Using hooves for high-resolution isotopic reconstruction of bovine dietary history

The objective of this study was to ascertain whether sequential sampling and isotopic analysis of bovine hooves could be used to reconstruct the dietary history of cattle. A controlled, on-farm experiment was conducted in which cattle were switched from a barley-based diet to an isotopically different diet incorporating maize, urea and seaweed (the isotopic spacing between diets was 13.6 per thousand for delta(13)C and 8.0 per thousand for delta(15)N) and maintained on that diet for 168 days. Postmortem sampling of the cleaned anterior wall of the lateral, left front claw was carried out on five individuals using a micro-drilling technique. From the first 60 mm of each claw, up to 41 samples with a spacing between them of less than 1 mm were collected. Bands were less than 1 mm deep and had a mean width of 1.2 mm. The hoof keratin showed a rapid increase followed by a slower increase in its delta(13)C and delta(15)N values following the diet switch, suggesting that C and N in hoof keratin originate from more than one pool. However, the response of the N isotope composition of the hoof was somewhat delayed compared with that of C. Estimated mean hoof growth rates for these cattle were 10.5 +/- 2.3 mm per month and 6.7 +/- 1.0 mm per month (+/-SD, n = 5) when receiving the barley-based transition diet and the maize-based experimental diet, respectively. These values are considerably higher than previous estimates obtained by visual methods and they suggest that diet may have a greater influence on hoof growth rates than seasonality. These results demonstrate that hooves are a suitable incremental tissue for high-resolution isotopic reconstruction of the dietary history of bovine animals.     

Oxygen isotopes in nitrate: new reference materials for 18O:17O:16O measurements and observations on nitrate-water equilibration

Despite a rapidly growing literature on analytical methods and field applications of O isotope-ratio measurements of NO(3)(-) in environmental studies, there is evidence that the reported data may not be comparable because reference materials with widely varying delta(18)O values have not been readily available. To address this problem, we prepared large quantities of two nitrate salts with contrasting O isotopic compositions for distribution as reference materials for O isotope-ratio measurements: USGS34 (KNO(3)) with low delta(18)O and USGS35 (NaNO(3)) with high delta(18)O and 'mass-independent' delta(17)O. The procedure used to produce USGS34 involved equilibration of HNO(3) with (18)O-depleted meteoric water. Nitric acid equilibration is proposed as a simple method for producing laboratory NO(3)(-) reference materials with a range of delta(18)O values and normal (mass-dependent) (18)O:(17)O:(16)O variation. Preliminary data indicate that the equilibrium O isotope-fractionation factor (alpha) between [NO(3)(-)] and H(2)O decreases with increasing temperature from 1.0215 at 22 degrees C to 1.0131 at 100 degrees C. USGS35 was purified from the nitrate ore deposits of the Atacama Desert in Chile and has a high (17)O:(18)O ratio owing to its atmospheric origin. These new reference materials, combined with previously distributed NO(3) (-) isotopic reference materials IAEA-N3 (=IAEA-NO-3) and USGS32, can be used to calibrate local laboratory reference materials for determining offset values, scale factors, and mass-independent effects on N and O isotope-ratio measurements in a wide variety of environmental NO(3)(-) samples. Preliminary analyses yield the following results (normalized with respect to VSMOW and SLAP, with reproducibilities of +/-0.2-0.3 per thousand, 1sigma): IAEA-N3 has delta(18)O = +25.6 per thousand and delta(17)O = +13.2 per thousand; USGS32 has delta(18)O = +25.7 per thousand; USGS34 has delta(18)O = -27.9 per thousand and delta(17)O = -14.8 per thousand; and USGS35 has delta(18)O = +57.5 per thousand and delta(17)O = +51.5 per thousand.     

Using dual-bacterial denitrification to improve delta15N determinations of nitrates containing mass-independent 17O

The bacterial denitrification method for isotopic analysis of nitrate using N(2)O generated from Pseudomonas aureofaciens may overestimate delta(15)N values by as much as 1-2 per thousand for samples containing atmospheric nitrate because of mass-independent (17)O variations in such samples. By analyzing such samples for delta(15)N and delta(18)O using the denitrifier Pseudomonas chlororaphis, one obtains nearly correct delta(15)N values because oxygen in N(2)O generated by P. chlororaphis is primarily derived from H(2)O. The difference between the apparent delta(15)N value determined with P. aureofaciens and that determined with P. chlororaphis, assuming mass-dependent oxygen isotopic fractionation, reflects the amount of mass-independent (17)O in a nitrate sample. By interspersing nitrate isotopic reference materials having substantially different delta(18)O values with samples, one can normalize oxygen isotope ratios and determine the fractions of oxygen in N(2)O derived from the nitrate and from water with each denitrifier. This information can be used to improve delta(15)N values of nitrates having excess (17)O. The same analyses also yield estimates of the magnitude of (17)O excess in the nitrate (expressed as Delta(17)O) that may be useful in some environmental studies. The 1-sigma uncertainties of delta(15)N, delta(18)O and Delta(17)O measurements are +/-0.2, +/-0.3 and +/-5 per thousand, respectively.     

Deuterium and oxygen-18 determination of microliter quantities of a water sample using an automated equilibrator

We describe a modified version of the equilibration method and a correction algorithm for isotope ratio measurements of small quantities of water samples. The deltaD and the delta(18)O of the same water sample can both be analyzed using an automated equilibrator with sample sizes as small as 50 microL. Conventional equilibration techniques generally require water samples of several microL. That limitation is attributable mainly to changes in the isotope ratio ((18)O/(16)O or D/H) of water samples during isotopic exchange between the equilibration gas (CO(2) or H(2)) and water, and therefore the technique for microL quantities of water requires mass-balance correction using the water/gas (CO(2) or H(2)) mole ratio to correct this isotopic effect. We quantitatively evaluate factors controlling the variability of the isotopic effect due to sample size. Theoretical consideration shows that a simple linear equation corrects for the effects without determining parameters such as isotope fractionation factors and water/gas mole ratios. Precisions (1-sigma) of 50-microL meteoric water samples whose isotopic compositions of -1.4 to -396.2 per thousand for deltaD are +/-0.5 to +/-0.6 per thousand, and of -0.37 to -51.37 per thousand for delta(18)O are +/-0.01 to +/-0.11 per thousand.     

Delta13C analyses of calcium carbonate: Comparison between the GasBench and elemental analyzer techniques

Measurements of stable carbon isotopic composition (delta13C) of carbonates or carbonate-rich soils are seldom performed in a continuous-flow isotope ratio mass spectrometer (IRMS) using an elemental analyzer (EA) as an online sample preparation device. Such analyses are routinely carried out with an external precision better than 0.1 per thousand using a GasBench II (GB) sample preparation device coupled online with a continuous-flow IRMS. In this paper, we report and compare delta13C analyses (86 total analyses) of calcium carbonates obtained by using both the GB and the EA. Using both techniques, the delta13C compositions of two in-house carbonate standards (MERCK carbonate and NR calcite) and ten selected carbonate-rich paleosol samples (of variable CaCO3 content) were analyzed, and data are reported in the VPDB scale calibrated against international standards, NBS 18 and 19. For the in-house standards analyzed by both techniques, a precision better than 0.08 per thousand is achieved. The analytical errors (1sigma) computed from multiple analyses of the delta13C of both the MERCK and NR obtained by the above two techniques are nearly identical. In general, the 1sigma (internal error) of paleosol analyses obtained in the GB is better than 0.06 per thousand, whereas that for the analyses in the EA (three repetitive analyses of the same sample) varies in the range 0.05-0.21 per thousand. However, for paleosols having more than 85% CaCO3, 1sigma is better than 0.15 per thousand (similar to the instrument precision), and in this case the delta13C(VPDB) of samples obtained by the GB is similar to that obtained by the EA. Our results suggest that the delta13C of pure calcium carbonate samples can also be analyzed using the EA technique.     

Clinical-scale investigation of stable isotopes in human blood: delta13C and delta15N from 406 patients at the Johns Hopkins Medical Institutions

Objective chemical biomarkers are needed in clinical studies of diet-related diseases to supplement subjective self-reporting methods. We report on several critical experiments for the development of clinically legitimate dietary stable isotope biomarkers within human blood. Our examination of human blood revealed the following: (1) Within blood clot and serum from anonymous individuals (201 males, 205 females) we observed: mean serum delta13C = -19.1 +/- 0.8 per thousand (standard deviation, SD); clot, -19.3 +/- 0.8 per thousand (SD); range = -15.8 per thousand to -23.4 per thousand. Highly statistically significant differences are observed between clot and serum, males and females for both clot and serum. For 15N (n = 206), mean serum = +8.8 +/- 0.5 per thousand (SD); clot +7.4 +/- 0.4 per thousand (SD); range = +6.3 per thousand to +10.5 per thousand. Blood serum is enriched in 15N relative to blood clot by +1.4 per thousand on average, which may reflect differing protein amino acid content. Serum nitrogen is statistically significantly different for males and females, however, clot shows no statistical difference. (2) Relative to clot, capillary blood is marginally different for 13C, but not 15N. Clot 13C is not significantly different from serum; however, it is depleted in 15N by 1.5 per thousand relative to serum. (3) We assessed the effect of blood additives (sodium fluoride and polymerized acrylamide resin) and laboratory process (autoclaving, freeze drying) commonly used to preserve or prepare venous blood. On average, no alteration in delta13C or delta15N is detected compared with unadulterated blood from the same individual. (4) Storage of blood with and without the additives described above for a period of up to 115 days exhibits statistically significant differences for 13C and 15N for sodium fluoride. However, storage for unadulterated blood and blood preserved with polymerized acrylamide resin does not change the delta13C or delta15N isotopic composition of the blood in a significant way. With these experiments, we gain a clinical context for future development of a stable isotope based dietary biomarker.     

Multiple stable isotope (18O, 13C, 15N and 34S) analysis of human hair to identify the recent migrants in a rural community in SW England

Relationships between recent migration and hair delta(18)O values were examined for 40 people living in a rural community in SW England. The isotopic contents of 35 'local' hair samples were compared with those of 5 recently arrived individuals (from Australia, Canada, Chile, Germany and the USA). The hair delta(18)O values of these 'visitors' were +7.9 (Omaha, USA), +11.2 (Jena, Germany), +12.1 (Osorno, Chile), +12.6 (Montreal, Canada) and +14.3 per thousand (Adelaide, Australia). The hair value for the USA visitor (+7.9 per thousand) fell outside the range for the 33 local adult residents, +10.5 to +14.3 per thousand (+12.7 +/- 0.8 per thousand). Hair delta(18)O values did not identify the individuals from Adelaide, Montreal and Osorno as 'visitors', but hair delta(13)C or delta(34)S data did. Combining the hair delta(18)O, delta(13)C and delta(34)S values using principal components analysis (two components explained 89% of the overall variation among the 40 subjects) helped to more clearly distinguish European from non-European individuals, indicating the existence of global overall isotope (geo-origin) relationships.     

Stable isotope natural abundance of nitrous oxide emitted from Antarctic tundra soils: effects of sea animal excrement depositions

Nitrous oxide (N2O), a greenhouse gas, is mainly emitted from soils during the nitrification and denitrification processes. N2O stable isotope investigations can help to characterize the N2O sources and N2O production mechanisms. N2O isotope measurements have been conducted for different types of global terrestrial ecosystems. However, no isotopic data of N2O emitted from Antarctic tundra ecosystems have been reported although the coastal ice-free tundra around Antarctic continent is the largest sea animal colony on the global scale. Here, we report for the first time stable isotope composition of N2O emitted from Antarctic sea animal colonies (including penguin, seal and skua colonies) and normal tundra soils using in situ field observations and laboratory incubations, and we have analyzed the effects of sea animal excrement depositions on stable isotope natural abundance of N2O. For all the field sites, the soil-emitted N2O was 15N- and 18O-depleted compared with N2O in local ambient air. The mean delta values of the soil-emitted N2O were delta15N = -13.5 +/- 3.2 per thousand and delta18O = 26.2 +/- 1.4 per thousand for the penguin colony, delta15N = -11.5 +/- 5.1 per thousand and delta18O = 26.4 +/- 3.5 per thousand for the skua colony and delta15N = -18.9 +/- 0.7 per thousand and delta18O = 28.8 +/- 1.3 per thousand for the seal colony. In the soil incubations, the isotopic composition of N2O was measured under N2 and under ambient air conditions. The soils incubated under the ambient air emitted very little N2O (2.93 microg N2O--N kg(-1)). Under N2 conditions, much more N2O was formed (9.74 microg N2O--N kg(-1)), and the mean delta15N and delta18O values of N2O were -19.1 +/- 8.0 per thousand and 21.3 +/- 4.3 per thousand, respectively, from penguin colony soils, and -17.0 +/- 4.2 per thousand and 20.6 +/- 3.5 per thousand, respectively, from seal colony soils. The data from in situ field observations and laboratory experiments point to denitrification as the predominant N2O source from Antarctic sea animal colonies.     

Development of a compound-specific isotope analysis method for acetone via 2,4-dinitrophenylhydrazine derivatization

A novel method has been developed for compound-specific isotope analysis for acetone via DNPH (2,4-dinitrophenylhydrazine) derivatization together with combined gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Acetone reagents were used to assess delta13C fractionation during the DNPH derivatization process. Reduplicate delta13C analyses were designed to evaluate the reproducibility of the derivatization, with an average error (1 standard deviation) of 0.17 +/- 0.05 per thousand, and average analytical error of 0.28 +/- 0.09 per thousand. The derivatization process introduces no isotopic fractionation for acetone (the average difference between the predicted and analytical delta13C values was 0.09 +/- 0.20 per thousand, within the precision limits of the GC/C/IRMS measurements), which permits computation of the delta13C values for the original underivatized acetone through a mass balance equation. Together with further studies of the carbon isotopic effect during the atmospheric acetone-sampling procedure, it will be possible to use DNPH derivatization for carbon isotope analysis of atmospheric acetone.     

Methods to reduce interference effects in thermal conversion elemental analyzer/continuous flow isotope ratio mass spectrometry delta18O measurements of nitrogen-containing compounds

On-line determination of the oxygen isotopic composition (delta(18)O value) in organic and inorganic samples is commonly performed using a thermal conversion elemental analyzer (TC-EA) linked to a continuous flow isotope ratio mass spectrometry (IRMS) system. Accurate delta(18)O analysis of N-containing compounds (like nitrates) by TC-EA-IRMS may be complicated because of interference of the N(2) peak on the m/z 30 signal of the CO peak. In this study we evaluated the effectiveness of two methods to overcome this interference which do not require any hardware modifications of standard TC-EA-IRMS systems. These methods were (1) reducing the amount of N(2) introduced into the ion source through He dilution of the N(2) peak and (2) an improved background correction on the CO m/z 30 sample peak integration.Our results show that He dilution is as effective as diverting the N(2) peak in order to eliminate this interference. We conclude that the He-dilution technique is a viable method for the delta(18)O analysis of nitrates and other N-containing samples (which are not routinely measured using He dilution) using TC-EA-IRMS, since it can easily be programmed in the standard software of IRMS systems. With the He-dilution technique delta(18)O values of the nitrate isotope standards USGS34, IAEA-N3 and USGS35 were measured using the shortest possible traceability chain to the VSMOW-SLAP scale, and the results were -28.1 +/- 0.1 per thousand, +25.5 +/- 0.1 per thousand and +57.5 +/- 0.2 per thousand, respectively. An improved background correction was also an effective method, but required manual correction of the raw data.     

A selective unsaturated hydrocarbon subtraction technique for stable carbon isotopic analysis of atmospheric methyl chloride, methyl bromide, and C2-C5 saturated hydrocarbons using continuous-flow isotope ratio mass spectrometry

Using continuous-flow isotope ratio mass spectrometry, we have developed a new analytical system which enables us to determine the stable carbon isotopic composition of CH3Cl, CH3Br, and C2-C5 saturated hydrocarbons in gas samples even if they contain substantial amounts of unsaturated hydrocarbons, using an I2O5 reagent for their selective subtraction. The analytical precision of the delta13C determinations is better than 0.5 per thousand for >300 pmolC injections and better than 5 per thousand for 20 pmolC injections. Using the system, delta13C values for CH3Cl and CH3Br were found in burning exhaust that contain a substantial quantity of unsaturated hydrocarbons. CH3Cl and CH3Br measured in exhaust from burning rice plants exhibit highly 13C-depleted values of -56.6 +/- 1.3 per thousand and -48.6 +/- 3.9 per thousand, respectively, while saturated hydrocarbons exhibit delta13C values (-26.4 to -28.9 per thousand) that are comparable with the total delta13C value of the parent material (rice plant; -28.0 per thousand). Using the system, we can determine the delta13C values of methyl halides and hydrocarbons in many kinds of gas samples.     

A gas chromatography/combustion/isotope ratio mass spectrometry system for high-precision delta13C measurements of atmospheric methane extracted from ice core samples

Past atmospheric composition can be reconstructed by the analysis of air enclosures in polar ice cores which archive ancient air in decadal to centennial resolution. Due to the different carbon isotopic signatures of different methane sources high-precision measurements of delta13CH4 in ice cores provide clues about the global methane cycle in the past. We developed a highly automated (continuous-flow) gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) technique for ice core samples of approximately 200 g. The methane is melt-extracted using a purge-and-trap method, then separated from the main air constituents, combusted and measured as CO2 by a conventional isotope ratio mass spectrometer. One CO2 working standard, one CH4 and two air reference gases are used to identify potential sources of isotope fractionation within the entire sample preparation process and to enhance the stability, reproducibility and accuracy of the measurement. After correction for gravitational fractionation, pre-industrial air samples from Greenland ice (1831 +/- 40 years) show a delta13C(VPDB) of -49.54 +/- 0.13 per thousand and Antarctic samples (1530 +/- 25 years) show a delta13C(VPDB) of -48.00 +/- 0.12 per thousand in good agreement with published data.     

Effects of acidification on carbon and nitrogen stable isotopes of benthic macrofauna from a tropical coral reef

Stable isotope analyses are widely used to determine trophic levels in ecological studies. We have investigated the effects of carbonate removal via acidification on the stable carbon and nitrogen isotopic composition of 33 species of tropical benthic macrofauna, and we report guidelines for standardizing this procedure for higher taxa in tropical coral reef ecosystems. Many tropical benthic invertebrates are small in size, and therefore body tissue isolation (separating organic carbon from inorganic structures) is difficult and time-consuming. Literature reviews of invertebrate studies show a lack of consistent procedures and guidelines for preparation techniques, especially for carbonate removal via acidification of whole individuals. We find that acidification decreases the delta(13)C values of samples containing carbonate, with shifts ranging from 0.21 to 3.20 per thousand, which can be related to CaCO(3) content (assessed by a carbonate proxy), justifying acid pre-treatment. Carbonate-containing taxa benefiting from acidification included Amphinomida, Terebellida (Annelida), Anomura, Brachyura, Caridea, Amphipoda, Tanaidacea (Arthropoda) and Edwardsiida (Cnidaria). The delta(13)C shifts of samples containing no carbonate varied up to 0.02 +/- 0.20 per thousand. As this induced delta(13)C shift was lower than the range of an average trophic level shift (0.5 to 1 per thousand), we conclude that acid pre-treatment is unnecessary. Carbonate-free taxa consisted of Eunicida, Phyllodocida (Annelida) and Mollusca. We note minimal impact of acidification on delta(15)N values except for Brachyura, which showed a shift of 0.83 +/- 0.46 per thousand, which is still lower than a single trophic level shift (2.9-3.8 per thousand). We conclude that for trophic level studies, both the delta(13)C and the delta(15)N of carbonate-rich macrofauna can be determined from the same acidified sample. Copyright (c) 2008 John Wiley & Sons, Ltd.     

The effect of growth rate on tissue-diet isotopic spacing in rapidly growing animals. An experimental study with Atlantic salmon (Salmo salar)

The difference in isotopic composition between a consumer's tissues and that of its diet is a critical aspect of the use of stable isotope analyses in ecological and palaeoecological studies. In a controlled feeding experiment with the Atlantic salmon, Salmo salar, we demonstrate for the first time that the value of tissue-diet isotope spacing in nitrogen in a growing animal is not constant, but varies inversely with growth rate. The value of tissue-diet isotopic spacing in N reflects N use efficiency. Thus, in salmon, growth rate is accompanied by, or requires, increased N use efficiency. The total range in tissue-diet isotopic spacing in N seen in the experimental population of 25 fish was 1 per thousand, approximately 50% of the total trophic shift. Mean equilibrium tissue-diet isotopic spacing (+/-standard deviation) in salmon averaged 2.3 per thousand (+/-0.3 per thousand) and 0.0 per thousand (+/-0.3 per thousand) for N in muscle and liver, respectively, and 2.1 per thousand (+/-0.1 per thousand) and 1.6 per thousand (+/-0.3 per thousand) for C in muscle and liver, respectively. Feeding with a mixed dietary source (wheat and fish-meal origin) resulted in tissue-diet isotopic fractionation in both C and N due to the differential digestibility of food components with distinct isotopic composition. The rate of change in isotopic composition of S. salar tissues was dominated by growth, but the estimated contribution of metabolic turnover to change in tissue N was relatively high for an ectothermic animal at ca. 20-40%. The estimated half-life for metabolic turnover of the tissue N pool was ca. 4 months in both muscle and liver tissue. This is the first study to demonstrate a direct relationship between tissue-diet isotopic spacing in N and growth rate and adds to the growing list of factors known to influence the level of isotopic separation between a consumer's tissue and that of its diet.     

Practical and theoretical considerations in the gas chromatography/combustion/isotope ratio mass spectrometry delta(13)C analysis of small polyfunctional compounds

Carbohydrates and proteins are among the most abundant naturally occurring biomolecules and so suitable methods for their reliable stable isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) are required. Due to the non-volatile nature of these compounds they require hydrolytic cleavage to their lower molecular weight subunits and derivatisation prior to GC/C/IRMS analysis. The addition of carbon to the molecules and any kinetic isotopic fractionation associated with derivatisation must be accounted for in order to provide meaningful stable isotope values and estimates of propagated errors. To illustrate these points amino acid trifluoroacetate/isopropyl esters and alditol acetates were prepared from authentic amino acids and monosaccharides, respectively. As predicted from the derivatisation reaction mechanisms, a kinetic isotope effect was observed which precludes direct calculation of delta(13)C values of the amino acids and monosaccharides by simple mass balance equations. This study shows that the kinetic isotope effect associated with derivatisation is both reproducible and robust, thereby allowing the use of correction factors. We show how correction factors can be determined and accurately account for the addition of derivative carbon. As a consequence of the addition of a molar excess of carbon and the existence of a kinetic isotope effect during derivatisation, errors associated with determined delta(13)C values must be assessed. We illustrate how such errors can be quantified (for monosaccharides +/-1.3 per thousand and for amino acids between +/-0.8 per thousand and +/-1.4 per thousand). With the magnitude of the errors for a given delta(13)C value of a monosaccharide or amino acid quantified, it is possible to make reliable interpretations of delta(13)C values, thereby validating the determination of delta(13)C values of amino acids as TFA/IP esters and monosaccharides as alditol acetates.     

Collection and storage of CO2 for 13C analysis: An application to separate soil CO2 efflux into root- and soil-derived components

Soil surface CO2 efflux is comprised of CO2 from (i) root respiration and rhizosphere microbes and (ii) heterotrophic respiration from the breakdown of soil organic matter (SOM). This efflux may be partitioned between these sources using delta13C measurements. To achieve this, continuous flow isotope ratio mass spectrometry can be used and, in conjunction with 10 mL septum-capped vials, large numbers of samples may be analysed using a Finnigan MAT Delta(plus)XP interfaced to a Gas Bench II. Here we describe a number of advances to facilitate such work, including: (i) a technique for monitoring mass spectrometer performance, (ii) improvements to sample storage, and (iii) a gas-handling system for incubating and sampling the CO2 derived from roots and soils. Mass spectrometer performance was monitored using an automated refillable vial. Compressed air analysed with this system had mean delta13C of -9.61 +/- 0.16 per thousand (+/- 1sigma, n = 28) collected over four runs. Heating the butyl rubber septa used to seal the vials at 105 degrees C for 12 h improved the sample storage. After air transportation over 12 days, the isotope composition of the CO2 at ambient concentrations was unchanged (before: -35.2 +/- 0.10 per thousand, n = 4; after: -35.3 +/- 0.10 per thousand, n = 15); without heat treatment of the septa the CO2 became slightly enriched (-35.0 +/- 0.14 per thousand, n = 15). The linearity of the Gas Bench II was found to decline above 8000 micromol CO2 mol(-1). To stay within a linear range and to allow the incubation of soil and root material we describe a gas-handling system based around a peristaltic pump. Finally, we demonstrate these methods by growing a C-4 grass (Guinea grass, Panicum maximum Jacq.) in a C-3 soil. Root respiration was found to contribute between 5 and 22% to the soil surface CO2 efflux. These methodologies will facilitate experiments aimed at measuring the isotopic composition of soil-derived CO2 across a range of ecological applications.     

Minimization of carbon addition during derivatization of monosaccharides for compound-specific delta13C analysis in environmental research

Little is known about the delta13C composition of monosaccharides representing the largest carbon reservoir in the biosphere. The main reason for this might be that monosaccharides have to be derivatized prior to gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analyses and that a large isotopic correction is necessary for the carbon that has to be added to the original molecule during derivatization, resulting in large uncertainty of the calculated delta13C values of individual monosaccharides. The amount of added derivatization carbon is twice (alditol acetates) or even three times (trimethylsilyl (TMS) derivatives) as high as the amount of the original monosaccharide carbon. In addition, isotope fractionation occurs during acetylation. Therefore, the objectives of this study were (i) to minimize carbon addition during derivatization for GC/C/IRMS measurements of monosaccharides in soil and sediment samples and (ii) to quantify improvements in accuracy and precision of the final results. Minimization of carbon addition was accomplished by derivatization with methylboronic acid (MBA) and TMS thereafter (MBA method). Monosaccharides derivatized with the MBA method instead of TMS reduced the number of added carbon atoms from 2.2-2.7 to 0.3-0.8 per sugar carbon atom. Although the precision of GC/C/IRMS measurements with both methods is comparable (about 0.3 per thousand), delta13C values of an internal standard indicated that the newly developed MBA method is about 2 per thousand more accurate than the TMS method. delta13C comparison between soil samples that differed only slightly in their bulk carbon isotope signature showed that the MBA method is better in proving these small differences on a significant level. Total precision of the whole MBA method including all analytical and calculation steps is better by a factor of almost three than the TMS method.     

Stable carbon and oxygen isotopic determination of sub-microgram quantities of CaCO3 to analyze individual foraminiferal shells

We have developed an analytical system to determine stable isotopic compositions (delta13C and delta18O) of sub-microgram quantities of CaCO3 for the purpose of analyzing individual foraminiferal shells, using continuous-flow isotope ratio mass spectrometry (CF-IRMS). The system consists of a micro-volume CaCO3 decomposition tube, stainless steel CO2 purification vacuum line with a quantity-regulating unit, helium-purged CO2 purification line, gas chromatograph, and a CF-IRMS system. By using this system, we can determine stable carbon and oxygen isotopic compositions as low as 0.2 microg of CaCO3, with standard deviations of +/-0.10 per thousand for delta13C and +/-0.18 per thousand for delta18O within a 4-h reaction time and 30-min analysis period.     

GasBench/isotope ratio mass spectrometry: a carbon isotope approach to detect exogenous CO(2) in sparkling drinks

A new procedure for the determination of carbon dioxide (CO(2)) (13)C/(12)C isotope ratios, using direct injection into a GasBench/isotope ratio mass spectrometry (GasBench/IRMS) system, has been developed to improve isotopic methods devoted to the study of the authenticity of sparkling drinks. Thirty-nine commercial sparkling drink samples from various origins were analyzed. Values of delta(13)C(cava) ranged from -20.30 per thousand to -23.63 per thousand, when C3 sugar addition was performed for a second alcoholic fermentation. Values of delta(13)C(water) ranged from -5.59 per thousand to -6.87 per thousand in the case of naturally carbonated water or water fortified with gas from the spring, and delta(13)C(water) ranged from -29.36 per thousand to -42.09 per thousand when industrial CO(2) was added. It has been demonstrated that the addition of C4 sugar to semi-sparkling wine (aguja) and industrial CO(2) addition to sparkling wine (cava) or water can be detected. The new procedure has advantages over existing methods in terms of analysis time and sample treatment. In addition, it is the first isotopic method developed that allows (13)C/(12)C determination directly from a liquid sample without previous CO(2) extraction. No significant isotopic fractionation was observed nor any influence by secondary compounds present in the liquid phase.     

Two new organic reference materials for delta13C and delta15N measurements and a new value for the delta13C of NBS 22 oil

Analytical grade L-glutamic acid is chemically stable and has a C/N mole ratio of 5, which is close to that of many of natural biological materials, such as blood and animal tissue. Two L-glutamic acid reference materials with substantially different 13C and 15N abundances have been prepared for use as organic reference materials for C and N isotopic measurements. USGS40 is analytical grade L-glutamic acid and has a delta13C value of -26.24 per thousand relative to VPDB and a delta15N value of -4.52 per thousand relative to N2 in air. USGS41 was prepared by dissolving analytical grade L-glutamic acid with L-glutamic acid enriched in 13C and 15N. USGS41 has a delta13C value of +37.76 per thousand and a delta15N value of +47.57 per thousand. The delta13C and delta15N values of both materials were measured against the international reference materials NBS 19 calcium carbonate (delta13C=+1.95 per thousand ), L-SVEC lithium carbonate (delta13C=-46.48 per thousand ), IAEA-N-1 ammonium sulfate (delta15N=0.43 per thousand ), and USGS32 potassium nitrate (delta15N=180 per thousand ) by on-line combustion continuous-flow and off-line dual-inlet isotope-ratio mass spectrometry. Both USGS40 and USGS41 are isotopically homogeneous; reproducibility of delta13C is better than 0.13 per thousand, and that of delta15N is better than 0.13 per thousand in 100-microg amounts. These two isotopic reference materials can be used for (i) calibrating local laboratory reference materials, and (ii) quantifying drift with time, mass-dependent fractionations, and isotope-ratio-scale contraction in the isotopic analysis of various biological materials. Isotopic results presented in this paper yield a delta13C value for NBS 22 oil of -29.91 per thousand, in contrast to the commonly accepted value of -29.78 per thousand for which off-line blank corrections probably have not been quantified satisfactorily.