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ORIGINAL PAPER

Raman spectroscopic analysis of human remainsfrom a seventh century cist burial on Anglesey, UK

Howell G. M. Edwards & Andrew S. Wilson &

Nik F. Nik Hassan & Andrew Davidson & Andrew Burnett

Received: 27 May 2006 /Revised: 17 August 2006 /Accepted: 18 August 2006 / Published online: 14 September 2006# Springer-Verlag 2006

Abstract Specimens from human remains exhibiting un-usual preservation excavated from a seventh century stonecist burial at Towyn y Capel in Anglesey, UK, have beenanalysed using Raman spectroscopy with near-infrared laser excitation at 1,064 and 785 nm. Specimens of hair and bone provided evidence for severe degradation and microbialcolonisation. The deposits within the stone cist showed that some microbially mediated compounds had been formed.Analysis of crystals found at the interface between the hair and the skeletal neck vertebrae revealed a mixture of newberyite and haematite, associated with decomposition products of the hair and bone. An interesting differentialdegradation was noted in the specimens analysed whichcould be related to the air-void and the presence of plant root inclusions into the stone cist. This is the first time that Raman spectroscopy has been used in the forensicarchaeological evaluation of burial remains in complexand dynamic environments.

Keywords Raman spectroscopy . Cist burial . Degradation .Hair . Bone . Newberyite . Forensic

Introduction

The analysis of human remains from archaeological andforensic contexts can provide important information about the conditions pertaining to the depositional environment and the effects of post-depositional change [ 1, 2]. Thesurvivability or otherwise of bone, teeth and other tissuessuch as hair, nail and skin is dependent upon a complex set of environmental parameters which include soil type, the pH of the burial matrix, moisture, the presence of metal salt and temperature [ 3]. Additionally, the method of burial canhave a marked effect on preservation. Many burials includetextile clothing or shrouds and other grave artifacts, whichcan also modify the role of the decomposition of soft tissues and can potentially give rise to a series of chemicalreactions that otherwise would not have occurred. Similarlythe presence or absence of a coffin or in this case the presence of a stone-lined cist grave will have a markedeffect on decomposition. Analysis of human remains provides detailed evidence for understanding mechanismsinfluencing decay in such complex environments [ 4 6].

Raman spectroscopy has been used successfully todemonstrate the molecular deterioration suffered by bio-materials and skeletal remains under a variety of different

environmental conditions at different sites. Skeletal remainsfrom a series of archaeological and forensic contexts [ 7, 8]have been investigated hitherto and conformational changesin protein configuration, fission of the cysteine disulphide bonds and the presence of associated mineralogical inclu-sions [ 7] have been ascertained from Raman spectroscopicstudies of hair, in particular. Special treatments applied tohuman skeletal remains have also been studied and canreveal evidence of different materials applied to the surfaceof bone as with limewash and pigment on material from a Sambaqui burial in Brazil [ 9]. Studies of frozen human and

Anal Bioanal Chem (2007) 387:821 828DOI 10.1007/s00216-006-0791-9

H. G. M. Edwards ( * ) : N. F. Nik Hassan : A. Burnett Molecular Spectroscopy Laboratory, University Analytical Centre,

Chemical and Forensic Sciences, University of Bradford,Bradford BD7 1DP, UK e-mail: [email protected]

A. S. WilsonDepartment of Archaeological Sciences and Biomedical Sciences,University of Bradford,Bradford BD7 1DP, UK

A. DavidsonGwynedd Archaeological Trust,Craig Beuno, Garth Road, Bangor,Gwynedd LL57 2RT, UK

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animal remains revealed that hair and feathers are morerobust and hence more likely to survive than skin and other soft tissue [ 10 12].

In the present study, we report a comprehensive Ramanspectroscopic investigation of human remains recoveredfrom a stone-lined cist grave in beach sand (Fig. 1) at Towyn y Capel, Anglesey, UK, dated to roughly A.D. 630

780. An unusual feature of the remains is the survival of hair and organic material and that the skeletal remains havesuffered differential degradation. Possible reasons for thisdifferential degradation are the particular microenviron-mental conditions brought about by burial within a stone-lined cist, which visually contains an invasion by plant root.This contrasts with other burials where the graves were backfilled and the body was covered with soil. One aspect of special interest within the burial was the accentuated bone degradation where the hair had been in intimatecontact with the skeletal remains. A detailed study of thedegradation suffered by these skeletal remains using Ramanspectroscopy provides not only novel information about molecular reactions in this burial environment but alsoserves to illustrate the fundamental role that Ramanspectroscopy can play in deciphering complex biomaterialdegradation in archaeology and forensic science.

Experimental

Specimens

During archaeological excavations in the sand dunes at Towyn y Capel, Anglesey, in 2003/2004, the remains of 23cist burials were excavated. The interiors of the cists weregenerally free of sand, and in only one instance was thevoid completely filled. In the remainder the depth of sandrarely covered the skeleton, and degraded roots were found

within the void. Two of the skeletons retained some hair,and one in particular, burial S105, an adult female, wasconsidered to be unusual and rather rare because of thelarge quantities of hair which survived. The cranium andupper portions of the vertebral column that were overlain by a large clump of matted hair were extremely fragileand degraded, in contrast to much of the remainder of the skeletal elements (Fig. 1). Samples of degraded bone, crystalline products and hair taken from specificlocations were submitted for Raman spectroscopic analysisin an attempt to reconstruct and understand the processesacting within the grave. Samples for analysis were asfollows:

1) Degraded (blackened and embrittled) cranial bonefragment: the grey-black colour is likely a function of various processes including degradation of the organicfraction of bone coupled with demineralisation as wellas other factors arising from the specific microenviron-

ment brought about by burial in a cist.2) Degraded (embrittled with matt appearance) hair.3) Crystalline products associated with the skeletal

remains.

a) A combination of translucent purple spiculatedcrystalline deposits with white solid material, foundinferior to the cranium and located adjacent tosurviving hair.

b) White powdery crystalline deposits considered to be bone-derived, but not intimately associated with bone surface and recovered from the upper torso.

c) White crystalline deposits (needle-like crystals)which were found to locally disrupt the degraded bone cortex of vertebral bodies.

4) Dark organic material: this powdery deposit distributedlargely across the torso of the burial and was found onotherwise intact bone surfaces. It likely represents bothdegraded (carbonised) soft tissue and/or degraded root material considering the presence of large quantities of plant roots within the void created by the cist.

5) Beach sand from the base of the cist burial, consolidated by body exudate and can be ascribed as a) yellow, b) black, c) quartz-like and d) grey components.

Raman spectroscopy

Raman spectra were obtained using a Bruker IFS 66instrument with a fibre Raman amplifier (FRA) 106 Ramanmodule attachment and Nd 3+ /YAG near-infrared excitationat 1,064 nm with a nominal power of 20 mW. Spectra wererecorded at 4 cm

1 resolution, with a 4,000-scan accumu-lation, and a spectral footprint of 100 m. Wavenumbers of sharp bands are accurate to better than 1 cm

1 . Spectra Fig. 1 Burial S105 in situ as excavated. Photo: Gwynedd Archaeo-logical Trust, 2003

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were also recorded using a Renishaw InVia confocalRaman microscope operating at 785 nm with charge-coupled device (CCD) array detector and a nominalmaximum power of 50 mW. As for all of our spectroscopicstudies with sensitive biomaterials, Raman spectra wereacquired using the lowest possible laser power that alloweda spectrum to be obtained. In most cases this was only1 mW or so at the sample. Also, the observation of a carbonspectrum can be assuredly taken not to be a carbonisationof sensitive organics by examination of the specimen beforeand after laser illumination. The spectral data were scannedfor the acquisition of up to 5 accumulations and 10-s laser exposure time with a spectral footprint of about 2 m (50objective lens). Spectroscopic analyses were performed inreplicates to provide reproducibility. Baseline correctionwas imposed on spectra using OPUS software prior to stack plotting.

Results and discussion

The results obtained are summarised in Table 1. Figure 2shows the Raman spectrum of degraded bone (cranialfragment) relative to modern human femoral bone for comparison. It is evident that degradation has occurred progressively. The organic signatures of collagen in particular are still observed but the intensities are reduceddue to leaching. The peak intensity of hydroxyapatite (PO)at 960 cm

1 is significantly reduced relative to organic peaks. The results suggest that the leaching of the mineralcomponent of bone has occurred faster than the organiccomponent in samples of degraded bone located inassociation with the hair. The presence of a carbonate peak near 1,070 cm

1 in the degraded bone is ascribed to theincorporation of carbonate (CO 3

2 ) from the burial matrixduring decomposition.

Raman spectra of the hair fibres show medium weak peaks at 1,628 and 1600 cm

1 and very weak peaks near 1,653, 1,265 and 1,164 cm

1 (Table 1). These bands areassigned to amide I and amide III disordered phase, and (CC) skeletal, respectively. Human hair is known tosurvive under a diverse range of environmental conditionscompared to other body proteins. Figure 3 shows the stack plot of Raman spectrum of these fibres along with thespectrum of modern human hair for comparison. It isevident from the result that the stability of the amidecomponents of hair appears greater, which is apparentlymore resistant to microbial and environmental attack thanthat of the disulphide linkage. The loss of protein structurein the fibres is clear and the peak assigned to (SS) modesnear 510 cm

1 is absent in comparison with modern humanhair. Breakdown of the protein structure and conversion of the structural disulphide linkages in cystine to cysteic acid

and intermediate states suggested that partial oxidation mayhave occurred. The extent of oxidative damage to hair through fission of the S S bond and formation of cysteicacid residues is in part responsible for the embrittlement of degraded hair fibres.

The peak near 1,600 cm1 , assigned to (C=O) and (C=C)

however, is increased in intensity and is possibly related to products of the breakdown of protein (amide) structure. Thechange of conformation in the protein structure is observedin the amide I and III peaks and is a feature of thedegradation of the keratin and keratin associated proteins.The -helical amide I mode near 1,650 cm

1 stronglyobserved in Raman spectrum of modern human hair, is veryweak or almost absent in the spectrum of the fibres. The peak is replaced by components arising from disorderedand random coil conformations near 1,628 cm

1 . Similar effects are also observed for the amide III component near 1,265 cm

1 which is reduced significantly in intensity.Change in the protein structure resulting from reactions anddecomposition processes in the burial environment isfrequently mediated by microbial activity and other factorssuch as temperature, humidity and pH of the deposit. Hair is very responsive to changes in relative humidity and theintimate contact of the hair with bone elements may have been in part responsible for the localised degradation of the bone. Fungi are known to be the major decomposers of hair in the burial environment [ 3]. Keratin is normally resistant to microbial attack but some fungi and actinomycetes candegrade the disulphide bonds and exploit keratin as a nutrient source [ 6]. The fact that hair has survived suggestsstrongly that the microbial activity has been limited; alsothere is no observation of (SH) modes in the Ramanspectra as expected for bacterial reduction of the S Slinkages.

Figure 4 shows the stack plot of the Raman spectra of the purple, white powdery and white crystallinedeposits recovered from the cranium, bone and vertebralcolumn, respectively. Raman spectrum of newberyite(MgHPO 4 3H 2 O) is also included for comparison purposes.This phosphate mineral was prevalent in human renalcalculi (kidney stones) [ 13] and bat guano [ 14] as well as ina decomposition product of ivory [ 15, 16]. Interestingly, allspectra of the crystalline deposits have closely matched thespectrum of newberyite; it should be noted that the peaksnear 984 and 870 cm

1 are diagnostic features of newberyite, assigned to symmetric (PO 4 ) and (P OH) modes,respectively. The formation of these crystals may be due tothe presence of salts, considering the proximity of the burialto the sea. It is possible newberyite was formed on thesurface of the degraded portions of bone because calciumloss in hydroxyapatite was not substituted by divalent magnesium ions (Mg 2+ ) [16]. The dissolution process of theMg 2+ component was favoured by the close contact of the

Anal Bioanal Chem (2007) 387:821 828 823

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external bone surface with the burial environment and possibly with invasion of the cist burial chamber withaquatic brines that are a source of magnesium ions [ 17].Traces of haematite (iron(III) oxide) at 412, 383, 279 and176 cm

1 , are also identified in all of the spectra of thecrystalline deposits (3a, 3b and 3c) and are likely to be

derived from beach sand. The white solid material (foundwith purple crystalline deposits) has bands corresponding toaragonite, the second most common polymorph of naturalcalcium carbonate (calcite is the most common). The bandsat 1,193 and 1,083 cm

1 represent the asymmetric andsymmetric stretch of carbonate, respectively, while the peak

Table 1 Raman spectral wavenumbers and vibrational assignments of specimens from burial S105


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at 702 cm1 represents the bend of the carbonate group. It

may either represent degraded shell from the beach sandsource (most bivalve animals and corals secrete aragonitefor their shells) or could be microbially mediated calciumleached from the degraded bone. Bone calcium and other mineral components provide a calcareous source for reaction with oxalic acid waste produced by lichen, fungalhyphae and root action. Microbial degradation is evidenced

by the peak near 1,480 cm1 in the spectrum of degraded

bone, assigned to calcium oxalate dihydrate (weddelite)(Fig. 2). The spectral features of both white powdery (3b)and vertebral white (3c) crystals are similar. In contrast, anincreased intensity of the peak near 520 cm

1 is observed inthe spectrum of the purple crystalline deposit (3a). Thedifference in relative peak intensities is significant fromthose of white crystalline and white powdery deposits

Fig. 2 Stack plot of a degraded bone (cranial fragment) and bmodern human femoral bone

Fig. 3 Stack plot of a degradedhair fibres and b modern humanhair


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(3b and 3c) and the peak near 520 cm1 , and is thought to be

associated with the occurrence of purple colour in the crystals(3a). Colour in post-mortem remains has previously beenattributed to a number of variables [ 18], including microbial

activity, phosphatic inclusions and breakdown products of haemoglobin such as porphyrins; the last of these haverecently been recognized as biomarkers of bacterial coloni-zation of geological matrices in ancient rocks [ 19].

Fig. 4 Stack plot of Ramanspectra of a purple (3a), b white powdery (3b) and c white crys-talline deposits (3c), andd newberyite

Fig. 5 Raman spectrum of white powdery crystallinedeposits (3b)


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It is presumed that the relatively shallow grave depth and proximity to the ocean will have exposed the remains torelatively high humidity conditions in the cist which mayhave accelerated the formation of crystalline products. Thisis supported by the fact that all three crystalline specimenswere hydrated as demonstrated by the presence of bands inthe region 3,000 3,500 cm 1 in the spectrum of white powdery crystalline deposits (3b), indicative of water

(Fig. 5): the bands are assigned to (OH). Organic signaturesare also observed in the region 2,900 3,000 cm 1 , assignedto (CH) and HPO 4

2 , in the spectrum of white powdery(3b) and white crystals (3c) but absent in the spectrum of purple crystals (3a). A very weak and broad peak near 1,650 cm

1 is also observed in the spectrum of white powdery crystals (3b) (Fig. 5), which could be assigned tothe -helical amide I and the (H O H). The organic

Fig. 6 Stack plot of Ramanspectra of consolidated beachsand components a yellow, b black, c quartz-like and d grey

Fig. 7 Raman spectrum of dark organic material


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components trapped in the crystalline deposits may haveleached from the bone whilst crystallization was taking place[20].

Raman spectra of the components of the consolidated beach sand, yellow, black, quartz-like and grey, are shownin Fig. 6 and their wavenumbers and vibrational assign-ments are given in Table 1. Although the beach sand had been consolidated by body exudates, evidence for the presence of intermediary organic decomposition product isnot manifested by Raman spectrum. Only the signature of carbon is identified. The yellow component is identified asaragonite, a marine carbonate made from coral and seashell.This comes as no surprise because of the location of the cist burial in beach sand. The black, grey and quartz-likecomponents are in fact all quartz, whose peak featured at 463 cm

1 . The difference in colour intensity can beexplained by the presence of carbon deposits (graphite)shown by the broad peaks at 1,320 cm

1 (D-band) and1,590 cm

1 (G-band), where the black component containsmore graphite, which could possibly arise from slate andshale deposits locally. On the other hand, the broad peaks of carbon are observed in the spectrum of dark material (4), issuspected to be of organic origin (Fig. 7). The greycomponent on the other hand, shows an additional weak peak at 960 cm

1 which corresponds to the symmetricstretching vibration of the PO 4

3 ion of hydroxyapatite, andmay be derived from the demineralized bone.

Conclusions

Our results highlight the fact that the localised microcli-mate within cist burials can markedly influence thedegradative processes acting on a body. A combinationof the void surrounding the body within these cist burialsand the fact that the cist burials were dug into a saline-rich beach sand environment were conducive to theformation of crystalline products. During putrefactivechange to the bulk of the body tissues, l iquefieddecomposition products seeped into the underlying sandof the grave base resulting in the consolidation of smalllumps of sand. The identification of newberyite in thecrystalline products is a clear indication that salts have been produced as a byproduct of the putrefaction andhave reacted with biological apatite. Although the purplecolourization has not been definitively assigned in thecurrent study, several possibilities can be proposed:microbial modification of the skeletal remains, porphyrindegradation products or a calcium ion phosphate. Variousdegradative processes have clearly acted upon theremains whilst in the cist. There is clear evidence for microbially mediated decomposition of hair and bone

with the formation of oxalates. Similarly, the hydratedcrystalline products identified, show that mineral has been leached from both bone and the calcareous fractionsof the beach sand within what has clearly been a moist environment.

Acknowledgements We thank the Malaysian Government for a

research studentship for NFNH, the Wellcome Trust for a ResearchFellowship to ASW (grant code: 024661). The skeletal remains wereexcavated by Gwynedd Archaeological Trust in conjunction with staff and students from the University of Central Lancashire.

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