- Email: [email protected]
Organic compounds preserved in a 2.9 million year old stalagmite from the Nullarbor Plain, Australia
Chemical Geology 279 (2010) 101–105
Contents lists available at ScienceDirect
Chemical Geology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c h e m g e o
Research paper
Organic compounds preserved in a 2.9 million year old stalagmite from the Nullarbor Plain, Australia Alison J. Blyth a,b,⁎, Jonathan S. Watson b,a, Jon Woodhead c, John Hellstrom c a b c
Department of Earth and Environmental Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK Planetary and Space Sciences Research Institute, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK School of Earth Sciences, University of Melbourne, VIC 3010, Australia
a r t i c l e
i n f o
Article history: Received 7 June 2010 Received in revised form 29 September 2010 Accepted 1 October 2010 Editor: J. Fein Keywords: Stalagmite Organic matter Lignin Vegetation Nullarbor Climate
a b s t r a c t A 2.9 Ma stalagmite from the Nullarbor Plain, South Australia was analysed for organic components. In contrast to previously analysed stalagmites, no significant lipid record was recovered. However, qualitative TMAH thermochemolysis of the residual organic matter recovered a range of compounds, including a number of phenols previously associated with lignin. Comparison of this sample with a range of modern stalagmite samples from different environments indicated that the phenolic record in stalagmites can reflect the overlying vegetation regime, and has potential in investigating palaeoenvironmental change in this climatically important region. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The Nullarbor Plain in South Australia (Fig. 1) is one of the largest karst regions in the world at ~240,000 km2, and contains a number of remarkable speleothems that have long been of interest to cave scientists in terms of both their great age and unusual dark colouration (Caldwell et al., 1982). The region is also of considerable importance to understanding past climatic changes as it has a strong sensitivity to Southern Ocean climate dynamics, and can be shown to have experienced a very different climate state in the past, with satellite-derived digital terrain data showing clear evidence of ancient river channels and other landforms indicative of higher rainfall. Fossil evidence also indicates that even when the climate was semi-arid, a more diverse vegetation regime was present (Prideaux et al., 2007). The development of stalagmite proxies from this area is of obvious value as they will provide a major southern hemisphere terrestrial archive to complement the regional and global records recovered from marine and ice cores. However, despite the importance of these stalagmites, they remain largely unstudied due to the lack of a reliable dating methodology, with the vast majority of samples being beyond the reach of the U–Th dating
techniques used in other stalagmite research (i.e. older than 500 ka). This issue has recently been resolved by the successful development of a U–Pb chronology (Woodhead et al., 2006), meaning we can begin to explore this potentially extremely valuable palaeoenvironmental archive. The use of organic matter in stalagmites as a palaeoenvironmental proxy is a growing field (for a review, see Blyth et al., 2008), with recent research indicating a connection between lipid biomarker content and both climate (Xie et al., 2003) and overlying vegetation regime (Blyth et al., 2007). Nullarbor stalagmites should be ideal for organic matter analyses as previous research has indicated that their dark colour may be due to unusually high levels of organics (Caldwell et al., 1982). A preliminary experiment with an undated sample showed a lipid biomarker signal comparable in concentration and constituent compounds to that from a modern African cave (Blyth, 2007; Blyth et al., 2008). This work therefore qualitatively analyses organic compounds recovered via a sequential organic extraction from a Nullarbor stalagmite with the aim of establishing the feasibility of organic analyses in these samples, and their potential for use in palaeoenvironmental reconstruction. 2. Methods and materials
⁎ Corresponding author. Present address: Department of Earth and Environmental Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK. Tel.: + 44 1908 659883; fax: + 44 1908 655151. E-mail address: [email protected] (A.J. Blyth). 0009-2541/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.chemgeo.2010.10.006
2.1. Site and sample The Nullarbor Plain is a flat-lying area where shallow marine limestones were exposed 15 million years ago due to a combination of
102
A.J. Blyth et al. / Chemical Geology 279 (2010) 101–105
Fig. 1. Map of Southern Australia showing the location of the cave.
uplift and global lowering of sea level. The current climate is arid to semi-arid, although it is clear from geomorphological evidence that this has not always been the case (Caldwell et al., 1982). The stalagmite analysed (1411-MO6) was collected in 2005 from ‘1411’ cave (lat 31.85; long 127.57), and has been dated via U–Pb techniques to 2.9 ± 0.1 Ma (disequilibrium corrected age), following the protocols laid out in Woodhead et al. (2006). Both the oldest and youngest parts of the stalagmite are within the error range for this date. This sample represents the end of a Pliocene high-humidity phase on the Nullarbor, based upon a geochronological survey of nearly 60 speleothem samples from a variety of caves across the region (Woodhead, unpublished data). Four subsamples of between 5 and 6 g of calcite were taken across the major coloured laminae (see Fig. 2). To remove any potential contamination from the collection or sectioning process, the outer 2 mm of each subsample was removed by digestion in cleaned 1 M hydrochloric acid (HCl); particular care was taken to remove white corrosion layers from the outer surface and from internal hiatuses. The samples were then washed in solvent cleaned ultra-pure MilliQ
Fig. 2. MO6 showing subsample location.
A.J. Blyth et al. / Chemical Geology 279 (2010) 101–105
water to remove excess acid, and successively sonicated in methanol and dichloromethane (DCM).
103
3. Results and discussion 3.1. Lipid results
2.2. Extraction of lipid biomarkers The cleaned samples were fully digested in 1 M HCl, and the solution was boiled under reflux for 2 h to maximise the release of bacterial lipids (Blyth et al., 2006; Huang et al., 2008). The lipids were extracted from the solution and prepared for analysis following the protocols laid out in Blyth et al. (2006). 5ß-Cholanic acid (Aldrich) was added at the reflux stage as an internal standard. The lipids were analysed on an Agilent Technologies 6890 gas chromatograph coupled to a 5973 mass spectrometer. Separation was performed on a DB-5MS column (30 m length, 0.25 mm internal diameter and 0.25 μm film thickness). Helium at a column flow rate of 1.1 ml min− 1 was used as the carrier gas. Injection was at a 10:1 split. The GC oven temperature was held for 1 min at 50 °C and then programmed at 5 °C min− 1 to 310 °C, the final temperature was held for 9 min. On extraction into DCM, a pink colouration of the solvent was apparent in two of the samples (MO6a, and more weakly, MO6d). This degraded overnight, suggesting instability or photosensitivity of the complex. A second sample from these two layers was therefore prepared and extracted as previously mentioned and an aliquot of the coloured solvent was removed and taken to the Mass Spectrometry Service in the Department of Chemistry at University College London, where it was analysed by MALDI-ToF analysis on a Micromass MALDI micro mx. Analysis was performed in both reflectron and linear modes operating in both positive and negative ionisations, covering mass-to-charge ratio from m/z = 700 to 6000 (reflectron mode) to m/ z = 6000–60,000 (linear mode). A number of different matrices were used in the analysis (CHCA (α-cyano-4-hydroxy cinnamic acid), SA (Sinapinic Acid) and DHB (2,5-dihydroxybenzoic acid)) in order to gain any molecular ion information.
2.3. Extraction and analysis of the residual organic matter After lipid extraction, an aliquot of each remaining acidified solution equivalent to 1 g of the original calcite sample was loaded onto a conditioned non-endcapped C18 solid phase extraction cartridge (IST Isolute) to recover the residual organic matter. SPE cartridges were pre-conditioned with hexane, DCM, methanol and 0.01 M HCl. Once the dissolved organic matter had adsorbed on the SPE cartridge any salts were removed by rinsing with 4 ml of 0.01 M HCl, the cartridge was then dried by flushing with helium. The organics were eluted from the column into clean vials with 4 ml of methanol, dried under nitrogen and rediluted in 50 μl of methanol. Approximately 10 μl of the sample in methanol was absorbed onto quartz wool in a quartz pyrolysis tube. The samples were left to dry overnight, before 10 μl of 25% tetramethylammonium hydroxide (TMAH) in methanol was added and allowed to dry overnight. TMAH was added in excess to ensure base conditions during thermochemolysis. Samples were heated (300 °C at a rate of 20 °C ms− 1 then held isothermally for 15 s) in a flow of helium using a CDS Pyroprobe 5000 fitted with a 1500 valve interface (CDS Analytical, Oxford, PA) and coupled to a gas chromatograph–mass spectrometer (GC–MS). GC–MS analysis was carried out using an Agilent Technologies 6890 gas chromatograph coupled to a 5973 mass spectrometer. Separation was performed on a S.G.E. (U.K.) BPX-5 column (30 m length, 0.25 mm internal diameter and 0.25 μm film thickness). Helium at a column flow rate of 1.1 ml min− 1 was used as the carrier gas. Injection was at a 5:1 split and injector temperature was 270 °C. The GC oven temperature was held for 1 min at 50 °C and then programmed at 5 °C min− 1 to 310 °C, the final temperature was held for 9 min.
Although a preliminary undated sample analysed via the same extraction method in 2006 showed a well preserved lipid signal (Blyth, 2007; Blyth et al., 2008), comparatively few lipid biomarkers were recovered from the current samples, with MO6b, MO6c, and MO6d containing only trace amounts of low molecular weight fatty acids below the contamination threshold associated with this method (0.3 μg for C16 n-alkanoic acid; Blyth et al., 2006). MO6a does appear to contain a genuine lipid signal with a total measurable lipid abundance of 1.31 μg/g calcite, composed of normal and branched saturated fatty acids (C14–C28, maximising at C16), a small number of 3-hydroxy acids (C12–C16), and n-alkanols (C14–C26, even only except for C19, maximising at C18). Other compounds routinely found in stalagmites from other environments such as sterols and n-alkanes were not observed. The lipid distribution in MO6a suggests a primarily microbial source based upon the dominance of the low molecular weight fatty acids and the presence of branched fatty acids and 3-hydroxy acids (Kaneda, 1991; Wakeham et al., 2003). However, a minor vegetation input is suggested by the higher molecular weight fatty acids and n-alkanols both of which have an even over odd carbon number predominance. The pink colouration observed on extraction of MO6a and more weakly in MO6d appears on analysis to be due to a species of iodine or bromine complex, although the precise formula is not currently identifiable. Only the use of CHCA matrix operating in reflectron positive mode yielded any possible molecular ion information, with the isotope pattern suggesting that the sample could be tribrominated. However, further investigation is required in order to confirm this and determine the molecular formula. The presence of halogens is consistent with previous samples from the Nullarbor that have previously been found to have unusually high iodine contents (Woodhead et al., 2007). Organic complexes of iodine and bromine can occur naturally through biological activity (Gribble, 2000), but have also been associated with biomass burning (e.g. Mano and Andreae, 1994; Andreae et al., 1996), and it is therefore possible that their presence in the stalagmite is indicative of an increased frequency of fire events during the period sampled. However, considerably more work on a wider range of samples is needed before this can be identified with confidence. 3.2. TMAH thermochemolysis Fig. 3 shows four composite selected ion chromatograms for subsample MO6a, representative for the stalagmite as a whole, identifying the peaks of the compounds of greatest environmental interest. Of the compounds from the p-courmaryl (P), guaiacyl (G) and syringyl (S) groups associated with a lignin source, 4-methoxybenzoic acid methyl ester (P6), 1-(4-methoxyphenyl)-2-methoxypropane (P23), 3,4-dimethoxytoluene (G2), 3,4-dimethoxybenzoic acid methyl ester (G6), 1,2,3-trimethoxybenzene (S1) and 3,4,5-trimethoxybenzoic acid methyl ester (S6) were present across all samples, and 4methoxybenzaldehyde (P4), 1,2-dimethoxybenzene (G1), 3,4dimethoxyacetophenone (G5), and 3,4,5-trimethoxytoluene (S2) were identifiable in one or more. Other compounds present include methyl esters of butanoic and pentanoic acids, which have previously been identified as general TMAH products (Frazier et al., 2003); 1,4dimethoxy benzene; 3-methyl- and 4-methyl-benzoic acid methyl esters; 2,5- and 2,6-dimethoxy toluene; a series of dimethyl benzoic acid methyl esters (2,6-; 2,5-; 2,4-; 2,3-; 3,5-, and 3,4-); 3-methoxy benzoic acid methyl ester; 3,5-benzoic acid methyl ester; cinnamic acid methyl ester; methyl-3-methoxy-4-methyl benzoic acid methyl ester; and a number of n-alkanoic acid methyl esters. Methylated carbohydrate and
104
A.J. Blyth et al. / Chemical Geology 279 (2010) 101–105
c
m/z 121 135 149
a
d
b
16.5 Retention time (min)
25 h
m/z 138 152 165 166
a g
d)
e
e) f) g) h)
b
f
Retention time (min)
13
a) b) c)
26
k m/z 168 182 226
I
i) j) k) l) m)
j
n) o) p) q)
18.5 Retention time (min)
m/z 131 133 177 221
3-methoxybenzoic acid 4-methoxybenzoic acid (P6) Methyl-3-methoxy-4-methyl benzoate 1-(4-methoxyphenyl)-2methoxypropane (P24) 3,4-dimethoxytoluene 2,5-dimethoxytoluene 3,5-dimethoxybenzoic acid 3,4-dimethoxybenzoic acid (G6) 1,2,3-trimethoxybenzene (S1) 3,4,5-trimethoxytoluene (S2) 3,4,5-trimethoxybenzoic acid (S6) 3,5-dimethylbenzoic acid 3-phenyl-2-propenoic acid (cinnamic acid) 4-methyl-1,3benzenedicarboxylic acid Unidentified isomer of n 1,2,4-benzenetricarboxylic acid 1,3,5-benzenetricarboxylic acid
29.5
n
o p
l
q
m
18.5 Retention time (min)
33
Fig. 3. Selected ion chromatograms from the TMAH analysis of MO6a, identifying the major compound peaks of environmental interest.
protein derivatives are also present, but have not been identified in this study due to the degree of coelution present. 3.3. Variation between samples Ratios in between ratios of the p-courmaryl, guaiacyl and syringyl groups (or their equivalent in cupric oxidation extractions) have previously been used to interpret the amount of input from angiosperms and gymnosperms and woody and non-woody plants (e.g. Wysocki et al., 2008). However, due to differences in products between cupric oxidation techniques and unlabelled TMAH thermochemolysis, it has been shown that the numerical parameters set with traditional lignin product ratios cannot be used to identify source vegetation regime when using the latter technique (Wysocki et al., 2008). Here therefore,
rather than using the compound ratios to assign source vegetation directly, we aim to investigate how the organic composition of our palaeo-samples compares to that of stalagmite material from known environments, by plotting the Nullarbor data against modern samples analysed in parallel (see Blyth and Watson, 2009, for discussion of the modern samples). Fig. 4 shows that when the normalised ratios of the p-courmaryl, guaiacyl and syringyl groups are plotted, the Nullarbor samples broadly group together. Within this grouping, subsamples A and C plot together with an S/G ratio of 0.4 and a P/G ratio of 1, whilst B and D fall further apart with a lower S/G ratio (0.33), and more divergent P/G ratios (1.2 and 0.7 respectively). This separation is interesting as subsamples B and C come from the same growth interval of black calcite, whilst A and D are of lighter calcite and separated by hiatuses. This suggests that the controls on the molecular composition of the organic
A.J. Blyth et al. / Chemical Geology 279 (2010) 101–105
0.6
Cold grassland (UK) Eucalypt scrub (Australia)
0.5
Peat (UK)
S/G
0.4
Agriculutral crops (Ethiopia) Arboreal scrub (Ethiopia)
0.3 Mediterranean grassland (Turkey) MO6a
0.2
105
should focus on the analysis of this organic matter, attempting to develop a more robust tool for recovering vegetational histories by exploiting the potential of both 13C labelled TMAH thermochemolysis, which can provide more detailed information on the source molecules, and ion selective mass spectrometry techniques to provide greater compound sensitivity. Acknowledgements
MO6b
0.1
MO6c MO6d
0 0
0.5
1
1.5
2
2.5
3
P/G Fig. 4. Plot showing the ratio of p-coumaryl (P)/guaiacyl (G) compounds against syringyl (S)/Guaiacyl (G) compounds, in the samples from MO6, and a range of modern samples from known environments (for separate discussion of modern samples, please see Blyth and Watson, 2009).
matter in the sample are not necessarily related to the controls on the calcite colour. On the plot the MO6 samples show most similarity to the modern stalagmites from warm grassland environments, with the samples from beneath Ethiopian cropland plotting within the Nullarbor sample grouping. The sample from beneath mediterranean grassland has lower ratios on both parameters than the Nullabor samples, but seems to fall in the same general area. This is in contrast to the samples from beneath arboreal environments but from comparable temperature regimes (for example the Ethiopian arboreal sample), which have distinctly lower (b0.1) S/G ratio. An interesting side point is the stalagmite from beneath Scottish peat, which has a particularly elevated S/G ratio (N0.5), and could be argued to fall within the same general grouping as the Nullarbor samples. As lignin ratios are conventionally used to distinguish between woody and non-woody plants, it is not unreasonable for peat and grass-related vegetation to have signatures more similar to each other than to those from wooded areas, even though the numerical parameters used in the interpretation of samples extracted via cupric oxidation cannot be applied. The results therefore indicate that there is a potential vegetation driven signal in the stalagmite lignin composition, illustrated by the clearly different groupings of samples from beneath grassland and wooded environments. 4. Conclusion Organic matter is extractable from the Nullarbor Plain stalagmites, and can be analysed at a molecular level. The results indicate that lipid analysis is not always feasible in these samples, although at this stage we cannot say whether this is due to a degradation of the signal in older samples, the lack of an original signal in some, but not all, samples, or the binding of the lipids into the bulk organic matter in some way that renders them unamenable to simple extraction. More promising is the analysis of the residual organic matter via thermochemolysis which produced results for all samples, and in comparison with modern stalagmites from known environments suggests that a vegetation driven trend may be present. We therefore suggest that future work
Dr. Lisa Harris of the Mass Spectrometry Facility, Department of Chemistry, UCL, undertook preliminary analyses of the pink colouration in M06a. Funding for this work was provided by the McDonald Institute for Archaeological Research at the University of Cambridge, The Open University, and a Leverhulme Early Career Fellowship to AJB. References Andreae, M.O., Atlas, E., Harris, G.W., Helas, G., de Kock, A., Koppmann, R., Maenhaut, W., Mano, S., Pollock, W.H., Ruldolph, J., Scharffe, D., Schebeske, G., Welling, M., 1996. Methyl halide emissions from savanna fires in southern Africa. Journal of Geophysical Research 101, 23603–23613. Blyth, A.J., 2007. Lipid biomarkers in speleothems. Unpublished PhD thesis, University of Newcastle Upon Tyne, UK. Blyth, A.J., Watson, J.S., 2009. Thermochemolysis of organic matter preserved in stalagmites: a preliminary study. Organic Geochemistry 40, 1029–1031. Blyth, A.J., Farrimond, P., Jones, M., 2006. An optimised method for the extraction and analysis of lipid biomarkers from stalagmites. Organic Geochemistry 37, 882–890. Blyth, A.J., Asrat, A., Baker, A., Gulliver, P., Leng, M., Genty, D., 2007. A new approach to detecting vegetation and land-use change: high resolution lipid biomarker records in stalagmites. Quaternary Research 68, 314–324. Blyth, A.J., Baker, A., Penkman, K.E.H., Collins, M.J., Gilmour, M.A., Moss, J.S., Genty, D., Drysdale, R., 2008. Molecular organic matter in speleothems as an environmental proxy. Quaternary Science Reviews 27, 905–921. Caldwell, J., Davey, A.G., Jennings, G.N., Spate, A.P., 1982. Colour in some Nullarbor Plain speleothems. Helictite 20, 3–10. Frazier, S.W., Nowack, K.O., Goins, K.M., Cannon, F.S., Kaplan, L.A., Hatcher, P.G., 2003. Characterisation of organic matter from natural waters using tetramethylammonium hydroxide thermochemolysis GC-MS. Journal of Analytical and Applied Pyrolysis 70, 99–128. Gribble, G.W., 2000. The natural production of organobromine compounds. Environmnetal Science and Pollution Research 7 (1), 37–47. Huang, X., Cui, J., Pu, Y., Huang, J., Xie, S., Blyth, A.J., 2008. Identifying ‘free’ and ‘bound’ lipid fractions in stalagmite samples: an example from Heshang Cave, Southern China. Applied Geochemistry 23, 2589–2595. Kaneda, T., 1991. Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance. Microbiological Reviews 55, 288–302. Mano, S., Andreae, M.O., 1994. Emission of methyl bromide from biomass burning. Science 263, 1225–1257. Prideaux, G.J., Long, J.A., Ayliffe, L.K., Hellstrom, J.C., Pillans, B., Boles, W.E., Hutchinson, M.N., Roberts, R.G., Cupper, M.L., Arnold, L.J., Devine, P.D., Warburton, N.M., 2007. An arid-adapted middle Pleistocene vertebrate fauna from south-central Australia. Nature 445, 422–425. Wakeham, S.G., Pease, T.K., Benner, R., 2003. Hydroxy fatty acids in marine dissolved organic matter as indicators of bacterial membrane material. Organic Geochemistry 34, 857–868. Woodhead, J., Hellstrom, J., Maas, R., Drysdale, R., Zanchetta, G., Devine, P., Taylor, E., 2006. U–Pb geochronology of speleothems by MC-ICPMS. Quaternary Geochronology 1, 208–221. Woodhead, J., Hellstrom, J., Hergt, J., Greig, A., Maas, R., 2007. Isotopic and elemental imaging of geological materials by laser ablation inductively coupled plasma mass spectrometry. Journal of Geostandards and Geoanalytical Research 31, 331–343. Wysocki, L.A., Filley, T.R., Bianchi, T.S., 2008. Comparison of two methods for the analysis of lignin in marine sediments: CuO oxidation versus tetramethylammonium hydroxide (TMAH) thermochemolysis. Organic Geochemistry 39, 1454–1461. Xie, S., YI, Y., Huang, J., Hu, C., Cai, Y., Collins, M., Baker, A., 2003. Lipid distribution in a subtropical southern China stalagmite as a record of soil ecosystem response to palaeoclimate change. Quaternary Research 60, 340–347.
Contents lists available at ScienceDirect
Chemical Geology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c h e m g e o
Research paper
Organic compounds preserved in a 2.9 million year old stalagmite from the Nullarbor Plain, Australia Alison J. Blyth a,b,⁎, Jonathan S. Watson b,a, Jon Woodhead c, John Hellstrom c a b c
Department of Earth and Environmental Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK Planetary and Space Sciences Research Institute, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK School of Earth Sciences, University of Melbourne, VIC 3010, Australia
a r t i c l e
i n f o
Article history: Received 7 June 2010 Received in revised form 29 September 2010 Accepted 1 October 2010 Editor: J. Fein Keywords: Stalagmite Organic matter Lignin Vegetation Nullarbor Climate
a b s t r a c t A 2.9 Ma stalagmite from the Nullarbor Plain, South Australia was analysed for organic components. In contrast to previously analysed stalagmites, no significant lipid record was recovered. However, qualitative TMAH thermochemolysis of the residual organic matter recovered a range of compounds, including a number of phenols previously associated with lignin. Comparison of this sample with a range of modern stalagmite samples from different environments indicated that the phenolic record in stalagmites can reflect the overlying vegetation regime, and has potential in investigating palaeoenvironmental change in this climatically important region. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The Nullarbor Plain in South Australia (Fig. 1) is one of the largest karst regions in the world at ~240,000 km2, and contains a number of remarkable speleothems that have long been of interest to cave scientists in terms of both their great age and unusual dark colouration (Caldwell et al., 1982). The region is also of considerable importance to understanding past climatic changes as it has a strong sensitivity to Southern Ocean climate dynamics, and can be shown to have experienced a very different climate state in the past, with satellite-derived digital terrain data showing clear evidence of ancient river channels and other landforms indicative of higher rainfall. Fossil evidence also indicates that even when the climate was semi-arid, a more diverse vegetation regime was present (Prideaux et al., 2007). The development of stalagmite proxies from this area is of obvious value as they will provide a major southern hemisphere terrestrial archive to complement the regional and global records recovered from marine and ice cores. However, despite the importance of these stalagmites, they remain largely unstudied due to the lack of a reliable dating methodology, with the vast majority of samples being beyond the reach of the U–Th dating
techniques used in other stalagmite research (i.e. older than 500 ka). This issue has recently been resolved by the successful development of a U–Pb chronology (Woodhead et al., 2006), meaning we can begin to explore this potentially extremely valuable palaeoenvironmental archive. The use of organic matter in stalagmites as a palaeoenvironmental proxy is a growing field (for a review, see Blyth et al., 2008), with recent research indicating a connection between lipid biomarker content and both climate (Xie et al., 2003) and overlying vegetation regime (Blyth et al., 2007). Nullarbor stalagmites should be ideal for organic matter analyses as previous research has indicated that their dark colour may be due to unusually high levels of organics (Caldwell et al., 1982). A preliminary experiment with an undated sample showed a lipid biomarker signal comparable in concentration and constituent compounds to that from a modern African cave (Blyth, 2007; Blyth et al., 2008). This work therefore qualitatively analyses organic compounds recovered via a sequential organic extraction from a Nullarbor stalagmite with the aim of establishing the feasibility of organic analyses in these samples, and their potential for use in palaeoenvironmental reconstruction. 2. Methods and materials
⁎ Corresponding author. Present address: Department of Earth and Environmental Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK. Tel.: + 44 1908 659883; fax: + 44 1908 655151. E-mail address: [email protected] (A.J. Blyth). 0009-2541/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.chemgeo.2010.10.006
2.1. Site and sample The Nullarbor Plain is a flat-lying area where shallow marine limestones were exposed 15 million years ago due to a combination of
102
A.J. Blyth et al. / Chemical Geology 279 (2010) 101–105
Fig. 1. Map of Southern Australia showing the location of the cave.
uplift and global lowering of sea level. The current climate is arid to semi-arid, although it is clear from geomorphological evidence that this has not always been the case (Caldwell et al., 1982). The stalagmite analysed (1411-MO6) was collected in 2005 from ‘1411’ cave (lat 31.85; long 127.57), and has been dated via U–Pb techniques to 2.9 ± 0.1 Ma (disequilibrium corrected age), following the protocols laid out in Woodhead et al. (2006). Both the oldest and youngest parts of the stalagmite are within the error range for this date. This sample represents the end of a Pliocene high-humidity phase on the Nullarbor, based upon a geochronological survey of nearly 60 speleothem samples from a variety of caves across the region (Woodhead, unpublished data). Four subsamples of between 5 and 6 g of calcite were taken across the major coloured laminae (see Fig. 2). To remove any potential contamination from the collection or sectioning process, the outer 2 mm of each subsample was removed by digestion in cleaned 1 M hydrochloric acid (HCl); particular care was taken to remove white corrosion layers from the outer surface and from internal hiatuses. The samples were then washed in solvent cleaned ultra-pure MilliQ
Fig. 2. MO6 showing subsample location.
A.J. Blyth et al. / Chemical Geology 279 (2010) 101–105
water to remove excess acid, and successively sonicated in methanol and dichloromethane (DCM).
103
3. Results and discussion 3.1. Lipid results
2.2. Extraction of lipid biomarkers The cleaned samples were fully digested in 1 M HCl, and the solution was boiled under reflux for 2 h to maximise the release of bacterial lipids (Blyth et al., 2006; Huang et al., 2008). The lipids were extracted from the solution and prepared for analysis following the protocols laid out in Blyth et al. (2006). 5ß-Cholanic acid (Aldrich) was added at the reflux stage as an internal standard. The lipids were analysed on an Agilent Technologies 6890 gas chromatograph coupled to a 5973 mass spectrometer. Separation was performed on a DB-5MS column (30 m length, 0.25 mm internal diameter and 0.25 μm film thickness). Helium at a column flow rate of 1.1 ml min− 1 was used as the carrier gas. Injection was at a 10:1 split. The GC oven temperature was held for 1 min at 50 °C and then programmed at 5 °C min− 1 to 310 °C, the final temperature was held for 9 min. On extraction into DCM, a pink colouration of the solvent was apparent in two of the samples (MO6a, and more weakly, MO6d). This degraded overnight, suggesting instability or photosensitivity of the complex. A second sample from these two layers was therefore prepared and extracted as previously mentioned and an aliquot of the coloured solvent was removed and taken to the Mass Spectrometry Service in the Department of Chemistry at University College London, where it was analysed by MALDI-ToF analysis on a Micromass MALDI micro mx. Analysis was performed in both reflectron and linear modes operating in both positive and negative ionisations, covering mass-to-charge ratio from m/z = 700 to 6000 (reflectron mode) to m/ z = 6000–60,000 (linear mode). A number of different matrices were used in the analysis (CHCA (α-cyano-4-hydroxy cinnamic acid), SA (Sinapinic Acid) and DHB (2,5-dihydroxybenzoic acid)) in order to gain any molecular ion information.
2.3. Extraction and analysis of the residual organic matter After lipid extraction, an aliquot of each remaining acidified solution equivalent to 1 g of the original calcite sample was loaded onto a conditioned non-endcapped C18 solid phase extraction cartridge (IST Isolute) to recover the residual organic matter. SPE cartridges were pre-conditioned with hexane, DCM, methanol and 0.01 M HCl. Once the dissolved organic matter had adsorbed on the SPE cartridge any salts were removed by rinsing with 4 ml of 0.01 M HCl, the cartridge was then dried by flushing with helium. The organics were eluted from the column into clean vials with 4 ml of methanol, dried under nitrogen and rediluted in 50 μl of methanol. Approximately 10 μl of the sample in methanol was absorbed onto quartz wool in a quartz pyrolysis tube. The samples were left to dry overnight, before 10 μl of 25% tetramethylammonium hydroxide (TMAH) in methanol was added and allowed to dry overnight. TMAH was added in excess to ensure base conditions during thermochemolysis. Samples were heated (300 °C at a rate of 20 °C ms− 1 then held isothermally for 15 s) in a flow of helium using a CDS Pyroprobe 5000 fitted with a 1500 valve interface (CDS Analytical, Oxford, PA) and coupled to a gas chromatograph–mass spectrometer (GC–MS). GC–MS analysis was carried out using an Agilent Technologies 6890 gas chromatograph coupled to a 5973 mass spectrometer. Separation was performed on a S.G.E. (U.K.) BPX-5 column (30 m length, 0.25 mm internal diameter and 0.25 μm film thickness). Helium at a column flow rate of 1.1 ml min− 1 was used as the carrier gas. Injection was at a 5:1 split and injector temperature was 270 °C. The GC oven temperature was held for 1 min at 50 °C and then programmed at 5 °C min− 1 to 310 °C, the final temperature was held for 9 min.
Although a preliminary undated sample analysed via the same extraction method in 2006 showed a well preserved lipid signal (Blyth, 2007; Blyth et al., 2008), comparatively few lipid biomarkers were recovered from the current samples, with MO6b, MO6c, and MO6d containing only trace amounts of low molecular weight fatty acids below the contamination threshold associated with this method (0.3 μg for C16 n-alkanoic acid; Blyth et al., 2006). MO6a does appear to contain a genuine lipid signal with a total measurable lipid abundance of 1.31 μg/g calcite, composed of normal and branched saturated fatty acids (C14–C28, maximising at C16), a small number of 3-hydroxy acids (C12–C16), and n-alkanols (C14–C26, even only except for C19, maximising at C18). Other compounds routinely found in stalagmites from other environments such as sterols and n-alkanes were not observed. The lipid distribution in MO6a suggests a primarily microbial source based upon the dominance of the low molecular weight fatty acids and the presence of branched fatty acids and 3-hydroxy acids (Kaneda, 1991; Wakeham et al., 2003). However, a minor vegetation input is suggested by the higher molecular weight fatty acids and n-alkanols both of which have an even over odd carbon number predominance. The pink colouration observed on extraction of MO6a and more weakly in MO6d appears on analysis to be due to a species of iodine or bromine complex, although the precise formula is not currently identifiable. Only the use of CHCA matrix operating in reflectron positive mode yielded any possible molecular ion information, with the isotope pattern suggesting that the sample could be tribrominated. However, further investigation is required in order to confirm this and determine the molecular formula. The presence of halogens is consistent with previous samples from the Nullarbor that have previously been found to have unusually high iodine contents (Woodhead et al., 2007). Organic complexes of iodine and bromine can occur naturally through biological activity (Gribble, 2000), but have also been associated with biomass burning (e.g. Mano and Andreae, 1994; Andreae et al., 1996), and it is therefore possible that their presence in the stalagmite is indicative of an increased frequency of fire events during the period sampled. However, considerably more work on a wider range of samples is needed before this can be identified with confidence. 3.2. TMAH thermochemolysis Fig. 3 shows four composite selected ion chromatograms for subsample MO6a, representative for the stalagmite as a whole, identifying the peaks of the compounds of greatest environmental interest. Of the compounds from the p-courmaryl (P), guaiacyl (G) and syringyl (S) groups associated with a lignin source, 4-methoxybenzoic acid methyl ester (P6), 1-(4-methoxyphenyl)-2-methoxypropane (P23), 3,4-dimethoxytoluene (G2), 3,4-dimethoxybenzoic acid methyl ester (G6), 1,2,3-trimethoxybenzene (S1) and 3,4,5-trimethoxybenzoic acid methyl ester (S6) were present across all samples, and 4methoxybenzaldehyde (P4), 1,2-dimethoxybenzene (G1), 3,4dimethoxyacetophenone (G5), and 3,4,5-trimethoxytoluene (S2) were identifiable in one or more. Other compounds present include methyl esters of butanoic and pentanoic acids, which have previously been identified as general TMAH products (Frazier et al., 2003); 1,4dimethoxy benzene; 3-methyl- and 4-methyl-benzoic acid methyl esters; 2,5- and 2,6-dimethoxy toluene; a series of dimethyl benzoic acid methyl esters (2,6-; 2,5-; 2,4-; 2,3-; 3,5-, and 3,4-); 3-methoxy benzoic acid methyl ester; 3,5-benzoic acid methyl ester; cinnamic acid methyl ester; methyl-3-methoxy-4-methyl benzoic acid methyl ester; and a number of n-alkanoic acid methyl esters. Methylated carbohydrate and
104
A.J. Blyth et al. / Chemical Geology 279 (2010) 101–105
c
m/z 121 135 149
a
d
b
16.5 Retention time (min)
25 h
m/z 138 152 165 166
a g
d)
e
e) f) g) h)
b
f
Retention time (min)
13
a) b) c)
26
k m/z 168 182 226
I
i) j) k) l) m)
j
n) o) p) q)
18.5 Retention time (min)
m/z 131 133 177 221
3-methoxybenzoic acid 4-methoxybenzoic acid (P6) Methyl-3-methoxy-4-methyl benzoate 1-(4-methoxyphenyl)-2methoxypropane (P24) 3,4-dimethoxytoluene 2,5-dimethoxytoluene 3,5-dimethoxybenzoic acid 3,4-dimethoxybenzoic acid (G6) 1,2,3-trimethoxybenzene (S1) 3,4,5-trimethoxytoluene (S2) 3,4,5-trimethoxybenzoic acid (S6) 3,5-dimethylbenzoic acid 3-phenyl-2-propenoic acid (cinnamic acid) 4-methyl-1,3benzenedicarboxylic acid Unidentified isomer of n 1,2,4-benzenetricarboxylic acid 1,3,5-benzenetricarboxylic acid
29.5
n
o p
l
q
m
18.5 Retention time (min)
33
Fig. 3. Selected ion chromatograms from the TMAH analysis of MO6a, identifying the major compound peaks of environmental interest.
protein derivatives are also present, but have not been identified in this study due to the degree of coelution present. 3.3. Variation between samples Ratios in between ratios of the p-courmaryl, guaiacyl and syringyl groups (or their equivalent in cupric oxidation extractions) have previously been used to interpret the amount of input from angiosperms and gymnosperms and woody and non-woody plants (e.g. Wysocki et al., 2008). However, due to differences in products between cupric oxidation techniques and unlabelled TMAH thermochemolysis, it has been shown that the numerical parameters set with traditional lignin product ratios cannot be used to identify source vegetation regime when using the latter technique (Wysocki et al., 2008). Here therefore,
rather than using the compound ratios to assign source vegetation directly, we aim to investigate how the organic composition of our palaeo-samples compares to that of stalagmite material from known environments, by plotting the Nullarbor data against modern samples analysed in parallel (see Blyth and Watson, 2009, for discussion of the modern samples). Fig. 4 shows that when the normalised ratios of the p-courmaryl, guaiacyl and syringyl groups are plotted, the Nullarbor samples broadly group together. Within this grouping, subsamples A and C plot together with an S/G ratio of 0.4 and a P/G ratio of 1, whilst B and D fall further apart with a lower S/G ratio (0.33), and more divergent P/G ratios (1.2 and 0.7 respectively). This separation is interesting as subsamples B and C come from the same growth interval of black calcite, whilst A and D are of lighter calcite and separated by hiatuses. This suggests that the controls on the molecular composition of the organic
A.J. Blyth et al. / Chemical Geology 279 (2010) 101–105
0.6
Cold grassland (UK) Eucalypt scrub (Australia)
0.5
Peat (UK)
S/G
0.4
Agriculutral crops (Ethiopia) Arboreal scrub (Ethiopia)
0.3 Mediterranean grassland (Turkey) MO6a
0.2
105
should focus on the analysis of this organic matter, attempting to develop a more robust tool for recovering vegetational histories by exploiting the potential of both 13C labelled TMAH thermochemolysis, which can provide more detailed information on the source molecules, and ion selective mass spectrometry techniques to provide greater compound sensitivity. Acknowledgements
MO6b
0.1
MO6c MO6d
0 0
0.5
1
1.5
2
2.5
3
P/G Fig. 4. Plot showing the ratio of p-coumaryl (P)/guaiacyl (G) compounds against syringyl (S)/Guaiacyl (G) compounds, in the samples from MO6, and a range of modern samples from known environments (for separate discussion of modern samples, please see Blyth and Watson, 2009).
matter in the sample are not necessarily related to the controls on the calcite colour. On the plot the MO6 samples show most similarity to the modern stalagmites from warm grassland environments, with the samples from beneath Ethiopian cropland plotting within the Nullarbor sample grouping. The sample from beneath mediterranean grassland has lower ratios on both parameters than the Nullabor samples, but seems to fall in the same general area. This is in contrast to the samples from beneath arboreal environments but from comparable temperature regimes (for example the Ethiopian arboreal sample), which have distinctly lower (b0.1) S/G ratio. An interesting side point is the stalagmite from beneath Scottish peat, which has a particularly elevated S/G ratio (N0.5), and could be argued to fall within the same general grouping as the Nullarbor samples. As lignin ratios are conventionally used to distinguish between woody and non-woody plants, it is not unreasonable for peat and grass-related vegetation to have signatures more similar to each other than to those from wooded areas, even though the numerical parameters used in the interpretation of samples extracted via cupric oxidation cannot be applied. The results therefore indicate that there is a potential vegetation driven signal in the stalagmite lignin composition, illustrated by the clearly different groupings of samples from beneath grassland and wooded environments. 4. Conclusion Organic matter is extractable from the Nullarbor Plain stalagmites, and can be analysed at a molecular level. The results indicate that lipid analysis is not always feasible in these samples, although at this stage we cannot say whether this is due to a degradation of the signal in older samples, the lack of an original signal in some, but not all, samples, or the binding of the lipids into the bulk organic matter in some way that renders them unamenable to simple extraction. More promising is the analysis of the residual organic matter via thermochemolysis which produced results for all samples, and in comparison with modern stalagmites from known environments suggests that a vegetation driven trend may be present. We therefore suggest that future work
Dr. Lisa Harris of the Mass Spectrometry Facility, Department of Chemistry, UCL, undertook preliminary analyses of the pink colouration in M06a. Funding for this work was provided by the McDonald Institute for Archaeological Research at the University of Cambridge, The Open University, and a Leverhulme Early Career Fellowship to AJB. References Andreae, M.O., Atlas, E., Harris, G.W., Helas, G., de Kock, A., Koppmann, R., Maenhaut, W., Mano, S., Pollock, W.H., Ruldolph, J., Scharffe, D., Schebeske, G., Welling, M., 1996. Methyl halide emissions from savanna fires in southern Africa. Journal of Geophysical Research 101, 23603–23613. Blyth, A.J., 2007. Lipid biomarkers in speleothems. Unpublished PhD thesis, University of Newcastle Upon Tyne, UK. Blyth, A.J., Watson, J.S., 2009. Thermochemolysis of organic matter preserved in stalagmites: a preliminary study. Organic Geochemistry 40, 1029–1031. Blyth, A.J., Farrimond, P., Jones, M., 2006. An optimised method for the extraction and analysis of lipid biomarkers from stalagmites. Organic Geochemistry 37, 882–890. Blyth, A.J., Asrat, A., Baker, A., Gulliver, P., Leng, M., Genty, D., 2007. A new approach to detecting vegetation and land-use change: high resolution lipid biomarker records in stalagmites. Quaternary Research 68, 314–324. Blyth, A.J., Baker, A., Penkman, K.E.H., Collins, M.J., Gilmour, M.A., Moss, J.S., Genty, D., Drysdale, R., 2008. Molecular organic matter in speleothems as an environmental proxy. Quaternary Science Reviews 27, 905–921. Caldwell, J., Davey, A.G., Jennings, G.N., Spate, A.P., 1982. Colour in some Nullarbor Plain speleothems. Helictite 20, 3–10. Frazier, S.W., Nowack, K.O., Goins, K.M., Cannon, F.S., Kaplan, L.A., Hatcher, P.G., 2003. Characterisation of organic matter from natural waters using tetramethylammonium hydroxide thermochemolysis GC-MS. Journal of Analytical and Applied Pyrolysis 70, 99–128. Gribble, G.W., 2000. The natural production of organobromine compounds. Environmnetal Science and Pollution Research 7 (1), 37–47. Huang, X., Cui, J., Pu, Y., Huang, J., Xie, S., Blyth, A.J., 2008. Identifying ‘free’ and ‘bound’ lipid fractions in stalagmite samples: an example from Heshang Cave, Southern China. Applied Geochemistry 23, 2589–2595. Kaneda, T., 1991. Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance. Microbiological Reviews 55, 288–302. Mano, S., Andreae, M.O., 1994. Emission of methyl bromide from biomass burning. Science 263, 1225–1257. Prideaux, G.J., Long, J.A., Ayliffe, L.K., Hellstrom, J.C., Pillans, B., Boles, W.E., Hutchinson, M.N., Roberts, R.G., Cupper, M.L., Arnold, L.J., Devine, P.D., Warburton, N.M., 2007. An arid-adapted middle Pleistocene vertebrate fauna from south-central Australia. Nature 445, 422–425. Wakeham, S.G., Pease, T.K., Benner, R., 2003. Hydroxy fatty acids in marine dissolved organic matter as indicators of bacterial membrane material. Organic Geochemistry 34, 857–868. Woodhead, J., Hellstrom, J., Maas, R., Drysdale, R., Zanchetta, G., Devine, P., Taylor, E., 2006. U–Pb geochronology of speleothems by MC-ICPMS. Quaternary Geochronology 1, 208–221. Woodhead, J., Hellstrom, J., Hergt, J., Greig, A., Maas, R., 2007. Isotopic and elemental imaging of geological materials by laser ablation inductively coupled plasma mass spectrometry. Journal of Geostandards and Geoanalytical Research 31, 331–343. Wysocki, L.A., Filley, T.R., Bianchi, T.S., 2008. Comparison of two methods for the analysis of lignin in marine sediments: CuO oxidation versus tetramethylammonium hydroxide (TMAH) thermochemolysis. Organic Geochemistry 39, 1454–1461. Xie, S., YI, Y., Huang, J., Hu, C., Cai, Y., Collins, M., Baker, A., 2003. Lipid distribution in a subtropical southern China stalagmite as a record of soil ecosystem response to palaeoclimate change. Quaternary Research 60, 340–347.