Geographical Discrimination of Honeysuckle (Lonicera japonica Thunb.) from China by Characterization of the Stable Isotope Ratio and Multielemental Analysis

A total of 117 honeysuckle (Lonicera japonica Thunb.) samples from four major regions of production in China, including Fengqiu in Henan, Pingyi in Shandong, Julu in Hebei, and Xiushan in Chongqing, were analyzed to determine their geographical origin. δ¹³C, δ¹⁵N, and δ¹⁸O values were determined by isotope ratio mass spectrometry (IRMS), and the contents of 18 elements (Fe, Mn, Cu, Zn, K, Ca, Mg, Pb, Cd, Cr, As, Hg, Se, Sr, Ni, Co, B, and Mo) were measured by inductively coupled plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS). Multivariate statistical analysis by analysis of variance (ANOVA), principal component analysis (PCA), and linear discriminant analysis (LDA) were performed. The results showed that there were very significant differences in the stable isotope ratios and elemental concentrations in honeysuckle based on geographical origin, with plants from each region having a unique fingerprint. Discriminant functions were established to distinguish the origin of honeysuckle using suitable indicators including Cd, Cr, As, Hg, Se, Co, Ni, Sr, Fe, δ¹³C, δ¹⁵N, and δ¹⁸O. Cross-validated cases of 95.7% were correctly classified.     

Detection of honey adulterations with sugar syrups by stable isotope mass spectrometry

A procedure is proposed for determining the quality of honey by measuring the isotope composition of carbon in the initial honey samples and their protein fractions by mass spectrometry. Seventeen samples of honey harvested in 2014 in various regions of Russia were investigated to identify falsifications with sugars or invert syrups. Using the data on the isotope ratios of δ¹³C of initial honey samples and protein fractions, the degree of adulteration of the test honey samples was determined. Concentration of sugar was used as a criterion of adulteration. According to the data obtained, in two samples of honey, the difference between the δ¹³C values in the protein fraction and the original honey was more than 1‰, indicating the dilution of these honey samples with cane sugar by more than 7%. It is shown that the isotope composition of honey is not only informative for detecting the adulteration of honey, but also can serve as a kind of marker for the geographical origin of honey.     

Mammalian DNA δ15N exhibits 40‰ intramolecular variation and is unresponsive to dietary protein level

We report the first high‐precision characterization of molecular and intramolecular δ¹⁵N of nucleosides derived from mammalian DNA. The influence of dietary protein level on brain amino acids and deoxyribonucleosides was determined to investigate whether high protein turnover would alter amino acid ¹⁵ N or ¹³ C values. Pregnant guinea pig dams were fed control diets, or high or low levels of dietary protein throughout gestation, and all pups were fed control diets. The cerebellar DNA of offspring was extracted at 2 and 120 days of life, nucleosides isolated and δ¹⁵N and δ¹³C values characterized. Mean diet δ¹⁵N was 0.45 ± 0.33‰, compared with cerebellar whole tissue and DNA δ¹⁵N = +4.1 ± 0.7‰ and ?4.5 ± 0.4‰, respectively. Cerebellar deoxythymidine (dT), deoxycytidine (dC), deoxyadenosine (dA), and deoxyguanosine (dG) δ¹⁵N were +1.4 ± 0.4, –2.1 ± 0.9, –7.2 ± 0.3, and ?10.4 ± 0.5‰, respectively. There were no changes in amino acid or deoxyribonucleoside δ¹⁵N values due to dietary protein level. Using known metabolic relationships, we developed equations to calculate the intramolecular δ¹⁵N values originating from aspartate (asp) in purines (pur) or pyrimidines (pyr), glutamine (glu), and glycine (gly) to be δ¹⁵N_ASP‐PUR, δ¹⁵N_ASP‐PYR, δ¹⁵N_GLN, and δ¹⁵N_GLY +11.9 ± 2.3‰, +7.0 ± 2.0‰, –9.1 ± 2.4‰, and ?31.8 ± 8.9‰, respectively. A subset of twelve amino acids from food and brain had mean δ¹⁵N values of 4.3 ± 3.2‰ and 13.8 ± 3.1‰, respectively, and δ¹⁵N values for gly and asp were 12.6 ± 2.2‰ and 15.2 ± 0.8‰, respectively. A separate isotope tracer study detected no significant turnover of cerebellar DNA in the first six months of life. The large negative δ¹⁵N difference between gly and cerebellar purine N at the gly (7) position implies either that there is a major isotope effect during DNA synthesis, or that in utero gly has a different isotope ratio during rapid growth and metabolism from that in adult life. Our data show that cerebellar nucleoside intramolecular δ¹⁵N values vary over more than 40‰ and are not influenced by dietary protein level or age. Copyright © 2011 John Wiley & Sons, Ltd.     

Error assessment of nitrogen and oxygen isotope ratios of nitrate as determined via the bacterial denitrification method

Currently, bacterial denitrification is becoming the accepted method for δ¹⁵N‐ and δ¹⁸O‐NO determination. However, proper correction methods with international references (USGS32, USGS34 and USGS35) are needed. As a consequence, it is important to realize that the corrected isotope values are derived from a combination of several other measurements with associated uncertainties. Therefore, it is necessary to consider the propagated uncertainty on the final isotope value. This study demonstrates how to correctly estimate the uncertainty on corrected δ¹⁵N‐ and δ¹⁸O‐NO values using a first‐order Taylor series approximation. The bacterial denitrification method errors from 33 batches of 561 surface water samples varied from 0.2 to 2.1‰ for δ¹⁵N‐NO and from 0.7 to 2.3‰ for δ¹⁸O‐NO, which is slightly wider than the machine error, which varied from 0.2 to 0.6‰ for δ¹⁵N‐N₂O and from 0.4 to 1.0‰ for δ¹⁸O‐N₂O. The overall uncertainties, which are composed of the machine error and the method error, for the 33 batches ranged from 0.3 to 2.2‰ for δ¹⁵N‐NO and from 0.8 to 2.5‰ for δ¹⁸O‐NO. In addition, the mean corrected δ¹⁵N and δ¹⁸O values of 132 KNO₃‐IWS (internal working standard) measurements were computed as 8.4 ± 1.0‰ and 25.1 ± 2.0‰, which is a slight underestimation for δ¹⁵N and overestimation for δ¹⁸O compared with the accepted values (δ¹⁵N = 9.9 ± 0.3‰ and δ¹⁸O = 24.0 ± 0.3‰). The overall uncertainty of the bacterial denitrification method allows the use of this method for source identification of NO. Copyright © 2010 John Wiley & Sons, Ltd.     

The effects of α-cellulose extraction and blue-stain fungus on retrospective studies of carbon and oxygen isotope variation in live and dead trees

Tree‐ring carbon and oxygen isotope ratios from live and recently dead trees may reveal important mechanisms of tree mortality. However, wood decay in dead trees may alter the δ¹³C and δ¹⁸O values of whole wood obscuring the isotopic signal associated with factors leading up to and including physiological death. We examined whole sapwood and α‐cellulose from live and dead specimens of ponderosa pine (Pinus ponderosa), one‐seed juniper (Juniperous monosperma), piñon pine (Pinus edulis) and white fir (Abies concolor), including those with fungal growth and beetle frass in the wood, to determine if α‐cellulose extraction is necessary for the accurate interpretation of isotopic compositions in the dead trees. We found that the offset between the δ¹³C or δ¹⁸O values of α‐cellulose and whole wood was the same for both live and dead trees across a large range of inter‐annual and regional climate differences. The method of α‐cellulose extraction, whether Leavitt‐Danzer or Standard Brendel modified for small samples, imparts significant differences in the δ¹³C (up to 0.4‰) and δ¹⁸O (up to 1.2‰) of α‐cellulose, as reported by other studies. There was no effect of beetle frass or blue‐stain fungus (Ophiostoma) on the δ¹³C and δ¹⁸O of whole wood or α‐cellulose. The relationships between whole wood and α‐cellulose δ¹³C for ponderosa, piñon and juniper yielded slopes of ~1, while the relationship between δ¹⁸O of whole wood and α‐cellulose was less clear. We conclude that there are few analytical or sampling obstacles to retrospective studies of isotopic patterns of tree mortality in forests of the western United States. Published in 2011 by John Wiley & Sons, Ltd.     

Discrimination between ginseng from Korea and China by light stable isotope analysis

Ginseng is a health food and traditional medicine highly valued in Asia. Ginseng from certain origins is higher valued than from other origins, so that a reliable method for differentiation of geographical origin is important for the economics of ginseng production. To discriminate between ginseng samples from South Korea and PR China, 29 samples have been analyzed for the isotopic composition of the elements H, C and N. The results showed δ(2)H values between -94 and -79‰, for δ(13)C -27.9 to -23.7‰ and for δ(15)N 1.3-5.4‰ for Chinese ginseng. Korean ginseng gave δ(2)H ratios between -91 and -69‰, δ(13)C ratios between -31.2 and -22.4‰ and δ(15)N ratios between -2.4 and +7‰. Despite the overlap between the values for individual isotopes, a combination of the isotope systems gave a reasonable differentiation between the two geographic origins. Especially the statistically significant difference in δ(2)H ratios facilitated the differentiation between Korean and Chinese ginseng samples.     

Diet discrimination factors are inversely related to δ15 N and δ13C values of food for fish under controlled conditions

A recent literature review reported negative relationships between diet discrimination factors (DDFs = X_fish – X_food; X = δ¹⁵N or δ¹³C) and the values of δ¹⁵N and δ¹³C in the food of wild organisms but there has been no laboratory‐based confirmation of these relationships to date. Laboratory reared guppies (Poecilia reticulata) fed a series of diets with a range of δ¹³C (?22.9 to ?6.6‰) and δ¹⁵N (6.5 to 1586‰) values were used to magnify diet‐tissue dynamics in order to calculate DDFs once the fish had achieved equilibrium with each of the diets. Values of DDFs range widely for δ¹⁵N (7.1 to ?849‰) and δ¹³C (1.1 to ?7.0‰) and showed a strong negative correlation with the stable isotope value in the food for δ¹⁵N (slope = ?0.59 ± 0.02, r² = 0.95) and δ¹³C (slope = ?0.56 ± 0.02, r² = 0.94). Based on these relationships, the magnitude of DDF change over environmentally relevant values of δ¹⁵N or δ¹³C would be significant and could confound the interpretation of stable isotopes in the environment. Using highly enriched experimental diets, our study adds to a growing number of studies that undermine the consistent trophic enrichment paradigm with results that demonstrate the currently poor mechanistic understanding of how DDFs arise. The results of our study highlight that the magnitude of the stable isotope values in prey must be considered when choosing DDF values. Future laboratory studies should therefore be directed at uncovering the mechanistic basis of DDFs and, like others before, we recommend the determination of diet‐dependent DDFs under laboratory conditions before modeling dietary proportions or calculating trophic positions. Copyright © 2010 John Wiley & Sons, Ltd.     

Precise and accurate compound-specific carbon and nitrogen isotope analysis of RDX by GC-IRMS

A new analytical method is presented for the compound-specific carbon and nitrogen isotope ratio analysis of a thermo-labile nitramine explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by gas chromatograph coupled to an isotope ratio mass spectrometer (GC-IRMS). Two main approaches were used to minimise thermal decomposition of the compound during gas chromatographic separation: programmed temperature vaporisation (PTV) as an injection technique and a high-temperature ramp rate during the GC run. δ¹⁵N and δ¹³C values of RDX measured by GC-IRMS and elemental analyser (EA)-IRMS were in good agreement within a standard deviation of 0.3‰ and 0.4‰ for nitrogen and carbon, respectively. Application of the method for the isotope analysis of RDX during alkaline hydrolysis at 50°C revealed isotope fractionation factors ε _carbon?=??7.8‰ and ε _nitrogen?=??5.3‰.     

An inexpensive, fast, and reliable method for vacuum extraction of soil and plant water for stable isotope analyses by mass spectrometry

The stable isotopes of water (hydrogen and oxygen isotopes) are of utmost interest in ecology and the geosciences. In many cases water has to be extracted directly from a matrix such as soil or plant tissue before isotopes can be analyzed by mass spectrometry. Currently, the most widely used technique for water is cryogenic vacuum extraction. We present a simple and inexpensive modification of this method and document tests conducted with soils of various grain size and tree core replicates taken on four occasions during 2010. The accuracies for sandy soils are between 0.4‰ and 3‰ over a range of 21‰ and 165‰ for δ¹⁸O and δ²H, respectively. Spiking tests with water of known isotope composition were conducted with soil and tree core samples; they indicate reliable precision after an extraction time of 15 min for sandy soils. For clayey soils and tree cores, the deviations were up to 0.63‰ and 4.7‰ for δ¹⁸O and δ²H, respectively. This indicates either that the extraction time should be extended or that mechanisms different from Rayleigh fractionation play a role. The modified protocol allows a fast and reliable extraction of large numbers of water samples from soil and plant material in preparation for stable isotope analyses. Copyright © 2011 John Wiley & Sons, Ltd.     

Measurements of nitrogen isotope composition of plants and surface soils along the altitudinal transect of the eastern slope of Mount Gongga in southwest China

The natural abundances of stable nitrogen isotopes in plants and soils have been viewed as recorders that can be used to reconstruct paleoclimate and ecological processes or to indicate the biogeochemical cycle of nitrogen in nature. This study systematically measured the nitrogen isotope composition (δ¹⁵N) in plants and surface soils along an altitudinal transect of elevation range of 1200 to 4500 m on the eastern slope of Mount Gongga in southwest China. The influences of photosynthetic pathways on plant δ¹⁵N as well as the effects of temperature and precipitation on δ¹⁵N altitudinal trends in plants and surface soils are discussed. Across this altitude transect, the δ¹⁵N values of C₃ and C₄ plants on Mount Gongga range between ?9.87‰ and 7.58‰ with a mean value of ?1.33‰, and between ?3.98‰ and 4.38‰ with a mean value of ?0.25‰, respectively. There is an evident δ¹⁵N difference between C₃ plants and C₄ plants. If, however, you only compare C₄ plants with those C₃ plants growing at the same altitudinal range, no significant difference in δ¹⁵N exists between them, suggesting that photosynthetic pathway does not have an influence on the plant δ¹⁵N values. In addition, we found that C₃, C₄ plants and surface soil (0–5 cm depth) all trend significantly towards more negative δ¹⁵N with increasing elevation. Furthermore, this study shows that the mean annual temperature and the mean annual precipitation positively and negatively correlate with δ¹⁵N in C₃ and C₄ plants, respectively. This indicates that precipitation and temperature are the main controlling factors of the δ¹⁵N variation in plants with altitude. We propose that lower δ¹⁵N values of plants and soils at higher altitude should be attributed to lower mineralization and lower net nitrification rates induced by low temperature and abundant rainfall. Copyright © 2010 John Wiley & Sons, Ltd.     

Does natural weathering change the stable isotope composition (2H, 13C, 15N, 18O and 34S) of cattle hair?

Stable isotope analysis of hair has found applications in many fields of science because it provides a temporally resolved, fairly stable isotopic archive of mammalian individuals. We investigated whether this hair archive is modified by natural weathering while attached to a living animal. We analyzed the tail switch hairs of one suckler cow, sampled seven times over a period of four annual summer pasture–winter stall feeding cycles. We compared relative isotope ratios (δ²H, δ¹³C, δ¹⁵N, δ¹⁸O and δ³⁴S) of sections of hair that grew simultaneously but were exposed to natural weathering conditions over different periods of time. Natural wear caused a loss of mass of approx. 0.13% day^–1, with no apparent effect of environmental conditions. Changes in δ²H, δ¹³C, δ¹⁵N and δ¹⁸O were below the detection limit, indicating that hair is a reliable archive for the isotopes of these elements. In contrast, δ³⁴S values increased during the grazing period by about 1 ‰, with exposure to UV radiation appearing to have a major influence on this result. The δ³⁴S values decreased during the subsequent stall period, probably due to abrasion. Seasonal variation in δ³⁴S may indicate alternating environments that differ in their weathering conditions. Copyright © 2011 John Wiley & Sons, Ltd.     

A new isolation procedure of nitrate from freshwater for nitrogen and oxygen isotope analysis

The nitrogen (δ¹⁵N) and oxygen isotope (δ¹⁸O) analysis of nitrate (NO₃^–) from aqueous samples can be used to determine nitrate sources and to study N transformation processes. For these purposes, several methods have been developed; however, none of them allows an accurate, fast and inexpensive analysis. Here, we present a new simple method for the isolation of nitrate, which is based on the different solubilities of inorganic salts in an acetone/hexane/water mixture. In this solvent, all major nitrate salts are soluble, whereas all other oxygen‐bearing compounds such as most inorganic carbonates, sulfates, and phosphates are not. Nitrate is first concentrated by freeze‐drying, dissolved in the ternary solvent and separated from insoluble compounds by centrifugation. Anhydrous barium nitrate is then precipitated in the supernatant solution by adding barium iodide. For δ¹⁸O analysis, dried Ba(NO₃)₂ samples are directly reduced in a high‐temperature conversion system to CO and measured on‐line using isotope ratio mass spectrometry (IRMS). For δ¹⁵N analysis, samples are combusted in an elemental analyzer (EA) coupled to an IRMS system. The method has been tested down to 20 µmol NO₃^– with a reproducibility (1SD) of 0.1‰ for nitrogen and 0.2–0.4‰ for oxygen isotopes. For nitrogen we observed a small consistent ¹⁵N enrichment of +0.2‰, probably due to an incomplete precipitation process and, for oxygen, a correction for the incorporation of water in the precipitated Ba(NO₃)₂ has to be applied. Apart from being robust, this method is highly efficient and low in cost. Copyright © 2011 John Wiley & Sons, Ltd.     

Stable isotopic analysis of pyrogenic organic matter in soils by liquid chromatography–isotope‐ratio mass spectrometry of benzene polycarboxylic acids

Pyrogenic organic matter (PyOM), the incomplete combustion product of organic materials, is considered stable in soils and represents a potentially important terrestrial sink for atmospheric carbon dioxide. One well‐established method of measuring PyOM in the environment is as benzene polycarboxylic acids (BPCAs), a compound‐specific method, which allows both qualitative and quantitative estimation of PyOM. Until now, stable isotope measurement of PyOM carbon involved measurement of the trimethylsilyl (TMS) or methyl (Me) polycarboxylic acid derivatives by gas chromatography–combustion–isotope ratio mass spectrometry (GC‐C‐IRMS). However, BPCA derivatives can contain as much as 150% derivative carbon, necessitating post‐analysis correction for the accurate measurement of δ¹³ C values, leading to increased measurement error. Here, we describe a method for δ¹³ C isotope ratio measurement and quantification of BPCAs from soil‐derived PyOM, based on ion‐exchange chromatography (IEC‐IRMS). The reproducibility of the δ¹³ C measurement of individual BPCAs by IEC‐IRMS was better than 0.35‰ (1σ). The δ¹³ C‐BPCA analysis of PyOM in soils, including at natural and artificially enriched ¹³ C‐abundance, produced accurate and precise δ¹³ C measurements. Analysis of samples that differed in δ¹³ C by as much as 900‰ revealed carryover of <1‰ between samples. The weighted sum of individual δ¹³ C‐BPCA measurements was correlated with previous isotopic measurements of whole PyOM, providing complementary information for bulk isotopic measurements. We discuss potential applications of δ¹³ C‐BPCA measurements, including the study of turnover rates of PyOM in soils and the partitioning of PyOM sources based on photosynthetic pathways. Copyright © 2011 John Wiley & Sons, Ltd.     

Continuous analysis of δ18O and δD values of water by diffusion sampling cavity ring‐down spectrometry: a novel sampling device for unattended field monitoring of precipitation,ground and surface waters

A novel sampling device suitable for continuous, unattended field monitoring of rapid isotopic changes in environmental waters is described. The device utilises diffusion through porous PTFE tubing to deliver water vapour continuously from a liquid water source for analysis of δ¹⁸O and δD values by Cavity Ring‐Down Spectrometry (CRDS). Separation of the analysed water vapour from non‐volatile dissolved and particulate contaminants in the liquid sample minimises spectral interferences associated with CRDS analyses of many aqueous samples. Comparison of isotopic data for a range of water samples analysed by Diffusion Sampling‐CRDS (DS‐CRDS) and Isotope Ratio Mass Spectrometry (IRMS) shows significant linear correlations between the two methods allowing for accurate standardisation of DS‐CRDS data. The internal precision for an integration period of 3 min (standard deviation (SD) = 0.1 ‰ and 0.3 ‰ for δ¹⁸O and δD values, respectively) is similar to analysis of water by CRDS using an autosampler to inject and evaporate discrete water samples. The isotopic effects of variable air temperature, water vapour concentration, water pumping rate and dissolved organic content were found to be either negligible or correctable by analysis of water standards. The DS‐CRDS system was used to analyse the O and H isotope composition in short‐lived rain events. Other applications where finely time resolved water isotope data may be of benefit include recharge/discharge in groundwater/river systems and infiltration‐related changes in cave drip water. Copyright © 2011 John Wiley & Sons, Ltd.     

A suite of sensitive chemical methods to determine the δ15N of ammonium, nitrate and total dissolved N in soil extracts

Natural ¹⁵N abundances (δ¹⁵N values) of different soil nitrogen pools deliver crucial information on the soil N cycle for the analysis of biogeochemical processes. Here we report on a complete suite of methods for sensitive δ¹⁵N analysis in soil extracts. A combined chemical reaction of vanadium(III) chloride (VCl₃) and sodium azide under acidic conditions is used to convert nitrate into N₂O, which is subsequently analyzed by purge‐and‐trap isotope ratio mass spectrometry (PTIRMS) with a cryo‐focusing unit. Coupled with preparation steps (microdiffusion for collection of ammonium, alkaline persulfate oxidation to convert total dissolved N (TDN) or ammonium into nitrate) this allows the determination of the δ¹⁵N values of nitrate, ammonium and total dissolved N (dissolved organic N, microbial biomass N) in soil extracts with the same basic protocol. The limits of quantification for δ¹⁵N analysis with a precision of 0.5‰ were 12.4 µM for ammonium, 23.7 µM for TDN, 16.5 µM for nitrate and 22.7 µM for nitrite. Copyright © 2010 John Wiley & Sons, Ltd.     

Compound-Specific Isotope Analysis of Amino Acid Labeling with Stable Isotope Nitrogen (15N) in Higher Plants

Individual free amino acid δ¹⁵N values in plant tissue reflect the metabolic pathways involved in their biosynthesis and catabolism and could thus aid understanding of environmental stress and anthropogenic effects on plant metabolism. In this study, compound-specific nitrogen isotope analysis of amino acid by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was carried out to determine individual free amino acid δ¹⁵N values. High correlations were observed between the δ¹⁵N values obtained by GC-C-IRMS and elemental analyzer-isotope ratio mass spectrometry (EA-IRMS) determinations, and the mean precision measured was better than 1 ‰. Cation-exchange chromatography was employed to purify the sample, and the difference between prior to and following passage through the resin was within 1 ‰. The amino acid δ¹⁵N values of plant leave samples following incubation in ¹⁵N-nitrate at different time points were determined. A typical foliar free amino acid ¹⁵N-enrichment pattern was found, and glutamine was the most rapidly labeled amino acid; other amino acids derived from the GS-GOGAT cycle were also enriched. The pyruvate family amino acids were labeled less quickly followed by the aromatic amino acids. This study highlighted that amino acid metabolism pathways had a major effect on the δ¹⁵N values. With the known amino acid metabolism pathways and δ¹⁵N values determined by the presented method, the influence of various external factors on the metabolic cycling of amino acid can be understood well.

     

Characterization of Vegetables by Stable Isotopic and Elemental Signatures

ABSTRACT

109 Romanian parsley, celery and parsnip root, cucumber, vegetable marrow, onion, and pepper samples were characterized by their stable isotope ratios and elemental concentrations in comparison to imported varieties. Organic and conventional agriculture practices were also compared as well as a differentiation between eggplant and peppers grown in greenhouses and those in the field. δ²H values were between ?70.8 and 5.6‰, while the δ¹⁸O values were between ?9.0 and 4.1‰. The δ¹⁵N values for white onions were from 2.8 to 11.1‰. For Romanian organic onions, the carbon isotopic values were lower (?29.2 to ?28.2‰) compared to conventional onions from other countries (?27.8‰ to ?23.8%). Linear discriminant analysis was used to characterize agricultural practices and the geographic origin of the vegetables.     

Source identification of nitrate by means of stable nitrogen isotopes in the river Daugava and loads of nitrogen to the Gulf of Riga

Nitrogen isotope ratio of nitrate provides a powerful tool to investigate nitrate sources and cycling mechanisms. Although the use of an isotope ratio method for ¹⁵N/¹⁴N allows identifying the nitrate sources in rivers by estimating a seasonal variation of N-NO₃ concentration, however, there are some restrictions. Nitrification, the conversion of NH₄⁺ to NO₃-, can proceed with significant nitrogen isotope fractionation, preferentially accumulating ¹⁴N in the produced NO₃-, and can make it difficult to identify the nitrate source with a high proportion of the isotope δ¹⁵N. However, the uptake and assimilation of NH₄⁺ and NO₃- have the capability of affecting isotopic compositions of riverine nitrogen compounds, and this may hinder the determination of whether the impact of the nitrate source with a high proportion of the isotope δ¹⁵N reduces. In addition, this study demonstrates that nitrate nitrogen concentration may correlate with δ¹⁵N_NO3 values both positively and negatively. Such correlations are the result of isotope effects during nitrogen transformation processes (e.g. nitrification and assimilation) and isotopic variability in the various nitrate sources. A comparison of NO₃- concentration and δ¹⁵N_NO3 can be used to further distinguish mixing from biological processing. However, in order to get a more precise answer regarding the nitrate sources, it would be useful to take both the data of nitrogen isotopes and data of oxygen isotopes present in nitrates.     

Identification and correction of spectral contamination in 2H/1H and 18O/16O measured in leaf, stem, and soil water

Plant water extracts typically contain organic materials that may cause spectral interference when using isotope ratio infrared spectroscopy (IRIS), resulting in errors in the measured isotope ratios. Manufacturers of IRIS instruments have developed post-processing software to identify the degree of contamination in water samples, and potentially correct the isotope ratios of water with known contaminants. Here, the correction method proposed by an IRIS manufacturer, Los Gatos Research, Inc., was employed and the results were compared with those obtained from isotope ratio mass spectrometry (IRMS). Deionized water was spiked with methanol and ethanol to create correction curves for δ(18)O and δ(2)H. The contamination effects of different sample types (leaf, stem, soil) and different species from agricultural fields, grasslands, and forests were compared. The average corrections in leaf samples ranged from 0.35 to 15.73‰ for δ(2)H and 0.28 to 9.27‰ for δ(18)O. The average corrections in stem samples ranged from 1.17 to 13.70‰ for δ(2)H and 0.47 to 7.97‰ for δ(18)O. There was no contamination observed in soil water. Cleaning plant samples with activated charcoal had minimal effects on the degree of spectral contamination, reducing the corrections, by on average, 0.44‰ for δ(2)H and 0.25‰ for δ(18)O. The correction method eliminated the discrepancies between IRMS and IRIS for δ(18)O, and greatly reduced the discrepancies for δ(2)H. The mean differences in isotope ratios between IRMS and the corrected IRIS method were 0.18‰ for δ(18)O, and -3.39‰ for δ(2)H. The inability to create an ethanol correction curve for δ(2)H probably caused the larger discrepancies. We conclude that ethanol and methanol are the primary compounds causing interference in IRIS analyzers, and that each individual analyzer will probably require customized correction curves.