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.     

Enhancing the understanding of earthworm feeding behaviour via the use of fatty acid delta13C values determined by gas chromatography-combustion-isotope ratio mass spectrometry

Litter-dwelling (epigeic) Lumbricus rubellus and soil-dwelling (endogeic) Allolobophora chlorotica earthworms were observed aggregating under C(3) (delta(13)C = -31.3 per thousand; delta(15)N = 10.7 per thousand) and C(4) (delta(13)C = -12.6 per thousand; delta(15)N = 7.5 per thousand) synthetic dung pats applied to a temperate grassland (delta(13)C = -30.3 per thousand; delta(15)N = 5.7 per thousand) in an experiment carried out for 372 days. Bulk delta(13)C values of earthworms collected from beneath either C(3) or C(4) dung after 28, 56, 112 and 372 days demonstrated that (i) L. rubellus beneath C(4) dung were significantly (13)C-enriched after 56 days (delta(13)C = -23.8 per thousand) and 112 days (delta(13)C = -22.4 per thousand) compared with those from C(3) dung treatments (56 days, delta(13)C = -26.5 per thousand; 112 days, delta(13)C = -27.0 per thousand), and (ii) A. chlorotica were 2.1 per thousand (13)C-enriched (delta(13)C = -24.2 per thousand) relative to those from C(3) dung (delta(13)C = -26.3 per thousand) treatments after 372 days. Bulk delta(15)N values did not suggest significant uptake of dung N by either species beneath C(3) or C(4) dung, but showed that the endogeic species (total mean delta(15)N = 3.3 per thousand) had higher delta(15)N values than the epigeic species (total mean delta(15)N = 5.4 per thousand). Although the two species exhibited similar fatty acid profiles, individual fatty acid delta(13)C values revealed extensive routing of dietary C into body tissue of L. rubellus, but minor incorporation into A. chlorotica. In particular, the direct incorporation of microbial biomarker fatty acids (iC(17:0), aC(17:0)) from (13)C-labelled dung in situ, the routing of dung C into de novo synthesised compounds (iC(20:4)(omega)(6),C(20:5)(omega)(3), and the assimilation of essential fatty acids ((C(18:1)(omega)(9), C(18:1)(omega(7), C(18:2)(omega(6), C(18:3)(omega)(3)) derived from dung, were determined.     

Identifying migratory Salmo trutta using carbon and nitrogen stable isotope ratios

Many Salmo trutta populations consist of non-anadromous (freshwater-resident) brown trout and anadromous (sea-run migratory) sea trout. Although adult brown trout and sea trout can usually be identified using differences in size and body colouration, it is not possible to easily identify eggs/alevins as the progeny of brown trout or sea trout. In this study we show that delta(13)C and delta(15)N, measured using a continuous flow isotope ratio mass spectrometer (CF-IRMS), can accurately identify fish eggs as the progeny of freshwater-resident (delta(13)C(egg) = -25.7 +/- 1.9 per thousand,delta(15)N(egg) = 9.2 +/- 1.8 per thousand) or migratory (delta(13)C(egg) = -19.9 +/- 1.1 per thousand, delta(15)N(egg) = 14. 3 +/- 1.5 per thousand) adult female Salmo trutta. Case studies show that stable isotope analysis is a more reliable technique for distinguishing anadromous adult fish than differentiation using morphological characteristics. For example, stable isotope analysis of brown trout from Loch Eck, Scotland, revealed that some individuals possessed delta(13)C and delta(15)N signatures indicative of marine feeding despite visual identification as freshwater-resident fish. It is most likely that these fish are misidentified sea trout although it possible that these fish may be brown trout that have adopted an estuarine feeding strategy to avoid interspecific competition for food within Loch Eck with salmon, powan and Arctic charr. Most stable isotope studies of fish ecology use terminal tissue sampling to provide sufficient biological material for isotopic analysis; however, our study suggests that adipose fin tissue could provide a comparable measure of delta(13)C and delta(15)N. Such a strategy would be invaluable when studying the trophic ecology or migration patterns of fish of high conservation value.     

Nitrogen balance and delta15N: why you're not what you eat during pregnancy

Carbon (13C/12C) and nitrogen (15N/14N) stable isotope ratios were longitudinally measured in human hair that reflected the period from pre-conception to delivery in 10 pregnant women. There was no significant change in the delta13C results, but all subjects showed a decrease in delta15N values (-0.3 to -1.1 per thousand) during gestation. The mechanisms causing this decrease in hair delta15N have not been fully elucidated. However, since the delta15N values of dietary nitrogen and urea nitrogen are significantly lower compared to maternal tissues, it is hypothesized that the increased utilization of dietary and urea nitrogen for tissue synthesis during pregnancy resulted in a reduction of the steady state diet to a body trophic level effect by approximately 0.5-1 per thousand. An inverse correlation (R2 = 0.67) between hair delta15N and weight gain was also found, suggesting that positive nitrogen balance results in a reduction of delta15N values independent of diet. These results indicate that delta15N measurements have the ability to monitor not only dietary inputs, but also the nitrogen balance of an organism. A potential application of this technique is the detection of fertility patterns in modern and ancient species that have tissues that linearly record stable isotope ratios through time.     

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.     

delta(13)C- and delta(18)O-values of glycerol of food fats

The average values of carbon and oxygen isotopic contents (delta(13)C and delta(18)O) of 36 glycerol samples from fats have been determined. The examined samples arise from many fats of animal and plant origin, as well as from the three Italian hard cheeses Parmigiano-Reggiano, Grana Padano and Trentingrana. The total (13)C content allows one to distinguish between glycerol from plants with the C-4 carbon fixation pathway (maize, mean delta(13)C = -14.4 per thousand) and that from plants with the C-3 pathway (mean delta(13)C = -30.7 per thousand). The delta(13)C-values of glycerols of animal origin seem to depend on the diet of the animal, as suggested by the mean values -29.6, -29.0 and -25.1 per thousand, respectively, observed for Parmigiano-Reggiano, Trentingrana and Grana Padano. Additionally, the mean total (18)O content of glycerol samples of vegetable origin is approximately 23.8 per thousand, while that from animal fat is 15.1 per thousand. However, the delta(18)O mean values relative to Parmigiano-Reggiano, Grana Padano and Trentingrana are 11.8, 16.0 and 13.8 per thousand, respectively. The combination of the (13)C and (18)O measurements relative to the fat glycerol of the three cheeses might be considered a potential criterion of authentication.     

Stable carbon and nitrogen isotope analysis of avian uric acid

We report results obtained using a new technique developed to measure the stable-isotope composition of uric acid isolated from bird excreta (guano). Results from a diet-switch feeding trial using zebra finches suggest that the delta(13)C of uric acid in the guano equilibrates with the diet of the bird within 3 days of a change in diet, while the equilibration time for delta(15)N may be longer. The average carbon isotope discrimination between uric acid and food before the diet switch was +0.34 +/- 1 per thousand (1sigma) while after the diet switch this increased slightly to +0.83 +/- 0.7 per thousand (1sigma). Nitrogen isotope discrimination was +1.3 +/- 0.3 per thousand (1sigma) and +0.3 +/- 0.3 per thousand (1sigma) before and after the diet switch; however, it is possible that the nitrogen isotope values did not fully equilibrate with diet switch over the course of the experiment. Analyses of other chemical fractions of the guano (organic residue after uric acid extraction and non-uric acid organics solubilised during extraction) suggest a total range of up to 3 per thousand for both delta(13)C and delta(15)N values in individual components of a single bulk guano sample. The analysis of natural samples from a range of terrestrial and marine species demonstrates that the technique yields isotopic compositions consistent with the known diets of the birds. The results from natural samples further demonstrate that multiple samples from the same species collected from the same location yield similar results, while different species from the same location exhibit a range of isotopic compositions indicative of different dietary preferences. Given that many samples of guano can be rapidly collected without any requirement to capture specimens for invasive sampling, the stable-isotope analysis of uric acid offers a new, simple and potentially powerful tool for studying avian ecology and metabolism.     

Assessing the use of delta(13)C natural abundance in separation of root and microbial respiration in a Danish beech (Fagus sylvatica L.) forest

Our understanding of forest biosphere-atmosphere interactions is fundamental for predicting forest ecosystem responses to climatic changes. Currently, however, our knowledge is incomplete partly due to inability to separate the major components of soil CO(2) effluxes, viz. root respiration, microbial decomposition of soil organic matter and microbial decomposition of litter material. In this study we examined whether the delta(13)C characteristics of solid organic matter and respired CO(2) from different soil-C components and root respiration in a Danish beech forest were useful to provide information on the root respiration contribution to total CO(2) effluxes. The delta(13)C isotopic analyses of CO(2) were performed using a FinniganMAT Delta(PLUS) isotope-ratio mass spectrometer coupled in continuous flow mode to a trace gas preparation-concentration unit (PreCon). Gas samples in 2-mL crimp seal vials were analysed in a fully automatic mode with an experimental standard error +/-0.11 per thousand. We observed that the CO(2) derived from root-free mineral soil horizons (A, B(W)) was more enriched in (13)C (delta(13)C range -21.6 to -21.2 per thousand ) compared with CO(2) derived from root-free humus layers (delta(13)C range -23.6 to -23.4 per thousand ). The CO(2) evolved from root respiration in isolated young beech plants revealed a value intermediate between those for the soil humus and mineral horizons, delta(13)C(root) = -22.2 per thousand, but was associated with great variability (SE +/- 1.0 per thousand ) due to plant-specific differences. delta(13)C of CO(2) from in situ below-ground respiration averaged -22.8 per thousand, intermediate between the values for the humus layer and root respiration, but variability was great (SE +/- 0.4 per thousand ) due to pronounced spatial patterns. Overall, we were unable to statistically separate the CO(2) of root respiration vs. soil organic matter decomposition based solely on delta(13)C signatures, yet the trend in the data suggests that root respiration contributed approximately 43% to total respiration. The vertical gradient in delta(13)C, however, might be a useful tool in partitioning respiration in different soil layers. The experiment also showed an unexpected (13)C-enrichment of CO(2) (>3.5 per thousand ) compared with the total-C signatures in the individual soil-C components. This may suggest that analyses of bulk samples are not representative for the C-pools actively undergoing decomposition.     

Relationship between soil organic C degradability and the evolution of the delta13C signature in profiles under permanent grassland

The main objective of this research was to investigate to what extent the potential C dynamics of soil organic matter (SOM) are related to the degree of 13C enrichment with increasing depth in soil profiles under permanent grassland. The evolution of the C content and the 13C natural abundance (delta13C value) of SOM were investigated in three soil profiles (0-40 cm depth) under permanent grassland of varying texture (a loamy sand, a loam and a clay loam soil). The delta13C value of the SOM showed a gradual increase with increasing depth and decreasing C content in the profiles, ranging from 1.9 per thousand (loamy sand soil), 2.9 per thousand (clay loam soil) and 4 per thousand (loam soil) in relation to the delta13C value of SOM at the surface. The relationship between the 13C enrichment and total organic C content at different depths in the profiles (down to 40 cm depth in the loam and clay loam soil, down to 25 cm depth in the loamy sand soil) could be well described by the Rayleigh equation. The enrichment factors epsilon, associated with the Rayleigh approximation of the data, ranged from -1.57 per thousand (clay loam soil) to -1.64 per thousand (loamy sand soil) and -1.91 per thousand (loam soil). The potential C dynamics in four depth intervals from the profiles (0-10, 10-20, 20-30 and 30-40 cm depth) were determined by means of an incubation experiment. The C decomposition rate constants from the four sampling depths in the profiles showed a significant, positive correlation (y = 0.21x + 0.018, R(2) = 0.66, p < 0.005) with the corresponding Deltadelta13C values (change of the delta13C value per depth increment). A better correlation was obtained when only the data from the upper 20 cm in the profiles (y = 0.21x + 0.019, R(2) = 0.78, p < 0.05) were considered. These results suggest that the Deltadelta13C values in the surface layers of profiles under permanent grassland may serve as an indicator of the potential degradability or the stability of the SOM (in terms of C decomposition rate constants).     

Nitrogen isotopic composition in hair protein is different in liver cirrhotic patients

The stable-isotopic composition of nitrogen (delta15N) or carbon (delta13C) of body tissues depends on the isotopic composition of food sources and on shifts due to isotopic fractionation during metabolism. As little is known about the effects of pathophysiological conditions we measured delta15N and delta13C values in hair and hair amino acids of patients with cirrhosis (n = 21) and compared the results with those of healthy subjects (n = 100) randomly selected from the 1987-1988 VERA German nutrition survey population. Cirrhosis was reflected in lower hair 15N abundances (6.7 vs. 9.9 per thousand delta15N; P < 0.001) whereas hair 13C abundances did not differ from healthy subjects (-19.4 vs. -19.6 per thousand 13C). Distinct patterns of delta15N and delta13C values were measured in hair amino acids. The delta15N values of phenylalanine were significantly higher in cirrhotics (P < 0.001). With the exception of isoleucine, threonine, and proline all other measured amino acids showed lower delta15N values than healthy subjects (P < 0.001). Lower hair delta15N values were associated with cirrhotic liver disease which suggests that under this condition the altered liver amino acid metabolism affects the nitrogen isotopic composition of the amino acids used for hair protein synthesis. It remains to be determined in controlled studies whether the altered nitrogen isotopic composition directly reflects the pathophysiological condition or is related to differences in dietary protein intake from plant or animal food sources.     

Detection of royal jelly adulteration using carbon and nitrogen stable isotope ratio analysis

Stable isotope ratios ((13)C/(12)C and (15)N/(14)N) were measured in royal jelly (RJ) samples by isotope ratio mass spectrometry (IRMS) to evaluate authenticity and adulteration. Carbon and nitrogen isotope contents (given as delta values relative to a standard, delta(13)C, delta(15)N) of RJ samples from various European origins and samples from commercial sources were analyzed. Uniform delta(13)C values from -26.7 to -24.9 per thousand were observed for authentic RJ from European origins. Values of delta(15)N ranged from -1.1 to 5.8 per thousand depending on the plant sources of nectars and pollen. High delta(13)C values of several commercial RJ samples from -20.8 to -13.3 per thousand indicated adulteration with high fructose corn syrup (HFCS) as a sugar source. Use of biotechnologically produced yeast powder as protein source for the adulterated samples was assumed as delta(15)N values were lower, as described for C(4) or CAM plant sources. RJ samples from authentic and from adulterated production were distinguished. The rapid and reliable method is suitable for urgent actual requirements in food monitoring.     

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.     

Vertical gradients of delta15N and delta18O in soil atmospheric N2O--temporal dynamics in a sandy soil

The greenhouse gas nitrous oxide (N(2)O) can be both formed and consumed by microbial processes in the soil. As these processes fractionate strongly in favour of (14)N and (16)O, delta(15)N and delta(18)O gradients of N(2)O in the soil profile may elucidate patterns of N(2)O formation, consumption or emission to the atmosphere. We present the first in situ data of such gradients over time for a mesic typic Haplaquod seeded with potatoes (Solanum tuberosum L.). On two adjacent fields in 2002 and 2003, topsoil N(2)O fluxes were measured and the soil atmosphere was regularly sampled for N(2)O concentrations, delta(15)N and delta(18)O signatures of N(2)O at depths of 18, 48 and 90 cm during approximately 400 days. During the entire sampling period, the N(2)O concentrations were the highest and the delta(15)N signatures the lowest in the subsoil (48 or 90 cm depth) as compared with the topsoil, indicating production of N(2)O in the subsoil. For delta(15)N, differences greater than 30 per thousand between topsoil and subsoil on the same date were regularly observed. The highest N(2)O concentration of 100385 microL m(-3) at 90 cm depth on 1 July 2003, was preceded by the lowest delta(15)N value of -43.5 per thousand one week earlier. This was followed by a 150-day general decrease of N(2)O concentrations at 90 cm depth to 1723 microL m(-3) and a simultaneous enrichment of delta(15)N to +7.1 per thousand, mostly without a significant topsoil flux. There was a negative logarithmic relationship between N(2)O concentration at 90 cm depth and its delta(15)N signature. This relationship indicated a delta(15)N signature of -40 to -45 per thousand during the production of N(2)O in the subsoil, and a subsequent enrichment during the consumption of N(2)O. We conclude that the isotopic signature of the N(2)O topsoil flux is the result of various processes of consumption and production at different depths in the soil profile. It is therefore not a reliable estimator for the overall delta(15)N signature of N(2)O in the soil atmosphere, nor for indirect losses of N(2)O to the environment. Therefore, these findings will pose a further challenge to ongoing efforts to draw up a global isotopic budget for N(2)O.     

On-line measurements of delta(15)N in biological fluids by a modified continuous-flow elemental analyzer with an isotope-ratio mass spectrometer

A modified continuous-flow elemental analyzer coupled to an isotope-ratio mass spectrometer (modified EA-IRMS) was tested for on-line delta(15)N measurement on urea solution and biological fluids (e.g. urine). The elemental analyzer configuration was adapted by adding a U-shaped cold trap and an X-pattern four-way valve for on-line trapping/venting of water from the liquid samples. Results indicate that the delta(15)N ratios show little variation (standard deviation (SD) = 0.05 per thousand) with a sample size above the equivalent N yield of 0.2 mg urea (0.092 mg N) when the mass spectrometer conditions were carefully optimized. By contrast, a significant logarithmic decrease in delta(15)N with sample size was observed but this can be offset by applying a linearity correction or blank correction when the sample size is between equivalent N yields of 0.05 and 0.2 mg urea. The blank corrected delta(15)N ratios give an overall precision of approximately 0.16 per thousand whereas the average precision for delta(15)N corrected using combined linearity and shift correction is 0.05 per thousand. The relatively large variation in blank corrected delta(15)N values may be attributed to the variability of the blank delta(15)N in the sequence. Therefore, the blank correction should be carefully performed in routine measurements. As a result, the linearity range of a modified EA-IRMS can be extended to a minimum sample size of 0.023 mg N. In addition, the reproducibility of the new system is good, as indicated by the precision (<0.2 per thousand) for a set of standards and unknowns. The data show that fluids containing nitrogen can be successfully analyzed in the modified EA-IRMS.     

Alteration of the carbon and nitrogen stable isotope composition of beef by substitution of grass silage with maize silage

This study investigated the effect of substituting grass silage (C3 photosynthetic plant product) with maize silage (C4 photosynthetic plant product) on the natural abundance carbon (delta13C) and nitrogen (delta15N) stable isotope composition of bovine muscle tissue. Forty-five continental crossbred heifers were assigned to one of three diets consisting of 3 kg of a barley-based concentrate plus grass silage, maize silage or an equal mixture (dry matter basis) of grass silage and maize silage, fed ad libitum, for 167 days. Substitution resulted in less negative delta13C values (P<0.001) in lipid-free muscle and in lipid, and also a lower delta15N (P<0.001) in lipid-free muscle. Feeding of maize silage was clearly reflected in the delta13C of muscle, with each 10% difference in the dietary C4 carbon intake resulting in a 0.9 to 1.0 per thousand shift of delta13C in lipid-free muscle and a 1.0 to 1.2 per thousand in lipid. Minimum detectable mean differences (95% confidence, power 0.80, n=15) in this experiment were about 0.5 per thousand and 1.0 per thousand for delta13C of lipid-free muscle and lipid, respectively, and about 0.5 per thousand for delta15N of lipid-free muscle. The power analysis presented here is useful for estimating minimum isotopic differences that can be detected between any two groups of beef samples with a given number of replicates. It is concluded that carbon stable isotope ratio analysis of meat can be used to quantify C3/C4 dietary constituents in beef production.     

Methods to adjust for the interference of N2O on delta13C and delta18O measurements of CO2 from soil mineralization

In this paper we present an overview of the present knowledge relating to methods that avoid interference of N2O on delta13C and delta18O measurements of CO2. The main focus of research to date has been on atmospheric samples. However, N2O is predominantly generated by soil processes. Isotope analyses related to soil trace gas emissions are often performed with continuous flow isotope ratio mass spectrometers, which do not necessarily have the high precision needed for atmospheric research. However, it was shown by using laboratory and field samples that a correction to obtain reliable delta13C and delta18O values is also required for a commercial continuous flow isotope ratio mass spectrometer. The capillary gas chromatography column of the original equipment was changed to a packed Porapak Q column. This adaptation resulted in an improved accuracy and precision of delta13C (standard deviation(Ghent): from 0.2 to 0.08 per thousand; standard deviation(Lincoln): from 0.2 to 0.13 per thousand) of CO2 for N2O/CO2 ratios up to 0.1. For delta18O there was an improvement for the standard deviation measured at Ghent University (0.13 to 0.08 per thousand) but not for the measurements at Lincoln University (0.08 to 0.23 per thousand). The benefits of using the packed Porapak Q column compared with the theoretical correction method meant that samples were not limited to small N(2)O concentrations, they did not require an extra N2O concentration measurement, and measurements were independent of the variable isotopic composition of N2O from soil.     

Isotopomeric characterization of N2O produced, consumed, and emitted by automobiles

Fossil fuel combustion is the second largest anthropogenic source of nitrous oxide (N2O) after agriculture. The estimated global N2O flux from combustion sources, as well as from other sources, still has a large uncertainty. Herein, we characterize automobile sources using N2O isotopomer ratios (nitrogen and oxygen isotope ratios and intramolecular site preference of 15N, SP) to assess their contributions to total global sources and to deconvolute complex production/consumption processes during combustion and subsequent catalytic treatments of exhaust. Car exhaust gases were sampled under running and idling state, and N2O isotopomer ratios were measured by mass spectrometry. The N2O directly emitted from an engine of a vehicle running at constant velocity had almost constant isotopomer ratios (delta15Nbulk = -28.7 +/- 1.2 per thousand, delta18O = 28.6 +/- 3.3 per thousand, and SP = 4.2 +/- 0.8 per thousand) irrespective of the velocity. After passing through catalytic converters, the isotopomer ratios showed an increase which varied with the temperature and the aging of the catalysts. The increase suggests that both production and consumption of N2O occur on the catalyst and that their rates can be comparable. It was noticed that in the idling state, the N2O emitted from a brand new car has higher isotopomer ratios than that from used cars, which indicate that technical improvements in catalytic converters can reduce the N2O from mobile combustion sources. On average, the isotopomeric signatures of N2O finally emitted from automobiles are not sensitive to running/idling states or to aging of the catalysts. Characteristic average isotopomer ratios of N2O from automobile sources are estimated at -4.9 +/- 8.2 per thousand, 43.5 +/- 13.9 per thousand, and 12.2 +/- 9.1 per thousand for delta15Nbulk, delta18O, and SP, respectively.     

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.     

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.     

Experimental and quantum-chemical studies of 1H, 13C and 15N NMR coordination shifts in Pd(II) and Pt(II) chloride complexes with methyl and phenyl derivatives of 2,2'-bipyridine and 1,10-phenanthroline

1H, 13C and 15N NMR studies of platinide(II) (M=Pd, Pt) chloride complexes with methyl and phenyl derivatives of 2,2'-bipyridine and 1,10-phenanthroline [LL=4,4'-dimethyl-2,2'-bipyridine (dmbpy); 4,4'-diphenyl-2,2'-bipyridine (dpbpy); 4,7-dimethyl-1,10-phenanthroline (dmphen); 4,7-diphenyl-1,10-phenanthroline (dpphen)] having a general [M(LL)Cl2] formula were performed and the respective chemical shifts (delta1H, delta13C, delta15N) reported. 1H high-frequency coordination shifts (Delta1Hcoord=delta1Hcomplex-delta1Hligand) were discussed in relation to the changes of diamagnetic contribution in the relevant 1H shielding constants. The comparison to literature data for similar [M(LL)(XX)], [M(LL)X2] and [M(LL)XY] coordination or organometallic compounds containing various auxiliary ligands revealed a large dependence of delta1H parameters on inductive and anisotropic effects. 15N low-frequency coordination shifts (Delta15Ncoord=delta 15Ncomplex-delta15Nligand) of ca 88-96 ppm for M=Pd and ca 103-111 ppm for M=Pt were attributed to both the decrease of the absolute value of paramagnetic contribution and the increase of the diamagnetic term in the expression for 15N shielding constants. The absolute magnitude of Delta15Ncoord parameter increased by ca 15 ppm upon Pd(II)-->Pt(II) transition and by ca 6-7 ppm following dmbpy-->dmphen or dpbpy-->dpphen ligand replacement; variations between analogous complexes containing methyl and phenyl ligands (dmbpy vs dpbpy; dmphen vs dpphen) did not exceed+/-1.5 ppm. Experimental 1H, 13C, 15N NMR chemical shifts were compared to those quantum-chemically calculated by B3LYP/LanL2DZ+6-31G**//B3LYP/LanL2DZ+6-31G*, both in vacuo and in DMSO or DMF solution.