Measurement of δ13C values of soil amino acids by GC–C–IRMS using trimethylsilylation: a critical assessment

In this study, we evaluated trimethylsilyl (TMS) derivatives as derivatization reagents for the compound-specific stable carbon isotope analysis of soil amino acids by gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS). We used non-proteinogenic amino acids to show that the extraction–derivatization–analysis procedure provides a reliable method to measure δ¹³C values of amino acids extracted from soil. However, we found a number of drawbacks that significantly increase the final total uncertainty. These include the following:

• production of multiple peaks for each amino acid, identified as di-, tri- and tetra-TMS derivatives;

• a number of TMS-carbon (TMS-C) atoms added lower than the stoichiometric one, possibly due to incomplete combustion;

• different TMS-C δ¹³C for di-, tri- and tetra-TMS derivatives.

For soil samples, only four amino acids (leucine, valine, threonine and serine) provide reliable δ¹³C values with a total average uncertainty of 1.3?‰. We conclude that trimethylsilyl derivatives are only suitable for determining the ¹³C incorporation in amino acids within experiments using ¹³C-labelled tracers but cannot be applied for amino acids with natural carbon isotope abundance until the drawbacks described here are overcome and the measured total uncertainty significantly decreased.     

The Effect of Diet Quality on δ13C and δ15N in the Tissues of Locusts,Locusta migratoria L

Abstract

Locust nymphs were raised from hatching to adult locusts on either seedling wheat (C₃) or maize (C₄), to determine whether relative enrichments/depletions of ¹⁵N and ¹³C within body tissues are influenced by diet. The maize contained less hexose sugars and protein per gram than wheat.

The isotopic spacing between the food and the whole insect was found to differ between the two diets. The lower quality maize diet showed an overall +5.1‰ enrichment in δ¹⁵N compared to + 2.8‰ for wheat, possibly due to increased fractionation due to protein recycling.

The maize diet resulted in increased depletion in lipid and trehalose and depletion in chitin relative to diet. The results for both δ¹⁵N and δ¹³C suggest that substrate recycling was occurring on the low quality maize diet. Therefore diet quality determines the enrichment/depletion in δ¹⁵N and δ¹³C within organisms.     

Spatial variations in larch needle and soil δ15N at a forest–grassland boundary in northern Mongolia

The spatial patterns of plant and soil δ¹⁵N and associated processes in the N cycle were investigated at a forest–grassland boundary in northern Mongolia. Needles of Larix sibirica Ledeb. and soils collected from two study areas were analysed to calculate the differences in δ¹⁵N between needle and soil (Δδ¹⁵N). Δδ¹⁵N showed a clear variation, ranging from ?8?‰ in the forest to ?2?‰ in the grassland boundary, and corresponded to the accumulation of organic layer. In the forest, the separation of available N produced in the soil with ¹⁵N-depleted N uptake by larch and ¹⁵N-enriched N immobilization by microorganisms was proposed to cause large Δδ¹⁵N, whereas in the grassland boundary, small Δδ¹⁵N was explained by the transport of the most available N into larch. The divergence of available N between larch and microorganisms in the soil, and the accumulation of diverged N in the organic layer control the variation in Δδ¹⁵N.     

A Sensitive Method for the Fast and Simultaneous Determination of δ15N- and δ18O-Values in Nitrate

A new method for on-line measurements of the isotopic abundances of N and O in nitrate using a Pyrolysis-GC-IRMS system is presented. The salt is heated to 1200°C together with graphite powder, the reaction products are separated by a GC column and allowed into an IRMS. The sample size could be reduced to 200 nmoles element for one measurement, which can be performed within 10 minutes with an accuracy of better than 1‰.     

Field-deployable measurements of free-living individuals to determine energy balance: fuel substrate usage through δ13C in breath CO2 and diet through hair δ13C and δ15N values

Carbon isotopes of breath CO₂ vary depending on diet and fuel substrate used. This study examined if exercise-induced δ¹³C-CO₂ changes in substrate utilization were distinguishable from baseline δ¹³C-CO₂ variations in a population with uncontrolled diet, and compared hair isotope values and food logs to develop an isotope model of diet. Study participants included nine women with diverse Body Mass Index (BMI), age, ancestry, exercise history, and diet. Breath samples were collected prior to and up to 12?h after a 5- or 10?K walk/run. Indirect calorimetry was measured with a smartphone-enabled mobile colorimetric device, and a field-deployable isotope analyzer measured breath δ¹³C-CO₂ values. Diet was assessed by food logs and δ¹³C, δ¹⁵N of hair samples. Post-exercise δ¹³C-CO₂ values increased by 0.54?±?1.09‰ (1 sd, n?=?9), implying enhanced carbohydrate burning, while early morning δ¹³C-CO₂ values were lower than daily averages (p?=?0.0043), indicating lipid burning during overnight fasting. Although diurnal δ¹³C-CO₂ variation (1.90?±?0.77‰) and participant baseline range (3.06‰) exceeded exercise-induced variation, temporal patterns distinguished exercise from dietary isotope effects. Hair δ¹³C and δ¹⁵N values were consistent with a new dietary isotope model. Notwithstanding the small number of participants, this study introduces a novel combination of techniques to directly monitor energy balance in free-living individuals.     

Variations of δ15N-Values and Hydrolyzable Amino Acids in Settling Particles in the Ocean

Abstract

The modification of nitrogen isotopic signals during particle sedimentation in the sea is of great interest for the use of sedimentary δ¹⁵N-values as a paleoceanographic tool. The effect of organic matter degradation on such modification was studied by analyzing nitrogen, hydrolyzable amino acids (THAA) and δ¹⁵N-values in a suit of marine settling particles collected from the Bay of Bengal, Indian Ocean, by using time-series sediment traps, and in underlying sediments. The flux of settling particles showed temporal variations which are related to the monsoons, the major climatic feature of this marine region. During high flux periods settling particles are enriched in nitrogenous material that is less degraded and exhibit higher δ¹⁵N-values than particles showing characteristics of degradation. At the sediment surface more than 95% of the settling particulate nitrogen is lost and the δ¹⁵N-values of the residual sedimentary nitrogen are higher than those of settling particles. The observed increase is interpreted to be due to fractionation during degradation of organic matter.     

Food Web Implications of δ13C and δ15N Variability over 370 km of the Regulated Colorado River USA

Abstract

Dual stable isotope analysis in the regulated Colorado River through Grand Canyon National Park, USA, revealed a food web that varied spatially through this arid biome. Down-river enrichment of δ¹³C data was detected across three trophic levels resulting in shifted food webs. Humpack chub δ¹³C and δ¹⁵N values from muscle plugs and fin clips did not differ significantly. Humpback chub and rainbow trout trophic position is positively correlated with standard length indicating an increase in piscivory by larger fishes. Recovery of the aquatic community from impoundment by Glen Canyon Dam and collecting refinements for stable isotope analysis within large rivers are discussed.     

The Effect of Diet Quality on δ(13)C and δ(15)N in the Tissues of Locusts, Locusta migratoria L

Abstract Locust nymphs were raised from hatching to adult locusts on either seedling wheat (C(3)) or maize (C(4)), to determine whether relative enrichments/depletions of (15)N and (13)C within body tissues are influenced by diet. The maize contained less hexose sugars and protein per gram than wheat. The isotopic spacing between the food and the whole insect was found to differ between the two diets. The lower quality maize diet showed an overall +5.1‰ enrichment in δ(15)N compared to + 2.8‰ for wheat, possibly due to increased fractionation due to protein recycling. The maize diet resulted in increased depletion in lipid and trehalose and depletion in chitin relative to diet. The results for both δ(15)N and δ(13)C suggest that substrate recycling was occurring on the low quality maize diet. Therefore diet quality determines the enrichment/depletion in δ(15)N and δ(13)C within organisms.     

Variations of δ(15)N-Values and Hydrolyzable Amino Acids in Settling Particles in the Ocean

Abstract The modification of nitrogen isotopic signals during particle sedimentation in the sea is of great interest for the use of sedimentary δ(15)N-values as a paleoceanographic tool. The effect of organic matter degradation on such modification was studied by analyzing nitrogen, hydrolyzable amino acids (THAA) and δ(15)N-values in a suit of marine settling particles collected from the Bay of Bengal, Indian Ocean, by using time-series sediment traps, and in underlying sediments. The flux of settling particles showed temporal variations which are related to the monsoons, the major climatic feature of this marine region. During high flux periods settling particles are enriched in nitrogenous material that is less degraded and exhibit higher δ(15)N-values than particles showing characteristics of degradation. At the sediment surface more than 95% of the settling particulate nitrogen is lost and the δ(15)N-values of the residual sedimentary nitrogen are higher than those of settling particles. The observed increase is interpreted to be due to fractionation during degradation of organic matter.     

First field-based observations of δ2H and δ18O values of event-based precipitation,rivers and other water bodies in the Dzungarian Gobi,SW Mongolia

For certain remote areas like Mongolia, field-based precipitation, surface and ground water isotopic data are scarce. So far no such data exist for the Mongolian Gobi desert, which hinders the understanding of isotopic fractionation processes in this extreme, arid region. We collected 26 event-based precipitation samples, 39 Bij river samples, and 75 samples from other water bodies in the Dzungarian Gobi in SW Mongolia over a period of 16 months for hydrogen and oxygen stable isotope analysis. δ²H and δ¹⁸O values in precipitation show high seasonal variation and cover an extreme range: 175?‰ for δ²H and 24?‰ for δ¹⁸O values. The calculated local meteoric water line (LMWL) shows the isotopic characteristics of precipitation in an arid region. Individual water samples fall into one of three groups: within, above or below the 95?% confidence interval of LMWL. Data presented provide a basis for future studies in this region.     

Determination of δ13 CV−PDB and δ15NAIR values of cocaine from a big seizure in Germany by stable isotope ratio mass spectrometry†

In this study, δ¹³C_{V? PDB} and δ¹⁵N_AIR values of 132 cocaine samples from a big seizure in Germany in 2002 were determined using elemental analyser isotope ratio mass spectrometry. The 1.2 tons of cocaine were packed in 1 kg packages and the cocaine bricks inside these packages showed certain logos. Twenty different logos could be identified. Results show a large variability among some samples, for δ¹⁵N_AIR values ranging from?17 to ?2 ‰. Furthermore, the possibility of linking samples with the same logo was checked. The results show that, in general, there is no relationship between the determined isotope ratio and a certain logo.     

Experimental plant for simultaneous production of 14N and 15N by 15N/14N exchange in NO,NO2–HNO3 system under pressure

An experimental study on ¹⁴N and ¹⁵N simultaneous separation using the chemical exchange in NO, NO₂–HNO₃ system under pressure is presented. The influence of the pressure and of the interstage 10 M HNO₃ flow rate on the separation of ¹⁴N and ¹⁵N was measured on a packed column with product and waste refluxers. At steady state and 1.8 atm (absolute), the isotopic concentration at the bottom of the separation column was 0.563 at% ¹⁵N, and in the top of the column was 0.159 at% ¹⁵N. The height equivalent to a theoretical plate and interstage 10 M HNO₃ flow rate values, obtained in these experimental conditions, allows the separation of ¹⁴N highly depleted of ¹⁵N and of ¹⁵N at 99 at% ¹⁵N concentration.     

On some empirical expressions for the contact values of the pair distribution functions and fluid—fluid phase separation in hard sphere mixtures

The fluid-fluid binodal of asymmetric hard sphere mixtures obtained from approximate expressions of the virial pressure is investigated. Also the behaviour of the Gibbs free energy following from particular combinations of standard expressions of the contact values of the pair distributions functions is examined. A recently proposed parametrization of the latter in the colloidal limit is then discussed and compared with existing simulation data for the binodal of the effective fluid.     

Seasonal variation in natural abundance of δ13C and 15N in Salicornia brachiata Roxb. populations from a coastal area of India

High and fluctuating salinity is characteristic for coastal salt marshes, which strongly affect the physiology of halophytes consequently resulting in changes in stable isotope distribution. The natural abundance of stable isotopes (δ¹³C and δ¹⁵N) of the halophyte plant Salicornia brachiata and physico-chemical characteristics of soils were analysed in order to investigate the relationship of stable isotope distribution in different populations in a growing period in the coastal area of Gujarat, India. Aboveground and belowground biomass of S. brachiata was collected from six different populations at five times (September 2014, November 2014, January 2015, March 2015 and May 2015). The δ¹³C values in aboveground (?30.8 to ?23.6?‰, average: ?26.6?±?0.4?‰) and belowground biomass (?30.0 to ?23.1?‰, average: ?26.3?±?0.4?‰) were similar. The δ¹³C values were positively correlated with soil salinity and Na concentration, and negatively correlated with soil mineral nitrogen. The δ¹⁵N values of aboveground (6.7–16.1?‰, average: 9.6?±?0.4?‰) were comparatively higher than belowground biomass (5.4–13.2?‰, average: 7.8?±?0.3?‰). The δ¹⁵N values were negatively correlated with soil available P. We conclude that the variation in δ¹³C values of S. brachiata was possibly caused by soil salinity (associated Na content) and N limitation which demonstrates the potential of δ¹³C as an indicator of stress in plants.     

Source attribution of agriculture-related deposition by using total nitrogen and δ15N in epiphytic lichen tissue,bark and deposition water samples in Germany

Compared with physico-chemical deposition measurement methods, lichens are able to identify the long-term overall effects of high N pollution concentrations in the air. In addition, the natural abundances of the stable isotope of N, ¹⁵N, are being widely used in research on N cycling in ecosystems. They can also be used as instruments for source attribution. In this study, epiphytic lichens were tested to determine whether their respective N content and δ¹⁵N ratios can be used to estimate N deposition rates and to locate various sources of N compounds. Epiphytic lichen and bark samples were collected from around various deposition measurement field stations at different sites in the western part of Germany. The N content of epiphytic lichens reflects the species-specific, agriculture-related circumstances of N deposition at various sites in Germany. At the same time, δ¹⁵N signatures of the different investigated epiphytic lichen species and bark samples are highly depleted in ¹⁵N under high ammonium deposition. The different surface types of lichens and barks exhibit different concentrations of N and δ¹⁵N ratios, despite being exposed to similar N deposition rates. The verification of highly negative δ¹⁵N ratios at sites with local and regional emitters shows that source attribution is possible by comparing different δ¹⁵N signatures in areas with a wide range of different N deposition types and the corresponding differences in δ¹⁵N among various source N pools. Especially nitrophytic lichens can support the on-site instrumentation measuring N deposition by qualification and quantification.     

Effect of different ratios of wheat to corn flour in the diet on the development and isotopic composition (δ13C, δ15N) of the red flour beetle Tribolium castaneum

The back-calculation of the diet is a common application of stable isotopes in animal ecology. The method is based on a predictable relation between the isotopic signature of the diet and the animal’s tissues. Frequently, the assumption of a constant difference in isotopic signatures (trophic shift) is made. Carbon isotopic ratios of C₃ and C₄ plants differ by approximately 10 ‰, making wheat (C₃-plant) and corn (C₄-plant) ideal materials for isotopic studies in nutritional ecology and especially for testing the back-calculation method. In this experiment, red flour beetles, Tribolium castaneum, were reared on wheat flour, corn flour and three different mixtures thereof, either in pure flour or with the addition of yeast inoculum or yeast grains. Development of T. castaneum on these experimental diets was monitored, and isotopic signatures of carbon and nitrogen in emerging adults were analysed. The values of trophic shift of C and N isotopes for wheat and corn flour were different, and the values for the mixtures did not correspond to those expected from a linear mixing model. The latter can be taken as an indication that the tiny larvae of T. castaneum may be capable of differentiating between particles of wheat and corn flour, making this animal model unsuitable for testing the back-calculation method.