Micromorphological and chemical elucidation of the degradation mechanisms of birch bark archaeological artefacts
© Orsini et al.; licensee Springer. 2015
Received: 13 June 2014
Accepted: 9 January 2015
Published: 20 January 2015
Since ancient times, the unique properties of birch barks (Betula genus) have made them a material of choice for producing both everyday-life and artistic objects. Yet archaeological birch bark artefacts are rare, and little is known about the chemical transformations undergone by bark (chemically composed mainly of suberin and triterpenes) in archaeological contexts. Understanding the chemical modifications induced by ageing is essential for selecting suitable preservation and conservation approaches. Thus, the main aim of this research is to assess the preservation and state of degradation of archaeological findings made of birch bark: a Neolithic bow case recovered from a melting ice patch in the Bernese Alps (Switzerland) and a waterlogged birch bark vessel discovered at Moossee Lake (Canton of Bern, Switzerland).
Scanning electron microscopy (SEM) and gas chromatography/mass spectrometry (GC/MS) were used to obtain information at micro-morphological and molecular levels on the state of degradation of the birch bark findings. GC/MS analysis followed two different sample preparations, alkaline hydrolysis and solvent extraction, in order to investigate respectively the hydrolysable and soluble constituents, and to test whether part of the suberin structure was depolymerised by the long period of burial.
Results and conclusions
SEM investigations on archaeological birch bark samples have shown that the extent of degradation of the microstructure is much higher in waterlogged birch bark than in birch bark preserved in ice. GC/MS analysis revealed that at a molecular level, the birch bark was quite well preserved. In both the archaeological environments, ice patch and lake water, various reactions had taken place leading to the depletion of reactive and sensitive compounds such as unsaturated acids and epoxy-compounds. In addition, archaeological birch bark had undergone depolymerization and oxidation reactions leading to the appearance of free suberin monomers and of oxidised triterpenes (betulone and lupenone). GC/MS data also seems to suggest that the birch bark preserved in the waterlogged site had a more pronounced degradation both in terms of oxidation and depolymerisation.
Birch bark is obtained from several Eurasian and North American birch trees of the genus Betula. The unique physical properties of these barks have made them the material of choice since ancient times for manufacturing artefacts both for the needs of daily life and artistic purposes. Birch bark can withstand changes in shape without breaking, is particularly waterproof, and has biocidal and antioxidant properties. These last features made it useful to past people for food preservation and pharmaceutical purposes. In addition, birch bark was largely used for the manufacture of tar [1,2]. Despite a presumable extensive utilization, at archaeological sites objects made out of birch bark are quite rare. Birch bark objects are preserved for long periods of time only under peculiar burial conditions, such as very dry conditions in arid or cold climates, or wet conditions in sediments and glaciers. In fact, birch bark artefacts used by Mesolithic hunter-gatherers were recovered in Germany and objects made by pre-contact hunter-gatherers were recovered from plateau contexts in Canada and at Star Carr (Yorkshire, UK) [3,4]. Horse-mounted nomads (7th to 14th centuries) from Mongolia also used birch bark artefacts .
As reported in the literature [6-13], birch bark contains especially high amounts of a waxy hydrophobic substance, called suberin, along with triterpenoids. Chemically the suberin is a bio-polyester and it has been studied and characterised using depolymerisation procedures. Several wet chemical techniques have been used for the depolymerisation of suberin by cleavage of the ester bonds, in order to analyse its monomeric sub-units. The depolymerisation pre-treatment is an essential step for the chemical characterization of this material. The depolymerisation is based on chemical reactions achieving the cleavage of ester bonds: alkaline hydrolysis, trans-esterification or reductive cleavage have been used. The most common procedures are the ester cleavage through alkaline methanolysis (NaOMe or CaO in MeOH) and alkaline hydrolysis using hydroalcoholic KOH [6,14-16]. The resulting depolymerisation products, following derivatisation reaction, can be analyzed and identified by gas chromatography coupled to mass spectrometry (GC/MS). The main suberin monomers are long-chain epoxy acids, long-chain α,ω-diacids, and long-chain ω-hydroxyacids, whose yield and variety are dependent on hydrolysis conditions [11,14]. Betulin and lupeol together with small amounts of lupenone, betulinic acid and betulone are known to be the main characteristic triterpenoids present in birch bark [9,10,12,13,17].
Little is known about the chemical transformations undergone by bark in archaeological contexts, nor about the chemical composition of the aged materials. To the best of our knowledge, this is the first analytical investigation done on such a subject. The study of the degradation processes undergone by suberin and triterpenes, and of the modification induced by ageing and different burial conditions in archaeological birch bark is essential so that optimal preservation and conservation strategies can be identified.
List of the samples used in this study
Location of find
Reference birch bark
Canton Valais, Switzerland
Birch bark, bow case (middle section), ice patch
Schnidejoch pass, Bernese Alps, Switzerland
Birch bark, bow case (lower section), ice patch
Schnidejoch pass, Bernese Alps, Switzerland
Birch bark, vessel, waterlogged site
Moossee Lake, Canton Bern, Switzerland
The anatomy and the morphology of the cell structure of the birch bark objects were observed by scanning electron microscopy (SEM). Gas chromatography/mass spectrometry (GC/MS) after alkaline hydrolysis, solvent extraction and trimethylsilylation was used to investigate the hydrolysable and soluble constituents and thus to determine the monomer composition of the suberin and the triterpenoid distribution. In addition, to test whether part of the suberin structure was depolymerised by the long period of burial, an analysis of the samples by direct extraction of the organic material with CH2Cl2/MeOH, without any alkaline treatments, was also performed.
Results and discussion
Suberized cells consist typically of a pectin rich middle lamellar layer and of a cellulose sometimes-lignified primary wall. The suberin occurs as a distinct lamella deposited on the inner face of the primary cell wall . The cell walls are stable, but the cell layers are becoming tangential separated due to natural weakness. The macroscopic separation of layers (delamination) is given by the different stability of the cell walls and appears in the area of the thin-walled cells or along the boundary between the thin and thick walled cells (Figure 2b). The cells of the cork have an elongated polygonal shape, measure between 60 to 120 μm, and rest horizontally.
In the sample from the waterlogged archaeological vessel (Sample 4), the cell walls have become very brittle and fragile, and many ruptures have occurred. In addition, the resin that fills the large birch cells, has almost completely disappeared (Figure 3c).
To get a clear picture on the chemical transformation mechanisms involving birch bark in archaeological contexts, a reference sample was comparatively studied. The obtained GC/MS profiles were in agreement with the literature [14-16,21,22] and enabled us to interpret and to critically evaluate the data obtained from the analysis of archaeological samples.
Compounds identified by GC/MS in reference and in archaeological birch bark
303, 259, 217, 187
312, 297, 282, 267, 253
317, 273, 201
331, 287, 215
338, 323, 308, 293
308, 293, 249, 219
313, 269, 145, 129, 117
338, 323, 308, 293
337, 293, 145, 129, 117
339, 295, 145, 129, 117
341, 297, 145, 129, 117
401, 385, 311, 217, 204, 147
384, 369, 145, 129, 117
α,ω- C16 dioic acid
415, 299, 217, 204, 147
442, 427, 411, 337, 217, 204, 147
isomer of ω-hydroxy-C18:1 acid
442, 427, 411, 337, 217, 204, 147
489, 383, 331, 317, 303, 289, 275
429, 413, 339, 217, 204, 147
412, 397, 145, 129, 117
α,ω- C18:1 dioic acid
456, 441, 366, 276
α,ω- C18 dioic acid
458, 443, 368, 278
515, 343, 329
9,10-dihydroxy- C18:1 acid
515, 425, 343, 329, 315
470, 455, 439, 365, 217, 204, 147
9,10-dihydroxy- C18 acid
517, 443,359, 345, 331, 303
457, 441, 367, 217, 204, 147
9-methoxy-10,18-dihydroxy- C18 acid*
547, 332, 317, 303
9,10,18- trihydroxy- C18 acid
605, 471, 390, 317, 303
9-methoxy-10-hydroxy- C18-1,18-dioic acid*
551,515, 441, 317, 303
9,10-dihydroxy- C18-1,18-dioic acid
619, 545, 373, 317, 303
485, 469, 395, 217, 204
α,ω- C22 dioic acid
499, 383, 217, 204
426, 411, 315, 207, 189
498, 483, 393, 369, 203, 189
422, 409, 203, 189
596, 496, 483, 393, 203, 189
600, 585, 512, 483, 203, 189
424, 409, 313, 245, 205
The comparison between the quantitative profile of the two archaeological samples conserved in the ice patch (Samples 2 and 3) reveals a very similar composition of the suberin fraction. The few differences between Samples 2 and 3 refer to the abundance of 9-methoxy-10,18-dihydroxy-C18 acid (peak 27), 9,10,18-trihydroxy-C18 acid (peak 28), 9-methoxy-10-hydroxy-C18-α,ω-dicarboxylic acid (peak 29) and ω-hydroxy-C22 acid (peak 31). The smaller amount of these compounds in Sample 3 seems to indicate a major degradation in the lower section of the archaeological object conserved in ice compared to the middle section of the same object.
% of free suberin-derived compounds in the extracts of samples 1, 2, 3 and 4
Suberin component (%)
ω-OH C18:1 A (15)
α,ω-C18:1 DIA (20)
ω-OH C20 A (26)
9,10,18-tri-OH C18 A (28)
ω-OH C22 A (31)
α,ω-C22 DIA (32)
Note that Sample 4, which was collected from the waterlogged site, shows a higher percentage of free suberin-derived compounds than the samples collected from the ice patch. This suggests a more degraded structure for Sample 4, as also highlighted by the results reported above and obtained after hydrolysis of the samples.
Materials and methods
All solvents were Carlo Erba (Milan, Italy) pesticide analysis grade. n-Hexadecane (internal standard, IS1), tridecanoic acid, (internal standard, IS2), hydrochloric acid (HCl), potassium hydroxide (KOH) and N,O-bis(trimethyl)silyltrifluoro-acetamide (BSTFA) containing 1% trimethylchlorosilane, were purchased from Sigma–Aldrich (Milan, Italy).
Reference and archaeological samples
Two Neolithic artefacts made of birch bark were investigated: a bow case recovered in a ice parch and a vessel recovered in a waterlogged environment (Figure 1). The bow case measures almost 2 meter in length and consists of three parts: an upper part that is the cover of the bow case, and a middle and a lower parts that are the body of the case. Originally the bow case body - middle and lower sections - consisted of one element, that has been separated either through usage or burial period in the ice. The bark used as reference material was collected from a Betula pendula tree in the district of Regensburg (Bavaria, Germany) in April 2011. Table 1 reports the list of reference and archaeological samples used in this study as well as information on the archaeological objects from which the samples were collected. Dendrochronology and Bayesian modelling of about 25 radiocarbon dates were used to calibrate the radio carbon dating.
Comparative visual examination of the microstructure of the radial section was carried out on each sample, using a Philips XL30 SEM scanning electron microscope. Neolithic birch bark samples from a melting ice patch and from a waterlogged burial site were compared with reference samples of 1-year-old birch bark.
Atmospheric freeze-drying at −20°C was used to remove the water from the samples. Samples were manually cut with a razor blade, and sections were mounted with conductive graphite adhesive (Leit-C after Göcke, Plano, Wetzlar, Germany) on an aluminium specimen stub. A conductive metal coating (AU) was applied by sputtering. The SEM examinations were carried out at a voltage of 10 kV and high vacuum (3 × 10−5 mbar).
Analysis of polymerised and soluble components
Sample (3–5 mg) was subjected to alkaline hydrolysis [23,24] by adding 1 mL of methanolic KOH (KOH in CH3OH (10 wt%)/KOH in H2O (10 wt%), 2:3), and heating at 60°C for 3 h. After hydrolysis, neutral organic components were extracted with n-hexane (3× 500 μL) and, after acidification with hydrochloric acid (10 M; to pH 2), the acidic organic components were extracted from the hydrolysate with diethyl ether (3× 500 μL). Aliquots of both fractions were evaporated to dryness under a gentle stream of nitrogen and subjected to trimethylsilylation. This was achieved by mixing the dried aliquots with an internal standard solution (5 μL of tridecanoic acid solution, 140 μg g−1) and derivatising with 20 μL of BSTFA (at 60°C, 30 min), using 150 μL iso-octane as the solvent. After adding 10 μL of n-hexadecane solution (80 μg g−1) as an internal standard for the injection, 2 μL of the solution were analyzed by GC/MS.
Analysis of extractable components
Sample (3–5 mg) was subjected to extraction in order to isolate the non-networked and soluble compounds from the birch bark. The samples were extracted three times with 500 μl of CH2Cl2/MeOH 3:1 (v/v) in an ultrasonic bath at 60°C . The extracts were dried under nitrogen and then subjected to derivatization with BSTFA for GC/MS analysis.
The gas chromatograph system 6890 N (Agilent Technologies, Palo Alto, CA, USA) was coupled with a 5973 mass selective detector (Agilent Technologies, Palo Alto, CA, USA) single-quadrupole mass spectrometer. For the gas chromatographic separation, an HP-5MS fused silica capillary column (5%diphenyl-95% dimethyl-polysiloxane, 30 m × 0.25 mm i.d., J&W Scientific Agilent Technologies, USA) with a de-activated silica pre-column (2 m × 0.32 mm i.d., J&W Scientific Agilent Technologies, USA) was used. The split-splitless injector was used in splitless mode at 320°C. The GC/MS parameters for the analysis of the different fractions were as follows: 80°C isothermal for 2 min, 10°C min−1 up 200°C and isothermal for 4 min, 6°C min−1 up 280°C and isothermal for 40 min. The carrier gas (He, purity 99.9995%) was used in the constant flow mode at 1.2 mL min−1.
Peak assignments were performed using mass spectra interpretation, comparison with mass spectral libraries (NIST 2.0), and with published mass spectra.
Scanning electron microscopy (SEM) and gas chromatography/mass spectrometry (GC/MS) were used to obtain information at a micro-morphological and molecular level on the preservation and the state of degradation of various birch bark archaeological findings.
Our SEM investigations on archaeological birch bark samples revealed that the extent of degradation of the microstructure was much higher in waterlogged birch bark than on birch bark preserved in ice. However, the two samples from birch bark bow case preserved in the same context of the ice patch, presented significant differences in deterioration at the cell level. In the sample collected from the lower area of the bow case, no evidence of degradation was visible; the cell walls were stable and intact. The sample collected from the middle section, however presented a significant degradation of thin non-suberized cell walls. This involves a great risk of macroscopical separation of the layers (delamination) and creates specific needs in terms of the conservation methodology. The reasons for the different state of preservation of the bow case is still unknown. It could be due to the different qualities of the raw material or repairs (newer parts), or differences in the burial conditions in the ice patch.
GC/MS analysis following two different sample preparations, alkaline hydrolysis and solvent extraction, respectively, was used to investigate the hydrolysable and soluble constituents and, to test whether part of the suberin structure was depolymerised by the long period of burial. GC/MS revealed that at a molecular level all the birch bark objects seemed to be quite well preserved. In the archaeological environments considered, various reactions had taken place leading to the depletion of more reactive and sensitive compounds such as unsaturated acids and epoxy-compounds. The archaeological birch bark underwent depolymerization and oxidation reactions leading to the appearance of free suberin monomers, and of oxidised triterpenes such as betulone and lupenone. In addition, GC/MS data seems to suggest that the birch bark preserved in the waterlogged site had a more pronounced degradation both in terms of oxidation and of depolymerisation. In fact, the presence of water may have favoured the hydrolysis reaction, i.e. depolymerisation, and the depletion of compounds bearing sensitive groups such as the epoxy groups via the opening of the ring. All that discussed above comes from considering the burial environments as the major factor that could have influenced the chemical alteration/degradation of the birch bark. However, we can not exclude that such differences pre-existed to the burial of the samples as both the pre-burial and burial history of the samples is unknown.
If confirmed by the planned analysis of a larger amount of samples, the preservation conditions of the birch bark objects suggest the possibility to perform drying treatments without previous consolidation and to limit consolidation locally avoiding full impregnation.
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