Stable isotope determination of ester and ether methyl moieties in plant methoxyl groups

Plant methoxyl groups of lignin and pectin have both distinct stable hydrogen isotope (δ(2)H) and carbon isotope (δ(13)C) values that can be used for studying environmental processes and for investigating the origin and authenticity of biomaterials. Up to now, the reported methods have been applied only to determine isotope values of the bulk plant methoxyl pool. In this work, we have applied several methods to distinguish between stable isotope ratios of methoxyl groups of pectin and the bulk plant methoxyl pool. Our results demonstrate that by applying alkaline hydrolysis to specifically cleave off the ester methyl moiety (pectin-like), we can distinguish δ(2)H and δ(13)C values of the pectin methoxyl pool from the bulk methoxyl pool. No measureable isotope discrimination was observed either during sample preparation or during analytical measurement. Furthermore, using this method, no major isotope difference in either the hydrogen or carbon isotope signature of the methoxyl groups of plant pectin and bulk matter from plant species such as leaves from trees, apples, carrots and potatoes was noted. We show the methanol released during alkaline hydrolysis of plant material and subsequently treated with hydriodic acid to be an excellent procedure to measure specifically and precisely the δ(13)C and δ(2)H isotope values of plant pectin-like methoxyl groups. This method is particularly advantageous when plant matter with a low methoxyl content has to be analysed.     

Non-linear dynamics of stable carbon and hydrogen isotope signatures based on a biological kinetic model of aerobic enzymatic methane oxidation

The non-linear dynamics of stable carbon and hydrogen isotope signatures during methane oxidation by the methanotrophic bacteria Methylosinus sporium strain 5 (NCIMB 11126) and Methylocaldum gracile strain 14 L (NCIMB 11912) under copper-rich (8.9 µM Cu²⁺), copper-limited (0.3 µM Cu²⁺) or copper-regular (1.1 µM Cu²⁺) conditions has been described mathematically. The model was calibrated by experimental data of methane quantities and carbon and hydrogen isotope signatures of methane measured previously in laboratory microcosms reported by Feisthauer et al. [1 Feisthauer S, Vogt C, Modrzynski J, Szlenkier M, Krüger M, Siegert M, Richnow HH. Different types of methane monooxygenases produce similar carbon and hydrogen isotope fractionation patterns during methane oxidation. Geochim Cosmochim Acta. 2011;75:1173–1184. doi: 10.1016/j.gca.2010.12.006[Crossref], [Web of Science ®] , [Google Scholar]] M. gracile initially oxidizes methane by a particulate methane monooxygenase and assimilates formaldehyde via the ribulose monophosphate pathway, whereas M. sporium expresses a soluble methane monooxygenase under copper-limited conditions and uses the serine pathway for carbon assimilation. The model shows that during methane solubilization dominant carbon and hydrogen isotope fractionation occurs. An increase of biomass due to growth of methanotrophs causes an increase of particulate or soluble monooxygenase that, in turn, decreases soluble methane concentration intensifying methane solubilization. The specific maximum rate of methane oxidation υ_m was proved to be equal to 4.0 and 1.3 mM mM^?1 h^?1 for M. sporium under copper-rich and copper-limited conditions, respectively, and 0.5 mM mM^?1 h^?1 for M. gracile. The model shows that methane oxidation cannot be described by traditional first-order kinetics. The kinetic isotope fractionation ceases when methane concentrations decrease close to the threshold value. Applicability of the non-linear model was confirmed by dynamics of carbon isotope signature for carbon dioxide that was depleted and later enriched in ¹³C. Contrasting to the common Rayleigh linear graph, the dynamic curves allow identifying inappropriate isotope data due to inaccurate substrate concentration analyses. The non-linear model pretty adequately described experimental data presented in the two-dimensional plot of hydrogen versus carbon stable isotope signatures.     

Stable isotopes determination in some Romanian fruit juices

The characterisation of 45 Romanian single-strength fruit juices (apples, pears, plums and grapes) collected from different Transylvanian areas by means of stable isotope approach are presented and discussed in this study. We measured ²H/¹H, ¹⁸O/¹⁶O ratios from water juice and ¹³C/¹²C from pulp and compared these results with those already reported in the literature for single-strength juices, in order to see how the geographical and climatic conditions of Transylvania and the meteorological peculiarities of the year 2010 influence the isotopic composition of the investigated fruit juices. The δ¹³C mean values that we found for apple pulp picked up from different Transylvanian areas show slight differences, probably due to the environmental conditions of the plants. No significant correlation either between the variety of apple or the geographical origin and δ¹³C value was established.     

Stable carbon and nitrogen isotope discrimination and turnover in a small-bodied insectivorous lizard

Laboratory experiments are useful for estimating the carbon and nitrogen isotope discrimination factors and turnover rates that are critical for drawing field-based inferences on consumer diets using stable isotopes. Although the utility of these discrimination factors is widely recognized, work in terrestrial systems has largely been limited to studies involving mammals and birds. In contrast, scant attention has been paid to the application of isotopic techniques to reptiles, despite their broad diversity in terms of numbers of species as well as their trophic roles. Here we estimate carbon and nitrogen isotope discrimination factors and turnover for the tree lizard (Urosaurus ornatus) using a diet-switch experiment. Lizards were collected from a C₄-dominated grassland and then switched to C₃-based diet (crickets) in the laboratory. We estimated discrimination by lizard claw tissue as Δ¹³C?=?1.2?±?0.1?‰ for carbon and Δ¹⁵N?=?0.7?±?0.1?‰ (mean?±?1 SE) for nitrogen, with 95?% turnover occurring after ～15.5 days. These estimates should be appropriate for use in trophic studies of U. ornatus, and possibly other related small-bodied insectivorous lizards.     

Stable isotope ratio method for the characterisation of the poultry house environment

Stable isotope analysis was applied to describe the poultry house environment. The poultry house indoor environment was selected for this study due to the relevant health problems in animals and their caretakers. Air quality parameters including temperature, relative humidity, airflow rate, NH₃, CO₂ and total suspended particles, as well as mean levels of total airborne bacteria and fungi count, were measured. Carbon isotope ratios (¹³C/¹²C) were obtained in size-segregated aerosol particles. The carbon (¹³C/¹²C) and nitrogen (¹⁵N/¹⁴N) isotope ratios were measured in feed, litter, scrapings from the ventilation system, feathers and eggs. Additionally, the distribution of δ¹³C and δ¹⁵N values in different tissues of the chicken was examined. The airborne bacteria and fungi extracted from the air filters collected from poultry farms were grown in the laboratory in media with known isotope values and measured for stable isotope ratios. Analysis of isotope fractionation between microorganisms and their media indicated the applicability of stable isotope analysis in bulk samples for the identification of source material. The analysed examples imply that stable isotope analysis can be used to examine the indoor environment along with its biology and ecology, and serve as an informative bioanalytical tool.     

Synthesis of deuterium-labelled fluorobenzoic acids to be used as internal standards in isotope dilution mass spectrometry

A method for the deuterium labelling of fluorobenzoic acids (FBAs) via acidic H/D exchange of the aromatic hydrogen atoms in concentrated D₂SO₄ is described. The synthesis is shown to be easy, fast, low-priced and without the use of catalysts or further purification. The use of at least double-deuterated FBAs as internal standards in organic isotope dilution mass spectrometry allows the determination of FBAs in complex matrices with highest possible accuracy in combination with a simplified analytical evaluation.     

Effect of waterlogging on carbon isotope discrimination during photosynthesis in Larix gmelinii

Soil moisture is a major factor controlling carbon isotope discrimination (Δ¹³C), which has been demonstrated to decrease under dry conditions in many studies; however, few studies on Δ¹³C under waterlogging condition have been conducted. In this study, a pot experiment was conducted with Larix gmelinii, a major larch species in the east Siberian Taiga, to investigate the effect of waterlogging on Δ¹³C during photosynthesis. Assimilation rate and Δ¹³C_RD (instantaneous Δ¹³C calculated with Rayleigh distillation equation) decreased drastically soon after waterlogging, followed by recovery in their values, which was caused by a change in stomatal conductance. Thereafter, assimilation rate decreased gradually, whereas Δ¹³C_RD decreased more gently. These results were thought to be caused by the decrease in both stomatal conductance and carboxylation. Our results indicate that extreme wet events may cause a decrease in Δ¹³C, which is important information for detecting flooding events in the past using tree-ring isotope analyses and for studying impacts of flooding on plants in areas where waterlogging might occur.     

Stable isotopes of H,C and N in mice bone collagen as a reflection of isotopically controlled food and water intake*

²H/¹H ratios in animal biomass reflect isotopic input from food and water. A 10-week controlled laboratory study raised 48 mice divided in two generations (8 mothers Mus musculus and their offspring). The mice were divided into four groups based on the combination of ²H, ¹³C, ¹⁵N-enriched and non-enriched food and water. Glycine, the most common amino acid in bone collagen, carried the ²H, ¹³C, ¹⁵N-isotopic spike in food. ANOVA data analysis indicated that hydrogen in food accounted for ～81?% of the hydrogen isotope inventory in collagen whereas drinking water hydrogen contributed ～17?%. Air humidity contributed an unspecified amount. Additionally, we monitored ¹³C and ¹⁵N-enrichment in bone collagen and found strong linear correlations with the ²H-enrichment. The experiments with food and water indicate two biosynthetic pathways, namely (i) de novo creation of non-essential amino acids using hydrogen from water, and (ii) the integration of essential and non-essential amino acids from food. The lower rate of isotope uptake in mothers’ collagen relative to their offspring indicates incomplete bone collagen turnover after ten weeks. The variance of hydrogen stable isotope ratios within the same cohort may limit its usefulness as a single sample proxy for archaeological or palaeoenvironmental research.     

Natural and anthropogenic variations in the Po river waters (northern Italy): insights from a multi-isotope approach

Po is the main Italian river and the δ¹⁸O and δ²H of its water reveal a similarity between the current meteoric fingerprint and that of the past represented by groundwater. As concerns the hydrochemisty, the Ca–HCO₃ facies remained constant over the last 50 year, and only nitrate significantly increased from less than 1?mg/L to more than 10?mg/L in the 1980s, and then attenuated to a value of 9?mg/L. Coherently, δ¹³C_DIC and δ³⁴S_SO4 are compatible with the weathering of the lithologies outcropping in the basin, while extremely variable δ¹⁵N_NO3 indicates contribution from pollutants released by urban, agricultural and zootechnical activities. This suggests that although the origin of the main constituents of the Po river water is geogenic, anthropogenic contributions are locally significant. Noteworthy, the associated aquifers have the same nitrogen isotopic signature of the Po river, but are characterized by significantly higher NO^– ₃ concentration. This implies that aquifers’ pollution is not ascribed to inflow of current river water, and that the attenuation of the nitrogen load recorded in the river is not occurring in the aquifers, due to their longer water residence time and delayed recovery from anthropogenic contamination.     

Ultra trace determination of fluorobenzoic acids in reservoir and ground water using isotope dilution gas chromatography mass spectrometry

The accurate ultra-trace analysis of six fluorobenzoic acids (FBAs) via isotope dilution gas chromatography mass spectrometry through their deuterated analogues is described. North Sea reservoir and ground water samples were spiked with six deuterated FBAs (dFBAs), enriched using solid-phase extraction (SPE) and analysed using GC/MS after derivatisation with BF ₃· MeOH. All FBAs were enriched and determined simultaneously. SPE allowed a 250-fold enrichment of the acids if 100 mL of sample volume was used. The method enables the determination of FBAs down to the range of 8–37 ng L ^?1 with recoveries between 66 % and 85 %. It uses low amounts of chemicals and is adaptable to larger and smaller sample volumes.     

A guide for proper utilisation of stable isotope reference materials*

Many scientific publications about stable isotope ratios suffer from flawed practices regarding calibration and normalisation of raw δ values in conjunction with prescribed δ values of reference materials. Violations of the identical treatment principle with regards to samples and standards (i.e. reference materials) and lack of adherence to SI-mandated and IUPAC-recommended nomenclature exacerbate the widespread problem of lackadaisical analytical practice and reporting. Science is supposed to strive for exactness, whereas ambiguity and jargon confound interdisciplinary communication. This contribution aims to expose typical misconceptions and avoidable errors and offers guidance toward the reproducible generation of isotope data, isotopic scale normalisation, and proper data reporting. We offer a comprehensive overview of sources of light stable isotope reference materials to best match sample matrices encountered by stable isotope practitioners with chemically similar reference materials.     

Tracing changes in the diet and habitat use of black-tailed godwits in Western France,using a stable isotope approach

Western France is at the crossroads of the migratory routes of two subspecies of black-tailed godwit, Limosa limosa. After leaving Iceland, the godwit L.l. islandica Icelandic black-tailed godwit (IBTG) winters on the coast of western Europe, while the continental black-tailed godwit (CBTG) L.l. limosa can stop in France when migrating between Iberia or Africa and their main breeding grounds in the Netherlands. In this study, we analysed δ¹⁵N and δ¹³C from flight feathers and whole blood throughout the non-breeding period to trace variations in habitat use for both subspecies on the western French coast. Adults and juveniles of IBTG adopt the same feeding habitats as soon as they arrive in the study area, progressively losing the Icelandic freshwater habitat signal, and becoming clearly restricted to marine habitats in winter. Some individuals begin to move locally to freshwater habitats, joining CBTG in a stopover at the end of the wintering period in preparation for northward migration.     

Partitioning of atmospheric carbon dioxide over Central Europe: insights from combined measurements of CO 2 mixing ratios and their carbon isotope composition

Regular measurements of atmospheric CO ₂ mixing ratios and their carbon isotope composition (¹³C/¹²C and ¹⁴C/¹²C ratios) performed between 2005 and 2009 at two sites of contrasting characteristics (Krakow and the remote mountain site Kasprowy Wierch) located in southern Poland were used to derive fossil fuel-related and biogenic contributions to the total CO ₂ load measured at both sites. Carbon dioxide present in the atmosphere, not coming from fossil fuel and biogenic sources, was considered ‘background’ CO ₂. In Krakow, the average contribution of fossil fuel CO ₂ was approximately 3.4%. The biogenic component was of the same magnitude. Both components revealed a distinct seasonality, with the fossil fuel component reaching maximum values during winter months and the biogenic component shifted in phase by approximately 6 months. The partitioning of the local CO ₂ budget for the Kasprowy Wierch site revealed large differences in the derived components: the fossil fuel component was approximately five times lower than that derived for Krakow, whereas the biogenic component was negative in summer, pointing to the importance of photosynthetic sink associated with extensive forests in the neighbourhood of the station. While the presented study has demonstrated the strength of combined measurements of CO ₂ mixing ratios and their carbon isotope signature as efficient tools for elucidating the partitioning of local atmospheric CO ₂ loads, it also showed the important role of the land cover and the presence of the soil in the footprint of the measurement location, which control the net biogenic surface CO ₂ fluxes.     

Determination of 13CO2/12CO2 Ratio by IRMS and NDIRS

Abstract

Breath tests using ¹³C-labelled substrates require the measurement of ¹³CO₂/¹²CO₂ ratio in breath gas samples. Next to isotope ratio mass spectrometry (IRMS), which is very sensitive but also complex and expensive, alternatively isotope selective nondispersive infrared spectrometry (NDIRS) can be used to determine the ¹³CO₂/¹²CO₂ ratio in expired breath. In this study we compared NDIRS- with IRMS-results to investigate whether the less expensive and very simply to operate NDIRS works as reliable as IRMS. For this purpose we applicated 1-¹³C-Phenylalanine to patients with advanced liver cirrhosis and healthy volunteers and took duplicated breath samples for IRMS and NDIRS at selected time points. Our data show a good correlation between these two methods for a small number of samples as required for simple breath tests. Longer series, where repeated measurements are required on the NDIRS instrument lead to a decreasing correlation. This indicates the superiority of IRMS concerning ¹³CO₂-kinetics over longer time periods.     

Determination of carbon isotope ratios of methanol and acetaldehyde in air samples by gas chromatography-isotope ratio mass spectrometry combined with headspace solid-phase microextraction

Isotopic signatures of atmospheric methanol and acetaldehyde have the potential to improve our ability to quantitatively assess their importance in atmospheric chemistry. However, isotopic measurements of atmospheric methanol and acetaldehyde and their individual source and sink processes have been limited. In this study, we examined gas chromatography-isotope ratio mass spectrometry combined with headspace solid-phase microextraction to measure the carbon isotope ratios of methanol and acetaldehyde in air samples. The method enabled us to determine carbon isotope ratios with a precision (1 standard deviation) of±0.6 ‰ for 20 ml of air sample containing more than 3 ppm of methanol and±0.7 ‰ for 20 ml of air sample containing more than 2 ppm of acetaldehyde. Moreover, the applicability of this method to determine isotope ratios of methanol and acetaldehyde emitted from detached plant leaves was demonstrated.     

Stable carbon isotopes in dissolved inorganic carbon: extraction and implications for quantifying the contributions from silicate and carbonate weathering in the Krishna River system during peak discharge

We present a comparative study of two offline methods, a newly developed method and an existing one, for the measurement of the stable carbon isotopic composition (δ¹³C) of dissolved inorganic carbon (DIC; δ¹³C_DIC) in natural waters. The measured δ¹³C_DIC values of different water samples, prepared from laboratory Na₂CO₃, ground and oceanic waters, and a laboratory carbonate isotope standard, are found to be accurate and reproducible to within 0.5 ‰\ (1σ). The extraction of CO₂ from water samples by these methods does not require pre-treatment or sample poisoning and can be applied to a variety of natural waters to address carbon cycling in the hydrosphere. In addition, we present a simple method (based on a two-end-member mixing model) to estimate the silicate-weathering contribution to DIC in a river system by using the concentration of DIC and its δ¹³C. This approach is tested with data from the Krishna River system as a case study, thereby quantifying the contribution of silicate and carbonate weathering to DIC, particularly during peak discharge.     

Development of non-lethal sampling of carbon and nitrogen stable isotope ratios in salmonids: effects of lipid and inorganic components of fins

The preferred tissue for analyses of fish stable isotope ratios for most researchers is muscle, the sampling of which typically requires the specimen to be sacrificed. The use of non-destructive methods in fish isotopic research has been increasing recently, but as yet is not a standard procedure. Previous studies have reported varying levels of success regarding the utility of non-lethally obtained stable isotope materials, e.g. fins, but none have accounted for the potential compounding effects of inorganic components of fin rays or lipids. Comparisons of carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope ratios of muscle with adipose and caudal fin of two salmonids, Atlantic salmon (Salmo salar L.) and brown trout (Salmo trutta L.), revealed that caudal fin can be used as a non-destructive surrogate for muscle in stable isotope analysis, but that adipose fin, where available, is a better proxy. The use of a published model to inexpensively counteract the confounding effect of lipids, which are depleted in ¹³C, greatly improved the relationship between fish muscle and fins. However, efforts to account for the inorganic components of fin rays were counterproductive and required twice the biomass of fins clipped from each fish. As this experiment was conducted on wild fish, controlled laboratory studies are required to confirm these field observations.     

Intraspecific variability of carbon isotope discrimination and its correlation with grain yield in safflower: prospects for selection in a Mediterranean climate

The goals of the present study were to obtain a first estimate of intraspecific variability of carbon isotope discrimination (Δ) in safflower, a thistle-like herbaceous plant, and to determine the statistical relationship between Δ and grain yield as well as its components in a collection of 45 accessions of different origins. Grain yield and aboveground biomass, harvest index, average grain weight, and Δ (measured on the bulk leaf organic matter) were investigated in experimental field conditions. A large variability was noted for all traits but a principal component analysis (PCA) allowed to identify several homogeneous groups of accessions. Average grain yield per plant varied between 1 and 39 g. Δ varied between 21.3 and 25.2?‰, i.e. a large variation of 3.9?‰. In our experiment, the variation of Δ was not significantly related to that of grain yield in the whole accession sample. However, we found contrasting trends for this relation within accession groups. These initial results motivate further experiments to assess more in depth correlation between Δ and yield in safflower and are encouraging regarding the possibility of using Δ as an effective selection index in safflower to obtain genotypes that efficiently consume water. This study also highlighted one accession that combines the two characters required in the Mediterranean regions, i.e. high yield performance and high water-use efficiency.     

Combined approach of isotope mass balance and hydrological water balance methods to constrain the sources of lake water as exemplified on the small dimictic lake Silbersee,northern Germany

Stable isotopes of hydrogen and oxygen are often used for water balance calculations of lakes. We present an approach combining the lake water balance with an isotope mass balance to constrain the sources and sinks of the water of a small dimictic lake subjected to eutrophication. Meteorological and hydraulic data in combination with measured isotope signatures of the different water compartments enabled to assess the degree of surface water/groundwater interaction and the amount of overland flow into the lake. Groundwater could be excluded as a lake water source, as its water level was always below the lake water level. In the absence of a channelled inflow, precipitation and overland flow were the remaining options, whereby the latter was only active during periods of exceptionally high rainfall. While the groundwater signatures adjacent to the lake showed an influence of lake water, the lake water balance itself indicated that the associated volumetric water loss to groundwater is rather negligible. In the present case, only a combined assessment of hydrological and isotopic data allowed for an accurate characterization of the studied lake and a quantification of its water sources and sinks, highlighting the importance of using more than one methodological approach for such a purpose.