How to Couple LC-IRMS with HRMS─A Proof-of-Concept Study.

Compound-specific stable isotope analysis (CSIA) is a unique analytical technique for determining small variations in isotope ratios of light isotopes in analytes from complex mixtures. A problem of CSIA using gas chromatography (GC) and liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) is that any structural information of the analytes is lost due to the processes involved in determining the isotope ratio. To obtain the isotopic composition of, for example, carbon from organic compounds, all carbon in each analyte is quantitatively converted to CO2. For GC-IRMS, open split GC-IRMS-MS couplings have been described that allow additional acquisition of structural information of analytes and interferences. Structural analysis using LC-IRMS is more difficult and requires additional technical and instrumental efforts. In this study, LC was combined for the first time with simultaneous analysis by IRMS and high-resolution mass spectrometry (HRMS), enabling the direct identification of unknown or coeluting species. We have thoroughly investigated and optimized the coupling and showed how technical problems, arising from instrumental conditions, can be overcome. To this end, it was successfully demonstrated that a consistent split ratio between IRMS and HRMS could be obtained using a variable postcolumn flow splitter. This coupling provided reproducible results in terms of resulting peak areas, isotope values, and retention time differences for the two mass spectrometer systems. To demonstrate the applicability of the coupling, we chose to address an important question regarding the purity of international isotope standards. In this context, we were able to confirm that the USGS41 reference material indeed contains substantial amounts of pyroglutamic acid as suggested previously in the literature. Moreover, the replacement material, USGS41a, still has significant amounts of pyroglutamic acid as impurity, rendering some caution necessary when using this material for isotopic calibration.

USGS44, a new high-purity calcium carbonate reference material for δ13 C measurements.

RATIONALE: The stable carbon isotopic (δ13 C) reference material (RM) LSVEC Li2 CO3 has been found to be unsuitable for δ13 C standardization work because its δ13 C value increases with exposure to atmospheric CO2 . A new CaCO3 RM, USGS44, has been prepared to alleviate this situation. METHODS: USGS44 was prepared from 8 kg of Merck high-purity CaCO3 . Two sets of δ13 C values of USGS44 were determined. The first set of values was determined by online combustion, continuous-flow (CF) isotope-ratio mass spectrometry (IRMS) of NBS 19 CaCO3 (δ13 CVPDB = +1.95 milliurey (mUr) exactly, where mUr = 0.001 = 1‰), and LSVEC Li2 CO3 (δ13 CVPDB = -46.6 mUr exactly), and normalized to the two-anchor δ13 CVPDB-LSVEC isotope-delta scale. The second set of values was obtained by dual-inlet (DI)-IRMS of CO2 evolved by reaction of H3 PO4 with carbonates, corrected for cross contamination, and normalized to the single-anchor δ13 CVPDB scale. RESULTS: USGS44 is stable and isotopically homogeneous to within 0.02 mUr in 100-µg amounts. It has a δ13 CVPDB-LSVEC value of -42.21 ± 0.05 mUr. Single-anchor δ13 CVPDB values of -42.08 ± 0.01 and -41.99 ± 0.02 mUr were determined by DI-IRMS with corrections for cross contamination. CONCLUSIONS: The new high-purity, well-homogenized calcium carbonate isotopic reference material USGS44 is stable and has a δ13 CVPDB-LSVEC value of -42.21 ± 0.05 mUr for both EA/IRMS and DI-IRMS measurements. As a carbonate relatively depleted in 13 C, it is intended for daily use as a secondary isotopic reference material to normalize stable carbon isotope delta measurements to the δ13 CVPDB-LSVEC scale. It is useful in quantifying drift with time, determining mass-dependent isotopic fractionation (linearity correction), and adjusting isotope-ratio-scale contraction. Due to its fine grain size (smaller than 63 µm), it is not suitable as a δ18 O reference material. A δ13 CVPDB-LSVEC value of -29.99 ± 0.05 mUr was determined for NBS 22 oil.

Spatial Microanalysis of Natural 13C/12C Abundance in Environmental Samples Using Laser Ablation-Isotope Ratio Mass Spectrometry.

The stable 13C/12C isotope composition usually varies among different organic materials due to isotope fractionation during biochemical synthesis and degradation processes. Here, we introduce a novel laser ablation-isotope ratio mass spectrometry (LA-IRMS) methodology that allows highly resolved spatial analysis of carbon isotope signatures in solid samples down to a spatial resolution of 10 µm. The presented instrumental setup includes in-house-designed exchangeable ablation cells (3.8 and 0.4 mL, respectively) and an improved sample gas transfer, which allow accurate δ13C measurements of an acryl plate standard down to 0.6 and 0.4 ng of ablated carbon, respectively (standard deviation 0.25‰). Initial testing on plant and soil samples confirmed that microheterogeneity of their natural 13C/12C abundance can now be mapped at a spatial resolution down to 10 µm. The respective δ13C values in soils with C3/C4 crop sequence history varied by up to 14‰ across a distance of less than 100 µm in soil aggregates, while being partly sorted along rhizosphere gradients of <300 µm from Miscanthus plant roots into the surrounding soil. These very first demonstrations point to the appearance of very small metabolic hotspots originating from different natural isotope discrimination processes, now traceable via LA-IRMS.

New Concepts for the Determination of Oxidation Efficiencies in Liquid Chromatography-Isotope Ratio Mass Spectrometry.

In liquid chromatography coupled to isotope ratio mass spectrometry (LC-IRMS), analytes are separated on an LC system and consecutively oxidized to CO2, which is required for the determination of compound-specific carbon isotope ratios. Oxidation is performed in an online reactor by sulfate radicals. Reaction conditions in the interface depend on the flow conditions determined by the LC method and the flow rates and concentrations of oxidation agent and phosphoric acid added in the interface. To determine accurate isotope ratios, a quantitative conversion of the carbon contained in the analyte to the CO2 measurement gas is a prerequisite. Oxidation efficiencies are not commonly evaluated during method development, although certain analytes are known to be difficult to be oxidized by sulfate radicals. For the assessment of the oxidation efficiency of the LC-IRMS system, three different approaches were evaluated. (1) Residual organic carbon in the eluent stream of the interface was determined to calculate oxidation yields depending on the initial analyte concentration. (2) The IRMS response was calibrated to an inorganic carbon reference material to determine oxidation efficiencies with the help of the IRMS as a detector. (3) The oxidation temperature was deliberately reduced while monitoring the δ13C and signal intensity. The common assumption that a linear relation of IRMS signal to analyte concentration is an indicator for complete oxidation in LC-IRMS could be disproved. All three approaches can be applied for future method development in LC-IRMS, monitoring of existing flow injection applications, as well as for verification of complete oxidation in established LC-IRMS methods.

Methyl sulfates as methoxy isotopic reference materials for δ13 C and δ2 H measurements.

RATIONALE: Stable hydrogen and carbon isotope ratios of methoxy groups (OCH3 ) of plant organic matter have many potential applications in biogeochemical, atmospheric and food research. So far, most of the analyses of plant methoxy groups by isotope ratio mass spectrometry have employed liquid iodomethane (CH3 I) as the reference material to normalise stable isotope measurements of these moieties to isotope-δ scales. However, comparisons of measurements of stable hydrogen and carbon isotopes of plant methoxy groups are still hindered by the lack of suitable reference materials. METHODS: We have investigated two methyl sulfate salts (HUBG1 and HUBG2), which exclusively contain carbon and hydrogen from one methoxy group, for their suitability as methoxy reference materials. Firstly, the stable hydrogen and carbon isotope values of the bulk compounds were calibrated against international reference substances by high-temperature conversion- and elemental analyser isotope ratio mass spectrometry (HTC- and EA-IRMS). In a second step these values were compared with values obtained by measurements using gas chromatography/isotope ratio mass spectrometry (GC/IRMS) where prior to analysis the methoxy groups were converted into gaseous iodomethane. RESULTS: The 2 H- and 13 C isotopic abundances of HUBG1 measured by HTC- and EA-IRMS and expressed as δ-values on the usual international scales are -144.5 ± 1.2 mUr (n = 30) and -50.31 ± 0.16 mUr (n = 14), respectively. For HUBG2 we obtained -102.0 ± 1.3 mUr (n = 32) and +1.60 ± 0.12 mUr (n = 16). Furthermore, the values obtained by GC/IRMS were in good agreement with the HTC- and EA-IRMS values. CONCLUSIONS: We suggest that both methyl sulfates are suitable reference materials for normalisation of isotope measurements of carbon of plant methoxy groups to isotope-δ scales and for inter-laboratory calibration. For stable hydrogen isotope measurements, we suggest that in addition to HUBG1 and HUBG2 additional reference materials are required to cover the full range of plant methoxy groups reported so far.

Preliminary assessment of stable nitrogen and oxygen isotopic composition of USGS51 and USGS52 nitrous oxide reference gases and perspectives on calibration needs.

RATIONALE: Despite a long history and growing interest in isotopic analyses of N2 O, there is a lack of isotopically characterized N2 O isotopic reference materials (standards) to enable normalization and reporting of isotope-delta values. Here we report the isotopic characterization of two pure N2 O gas reference materials, USGS51 and USGS52, which are now available for laboratory calibration (https://isotopes.usgs.gov/lab/referencematerials.html). METHODS: A total of 400 sealed borosilicate glass tubes of each N2 O reference gas were prepared from a single gas filling of a high vacuum line. We demonstrated isotopic homogeneity via dual-inlet isotope-ratio mass spectrometry. Isotopic analyses of these reference materials were obtained from eight laboratories to evaluate interlaboratory variation and provide preliminary isotopic characterization of their δ15 N, δ18 O, δ15 Nα , δ15 Nß and site preference (SP ) values. RESULTS: The isotopic homogeneity of both USGS51 and USGS52 was demonstrated by one-sigma standard deviations associated with the determinations of their δ15 N, δ18 O, δ15 Nα , δ15 Nß and SP values of 0.12 mUr or better. The one-sigma standard deviations of SP measurements of USGS51 and USGS52 reported by eight laboratories participating in the interlaboratory comparison were 1.27 and 1.78 mUr, respectively. CONCLUSIONS: The agreement of isotope-delta values obtained in the interlaboratory comparison was not sufficient to provide reliable accurate isotope measurement values for USGS51 and USGS52. We propose that provisional values for the isotopic composition of USGS51 and USGS52 determined at the Tokyo Institute of Technology can be adopted for normalizing and reporting sample data until further refinements are achieved through additional calibration efforts.

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).

RATIONALE: Accurate hydrogen isotopic analysis of halogen- and sulfur-bearing organics has not been possible with traditional high-temperature conversion (HTC) because the formation of hydrogen-bearing reaction products other than molecular hydrogen (H2 ) is responsible for non-quantitative H2 yields and possible hydrogen isotopic fractionation. Our previously introduced, new chromium-based EA-Cr/HTC-IRMS (Elemental Analyzer-Chromium/High-Temperature Conversion Isotope Ratio Mass Spectrometry) technique focused primarily on nitrogen-bearing compounds. Several technical and analytical issues concerning halogen- and sulfur-bearing samples, however, remained unresolved and required further refinement of the reactor systems. METHODS: The EA-Cr/HTC reactor was substantially modified for the conversion of halogen- and sulfur-bearing samples. The performance of the novel conversion setup for solid and liquid samples was monitored and optimized using a simultaneously operating dual-detection system of IRMS and ion trap MS. The method with several variants in the reactor, including the addition of manganese metal chips, was evaluated in three laboratories using EA-Cr/HTC-IRMS (on-line method) and compared with traditional uranium-reduction-based conversion combined with manual dual-inlet IRMS analysis (off-line method) in one laboratory. RESULTS: The modified EA-Cr/HTC reactor setup showed an overall H2 -recovery of more than 96% for all halogen- and sulfur-bearing organic compounds. All results were successfully normalized via two-point calibration with VSMOW-SLAP reference waters. Precise and accurate hydrogen isotopic analysis was achieved for a variety of organics containing F-, Cl-, Br-, I-, and S-bearing heteroelements. The robust nature of the on-line EA-Cr/HTC technique was demonstrated by a series of 196 consecutive measurements with a single reactor filling. CONCLUSIONS: The optimized EA-Cr/HTC reactor design can be implemented in existing analytical equipment using commercially available material and is universally applicable for both heteroelement-bearing and heteroelement-free organic-compound classes. The sensitivity and simplicity of the on-line EA-Cr/HTC-IRMS technique provide a much needed tool for routine hydrogen-isotope source tracing of organic contaminants in the environment. Copyright © 2016 John Wiley & Sons, Ltd.

Reassessment of the NH4 NO3 thermal decomposition technique for calibration of the N2 O isotopic composition.

RATIONALE: In the last few years, the study of N2 O site-specific nitrogen isotope composition has been established as a powerful technique to disentangle N2 O emission pathways. This trend has been accelerated by significant analytical progress in the field of isotope ratio mass spectrometry (IRMS) and more recently quantum cascade laser absorption spectroscopy (QCLAS). METHODS: The ammonium nitrate (NH4 NO3 ) decomposition technique provides a strategy to scale the 15 N site-specific (SP ≡ Î´15 Nα - δ15 Nß ) and bulk (δ15 Nbulk  = (δ15 Nα  + Î´15 Nß )/2) isotopic composition of N2 O against the international standard for the 15 N/14 N isotope ratio (AIR-N2 ). Within the current project 15 N fractionation effects during thermal decomposition of NH4 NO3 on the N2 O site preference were studied using static and dynamic decomposition techniques. RESULTS: The validity of the NH4 NO3 decomposition technique to link NH4+ and NO3- moiety-specific δ15 N analysis by IRMS to the site-specific nitrogen isotopic composition of N2 O was confirmed. However, the accuracy of this approach for the calibration of δ15 Nα and δ15 Nß values was found to be limited by non-quantitative NH4 NO3 decomposition in combination with substantially different isotope enrichment factors for the conversion of the NO3- or NH4+ nitrogen atom into the α or ß position of the N2 O molecule. CONCLUSIONS: The study reveals that the completeness and reproducibility of the NH4 NO3 decomposition reaction currently confine the anchoring of N2 O site-specific isotopic composition to the international isotope ratio scale AIR-N2 . The authors suggest establishing a set of N2 O isotope reference materials with appropriate site-specific isotopic composition, as community standards, to improve inter-laboratory compatibility. Copyright © 2016 John Wiley & Sons, Ltd.

Automated simultaneous measurement of the δ(13) C and δ(2) H values of methane and the δ(13) C and δ(18) O values of carbon dioxide in flask air samples using a new multi cryo-trap/gas chromatography/isotope ratio mass spectrometry system.

RATIONALE: The isotopic composition of greenhouse gases helps to constrain global budgets and to study sink and source processes. We present a new system for high-precision stable isotope measurements of carbon, hydrogen and oxygen in atmospheric methane and carbon dioxide. The design is intended for analyzing flask air samples from existing sampling programs without the need for extra sample air for methane analysis. METHODS: CO2 and CH4 isotopes are measured simultaneously using two isotope ratio mass spectrometers, one for the analysis of δ(13) C and δ(18) O values and the second one for δ(2) H values. The inlet carousel delivers air from 16 sample positions (glass flasks 1-5 L and high-pressure cylinders). Three 10-port valves take aliquots from the sample stream. CH4 from 100-mL air aliquots is preconcentrated in 0.8-mL sample loops using a new cryo-trap system. A precisely calibrated working reference air is used in parallel with the sample according to the Principle of Identical Treatment. RESULTS: It takes about 36 hours for a fully calibrated analysis of a complete carousel including extractions of four working reference and one quality control reference air. Long-term precision values, as obtained from the quality control reference gas since 2012, account for 0.04 ‰ (δ(13) C values of CO2 ), 0.07 ‰ (δ(18) O values of CO2 ), 0.11 ‰ (δ(13) C values of CH4 ) and 1.0 ‰ (δ(2) H values of CH4 ). Within a single day, the system exhibits a typical methane δ(13) C standard deviation (1σ) of 0.06 ‰ for 10 repeated measurements. CONCLUSIONS: The system has been in routine operation at the MPI-BGC since 2012. Consistency of the data and compatibility with results from other laboratories at a high precision level are of utmost importance. A high sample throughput and reliability of operation are important achievements of the presented system to cope with the large number of air samples to be analyzed. Copyright © 2016 John Wiley & Sons, Ltd.

Organic Reference Materials for Hydrogen, Carbon, and Nitrogen Stable Isotope-Ratio Measurements: Caffeines, n-Alkanes, Fatty Acid Methyl Esters, Glycines, L-Valines, Polyethylenes, and Oils.

An international project developed, quality-tested, and determined isotope-δ values of 19 new organic reference materials (RMs) for hydrogen, carbon, and nitrogen stable isotope-ratio measurements, in addition to analyzing pre-existing RMs NBS 22 (oil), IAEA-CH-7 (polyethylene foil), and IAEA-600 (caffeine). These new RMs enable users to normalize measurements of samples to isotope-δ scales. The RMs span a range of δ(2)H(VSMOW-SLAP) values from -210.8 to +397.0 mUr or ‰, for δ(13)C(VPDB-LSVEC) from -40.81 to +0.49 mUr and for δ(15)N(Air) from -5.21 to +61.53 mUr. Many of the new RMs are amenable to gas and liquid chromatography. The RMs include triads of isotopically contrasting caffeines, C16 n-alkanes, n-C20-fatty acid methyl esters (FAMEs), glycines, and l-valines, together with polyethylene powder and string, one n-C17-FAME, a vacuum oil (NBS 22a) to replace NBS 22 oil, and a (2)H-enriched vacuum oil. A total of 11 laboratories from 7 countries used multiple analytical approaches and instrumentation for 2-point isotopic normalization against international primary measurement standards. The use of reference waters in silver tubes allowed direct normalization of δ(2)H values of organic materials against isotopic reference waters following the principle of identical treatment. Bayesian statistical analysis yielded the mean values reported here. New RMs are numbered from USGS61 through USGS78, in addition to NBS 22a. Because of exchangeable hydrogen, amino acid RMs currently are recommended only for carbon- and nitrogen-isotope measurements. Some amino acids contain (13)C and carbon-bound organic (2)H-enrichments at different molecular sites to provide RMs for potential site-specific isotopic analysis in future studies.

A new organic reference material, l-glutamic acid, USGS41a, for δ(13) C and δ(15) N measurements - a replacement for USGS41.

RATIONALE: The widely used l-glutamic acid isotopic reference material USGS41, enriched in both (13) C and (15) N, is nearly exhausted. A new material, USGS41a, has been prepared as a replacement for USGS41. METHODS: USGS41a was prepared by dissolving analytical grade l-glutamic acid enriched in (13) C and (15) N together with l-glutamic acid of normal isotopic composition. The δ(13) C and δ(15) N values of USGS41a were directly or indirectly normalized with the international reference materials NBS 19 calcium carbonate (δ(13) CVPDB = +1.95 mUr, where milliurey = 0.001 = 1 ‰), LSVEC lithium carbonate (δ(13) CVPDB = -46.6 mUr), and IAEA-N-1 ammonium sulfate (δ(15) NAir = +0.43 mUr) and USGS32 potassium nitrate (δ(15) N = +180 mUr exactly) by on-line combustion, continuous-flow isotope-ratio mass spectrometry, and off-line dual-inlet isotope-ratio mass spectrometry. RESULTS: USGS41a is isotopically homogeneous; the reproducibility of δ(13) C and δ(15) N is better than 0.07 mUr and 0.09 mUr, respectively, in 200-µg amounts. It has a δ(13) C value of +36.55 mUr relative to VPDB and a δ(15) N value of +47.55 mUr relative to N2 in air. USGS41 was found to be hydroscopic, probably due to the presence of pyroglutamic acid. Experimental results indicate that the chemical purity of USGS41a is substantially better than that of USGS41. CONCLUSIONS: The new isotopic reference material USGS41a can be used with USGS40 (having a δ(13) CVPDB value of -26.39 mUr and a δ(15) NAir value of -4.52 mUr) for (i) analyzing local laboratory isotopic reference materials, and (ii) quantifying drift with time, mass-dependent isotopic fractionation, and isotope-ratio-scale contraction for isotopic analysis of biological and organic materials. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.

Isotopic disproportionation during hydrogen isotopic analysis of nitrogen-bearing organic compounds.

RATIONALE: High-precision hydrogen isotope ratio analysis of nitrogen-bearing organic materials using high-temperature conversion (HTC) techniques has proven troublesome in the past. Formation of reaction products other than molecular hydrogen (H(2)) has been suspected as a possible cause of incomplete H(2) yield and hydrogen isotopic fractionation. METHODS: The classical HTC reactor setup and a modified version including elemental chromium, both operated at temperatures in excess of 1400 °C, have been compared using a selection of nitrogen-bearing organic compounds, including caffeine. A focus of the experiments was to avoid or suppress hydrogen cyanide (HCN) formation and to reach quantitative H(2) yields. The technique also was optimized to provide acceptable sample throughput. RESULTS: The classical HTC reaction of a number of selected compounds exhibited H(2) yields from 60 to 90 %. Yields close to 100 % were measured for the experiments with the chromium-enhanced reactor. The δ(2)H values also were substantially different between the two types of experiments. For the majority of the compounds studied, a highly significant relationship was observed between the amount of missing H(2) and the number of nitrogen atoms in the molecules, suggesting the pyrolytic formation of HCN as a byproduct. A similar linear relationship was found between the amount of missing H(2) and the observed hydrogen isotopic result, reflecting isotopic fractionation. CONCLUSIONS: The classical HTC technique to produce H(2) from organic materials using high temperatures in the presence of glassy carbon is not suitable for nitrogen-bearing compounds. Adding chromium to the reaction zone improves the yield to 100 % in most cases. The initial formation of HCN is accompanied by a strong hydrogen isotope effect, with the observed hydrogen isotope results on H(2) being substantially shifted to more negative δ(2)H values. The reaction can be understood as an initial disproportionation leading to H(2) and HCN with the HCN-hydrogen systematically enriched in (2)H by more than 50 ‰. In the reaction of HCN with chromium, H(2) and chromium-containing solid residues are formed quantitatively.

On-line hydrogen-isotope measurements of organic samples using elemental chromium: an extension for high temperature elemental-analyzer techniques.

The high temperature conversion (HTC) technique using an elemental analyzer with a glassy carbon tube and filling (temperature conversion/elemental analysis, TC/EA) is a widely used method for hydrogen isotopic analysis of water and many solid and liquid organic samples with analysis by isotope-ratio mass spectrometry (IRMS). However, the TC/EA IRMS method may produce inaccurate δ(2)H results, with values deviating by more than 20 mUr (milliurey = 0.001 = 1‰) from the true value for some materials. We show that a single-oven, chromium-filled elemental analyzer coupled to an IRMS substantially improves the measurement quality and reliability for hydrogen isotopic compositions of organic substances (Cr-EA method). Hot chromium maximizes the yield of molecular hydrogen in a helium carrier gas by irreversibly and quantitatively scavenging all reactive elements except hydrogen. In contrast, under TC/EA conditions, heteroelements like nitrogen or chlorine (and other halogens) can form hydrogen cyanide (HCN) or hydrogen chloride (HCl) and this can cause isotopic fractionation. The Cr-EA technique thus expands the analytical possibilities for on-line hydrogen-isotope measurements of organic samples significantly. This method yielded reproducibility values (1-sigma) for δ(2)H measurements on water and caffeine samples of better than 1.0 and 0.5 mUr, respectively. To overcome handling problems with water as the principal calibration anchor for hydrogen isotopic measurements, we have employed an effective and simple strategy using reference waters or other liquids sealed in silver-tube segments. These crimped silver tubes can be employed in both the Cr-EA and TC/EA techniques. They simplify considerably the normalization of hydrogen-isotope measurement data to the VSMOW-SLAP (Vienna Standard Mean Ocean Water-Standard Light Antarctic Precipitation) scale, and their use improves accuracy of the data by eliminating evaporative loss and associated isotopic fractionation while handling water as a bulk sample. The calibration of organic samples, commonly having high δ(2)H values, will benefit from the availability of suitably (2)H-enriched reference waters, extending the VSMOW-SLAP scale above zero.

Drought impact on water use efficiency and intra-annual density fluctuations in Erica arborea on Elba (Italy).

Erica arborea (L) is a widespread Mediterranean species, able to cope with water stress and colonize semiarid environments. The eco-physiological plasticity of this species was evaluated by studying plants growing at two sites with different soil moistures on the island of Elba (Italy), through dendrochronological, wood-anatomical analyses and stable isotopes measurements. Intra-annual density fluctuations (IADFs) were abundant in tree rings, and were identified as the key parameter to understand site-specific plant responses to water stress. Our findings showed that the formation of IADFs is mainly related to the high temperature, precipitation patterns and probably to soil water availability, which differs at the selected study sites. The recorded increase in the (13) C-derived intrinsic water use efficiency at the IADFs level was linked to reduced water loss rather than to increasing C assimilation. The variation in vessel size and the different absolute values of δ(18) O among trees growing at the two study sites underlined possible differences in stomatal control of water loss and possible differences in sources of water uptake. This approach not only helped monitor seasonal environmental differences through tree-ring width, but also added valuable information on E. arborea responses to drought and their ecological implications for Mediterranean vegetation dynamics.

A common fungal associate of the spruce bark beetle metabolizes the stilbene defenses of Norway spruce.

Norway spruce (Picea abies) forests suffer periodic fatal attacks by the bark beetle Ips typographus and its fungal associate, Ceratocystis polonica. Norway spruce protects itself against fungal and bark beetle invasion by the production of terpenoid resins, but it is unclear whether resins or other defenses are effective against the fungus. We investigated stilbenes, a group of phenolic compounds found in Norway spruce bark with a diaryl-ethene skeleton with known antifungal properties. During C. polonica infection, stilbene biosynthesis was up-regulated, as evidenced by elevated transcript levels of stilbene synthase genes. However, stilbene concentrations actually declined during infection, and this was due to fungal metabolism. C. polonica converted stilbenes to ring-opened, deglycosylated, and dimeric products. Chromatographic separation of C. polonica protein extracts confirmed that these metabolites arose from specific fungal enzyme activities. Comparison of C. polonica strains showed that rapid conversion of host phenolics is associated with higher virulence. C. polonica is so well adapted to its host's chemical defenses that it is even able to use host phenolic compounds as its sole carbon source.

Stable isotope deltas: tiny, yet robust signatures in nature.

Although most of them are relatively small, stable isotope deltas of naturally occurring substances are robust and enable workers in anthropology, atmospheric sciences, biology, chemistry, environmental sciences, food and drug authentication, forensic science, geochemistry, geology, oceanography, and paleoclimatology to study a variety of topics. Two fundamental processes explain the stable isotope deltas measured in most terrestrial systems: isotopic fractionation and isotope mixing. Isotopic fractionation is the result of equilibrium or kinetic physicochemical processes that fractionate isotopes because of small differences in physical or chemical properties of molecular species having different isotopes. It is shown that the mixing of radioactive and stable isotope end members can be modelled to provide information on many natural processes, including (14)C abundances in the modern atmosphere and the stable hydrogen and oxygen isotopic compositions of the oceans during glacial and interglacial times. The calculation of mixing fractions using isotope balance equations with isotope deltas can be substantially in error when substances with high concentrations of heavy isotopes (e.g. (13)C, (2)H, and (18)O ) are mixed. In such cases, calculations using mole fractions are preferred as they produce accurate mixing fractions. Isotope deltas are dimensionless quantities. In the International System of Units (SI), these quantities have the unit 1 and the usual list of prefixes is not applicable. To overcome traditional limitations with expressing orders of magnitude differences in isotope deltas, we propose the term urey (symbol Ur), after Harold C. Urey, for the unit 1. In such a manner, an isotope delta value expressed traditionally as-25 per mil can be written as-25 mUr (or-2.5 cUr or-0.25 dUr; the use of any SI prefix is possible). Likewise, very small isotopic differences often expressed in per meg 'units' are easily included (e.g. either+0.015 ‰ or+15 per meg can be written as+15 µUr.