FT-Raman spectroscopy of avian mummified tissue of archaeological relevance

Spectrochimica Acta Part A 55 (1999) 2691 – 2703 www.elsevier.nl/locate/saa

FT-Raman spectroscopy of avian mummified tissue of archaeological relevance H.G.M. Edwards a,*, D.W. Farwell a, D.D. Wynn-Williams b a b

Chemical and Forensic Sciences, Uni6ersity of Bradford, Bradford BD7 1DP, UK British Antarctic Sur6ey, High Cross, Madingley Road, Cambridge CB3 0ET, UK

Received 12 January 1999; received in revised form 16 April 1999; accepted 19 April 1999

Abstract There are frequent reports of mummified seals in the cold deserts of Antarctica. However, a mummified penguin found over 100 miles from the coast and at an altitude of 600 m is most unusual. The non-intrusive FT laser Raman spectra of untreated mummified tissues from this bird are presented; the specimen has three distinct types of tissue clearly identifiable in a limb, namely the bone, claw and skin, along with feathers from the carcass. The unusually fine preservation of these materials in an Antarctic cold desert climate means that bacterial degradation has been arrested and affords an opportunity to compare the molecular deterioration of the keratotic or collagenic components of the avian ice-mummified tissue with those from the skin of human ice-mummies. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Raman spectroscopy; Mummified tissue; Keratin; Penguin; Antarctic

1. Introduction Although the Raman spectra of keratins and mammalian keratotic tissues have been reported previously, there has been little work in the literature on aged keratotic biopolymers. The reason for this is clear; the survival of soft tissue is critically dependent on the minimisation of bacterial attack either through the operation of specific environmental conditions or through the action of * Corresponding author. Tel.: + 44-1274-233787; fax: +441274-235350. E-mail address: [email protected] (H.G.M. Edwards)

applied preservatives. The keratins are a group of structural fibrous proteins which occur in man as the ectodermal tissues, hair, nail and epidermis (stratum corneum), whereas in other mammals, birds and reptiles they are also found in claws, beaks, scales and feathers. In contrast to other structural proteins such as collagen and elastin found in bones and teeth, the keratins contain the sulfur-rich amino-acid, cysteine, and their resistance towards proteolytic enzymes is ascribed to the presence of disulfide links (–S–S–) between cysteine residues forming cystine. Local structural conformation studies [1–3] of the disulfide links in keratins have provided a rich source of detail in the Raman spectra of these

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materials through observation of the n(SS) stretching band(s) near 500 cm − 1. In our own laboratories, we have studied the Raman spectra of the human keratotic biopolymers, skin, hair and nail [4] and have reported studies of the degradation and modification of skin tissue in the form of callus, veruccae and psoriasis [5– 7]; the former arises in load-bearing areas of the body such as the soles of the feet, where the stratum corneum is thickened, whereas the latter is an extremely painful affliction which occurs over widespread regions of the body accompanied by sores and skin lesions. In studies of callus and psoriatic skin materials, it has been demonstrated clearly from Raman spectroscopic studies that delipidization mechanisms are responsible for both conditions [8]. From a previous study [9,10] of the stratum corneum and dermis of the ‘Ice-man’, a 5200-year old sample of mummified human keratotic tissue recovered from glacial interment at an elevation of 3200 m in the Austrian Tyrol, it was shown that, whereas the dermal tissue was identical with that of modern freeze-dried samples, the stratum corneum was different in the two cases; the lipid conformation was essentially intact, with some evidence for oxidation of the n(CC) features, but in contrast the proteinaceous component had been degraded, possibly as a result of bacterial action over a short period before mummification took place. A major problem facing spectroscopists is the shortage of specimens of keratotic soft tissue from humans or animals which have survived bacteriological decay processes, and which have not been modified by the application of exogeneous chemicals. The extensive collections of Egyptian mummies which exist in museums, for example, have been preserved with varying degrees of success through the use of resins, oils, bitumens and desiccants such as natron; any identifiable changes in the molecular composition of the tissues are hence not solely attributable to survival capabilities of their different components. In this respect, the recent discovery of ice-mummies affords a possibly unique source of tissue specimens for spectroscopic analysis; however, other considerations, including political and national sensitivity,

normally apply which mean that specimens cannot be accessed for study. Thus, to date only the Alpine Ice-man and the Qilakitsoq human icemummies have been studied spectroscopically. There are similarly few examples of ice-mummified animals known. In the present study, we report for the first time the FT-Raman spectra of tissues from a mummified penguin from the Antarctic region. The survival of these remains in a xeric Antarctic climate is characteristic, since bacterial and chemical degradation which would normally occur in more temperate latitudes are minimised by desiccation. Four tissues have been examined in detail, namely the claw, skin and feathers, which are keratotic materials, and the bones which contain protein (collagen, elastin) in an inorganic hydroxyapatite matrix [11,12]. It is valuable to compare the spectra of the mummified animal remains with those from other archaeological tissue to see if the survival of the protein has been successful. It must be stressed here that the objective of the present study is the characterisation of tissue changes in ice-mummified specimens, with a comparison being made between human and animal specimens; this comparison focusses attention on the survivability of tissue components under sub-zero environments — it is not intended to be a comparison per se between modern and ancient avian specimens. The value of the non-destructive capability of FT-Raman spectroscopy for the examination of biological archaeological materials including interred remains and artefacts, including bone, human hair and animal horn has been already illustrated in recent work from our laboratories [13–15].

2. Experimental

2.1. Samples During the 1992–93 Antarctic summer season, a British Antarctic Survey geological field party on Alexander Island found a mummified penguin on Offset Ridge, Alexander Island (71° 41%S, 68° 42%W) on North-facing rock at an altitude of 600 m between the Venus and Neptune Glaciers inland from the George VI Sound ice-shelf, locality

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ref. KG.4604, 15 km W of Triton Point, Mount Olrig–Gateway Pass [16]. The remains were found 5 miles inland from the coast of the iceshelf and the nearest open water is over 110 miles to the north. The region has a continental Antarctic climatic regime with strong katabatic winds. The specimen is a mature Adelie penguin (Plate 1a), intact and mummified with extensive green colonization of the white breast feathers by microalgae. The remains were stored and transported at − 20°C for microbiological and spectroscopic study at BAS, Cambridge. Although mummified seals are found frequently in the McMurdo Dry Valleys region of Antarctica as a result of their disorientation leading them inland [17], the penguin specimen is unique both in itself and in its location over 110 miles from open water. Also, current international legislation now prohibits the removal of such specimens so, such a unique specimen in a remarkable state of presentation provides an excellent opportunity to study the constraints of biodeterioration of homokilothermic tissue in a cold desert environment and especially the results of localised colonization by organisms in a nitrogen-limited habitat.

2.2. FT-Raman spectroscopic analysis FT-Raman spectra were recorded using a Bruker IFS66 spectrometer with FRA106 Raman module attachment. The excitation source was a Nd–YAG laser operating at 1064 nm with a nominal power of 100 mW focussed to a 100 mm diameter spot at the tissue sample. Typically, 2000 scans were collected at 4 cm − 1 spectral resolution over the wavenumber range, 100–3500 cm − 1. The observed band wavenumbers, calibrated against the internal laser frequency, were correct to better than 9 1 cm − 1 and the spectral intensities were subjected to a white-light correction. Five replicates were obtained from each tissue sample; the variation from one sampling spot to another for the same tissue was within about 9 5% of band intensity, for sharp, spectral features.

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3. Results and discussion Stack-plotted FT-Raman spectra of the mummified claw, skin, bone and feathers (Plate 1b) are shown in Figs. 1–4 over the wavenumber ranges, 100–1800 and 2600–3500 cm − 1 with examples from contemporary equivalents for comparison. In the case of the mummified skin sample, the Raman spectrum of the Alpine Ice-man is also shown. The key molecular assignments and listings of observed band wavenumbers are shown in Tables 1–4. In Table 1 the spectral bands from the penguin bone sample are compared with the Raman spectra of bone from an artefact fashioned from cow bone and a modern child’s exfoliate deciduous tooth [12,18]. Table 2 gives a comparison of the mummified penguin claw and spectra recorded of a human nail and cat’s claw. Table 3 compares the mummified skin from the penguin with specimens from the Alpine ice-man and contemporary freeze-dried human skin. Table 4 gives the wavenumbers and spectroscopic assignments of penguin feathers in comparison with contemporary pigeon feather specimens and a bovine keratin extract. The good quality of the Raman spectra recorded from the ice-mummified samples may be contrasted with the Raman spectra of other archaeological biomaterials from warmer locations. These often display fluorescence backgrounds and thermal effects ascribed to sample discoloration. The high signal-to-noise levels obtained from the mummified penguin material facilitate comparison with contemporary biomaterials such as claws, horn, bone, tooth and skin [19]. Fig. 1 compares the Raman spectra of the bone from the mummified penguin foot, with specimens of cow bone and contemporary deciduous human tooth. The spectra are presented in two wavenumber regions, namely 2600–3600 cm − 1 and 300– 1800 cm − 1 for ease of comparison of the n(CH) modes and skeletal or functionality modes, respectively. Fig. 2 contrasts similarly the penguin claw, a contemporary cat’s claw and human nail. Fig. 3 compares Raman spectra of the mummified skin of the penguin with that of the Alpine iceman, a rare example of mummified human skin.

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Plate 1. a. Mummified penguin remains from Antarctic ‘cold desert’ region. b. Close-up of foot from mummified remains in Plate 1a, showing bone, skin and keratotic claw features.

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Fig. 1. FT-Raman spectra of: top, penguin tarsal bone; middle, contemporary bovine bone; and bottom, contemporary deciduous human tooth; 1064 nm excitation, 4 cm − 1 spectral resolution, 2000 scans accumulated. a, wavenumber region, 2600 – 3600 cm − 1; b, wavenumber region, 300–1800 cm − 1.

These are compared with the corresponding spectrum for contemporary desiccated human stratum corneum skin. In Fig. 4, the Raman spectra of penguin feathers are compared with those of con-

temporary pigeon feathers and a bovine keratin extract. Fig. 1 and Table 1 show the close similarities between the Raman spectra of the mummified

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Fig. 2. FT-Raman spectra of: top, penguin claw; middle, contemporary human nail; and bottom, contemporary cat claw; conditions as for Fig. 1. a, wavenumber region 2600–3600 cm − 1; b, wavenumber region, 300 – 1800 cm − 1.

penguin foot and those of contemporary animal bone; the spectrum of human tooth is also included here because of the content of collagen and of inorganic phosphatic matrix, hydroxyapatite, in the tooth. The interesting result from Table 1

and Fig. 1 is the similarity in band wavenumber positions of mummified calciferous tissue and its contemporary counterparts. In the n(CH) stretching region the overall band profile is very similar for all three specimens, but the 3060 cm − 1 feature

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Fig. 3. FT-Raman spectra of: top contemporary human skin; middle, mummified skin of the Alpine iceman; and bottom, mummified penguin skin; conditions as for Fig. 1, except for the spectrum of the Alpine iceman, which was obtained from 20 000 accumulations. a, wavenumber region 2600–3600 cm − 1; b, wavenumber region, 300 – 1800 cm − 1.

ascribed to n(CCH) olefinic stretching is perhaps rather reduced in intensity for the mummified tissue. This can be explained by bacterial or aerial oxidation of lipid-like CC moieties in the ancient

tissue. However, a significant proportion of these moieties must still remain intact in the mummified specimen. The mummified avian tissue also has a broad feature centred near 3200 cm − 1 which can

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Fig. 4. FT-Raman spectra of: top, penguin feather; middle, contemporary pigeon feather; and bottom, sample of extracted bovine keratin powder; conditions as for Fig. 1. a, wavenumber region 2600 – 3600 cm − 1; b, wavenumber region, 300 – 1800 cm − 1.

be ascribed to n(OH) and n(NH) hydrogenbonded modes; this is stronger in the avian tissue than it is in its counterparts. A significant difference between the mummified tissue and its contemporary analogues is observed

in the n(CONH) amide I stretching region; instead of a medium intensity, asymmetric band at 1665 cm − 1 characteristic of helical protein chain conformations, we now observe two bands of medium intensity at 1670 and 1602 cm − 1. This

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Table 1 Wavenumbers and molecular assignments of FT-Raman spectra of bone samples of penguin foot bone, cow bone and contemporary tootha Wavenumbers, n (cm−1) Penguin bone (this work) 3224 3060 2973 2937 2886 1670

mw, br vw mw, sh s mw, sh m, br

1602 1449 1426 1270

m, br m w, sh mw, br

1072 m 1040 w 1003 w 963 855 818 589

s w w mw, br

460 w, sh 432 m a

Molecular assignment Bovine bone Ref. [13]

3061 w 2973 mw, sh 2940 s 2884, mw, sh 1665 m 1647 mw 1451 1427 1275 1246 1070

mw w, sh mw mw m

1042 w, sh 1003 w 959 s 855 vw 607 w 585 m 462 mw, sh 430 m

Human tooth Ref. [11]

2973 2941 2886 1664

w, sh m mw mw, br

1451 mw 1260 w 1248 w 1070 mw 1045 mw, br 1003 w 960 s 855 vw 607 w 591 mw 581 mw 430 mw

n(NH) n(CCH) olefinic n(CH3) symmetric n(CH2) symmetric n(CH2) asymmetric n(CONH) amide I n(CC) n(CO); n(CC) aromatic d(CH2) scissors d(CH3) d(NH) d(NH) amide III disordered 3− n(CO), CO2− asymmetric 3 ; n(PO), PO4 n(PO); n(CC) n(PO), out-of-plane, phosphate n(CC) aromatic ring stretching n(PO) symmetric d(CCH) aromatic d(PO) phosphate d(PO) phosphate d(PO) phosphate d(PO) phosphate n(SiO) silicate

n, stretching; d, deformation; r, rocking; s, strong; m, medium; w, weak; v, very; sh, shoulder; br, broad.

situation is held to be consistent with a degeneration of organic tissue in the penguin bone. This effect is also evident in the Raman spectra of the skin. Another significant difference between the bone specimens is seen in the ratio of the organic– inorganic components. In the mummified specimen this ratio is significantly larger than in contemporary bone or tooth, i.e. the n(PO) symmetric stretching mode of the phosphatic matrix is of lower intensity relative to the organic keratotic content of the specimen. Since we cannot envisage a process whereby the phosphatic matrix is degraded relative to the keratin, we conclude that the protein content has not been destroyed, but merely changed in conformation. The retention of the n(PO) and d(PO2) modes in the mummified specimen is also noteworthy as these are sensitive to the incorporation of inorganic materials into the phosphatic matrix; in this case we cannot defin-

itely ascribe a feature due to a n(SiO) of a silicaceous incorporation into the inorganic matrix. The onset of a fossilisation process is not evident in the penguin bone but has been noted in the spectra of mediaeval teeth, Romano–British human bone and in the Raman spectra of dinosaur and prehistoric shark teeth [11]. This confirms the unique preservation of the Antarctic material over others which have been studied to date. Examination of the spectra of the claw from the mummified penguin and contemporary animal claw and human nail shows a similar pattern of behaviour to that of the bone specimen. However, claw is a pure keratotic tissue and not a keratotic component encased in an inorganic phosphatic matrix as in the case of bone. The spectra of penguin claw and contemporary cat’s claw have some similarity, both in band intensity and in wavenumbers. However, we can conclude that

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Table 2 Wavenumbers and molecular assignments of FT-Raman spectra of penguin claw, cat claw and human naila Wavenumbers, n (cm−1) Penguin claw (this work)

Human nail Ref. [4]

Cat claw Ref. [3]

Molecular assignment

3059 2967 2936 2875 2729

3060 2968 2931 2874 2733 1727 1666 1654

3061 2968 2932 2875 2730

n(CCH) olefinic n(CH3) symmetric n(CH2) symmetric n(CH2) asymmetric n(CH3–C) n(CO) n(CONH) amide I n(CONH) amide I, a-helix n(CONH) amide I, disorder n(CO); n(CC) n(CC) aromatic d(NH), n(CN) amide II d(CH2) scissors d(CH3) d(CH) d(CH2) d(NH) n(CC) n(CC) n(CC) skeletal, trans conformation n(CC) skeletal, random conformation n(CC) skeletal, cis configuration n(CC) aromatic ring r(CH3); d(CCH) olefinic r(CH3) terminal, n(CC), a-helix d(CCH) aromatic d(CCH) n(CS) n(CS) n(SS)

w m, sh ms mw w

1669 mw, br

w m, sh s mw w mw ms mw

w m, sh s mw w

1657 ms 1615 m

1601 mw 1585 ms 1448 1421 1340 1316 1268

m mw w w mw, br

1449 ms 1341 m 1310 m

1127 mw 1087 mw 1031 w 1003 mw 963 mw – 852 w

1003 m 935 858 mw

644 vw –

643 w 622 w 513 m, br

427 mw, br

–

a

1555 w 1448 ms 1416 mw 1338 m 1316 mw 1265 mw, br 1207 w 1176 w 1126 w 1086 w 1030 w 1003 w 956 vw 936 w 852 mw 829 mw, sh 643 mw 621 w 508 m, br 472 vw –

n(SiO) silicate

n, stretching; d, deformation; r, rocking; s, strong; m, medium; w, weak; v, very; sh, shoulder; br, broad.

there has again been significant keratotic degradation in penguin during seasonal moistening with snow-melt. The presence of a silicaceous incorporation at 427 cm − 1 is now noticed in the upper spectrum of Fig. 2b, which indicates the onset of matrix incorporation, unlike the situation which was found in the bone sample. Comparison of the skin samples in Table 3 and Fig. 3 clearly show that there is some similarity between the mummified human skin and the penguin but both differ from contemporary human skin, the outer region of which, the stratum

corneum, is a mixture of keratins, lipids and water. The penguin skin may be seen to be only partially degraded compared with the Iceman as some olefinic n(CCH) modes still remain, e.g. the band at 3060 cm − 1, and the d(CH2) modes near 1427 and 1337 cm − 1 although broad, are still present. The strong aliphatic n(CH) lipid modes in the contemporary and ancient human skin are clearly not replicated in the avian specimen. Similarities between the mummified penguin and mummified human skin samples indicate that the degradation suffered by the Iceman specimen has

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Table 3 Wavenumbers and molecular assignments of FT-Raman spectra of mummified skin samples from Antarctic penguin and Alpine ice-mana Wavenumbers, n (cm−1) Penguin skin (this work) 3170 3053 2970 2932 2884

vw w mw, sh ms m, sh

2850 w, sh 1653 m 1602 mw 1448 1427 1337 1297

m mw, sh w mw, br

1127 w

1030 w 1003 w 940 w

Molecular assignment Ice-man skin Ref. [9,10]

Contemporary skin Ref. [4–6]

3060 vw 2932 2895 2873 2853 1655 1597 1460 1441

m m, br m m w, br m, br w, sh ms

1296 ms 1128 1085 1062 1028 1001

w w mw w w

2893 2873 2852 1652 1602

ms m m s w

1438 s

1296 1274 1126 1082 1062 1031 1003

ms mw mw mw mw mw mw

644 w 623 w 526 mw 438 w a

n(NH) amide n(C=CH) olefinic n(CH3) symmetric n(CH2) symmetric n(CH2) asymmetric n(CH2) n(CH2) asymmetric n(CONH) amide I n(CONH); n(C =C) d(CH2) d(CH2) d(CH2) d(CH2) d(NH) amide d(NH) n(CC) n(CC) n(CC) n(CC) n(CC) aromatic r(CH2) n(CS) n(CS) n(SS) n(SiO) silicate

n, stretching; d, deformation; r, rocking; s, strong; m, medium; w, weak; v, very; sh, shoulder; br, broad.

also occurred for the Antarctic avian specimen, but to a lesser extent and this perhaps reflects a differential exposure of the mummified specimens to the environment. Again, it is the keratotic component which has been affected more significantly and which is therefore inferred to be more labile. Comparison of the avian feathers of the mummified penguin and those of a contemporary pigeon [3] show a close resemblance (Fig. 4). In particular, the top and middle spectra in Fig. 4b, which gives the 300 – 1800 cm − 1 spectral regions of the penguin and pigeon feathers, respectively, are seen to be remarkably similar; it is to be concluded that there has been but little degradation of the feather keratin during ice-mummification in the Antarctic. Both spectra are similar to

the spectrum of bovine keratin which is also shown in Table 4 and Fig. 4 for comparison. The similarities between the penguin and contemporary feather and extracted keratinous material again highlight the minimal degradation which has occurred under Antarctic conditions. A point worthy of comment is the observation of the broad Raman bands in the neighbourhood of 500 cm − 1, assigned to n(SS) stretching of the cystine CSSC links. Some conformational information is obtained from these bands [1–3] in terms of trans and gauche structures. Hence, the 526 cm − 1 band in contemporary skin probably arises from a gauche–gauche–trans conformation, whereas the features in the avian feather specimens in Table 4 are best described on the basis of a gauche– gauche–gauche conformation.

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Table 4 Wavenumbers and molecular assignments of FT-Raman spectra of penguin feathers and contemporary avian feathers, with keratin extract for comparisona Wavenumbers, n (cm−1)

Molecular assignments

Penguin feather (this work)

Pigeon feather Ref. [4]

Keratin extract (bovine) (this work)

3056 2973 mw, sh

3060 w

3060 w 2972 mw, sh

2933 ms 2886 mw, sh 1666 ms 1608 mw, sh

2960 2933 2880 1665 1615 1608 1550

mw ms m ms mw mw w

"

2932 ms 2876 m, sh 1669 ms

n(C= CH) olefinic n(CH3) symmetric n(CH3) n(CH2) symmetric n(CH2) asymmetric n(CONH) amide I

1616 mw

n(CONH); n(C =C)

1524 mw 1451 s 1427 w, sh

1340 1315 1290 1246

w w w m, br

1157 mw 1126 w 1096 vw 1032 vw 1004 m 958 w 881 vw 855 w 829 vw 752 w

515 mw, br 419 w, br

a

1508 mw 1449 s 1414 mw 1395 w 1342 w 1317 w 1270 w 1241 mw 1210 w 1174 w 1154 1126 mw 1095 w 1060 w 1030 w 1003 m 956 w 880 w 853 w 828 w 756 w 643 621 512 mw

1448 s 1427 w, sh

1340 w 1317 w 1244 m, br 1174 w 1126 w 1090 w, br 1032 w 1003 m 958 w 890 vw 853 w 829 w 758 w

512 mw, br 410 w, br

d(NH), n(CN) n(C= C) carotenoid n(C=C) carotenoid n(CH2) n(CH2) n(CH2) d(CH3) symmetric n(CH2) d(CH2) n(CN) amide III d(CH2) wagging d(CH2) n(CC) n(CC); d(OH) n(CC) skeletal, trans conformation n(CC) skeletal n(CC) skeletal n(CC) skeletal, cis conformation n(CC) aromatic ring r(CH3); d(CCH) r(CH2) d(CCH) aromatic d(CCH) r(CH2) in-phase n(CS) n(CS) n(SS)

n, stretching; d, deformation; r, rocking; s, strong; m, medium; w, weak; v, very; sh, shoulder; br, broad.

In summary, the first Raman spectroscopic analysis of mummified avian tissues have revealed that the bone, claw and skin suffer keratotic degradation with time. This is probably the result of aerial oxidation, resulting in protein damage with consequent change of protein conformation. In contrast, however, there is little observable

effect on the feather keratins. Even low-temperature desiccation of mummified human skin [20] demonstrates only a slow keratotic degeneration. In the present work, we have compared objectively the Raman spectra of ice-mummified human and avian skin; the alpine Iceman was selected for this comparison since his body was

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not clothed on discovery unlike the Qilakitsoq mummies [20] which had been buried with protective clothing. Predictably also, the extent of the degeneration process for the 5200 year old human specimen is significantly greater than that of the avian mummy from the Antarctic, whose age is at present indeterminate. It would be a natural extension of this work to apply FT-Raman spectroscopy to the bone, teeth and nail of ice-mummified humans as an addition to the other techniques used for characterisation of archaeological animal remains.

Acknowledgements We are grateful to Dr S.R.A. Kelly for presenting us with the mummified penguin specimen and to the British Antarctic Survey, Cambridge, for support of the Mars Glacier biological field party of 1992–3.

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[5] H.G.M. Edwards, A.C. Williams, B.W. Barry, J. Mol. Struct. 347 (1995) 379. [6] H.G.M. Edwards, B.W. Barry, A.C. Williams, J. Mol. Struct. 347 (1995) 379. [7] E.E. Lawson, H.G.M. Edwards, A.C. Williams, B.W. Barry, J. Drug Target. 5 (1998) 343. [8] H.G.M. Edwards, B.W. Barry, A.C. Williams, in: R.S. Armstrong (Ed.), Proceedings of the First Australian Conference on Vibrational Spectroscopy, University Press, Sydney, Sydney, Australia, 1995, p. 26. [9] A.C. Williams, H.G.M. Edwards, B.W. Barry, Biochim. Biophys. Acta 1246 (1995) 98. [10] H.G.M. Edwards, D.W. Farwell, A.C. Williams, B.W. Barry, F. Rull, J. Chem. Soc. Faraday Trans. 91 (1995) 3883. [11] H.G.M. Edwards, D.W. Farwell, A.C. Williams, Biospectroscopy 1 (1995) 29. [12] M.T. Kirchner, H.G.M. Edwards, D. Lucy, A.M. Pollard, J. Raman Spectrosc. 28 (1997) 171. [13] H.G.M. Edwards, D.W. Farwell, C.A. Roberts, A.C. Williams, in: J.E. Bertie, H. Wieser (Eds.), Proceedings of the Ninth International Conference on Fourier-Transform Spectroscopy, SPIE Publishing, Washington, Calgary, Canada, 2089, 1993, p. 256. [14] H.G.M. Edwards, D.W. Farwell, Spectrochim. Acta Part A 51 (1995) 2073. [15] H.G.M. Edwards, D.W. Farwell, T. Seddon, J.K.F. Tait, J. Raman Spectros. 26 (1995) 623. [16] S.R.A. Kelly, Personal communication. [17] W. Dort, The mummified seals of Southern Victoria Land, Antarctica, in: B.C. Parker (Ed.), Terrestrial Biology III, American Geophysical Union, Washington. [18] H.G.M. Edwards, D.W. Farwell, J.M. Holder, E.E. Lawson, Spectrochim. Acta Part A 53 (1997) 2403. [19] M. Gniadecka, O. Faurskov Nielsen, D.H. Christensen, H.C. Wulf, J. Invest. Dermatol. 110 (1998) 393. [20] M. Gniadecka, H.G.M. Edwards, J.P. Hart-Hansen, O.F. Nielsen, D.H. Christensen, S.E. Guillen, H.C. Wulf, J. Raman Spectrosc. 28 (1997) 179.