarones Rivers (Baeza et al. 2014; García et al.

Fig. 3
Fig. 3 Artistic representation of Chinchorro people painting a corpse with Mn

Fig. 4
Fig. 4 Manganese concentration (ppm) in ancient Andean mummy ribs by age group.Boxes indicate 25% and 75% quartiles with a median line; whiskers denote the upper and lower ranges of Mn concentrations.The square in the box depicts the mean Mn concentration and crosses indicate outliers.The color-filled dots are data points fitted to a normal distribution

Table 2
Summary of the descriptive statistics of all valid cases (n = 68), and excluding one outlier