Distribution and Chemical Speciation of Arsenic in Ancient HumanHair Using Synchrotron RadiationIoanna Kakoulli,*,†,‡ Sergey V. Prikhodko,† Christian Fischer,†,‡ Marianne Cilluffo,§ Mauricio Uribe,∥

Hans A. Bechtel,⊥ Sirine C. Fakra,⊥ and Matthew A. Marcus⊥

†Materials Science and Engineering Department, University of California Los Angeles, PO Box 951595, Engineering V, Los Angeles,CA 90095-1595, United States‡Cotsen Institute of Archaeology, University of California Los Angeles, A210 Fowler Building, Los Angeles, CA 90095-1510, UnitedStates§Department of Integrative Biology and Physiology, 1031 Terasaki Life Sciences Building, PO Box 957239, University of CaliforniaLos Angeles, CA 90095-7230, United States∥Facultad de Ciencias Sociales de la Universidad de Chile, Av. Capitan Ignacio Carrera Pinto N°1045, Nunoa, Santiago de Chile,Chile⊥Advanced Light Source, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS 6-2100, Berkeley, CA 94720-8226, UnitedStates

*S Supporting Information

ABSTRACT: Pre-Columbian populations that inhabited theTarapaca mid river valley in the Atacama Desert in Chileduring the Middle Horizon and Late Intermediate Period (AD500−1450) show patterns of chronic poisoning due toexposure to geogenic arsenic. Exposure of these people toarsenic was assessed using synchrotron-based elemental X-rayfluorescence mapping, X-ray absorption spectroscopy, X-raydiffraction and Fourier transform infrared spectromicroscopymeasurements on ancient human hair. These combinedtechniques of high sensitivity and specificity enabled thediscrimination between endogenous and exogenous processesthat has been an analytical challenge for archeological studiesand criminal investigations in which hair is used as a proxy ofpremortem metabolism. The high concentration of arsenic mainly in the form of inorganic As(III) and As(V) detected in the hairsuggests chronic arsenicism through ingestion of As-polluted water rather than external contamination by the deposition of heavymetals due to metallophilic soil microbes or diffusion of arsenic from the soil. A decrease in arsenic concentration from theproximal to the distal end of the hair shaft analyzed may indicate a change in the diet due to mobility, though chemical ormicrobiologically induced processes during burial cannot be entirely ruled out.

Human hair is a widely accepted biomonitor intoxicology.1,2 The chemical and physical attributes that

provide hair with its robust structure facilitate its survival inadverse environmental conditions, while its biological for-mation and constant growth-rate (roughly 1 cm per month)make it useful as a biosensor of recent life history.3 Bone andother tissues change throughout life, generating an averagebiogenic response over the remodeling timeframe. In contrast,once hair is keratinized, it no longer remodels and thereforeoffers superior chronological resolution.3 In chronic exposureto toxic environments, hair analysis can therefore provide alongitudinal time-resolved signal.4 Furthermore, arsenic andother toxic elements can be transported through the blood-stream to the hair fiber. The α-keratin in hair contains abundantthiol groups in its cysteine residues, which readily react witharsenic. Thus, the concentration of As in hair is much higher

than in other human biological tissues.5,6 Consequently, owingto its intrinsic properties, hair provides an excellent bioresourcein forensics and biological archeology.Previous studies on hair analysis combined with paleopatho-

logical investigations of ancient pre-Columbian populations innorthern Chile, from the Chinchorro culture to the Incas,7−11

have indicated high levels of arsenic in both highland andcoastal people, most likely as a result of long exposure togeogenic toxic contaminants through volcanism and weatheringof arsenic-containing geological formations.5,8−15 However,discriminating endogenous arsenic in hair (by ingestion) fromexternal contamination has not always been possible and

Received: August 8, 2013Accepted: December 10, 2013

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remains an analytical challenge.5,16 This is particularly true forarcheological and historic hair that undergoes diageneticalterations during burial17 or subsequent conservation treat-ments involving the application of heavy metals aspesticides.4,16,18 Combined analytical methods enabling thedetection, spatial distribution, and speciation of arsenic on thesame hair while requiring minimal sample preparation, offer aholistic approach for assessing As-exposure and discriminatingbetween endogenous and exogenous processes.4

■ EXPERIMENTAL SECTIONHere we report synchrotron-based X-ray and Fourier transforminfrared spectromicroscopy, field emission gun (FEG) variablepressure scanning electron microscopy (VPSEM), and energydispersive X-ray spectroscopy (EDS) measurements, testing thehypothesis of arsenicism (chronic arsenic poisoning) amongancient populations in northern Chile.4,8−10,12,16 μXRFmapping was first performed to obtain elemental distributionmaps on the samples. Specific spots of interest were thenselected on which to perform μXANES for chemical speciationand μXRD for identification of minerals. Complementaryanalyses on hair transverse thin sections (5 μm) wereperformed using synchrotron-based Fourier transform infrared(SR-μFTIR) spectroscopy. By combining the synchrotrontechniques with FEGVPSEM-EDS, in situ submicrometermorphological characterization and topographic mappingwere achieved with simultaneous spatially resolved elementalidentification of the major and minor elements at the surfaceand subsurface of the hair, allowing accurate correlations withthe synchrotron-based analytical data. Owing to the variablepressure capabilities of the FEGVPSEM, these insulatingsamples were analyzed without the need of any coating whilemaintaining the physical and chemical integrity and attributesof the specimens. We analyzed scalp hair from a 1000 to 1500-year-old mummy (Figure 1A) recovered from TR40-A, a pre-

Columbian cemetery in the Tarapaca Valley in 2005 and keptin an archival-quality storage facility. To characterize arsenicand other trace elements on the surface and inside the hair,μXRF elemental distribution maps were obtained on large areas(several mm) from five longitudinal segments along the entirehair length, approximately 25 cm from the root (proximal end)to the tip (distal end) (Figure 1B). For each area mapped, the

relative transverse profile distribution of arsenic was deduced bycomparison to a theoretical model, taking into considerationthe cylindrical geometry of the hair (see section 3.2 of theSupporting Information). The arsenic oxidation state wasdetermined using As K-edge μXANES, while μXRD measure-ments combined with VPSEM-EDS phase maps were used toidentify mineralogical phases associated with the burial soil.Complementary analyses on hair transverse thin sections (5μm) to assess the degree of preservation of the hair wereperformed using μFTIR spectromicroscopy.The analytical protocol followed in this study has several

advantages over other conventional methods employed forarsenic-exposure studies, particularly for archeological andhistorical hair samples.9,10,18,19 Since the use of synchrotron-based μFTIR, μXRF, and μXANES does not require elaboratesample preparation or extraction methods as in the case ofchromatographic techniques,5 the risk of chemical changes20

during analysis within the sample is minimized when carefullymonitored. In addition, the variable pressure capabilities of theFEGVPSEM enable the analysis of these insulating sampleswithout the need of any conductive coating maintaining theirphysicochemical integrity. This further allows the reuse of thesamples for subsequent analyses. In this research, thecombination of these virtually nondestructive techniquesallowed morphological and elemental spatially resolvedcharacterization and chemical speciation in the same regionsof the hair.

■ RESULTS AND DISCUSSION

Micromorphological characterization of the untreated hairsegments using VPSEM indicated at first that the hair waswell-preserved with evidence of well-maintained nits andunderwent little taphonomic change (Figure 2).SR-FTIR spectromicroscopy on 5 μm transverse sections

(Figure 3), however, showed partial delipidization of theinternal structure primarily affecting the lipids in the cortex,most likely as a result of postdepositional processes andmicrobial activity.21,22 The degradation of the lipids backbonein the cortex was evidenced by the decrease in the ratio

Figure 1. (A) Mummy bundle (Locus 9) from the TR40-A cemeteryin the Tarapaca Valley in northern Chile. (B) Five hair segments fromthis mummy (∼4−5 mm each), cut along the entire length of a hairshaft at 5 cm intervals from the root (proximal end) to the tip (distalend). Photo of mummy 2008, Tarapaca Valley Archaeological Project,Ioanna Kakoulli.

Figure 2. FEGVPSEM micrographs showing the intact preservation ofthe external morphology of the hair, clearly showing the imbricatescale pattern of (A and B) the cuticle and (C) a club-shaped root(proximal end). Lice infested this mummy and (D) a well-preservednit is visible on this hair strand.

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between the asymmetric CH3 and CH2 stretch at ∼2960 cm−1

and ∼2930 cm−1 respectively. This ratio is similar among allhair segments (Figure 3C) and therefore excludes preferentialoxidation along the length of the hair. This is further supportedby evidence of consistent oxidative damage23 at the proximal,middle, and distal end with the appearance of an absorptionband at 1040 cm−1, characteristic of the formation of cysteicacid (Figure 3C).Speciation analysis of sulfur on the hair segments using S K-

edge μXANES revealed mostly the presence of organic sulfurassociated with structural proteins as well as inorganic sulfur inthe form of sulfides and sulfates. EDS measurements on theunwashed hair cuticle showed the presence of S, K, Ca, Cl, Fe,Si, and Na, while μXRF also identified traces of Zn, As, and Ti.Soil data using a portable XRF (above Z = 13, due to thedetection limit of pXRF) compared to the major and minorelements identified with the EDS. In addition, traces of arsenic(∼30 ppm) untraceable by EDS were also detected.Additional measurements using EDS elemental maps

combined with μXRF, μXANES, and μXRD data allowed theidentification and mapping of particular salts and mineralsattached to the hair cuticle, such as halite, pyrite, pyrrhotite, andquartz. These results are consistent with XRF and XRD data ofthe burial enveloping soil, pointing to a relatively immaturesandy soil consisting of quartz, feldspars, and rock fragmentswith minor contributions from clay and iron-rich minerals. Thesalts identified in the soil are responsible for the relatively goodpreservation of human hair and human tissues and are oftenfound in association with caliche, a characteristic deposit of theAtacama Desert that reflects its hyperaridity and the influenceof a dense coastal fog known as camanchaca.24

μXRF mapping of arsenic at an incident energy below the PbL3-edge showed As in all five hair segments analyzed (Figure 4),with at least 150 ppm of total arsenic concentration in the hairmatrix (see section 3.1 of the Supporting Information).This concentration is 2 orders of magnitude greater than

normal human levels in unexposed individuals estimated to lessthan 1 ppm in hair,5,25 which was also confirmed by theundetectable arsenic in the control sample (of modern hairfrom an individual who has never been exposed to arseniccontaminated environments) (Figure 5).

Although the qualitative arsenic distribution appears highercloser to the center in all hair segments (Figure 4, B, F, J, N,and R), the distribution is consistent with a uniformconcentration because the maximum thickness is at themidpoint of the cylindrically shaped hair. A lineout for arsenicprofile in hair segment 3 (Figure 6) shows a good fit to thesimple model of a uniform cylinder. Zn on the other hand(Figure 4D, H, L, P, and T and Figure 6) (see section 3.2 of theSupporting Information), an element that is mainly associatedwith diet and the third most abundant element in the hair of

Figure 3. Transverse section of hair from (A) the middle of the shaftand identification and spatial distribution of the absorption band at∼1660 cm−1 (Amide I), characteristic of (B) proteins. (C) FT-IRspectra from the proximal (blue), middle (green), and distal (red) endof the hair. All three spectra show absorption at ∼1040 cm−1

corresponding to the formation of cysteic acid as a result of oxidativedamage to the hair. The spectra also show vibrational bands for amideI and II and −CH3 and −CH2 bands, characteristic of proteins andlipids, respectively.

Figure 4. FEGVPSEM micrographs (A, E, I, M, Q) showing theexternal morphology of the hair and soil debris on five segments 1 to 5from top to bottom (see also Figure1B). Corresponding μXRF mapsof arsenic (B, F, J, N, and R), sulfur (C, G, K, O, and S), and zinc (D,H, L, P, and T) on the same hair segment locations (see theSupporting Information). A uniform radial distribution of arsenic,rather than coating the surface can be observed. Intensity scale barsshow XRF counts in arbitrary units. Scale bars: 50 μm.

Figure 5. Average as K-edge μXANES spectra of (a) mummy hairsegment 3 compared to (b) modern hair showing that modern hair hasno detectable As. (c) Signal from the modern hair with a cubicpolynomial subtracted off and multiplied by 10, to demonstrate thelack of an edge jump. All spectra are displayed on the same verticalscale.

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the mummy after S (Figure 4C, G, K, O, and S) and As(external contaminant) (Figure 4B, F, J, N, and R and Figure6], shows a very different profile with uneven distribution andhigher concentration in the outer part of the hair.Arsenic K-edge μXANES revealed the presence of trivalent

and pentavalent arsenic (Figure 7A). Arsenic species, bothAs(III) and As(V), were largely in the form of inorganic arsenicand to a lesser extent as organo-arsenic compounds. Thepredominant species identified in the hair samples was As(III)bound to S, either as As2S3 or AsCysteine with 27 ± 5%As(V)O4

3−, although inorganic arsenic in surface and ground-water sources is mainly composed of arsenate (As(V)O4

3‑).5

These findings are closely correlated with data from publishedarsenic speciation analyses in human hair from modernepidemiological studies in the Atacama Desert.5 In consid-eration that the chronic arsenic poisoning is through ingestionof arsenic-contaminated drinking water, the higher amounts ofAs(III) species detected in the hair may be associated with theinclusion processes (biotransformation) of arsenic in the hairand the bioavailability of arsenic in the water.5,26 However, timeof exposure and age of the individuals as well as additionalquantities of As(III) contained in vegetables and other edibleplants consumed may have contributed to the assimilation of ahigher arsenite level.5

Further comparison with studies on modern populations inrural areas of the Atacama Desert suggest that the presence ofAs(III) in the hair can be regarded as a good indicator forchronic arsenic exposure through the consumption ofcontaminated superficial and groundwater.5,11 The AtacamaDesert and the surrounding region of northern Chile is an

arsenic-rich geo-environment. We considered that the arseniccontained in tertiary-quaternary sediments, soils, and arsenic-containing metallic sulfides in the Andean highlands is leachedout through geochemical processes and transported intoregional rivers in the mid valleys of the Atacama Desert usedas a drinking water source and to irrigate edible plants. Eventoday, while arsenic is removed from the water supply of majorcities in Chile, rural areas in the Atacama Desert are stillexposed to arsenic polluted drinking water sources.5,11,27,28 Theconsumption of arsenic-contaminated water is the mostcommon and major cause of arsenicism.5 Despite the overalllimited bioavailability of arsenic through drinking water, thechronic consumption can cause severe health effects to theexposed population, often accompanied by skin manifestationssuch as those observed on some of the mummies investigatedhere (Figure S1 of the Supporting Information).1,15,27 Thedistribution and chemical speciation of arsenic along the hairshaft further supports biogenic incorporation, induced mainlyby ingestion of arsenic-contaminated water.8−12,14,15,29

Quantitative measurements of the arsenic concentrationwithin the different hair segments have further indicated adecrease of arsenic concentration toward the distal end (Figure7B). This may indicate (1) depletion of arsenic due to chemical

Figure 6. X-ray fluorescence emission signal for (A) transverse profilesof As (upper) and Zn (lower) in hair segment 3 and (B) microfocusedX-ray fluorescence maps of As and Zn from which the lineouts in (A)were extracted. In (A) the upper curve, the dashed line with symbolsrepresents the data while the solid line is the fit to eq 2 (see section 3.2of the Supporting Information). The lower curve represents the datafor Zn along the same transect as for As. In (B), the arrows show thetransect plotted, and the width of the rectangles show the transversewidth over which pixel values were averaged in order to reduce noise.

Figure 7. (A) Average As K-edge μXANES spectra collected fromvarious spots on 5 hair segments along the entire hair shaft from theproximal (seg. 1) to the distal end (seg. 5). As (III) and As (V) arepresent in all hair segments analyzed. (B) Average arsenic counts oneach segment, showing an almost 4× decrease in arsenic content in thedistal end (tip) of the hair.

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and/or microbiological processes both in life and post-mortem30 or (2) a change in the diet due to mobility.10,31

■ CONCLUSIONSThe characterization of the longitudinal and transverse profilesof arsenic in the hair and its oxidation state was essential toidentify the origin and toxicity of this poisonous metalloid,reflecting a retrospective time span along the hair growth.4,21

These data helped to discriminate from exogenous contami-nation induced by metallophillic bacteria as well as environ-mental heavy metal pollutant diffusion mechanisms based onphysical contact during burial, as (1) the arsenic concentrationin the burial enveloping soil does not exceed 30 ppm, aconcentration consistent with uncontaminated sandy soils32

and (2) the mummy investigated was wrapped in a bundle andnot in direct contact with soil. Our data support a previoushypothesis of the existence of a native population circa AD500−1450 in the Tarapaca Valley that has been adapted to localgeopolitical, social, and economic conditions33 and who livedand moved along the mid river valleys. This hypothesis is alsosupported by the observed reduction in the arsenicconcentration from the proximal to the distal end of the hairthat could be due to a change in diet due to mobility, thoughchemical and/or microbiologically induced processes cannot beentirely ruled out. The protocol for the scientific method usedin this study,34 demonstrating high sensitivity and highspecificity in toxicity assessment and geographic mobility, wascentral to overcoming analytical challenges20 and can bebroadly applied to forensic investigations and environmentalmonitoring programs for which heavy metal quantification andsourcing is necessary.

■ ASSOCIATED CONTENT*S Supporting InformationFurther content on materials and methods, sample preparation,hair geometry, and element concentration profiles used tosupport hypotheses made in this study. One additionalexperimental figure connected to analytical and characterizationdata (also referenced in the article). This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: kakoulli@ucla.edu.Author ContributionsThe manuscript was written through contributions of allauthors. All authors have given approval to the final version ofthe manuscript. All authors contributed equally.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank the Consejo de Monumentos Nacionales de Chile forsite access and permissions for sampling and analysis and RanBoytner and Maria Cecilia Lozada codirectors of the Tarapaca Valley Archaeological Project for providing information on thearchaeology and ethnography of the area. The operations of theAdvanced Light Source at Lawrence Berkeley NationalLaboratory are supported by the Director, Office of Science,Office of Basic Energy Sciences, U.S. Department of Energyunder contract number DE-AC02-05CH11231. FEGVPSEM-EDS analysis was conducted at the Molecular and Nano

Archaeology Laboratory at UCLA on the FEI Nova NanoSEM230 purchased with NSF award no. 0813649. Travel funding tothe synchrotron facility was provided by the Senate FacultyAwards at the University of California Los Angeles (UCLA).Physical microsamples used for the analysis and analytical dataare stored and accessed through the Molecular and NanoArchaeology Laboratory, UCLA.

■ REFERENCES(1) Xia, Y. J.; Wade, T. J.; Wu, K. G.; Li, Y. H.; Ning, Z. X.; Le, X. C.;He, X. Z.; Chen, B. F.; Feng, Y.; Mumford, J. L. Int. J. Environ. Res.Public Health 2009, 6 (3), 1010−1025.(2) Chevallier, P.; Ricordel, I.; Meyer, G. X-Ray Spectrom. 2006, 35,125−130. Nicolis, I.; Curis, E.; Deschamps, P.; Benazeth, S. Biochimie2009, 91, 1260−1267.(3) Wilson, A. S. In Hair in Toxicology: An Important Biomonitor;Tobin, D. J., Ed.; Royal Society of Chemistry: Cambridge, 2005; pp321−345.(4) Kempson, I. M.; Henry, D. A. Angew. Chem., Int. Ed. 2010, 49,4237−4240.(5) Yanez, J.; Fierro, V.; Mansilla, H.; Figueroa, L.; Cornejo, L.;Barnes, R. M. J. Environ. Monit. 2005, 7, 1335−1341.(6) Paswan, S. Studying the arsenic absorption by keratin proteinextracted from human hair. Bachelor of Technology Thesis, NationalInstitute of Technology Rourkela, 2012.(7) Allison, M. J. In Human Mummies; Spindler, K., Ed.; Springer-Verlag/Wien: New York, 1996; pp 125−129.(8) Allison, M. J. Chronic arsenic poisoning in South AmericanMummies. Annual Meeting of the International Academy of Pathology,Washington D.C., March 1996.(9) Arriaza, B.; Amarasiriwardena, D.; Cornejo, L.; Standen, V.;Byrne, S.; Bartkus, L.; Bandak, B. J. Archaeol. Sci. 2010, 37, 1274−1278.(10) Byrne, S.; Amarasiriwardena, D.; Bandak, B.; Bartkus, L.; Kane,J.; Jones, J.; Yanez, J.; Arriaza, B.; Cornejo, L. Microchem. J. 2010, 94,28−35.(11) Figueroa, L. Arica Inserta en una Region Arsenical: El Arsenico enAmbiente que la Afecta y 45 Siglos de Arsenicismo Cronico; Universidadde Tarapaca: Arica, Chile, 2001. Figueroa, L.; Bazmilic, B.; Allison, M.;Gonzales, M. Chungara 1988, 21, 33−42.(12) Arriaza, B. T. Chungara, Revista de Antropologıa Chilena 2005,37, 255−260.(13) Leybourne, M. I.; Cameron, E. M. Chem. Geol. 2008, 247, 208−228.(14) Pringle, H. Science 2009, 324, 1130−1130.(15) Rahman, M.; Ng, J.; Naidu, R. Environ. Geochem. Health 2009,31, 189−200.(16) Kempson, I. M.; Henry, D.; Francis, J. J. Synchrotron. Radiat.2009, 16, 422−427.(17) Wilson, A. S.; Dodson, H. I.; Janaway, R. C.; Pollard, A. M.;Tobin, D. J. Archaeometry 2010, 52, 467−481. Bianucci, R.; Jeziorska,M.; Lallo, R.; Mattutino, G.; Massimelli, M.; Phillips, G.; Appenzeller,O. PLoS ONE 2008, 3, e2053. Phillips, G.; Reith, F.; Qualls, C.; Ali, A.-M.; Spilde, M.; Appenzeller, O. PLoS ONE 2010, 5, e9335.(18) Kintz, P.; Ginet, M.; Cirimele, V. J. Anal. Toxicol. 2006, 30,621−623. Kintz, P.; Ginet, M.; Marques, N.; Cirimele, V. Forensic Sci.Int. 2007, 170, 204−206.(19) Forshufvud, S.; Smith, H.; Wassen, A. Nature 1961, 192, 103−105. Kintz, P.; Goulle, J. P.; Fornes, P.; Ludes, B. J. Anal. Toxicol. 2002,26, 584−585.(20) Pezzotti, G.; Sakakura, S. J. Biomed. Mater. Res. 2003, A, 230−236.(21) Wilson, A. In Soil Analysis in Forensic Taphonomy: Chemical andBiological Effects of Buried Human Remains, Tibbett, M.; Carter, D. O.,Eds.; Taylor & Francis Group: Boca Raton, FL, 2008.(22) Wilson, A. S.; Dodson, H. I.; Janaway, R.; Pollard, A.; Tobin, D.Br. J. Dermatol. 2007, 157, 450−457.

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(23) Lubec, G.; Zimmerman, M. R.; Teschler-Nicola, M.; Stocch, V.;Aufderheide, A. C. Free Radical Res. 1997, 26, 457−462.(24) Berger, I. A.; Cooke, R. U. Earth Surf. Processes Landforms 1997,22, 581−600. Clarke, J. D. A. Geomorphology 2006, 73, 101−114.Duncan, P. B.; Morrison, R. D.; Vavricka, E. Environ. Forensics 2005, 6,205−215. Katz, H. R. Miner. Deposita 1967, 2, 131−134. Kholodov, V.N. Lithol. Miner. Resour. 2007, 42, 246−256. Rech, J. A.; Currie, B. S.;Shullenberger, E. D.; Dunagan, S. P.; Jordan, T. E.; Blanco, N.;Tomlinson, A. J.; Rowe, H. D.; Houston, J. Earth Planet. Sci. Lett.2010, 292, 371−382.(25) Smith, H. J. Forensic Sci. Soc. 1964, 4, 192−199.(26) Steely, S.; Amarasiriwardena, D.; Jones, J.; Yanez, J.Microchem. J.2007, 86, 235−240.(27) Smith, A. H.; Arroyo, A. P.; Mazumder, D. N. G.; Kosnett, M. J.;Hernandez, A. L.; Beeris, M.; Smith, M. M.; Moore, L. E. Environ.Health Perspect. 2000, 108, 617−620.(28) Smith, A. H.; Lopipero, P. A.; Bates, M. N.; Steinmaus, C. M.Science 2002, 296, 2145−2146.(29) Smith, E.; Kempson, I.; Juhasz, A. L.; Weber, J.; Skinner, W. M.;Grafe, M. Chemosphere 2009, 76, 529−535.(30) Muller, D.; Medigue, C.; Koechler, S.; Barbe, V.; Barakat, M.;Talla, E.; Bonnefoy, V.; Krin, E.; Arsene-Ploetze, F.; Carapito, C. PLoSGenetics 3 2007, e53. Hindmarsh, J. T. Clinical Biochemistry 2002, 35,1−11.(31) Knudson, K. J.; Price, T. D. Am. J. Phys. Anthropol. 2007, 132,25−39. Sturgis, R. The Macmillan Company: New York, 1905, 2.(32) Mandal, B. K.; Suzuki, K. T. Talanta 2002, 58, 201−235.(33) Uribe, R. M. In Esferas de Interaccion Prehistoricas Y FronterasNacionales Modernas: Los Andes Sur Centrales; Lechtman, H., Ed.;Instituto de Estudios Peruanos, Lima (IEP) and the Institute ofAndean Research, New York (IAR): Lima and New York, 2006.(34) Kempson, I. M.; Kirkbride, K. P.; Skinner, W. M.; Coumbaros, J.Talanta 2005, 67, 286−303. Martin, R. R.; Kempson, I. M.; Naftel, S.J.; Skinner, W. M. Chemosphere 2005, 58, 1385−1390.

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S-1

Supporting Information for

Distribution and chemical speciation of arsenic in

ancient human hair using synchrotron radiation

Ioanna Kakoulli*, Sergey V. Prikhodko, Christian Fischer, Marianne Cilluffo, Mauricio Uribe,

Hans A. Bechtel, Sirine C. Fakra, Matthew A. Marcus

*Correspondence to: kakoulli@ucla.edu

This PDF file includes:

Materials and Methods

Supporting Figures: Fig. S1

References

S-2

SUPPORTING INFORMATION

Materials and Methods

1. Portable non-invasive and non-destructive methods Prior to micro-sampling, the entire mummy bundle and the burial enveloping soil were

analyzed in situ at multiple scales non-invasively using broadband digital and multispectral

imaging technology (MSI) combined with portable X-ray fluorescence (pXRF) spectroscopy and

portable ultraviolet-visible-near infrared (pUV/Vis/NIR) diffuse reflectance spectroscopy.

1.1. Multispectral Imaging (MSI) MSI1 records simultaneous spectral and spatial information of an object from which composite

images can be obtained highlighting the difference in the reflectance behavior characteristic to

each individual material. Here we have used the portable MuSIS™ HS multi-spectral imaging

system which acquires digital images at specific bandwidths using optical filters which control

33 spectral bands in the range of 400-1000 nm and full spectra per image pixel were calculated

from the captured spectral images. RGB images were obtained by combining the incoming

radiation intensity values from images in the green, red and blue regions. The grey level (0-255)

of each pixel of the image represents the intensity of reflected light at a specific wavelength. We

have recorded images and spectra of % diffuse reflectance and UV-induced fluorescence based

on the type of illumination used (halogen or ultraviolet).

1.2. Portable X-ray Fluorescence (pXRF) spectroscopy Portable X-ray fluorescence spectroscopy is a well-established non-invasive technique in

archaeology widely used for the elemental characterization of materials.2 In this project, pXRF

was used for preliminary characterization of the burial enveloping soil and soil encrustations

firmly attached to the mummy bundles, enabling direct and indirect comparisons of the different

S-3

burial chemical environments. A Bruker Tracer III-V (Si-PIN detector) handheld XRF

instrument was used with capabilities at detecting elements with Z≥13 when operated under

vacuum (~5 torr). Spectra were collected – under vacuum – at 40Kev and 1.35mA for 120

seconds acquisition time.

1.3. Portable Ultraviolet-Visible-Near Infrared (pUV/Vis/NIR) diffuse reflectance spectroscopy

Non-invasive portable diffuse reflectance UV/Vis/NIR spectroscopy was used to characterize

both organic and inorganic materials without having to extract a sample.3 This project used the

FieldSpec® 3 by Analytical Spectral Devices Inc (ASD), with high spectral resolution (3 nm @

700 nm and 10 nm @ 1400/ 2100 nm) and wide spectral range between 350-2500 nm. The

flexible spot size analyzer for contact analysis facilitated the systematic study of specific areas.

The high spectral and spatial resolution of the spectrometer was particularly useful for the

identification of different minerals in the soil and attached to the mummy bundles, as well as,

proteins and lipids in the skin due to its high sensitivity to both the electron transitions in the

visible part of the spectrum and the overtones from the organic molecules in the near infrared.

1.4. Results from in situ analyses In situ preliminary analyses were pivotal to help differentiate postmortem artifacts and support

data suggesting chronic arsenic poisoning. Investigations of the skin using MSI revealed

discolorations with hyper- and hypopigmentation (Figure S1) commonly encountered in

populations with chronic arsenic exposure.4 Skin areas with white protruding pustule

manifestations were analyzed by UV/Vis/NIR spectroscopy and µRS, and have indicated

increased epidermal keratin formation, which support the hypothesis of chronic arsenicism.5

Additional skin areas with similar signatures of elevated keratin suggesting possible poisoning

S-4

induced by arsenic were identified in the soles of the feet and toes. Other skin topical signs with

characteristic micro-roughness related more to post mortem effects and biofilm formations.

2. Laboratory microanalytical methods

2.1. Variable Pressure Scanning Electron Microscopy coupled with Energy Dispersive X-ray Spectroscopy ((VPSEM-EDS)

Field emission gun (FEG) variable pressure scanning electron microscopy (VPSEM) coupled

with energy dispersive X-ray spectroscopy (EDS)6 provided a powerful system (the

FEGVPSEM-EDS) that enabled the study of small objects and samples without any alteration of

their morphology and structure. This instrument takes full advantage of the high spatial

resolution offered by the FEGVPSEM for non-invasive and non-destructive in situ nanoscale

morphological and topographic mapping, and the micro-probing of the EDS for simultaneous

spatially resolved elemental characterization of materials.7,8 In addition, the variable pressure

capabilities of the SEM allows the analysis of hydrated or insulating samples and facilitates

dynamic experiments of the behavior of materials’ performance (physicochemical integrity) and

deterioration processes (physical and chemical reactions) under variable conditions.8 In this

study, human hair and skin specimens were primarily analyzed without any special preparation

as the analytical techniques used allowed the samples to be retrieved and re-used for subsequent

analyses. Cross sections and thin sections were also analyzed as they provided supplementary

information on the internal structure and composition of materials. For VPSEM-EDS analysis

two different instruments were used: 1) a LEO 1430 VP instrument coupled with an Oxford

Instruments EDS detector and 2) a FEI Nova NanoSEM 230 coupled with a Noran EDS detector

from ThermoFisher Scientific. Additional micro-Raman spectroscopic analyses on the same

specimens were carried out by Bruker Optics, Inc. using a Senterra Raman spectrometer and

1064 nm laser excitation source.

S-5

2.2. Thin section preparation for histological analyses Thin sections were prepared by first embedding the sample in LR White Resin then cut with an

ultramicrotome to a thickness of 5 µm and stained using 1% toluidine blue in 1% sodium borate

and standard Hematoxylin and eosin. LR White is a very low viscosity polar monomer

polyhydroxylated aromatic acrylic resin suitable for both light and electron microscopy. The

resin was cured by UV light.

3. Synchrotron-based x-ray microprobe

Micro-focused X-ray fluorescence (µXRF) mapping, micro X-ray absorption near edge

structure (µXANES) spectroscopy and micro X-ray diffraction (µXRD) were performed at

beamline 10.3.2 of the Advanced Light Source (ALS), Lawrence Berkeley National Lab,

Berkeley, CA.9 µXRF maps were acquired at 3.9, 10, 13.025 and 13.085 keV incident energies

to facilitate detection of elements between S and K, Ca-Zn, As and Pb, respectively. Since Pb Lα

and As Kα fluorescence emission lines are nearly coincident, detection of both elements required

mapping above and below the Pb L3-edge and taking the difference of the signals. Though there

was not evidence of Pb, at energy below the Pb L3-edge, there will be no Pb background.

Furthermore, in this beamline, higher harmonics are undetectable at this energy because the

bend-magnet source provides almost no flux at 35keV and the mirrors don't reflect at that

energy. The pixel size for mapping and spectroscopy ranged from 3-5µm and the beam size

from 3-7µm. Maps and spectra were recorded with a 7-element Ge solid-state detector

(Canberra). As K-edge spectra were calibrated using NaHAsO4 (white line set at 11873.7eV). As

XANES spectra from each spot were too noisy for reliable fitting, so spectra from all spots were

averaged together. This average spectrum was then pre-edge background subtracted, post-edge

normalized and fitted to least-square linear combinations (non-negative loadings) of reference

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spectra following standard procedures described elsewhere.10 The species, which came out in the

fits were As2S3, aqueous arsenite and AsCysteine. All data processing was carried out with a

suite of custom LabView programs available at the beamline. Micro-XRD patterns were

recorded with a 1024x1024 pixels Bruker SMART6000 CCD camera at an incident energy of

17keV and a beam size of 16 µm (H) x 7 µm (V). The two-dimensional patterns were calibrated

with Alumina (Al2O3) and radially integrated to one dimensional XRD profiles using Fit2D

software.11

3.1. Arsenic detection limit Using the analytical setup of ALS beamline 10.3.2 the amount of As that could be measured

was on the order of 1016 atoms of As per cm2. If we consider an average thickness of hair at 80

microns and the density of hair as 1, the detectable concentration (Cd) can be estimated as about

150 ppm.

No As was detected in a modern human hair from an individual with no prior exposure to

arsenic was analyzed as control. Figure 5 (main text) shows the absence of arsenic in the modern

hair.

3.2. Hair geometry and concentration profiles Assuming that the beam size b and the absorption of fluorescent and incident X-rays are

negligible, the output signal at any point in a µXRF map will be simply proportional to the total

amount of the detected element illuminated by the beam. Thus, the signal will be proportional to

the concentration in the hair, projected onto a plane perpendicular to the probe beam. Two cases

are then considered:

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Case 1. The elemental concentration is uniform throughout the volume of the hair, which we

consider as cylinder of radius R. In this case, the signal is proportional to the thickness of the hair

at the probe position. If the probe is at a distance x from the center of the hair, then the path

length is given by

(1)

For As, we find that the observed transverse profile, averaged over a short length of hair,

follows this formula fairly well Figure 6A curve 1 (main text).

Case 2. The element is present only at the surface, assuming a surface coating. In the limit of

a thin coating, the intensity may be obtained by differentiating (1) with respect to R,

(2)

This function has maxima at because the maximum number of atoms is illuminated

when the beam just skims the surface. The Zn intensity profile displayed in Figure 6A curve 2

(main text) shows this effect in a qualitative sense.

If we now take into consideration that the beam has a finite size we then have to convolve I(x)

with the beam profile, an operation which rounds off the features as shown in figure 5 in Martin

et al.12

Based on the above, the profile of the Zn distribution (Figure 6B) (main text) in the hair

analyzed looks qualitatively like curve 2 in Figure 6A (main text), whereas As looks a bit

brighter toward the center and has a profile corresponding to curve 1 in Figure 6A (main text).

2max 1 ( / ) | |( )

0 | |I x R x RI x

x R

− ≤= >

2/ 1 ( / ) | |( )0 | |

C x R x RI xx R

− ≤= >

x R= ±

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4. Synchrotron-based Fourier transform infrared spectromicroscopy Additional analyses were performed on hair thin sections using Fourier transform infrared

spectromicroscopy (FTIR). FTIR measurements were performed at the ALS infrared beamline

1.4.3, which offers spatial mapping of molecular signatures with a diffraction-limited spot size of

3-10 µm.13

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Supporting Figures

Figure S1. Monochromatic and composite images of spectral cube of Locus 9 from cemetery TR-40A. The images show part of the face of the mummy at different wavelengths enhancing features of the skin related to the antemortem pathologies and diagenetic alterations during burial. Images A, B and C were taken at λmax of 600 nm, 800 nm and 1000 nm respectively. Based on the differential absorption and reflection of light, features such as hyper and hypopigmentations not visible to the naked eye can be observed. These details are further enhanced through recombination of monochromatic images at different wavelengths to form composite images. D is a normal RGB image; E is a false-color trichromatic ultraviolet reflectance image (where the red channel has been replaced by the green, the green by the blue and the blue by an ultraviolet image with (λmax=370 nm) and F a false-color image combining ultraviolet (λmax=370 nm), visible ((λmax=600 nm) and infrared (λmax=900 nm) information. The scale is 2 cm.

S-10

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