Three wood isotopic reference materials for δ2H and δ13C measurements of plant methoxy groups
Introduction
Methoxy groups (OCH3) of lignin and pectin constitute a significant fraction of the biospheric C1 methyl (CH3) pool of plant origin accounting for ~4–6% of wood dry mass (Galbally and Kirstine, 2002; Keppler et al., 2004) and ~1–3% of leaf dry mass (McRoberts et al., 2015). In the past 15 years several studies have shown that methoxy groups of plants have both distinct stable hydrogen (δ2HOCH3) and carbon (δ13COCH3) isotope values (Keppler et al., 2004) with reported δ2HOCH3 and δ13COCH3 values ranging from −149 mUr (Greule et al., 2010) to −405 mUr (Anhäuser et al., 2018) and −7.1 mUr (Greule et al., 2010) to −77.2 mUr (Keppler et al., 2004), respectively. These specific δ2H and δ13C isotope patterns have considerable potential for application as tools for investigations in biogeochemical, atmospheric, paleoclimatic and food research (Greule et al., 2019). For a more detailed overview of its applications in environmental research we refer to previous studies by Anhäuser et al. (2014), Feakins et al. (2013a), Greule et al., 2012, Greule et al., 2010, Hepp et al. (2017), Keppler et al. (2004) and Lee et al. (2019). Please also note that we follow the suggestion by Brand and Coplen (2012) and express isotope delta values in milli-Urey [mUr] (after Urey, 1948) instead of per mil [‰] (1 mUr = 1‰).
Most measurements of site-specific isotopic abundances of organic molecules are accomplished by nuclear magnetic resonance (NMR) spectroscopy, which requires relatively large masses of chemically pure samples. This complicates the application of this technique to most natural samples. By combining a long-established technique for the derivatization of plant methoxy groups to gaseous iodomethane (CH3I) using hydriodic acid (Zeisel, 1885) with conventional methods for continuous flow isotope ratio mass spectrometry (CF-IRMS), it is possible to determine the 13C and 2H isotope compositions of plant methoxy groups with high precision and no apparent isotopic fractionation on milligram quantities of bulk tissue (Greule et al., 2009, Greule et al., 2008).
Stable isotope analyses require reference materials to normalize stable isotope ratios on the respective δ-scale (Brand et al., 2014; Carter and Barwick, 2011; Meier-Augenstein and Schimmelmann, 2019; Werner and Brand, 2001). Highest accuracy is achieved when workflows adhere to the principle of “identical treatment of sample and reference material” (IT) in preparation and analysis, which requires that samples are chemically analogous to standards of known isotopic composition (Meier-Augenstein and Schimmelmann, 2019; Schimmelmann et al., 2016; Werner and Brand, 2001). When selecting reference materials for IRMS analyses further criteria have to be considered as outlined in detail by several previous studies, such as isotopic homogeneity, unchanging stable isotopic composition over time, chemical similarity to the samples, easy preparation, storage and handling as well as no health risk (Brand et al., 2014; Carter and Barwick, 2011; Meier-Augenstein and Schimmelmann, 2019; Schimmelmann et al., 2016; Werner and Brand, 2001). Most published stable isotope values of methoxy groups analyzed by IRMS were normalized to the respective δ-scale using liquid CH3I as the reference material. However, this practice violates the above principles because liquid CH3I, unlike the sample, is typically not heated at 130 °C with HI for derivatization (Greule et al., 2019). Moreover, δ2H and δ13C values of commercially available CH3I have restricted ranges (δ2H: −66 to −179 mUr; δ13C: −46 to −70 mUr) (Feakins et al., 2013b; Greule et al., 2019; Keppler et al., 2007) that do not bracket typical δ2HOCH3 and δ13COCH3 values of plant methoxy groups.
For these reasons, we have recently investigated two methyl sulfate salts (HUBG1 and HUBG2), for their suitability to serve as methoxy reference materials (Greule et al., 2019). These methyl sulfate salts have been demonstrated to be highly suitable to serve as reference materials to normalize δ13COCH3 values, as they are treated in an identical manner as the sample and span a relatively wide range of δ13C values (HUBG1: −50.31 ± 0.16 mUr; HUBG2: +1.60 ± 0.12 mUr) covering most of the natural δ13COCH3 values of terrestrial plant methoxy groups that have been reported so far (Greule et al., 2019). However, these methyl sulfate salts have a restricted range of δ2HOCH3 values (HUBG1: −144.5 ± 1.2 mUr; HUBG2: −102.0 ± 1.3 mUr) and thus are of limited use for normalizing δ2HOCH3 values of plant methoxy groups, which have δ2HOCH3 values in the range of −150 mUr to −300 mUr (Anhäuser et al., 2015; Greule et al., 2015, Greule et al., 2012; Keppler and Hamilton, 2008; Riechelmann et al., 2017). To properly bracket δ2H values of plant methoxy groups, additional reference materials, with lower δ2HOCH3 values, are needed. To meet the requirements for stable isotope analysis (Brand et al., 2014; Carter and Barwick, 2011; Schimmelmann et al., 2016; Werner and Brand, 2001), these reference materials must be available in sufficiently large quantities to be applied on a long-term basis and be readily distributable for inter-laboratory comparisons.
To fulfill this need, we have investigated the suitability of three woods from different geographical locations as long-term reference materials for normalization of δ2HOCH3 values. Wood contains relatively high concentrations of methoxy groups in the range of 4–6% (dry weight basis), primarily originating from lignin. The δ2H values of these lignin methoxy groups are highly depleted in 2H compared to the δ2H values of the source water due to a large, uniform apparent biosynthetic isotopic fractionation of −213 ± 17 mUr (Anhäuser et al., 2017b; Keppler et al., 2007). Since the stable hydrogen isotope values of tree source waters generally range from ~0 to −150 mUr, depending on the geographical origin, δ2HOCH3 values of wood lignin can be found in the range of −200 to −350 mUr. Hence, woods from different climatic and geographic regions are ideally suited to serve as reference materials for plant methoxy groups. Wood meets additional requirements for a suitable reference material because it is a non-toxic, solid, stable substance amenable to long-term storage and safe distribution, and can be treated identically to the samples when analyzing stable isotope signatures of methoxy groups.
Here we describe the preparation and analysis of three wood samples (HUBG3, HUBG4 and HUBG5) and make them available as interlaboratory reference materials for δ2HOCH3 and δ13COCH3 measurements of plant methoxy groups. We detail the procedures (Fig. 1) used to verify the homogenization of these materials, and the techniques used to determine their δ2HOCH3 and δ13COCH3 values with high confidence.
Section snippets
Wood samples
Slices of three tree specimens of different species were collected from three different locations spanning the globe. Details on their origin, mass, and methoxy content are given in Table 1.
Homogenization of wood samples
Prior to the homogenization of wood samples dried at ambient temperature, the bark of each tree slice was removed. Abundant sawdust from each sample was generated by repeatedly slicing each sample with a bench saw. The sawdust of each sample was collected in a barrel and thoroughly mixed by stirring and
Quantitative conversion of methoxy groups from the three wood samples to iodomethane
First, methoxy groups from subsamples of the three homogenized wood slices (for detailed description please see Method Section 2.3.1) with measured methoxy content of 4.7, 5.4 and 5.1% for birch, beech and tineo, respectively were quantitatively converted to CH3I. Four aliquots of each wood sample were generated and stored in borosilicate glass break-seals. To verify that aliquots of CH3I were isotopically homogenized among vacuum line chambers, the δ13C values of two break-seals from disparate
Conclusion
We suggest that the investigated three wood reference materials HUBG3–5 are highly suited for normalization of δ2HOCH3 and δ13COCH3 values of plant origin. Moreover, they are ideal for long-term usage and inter-laboratory comparison. The δ2HOCH3 values of the three wood samples span a relatively wide range (~−280 to −190 mUr) suitable for normalization of δ2HOCH3 values of most plant samples that have been reported so far. δ13COCH3 values of the two wood samples (HUBG3–4) are restricted in
Source for methoxy reference material
HUBG3, HUBG4, and HUBG5 methoxy wood isotopic reference materials as well as HUBG1 and HUBG2 methyl sulfate salts are available upon request from:
University Heidelberg, Institute of Earth Sciences, Biogeochemistry Group, INF 236, 69120 Heidelberg, Germany.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank Axel Groh from the company Schorn & Groh veneers for providing slices of birch and tineo wood, the carpentry of the youth institution “Stift Sunnisheim” and Hendric Glatting for the homogenization of the wood samples and Timo Schreiter for measurements of methoxy quantification. We also thank two anonymous reviewers for carefully reading the manuscript and suggesting substantial improvements. This study was supported by the German Research Foundation DFG (KE 884/6-3, KE 884/8-2).
References (43)
- et al.
D/H ratios of methoxyl groups of the sedimentary organic matter of Lake Holzmaar (Eifel, Germany): a potential palaeoclimate/-hydrology proxy
Geochim. Cosmochim. Acta
(2014) - et al.
Stable hydrogen isotope values of lignin methoxyl groups of four tree species across Germany and their implication for temperature reconstruction
Sci. Total Environ.
(2017) - et al.
Mean annual temperatures of mid-latitude regions derived from δ2H values of wood lignin methoxyl groups and its implications for paleoclimate studies
Sci. Total Environ.
(2017) - et al.
Earliest Eocene cold period and polar amplification – insights from δ2H values of lignin methoxyl groups of mummified wood
Palaeogeogr. Palaeoclimatol. Palaeoecol.
(2018) - et al.
Lignin methoxyl hydrogen isotope ratios in a coastal ecosystem
Geochim. Cosmochim. Acta
(2013) - et al.
Stable hydrogen-isotope analysis of methyl chloride emitted from heated halophytic plants
Atmos. Environ.
(2012) - et al.
Late Quaternary relative humidity changes from Mt. Kilimanjaro, based on a coupled 2H-18O biomarker paleohygrometer approach
Quat. Int.
(2017) - et al.
Three whole-wood isotopic reference materials, USGS54, USGS55, and USGS56, for δ2H, δ18O, δ13C, and δ15N measurements
Chem. Geol.
(2016) - et al.
Climate signal in d13C of wood lignin methoxyl groups from high-elevation alpine larch trees
Palaeogeogr. Palaeoclimatol. Palaeoecol.
(2016) - et al.
Stable hydrogen and carbon isotope ratios of methoxyl groups during plant litter degradation
Isot. Environ. Health Stud.
(2015)
IAEA stable isotope reference materials: addressing the needs of atmospheric greenhouse gas monitoring
Stable isotope deltas: tiny, yet robust signatures in nature
Isot. Environ. Health Stud.
Assessment of international reference materials for isotope-ratio analysis (IUPAC technical report)
Pure Appl. Chem.
Improved precision of coupled δ13C and δ15N measurements from single samples using an elemental analyzer/isotope ratio mass spectrometer combination with a post-column six-port valve and selective CO2 trapping; improved halide robustness of the combustion
Rapid Commun. Mass Spectrom.
Good Practice Guide for Isotope Ratio Mass Spectrometry
Analytical challenges in the quantitative determination of 2H/1H ratios of methyl iodide
Rapid Commun. Mass Spectrom.
The production of methanol by flowering plants and the global cycle of methanol
J. Atmos. Chem.
Continuous flow 2H/1H and 18O/16O analysis of water samples with dual inlet precision
Rapid Commun. Mass Spectrom.
On-line hydrogen-isotope measurements of organic samples using elemental chromium—an extension for high temperature elemental-analyzer techniques
Anal. Chem.
Optimization of on-line hydrogen stable isotope-ratio measurements of halogen- and sulfur-bearing organic compounds using elemental analyzer-chromium/high-temperature conversion-isotope-ratio mass spectrometry (EA-Cr/HTC-IRMS)
Rapid Commun. Mass Spectrom.
Carbon, hydrogen and oxygen stable isotope ratios of whole wood, cellulose and lignin methoxyl groups of Picea abies as climate proxies
Rapid Commun. Mass Spectrom.
Cited by (17)
Tree-ring stable isotopes in cellulose and lignin methoxy groups reveal different age-related behaviour
2024, Quaternary InternationalAnisole-based assessment for the quantification of methoxyl groups in lignin-rich biomass
2023, Organic GeochemistryPermafrost degradation alters the environmental signals recorded in tree-ring lignin methoxy group δ<sup>2</sup>H in northeastern China
2023, Science of the Total EnvironmentAltitude-specific differences in tree-ring δ<sup>2</sup>H records of wood lignin methoxy in the Qinling mountains, central China
2023, Quaternary Science ReviewsCitation Excerpt :These two homogenized tree ring samples of different species, Larix gmelinii (LG, −307.5 ± 2.3‰) and Cryptomeria fortunei (CF, −210.1 ± 2.5‰), were collected from northeastern (permafrost area) and southwestern (Sichuan Basin) China. The δ2H values of the two working standards were measured by the stable isotope lab of the Biogeochemistry research group at Heidelberg University against the methoxy group reference materials HUBG 2 & 3 (refer to Greule et al., 2020, 2021). Climate data (including monthly air temperature and total precipitation) from the three meteorological stations (TB, MX, and FP) were obtained from the Chinese Meteorological Data Service Center.
Tree-ring δ<sup>2</sup>H records of lignin methoxy indicate spring temperature changes since 20th century in the Qinling Mountains, China
2022, DendrochronologiaCitation Excerpt :Two homogenized wood samples Larix gmelinii (LG) and Cryptomeria fortunei Hooibrenk (CFH) collected from Northeastern (permafrost area) and Southwest (Sichuan Basin) China were applied as in-house standards to normalize the isotope measurements. The two homogenized wood samples have been measured by the stable isotope lab of the Biogeochemistry research group at Heidelberg University against the methoxy group reference materials HUBG 2 & 3 (Greule et al., 2020, 2021) and showed δ2HLM values of − 307.5 ± 2.3 ‰ and − 210.1 ± 2.5 ‰ for LG and CFH, respectively. The temporal trends of the annually resolved δ2HLM chronologies were investigated by linear regression.
Measurements and applications of δ<sup>2</sup>H values of wood lignin methoxy groups for paleoclimatic studies
2021, Quaternary Science ReviewsCitation Excerpt :Three additional wood samples (USGS54–56) were recently introduced as RMs for δ2H, δ18O, δ13C, and δ15N bulk measurements (Qi et al., 2016). The reported δ2HLM values of these materials were in the range of −319 mUr to −178 mUr (Greule et al., 2020). In this study the newly available RMs HUBG1 & 3 were used for measurement of wood samples from European mid-latitude and polar regions in order to establish a reliable data set as a basis for future application to paleoclimatic and authenticity studies.