Temporary Biological N Cońservation by Winter Oilseed Turnip (Brassica Rapa L.) and its Influence on Following Crops and N-Percolation

Abstract

As shown in field plot trials with application of ¹⁵N on loamy sand (albic luvisol), winter oilseed turnip (Brassica rapa L.; ssp. oleifera (Dc.) METZG.) incorporated considerable N amounts during the winter period. By this a reduction of N percolation into deeper soil layers was achieved. After mulching, about 30% of the catch crop N was taken up by the following crop, maize (Zea mays L., cv. ‘BEKENOVA’). Maize covered 11% of its total N demand deriving from the catch crop.     

The Fate of [15N]Ammonium and [15N]Nitrate in the Soil of a 140-Year-Old Spruce Stand (Picea Abies) in the Fichtelgebirge (NE-Bavaria)

Abstract

A ¹⁵N tracer-experiment was carried out in a 140-year-old spruce stand (Picea abies (L.) Karst.) in the Fichtelgebirge (NE-Bavaria, Germany). Highly enriched (98 at%) [¹⁵N]ammonium and [¹⁵N]nitrate were applied as tracers by simulation of a deposition of 41.3 mol N ha^?1 with 11 water m^?2. To examine seasonal variations of uptake by spruce and understorey vegetation, different plots were labelled in spring, summer and autumn 1994.

One aim of the present study was to perfect a method of preparation of soil extracts for isotope ratio mass spectrometry (IRMS) measurements. Ammonium and nitrate from soil extracts were prepared for IRMS measurements by steam distillation and subsequent freeze drying. Additionally, tracer distribution and transformations in the soil nitrogen pools were examined. Ammonium, nitrate and total nitrogen were examined in the organic layer and the upper 10 cm of the mineral soil during 3 months after the first tracer application in spring 1994.

In July 1994, three months after tracer application, 40% of the [¹⁵N]ammonium label and 29% of the [¹⁵N]nitrate label, respectively, were recovered in the total N pool of the investigated soil horizons. In the organic layer the L/Of horizon retained most of the recovered tracers. Nitrification, immobilisation and mineralisation occurred even under the conditions of high soil acidity at the study site.     

Nitrogen-15 Leaching Under Temporarily Weedy Maize

Abstract

In pot experiments the effect of a temporary infestation of maize by Chenopodium album L. on water and nitrogen leaching after a simulated strong rainfall was tested for a silty loam and a loamy sand. The results show that the leaching of ¹⁵N under the initial stage of maize can be reduced considerably by a temporarily weed infestation. Neither yield of maize nor uptake of nitrogen until corn maturity were reduced on both a silty loam and a loamy sand.     

The Influence of Ammonium on Nitrate Uptake and Assimilation in 2-Year-Old Ash and Oak Trees - A Tracer-Study with 15N

Abstract

Interactions between ammonium and nitrate as competitive N sources depend on various biotic and abiotic factors. The preference for one of these N sources and the influence of ammonium on nitrate uptake and nitrate reductase activity was investigated in a ¹⁵N labelling experiment using 2-year-old potted plants of ash (Fraxinus excelsior L.) and oak (Quercus robur L.) under greenhouse conditions.

Seedlings of both tree species use ammonium and nitrate in equal amounts when both N forms are supplied in a 1:1 ratio (1.5 mM NH₄ ⁺ + 1.5 mM NO₃ ^?), although there is a slight tendency that ammonium is preferred. In both species total N uptake is higher if ammonium and nitrate are supplied simultaneously when compared with uptake of nitrate alone (3 mM nitrate). If nitrate is the sole N source N uptake is only half as high as if ammonium and nitrate are supplied in a ratio of 1:1.

The distribution of nitrate reductase between shoot and roots is not influenced by the N-form: nitrate reductase activity is always highest in the roots of both species under the conditions of this experiment.

Xylem sap analyses showed that both species transport higher concentrations of amino acids than of nitrate from the roots to the shoot. The amino acid composition is independent of the type of N source. Furthermore, ash trees contain more nitrate in the xylem sap than oak trees, reflecting the higher N uptake and the higher nitrate reductase activity in the leaves of this species.     

Lysimeter Investigations on the Effect of Winter Catch Crops and Weeded Fallow on the N-Dynamics in a Sandy Treposol Soil of Northeast Germany

Abstract

Lysimeter experiments (soil: sandy treposol, from the region “Havelländisches Luch”, Brandenburg, Germany) with application of ¹⁵N labelled fertilizer (80 kg N per ha as ¹⁵NH₄ ¹⁵NO₃, 10 at.-%¹⁵N exc.; for simulating mineralization in the early autumn period) were carried out to determine to what extent the amount of mineral- N was temporary conserved by winter catch crops, taken up subsequently in the vegetation periods by following crops, taken by subsequently in the vegetation periods by following crops, or percolated in the leaching water, respectively. The results were as follows:

1) Until winter or spring respectively, the catch crop uptake rates of applied mineral-N were 32% for phacelia (Phacelia tanacetifolia BENTH.), 25% for winter rape (Brassica napus L. cv. ‘AKELA’), and 16% for white mustard (Sinapis alba L.).

2) In the year after, following maize incorporated from 2.1 to 4.5% of the fertilizerborne N. The following plant community of fallow took up from 0.2 to 0.5% N originating from the fertilizer-N.

3) In comparison with the catch crops, N-leaching losses under fallow conditions were highest and equivalent to 17% of the applied fertilizer-N amount. In contrast to 3% of white mustard, phacelia and winter rape reduced N-leaching losses to 0.2 and 0.3% of the applied fertilizer-N amount.

4) In spring of the first year after the beginning of investigations, N-leaching losses were highest under fallow conditions and white mustard cultivation. Thus, the amounts of nitrate losses would exceed the EU limit for drinking water.

5) Three years after the investigations had been started, 10% (white mustard) and 20% (fallow) of the applied fertilizer-N was still found in th lysimeter soil.     

Split-root labelling to investigate 15N rhizodeposition by Pinus sylvestris and Picea abies*

We investigated the transfer of ¹⁵N into the soil via ¹⁵N uptake and release by tree roots, which involves the principles of the split-root technique. One half of the root system received an injection of (¹⁵NH₄)₂SO₄ and the other half equivalent amounts of (NH₄)₂SO₄ at ¹⁵N natural abundance level. ¹⁵N was transferred from one side of the root system (¹⁵N side) to the other side (¹⁴N side) and released into the soil. The method was conducted with Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies [L.] Karst). Two concentration levels of (NH₄)₂SO₄ were used, corresponding with annual N deposition in the Netherlands (30 kg N ha^–1) and a twelfth of that (2.5 kg N ha^?1). Samples were taken 3 and 6 weeks after labelling and divided into needles?+?stem, roots, rhizosphere and bulk soil. Already 3 weeks after labelling, Scots pine took up 23.7?% of the low and 9.1?% of the high amounts of ¹⁵N, while Norway spruce took up 21.5 and 32.1?%, respectively. Both species transported proportions of ¹⁵N to the rhizosphere (0.1–0.2?%) and bulk soil (0.3–0.9?%). The method is a useful tool to investigate the fate of root-derived N in soils, for example, for the formation of stable forms of soil organic matter.     

A Dual 13C and 15N Long Term Labelling Technique to Investigate Uptake and Translocation of C and N in Beech (Fagus sylvatica L.)

Abstract

A continuous dual ¹³CO₂ and ¹⁵NH₄ ¹⁵NO₃ labelling experimental set-up is presented that was used to investigate the C and N uptake and allocation within 3-year old beech (Fagus sylvatica L.) during one growing season. The C and N allocation pattern was determined after six, twelve and eighteen weeks of growth. The carbon uptake was distinctly different in the three phases examined: The first six weeks after budbreak were dedicated to leaf growth with a R/S (root to shoot) ratio of 0.14 for the new carbon. The second growth phase showed a balanced R/S ratio of C allocation and after week 13, the root compartment was the main carbon sink (R/S = 6.97).

Nitrogen allocation was more basipetal as compared to carbon. In the second growth phase, R/S of N_new was 5.57 but fell to 3.54 for the third growth phase probably due to formation of reserves in buds and stem.     

Investigations on the Nitrogen Metabolism of Forest Trees by Mathematical Modelling of Natural Isotope Ratios

Abstract

Based on an experimental investigation of nitrogen concentrations and δ¹⁵N-values in different parts of Picea abies (L.) trees and soil samples and of their dependence on the age of plant parts and on damaging stress, mathematical models which represent main features of nitrogen turnover in the forest tree - soil system and associated isotope variations are developed. These models consider parts of 3 cycles of nitrogen: (1) transport of soluble nitrogen within trees, (2) turnover of protein nitrogen in different plant parts, and (3) the exchange of N between tree and soil via litter and the uptake of mineralized soil N by plant roots. Isotope effects of protein synthesis and mineralization of organic soil N are included. The models give a qualitative explanation of the data and lead to a better understanding of isotopic variations as indicators of forest damage.     

Natural abundances of 15N and 13C in leaves of some N2-fixing and non-N2-fixing trees and shrubs in Syria

A survey study was conducted on man-made plantations located at two different areas in the arid region of Syria to determine the variations in natural abundances of the ¹⁵N and ¹³C isotopes in leaves of several woody legume and non-legume species, and to better understand the consequence of such variations on nitrogen fixation and carbon assimilation. In the first study area (non-saline soil), the δ¹⁵N values in four legume species (Acacia cyanophylla,?1.73 ‰ Acacia farnesiana,?0.55 ‰ Prosopis juliflora,?1.64 ‰; and Medicago arborea,+1.6 \textperthousand) and one actinorhizal plant (Elaeagnus angustifolia,?0.46 to?2.1 ‰) were found to be close to that of the atmospheric value pointing to a major contribution of N₂ fixing in these species; whereas, δ¹⁵N values of the non-fixing plant species were highly positive. δ¹³C ‰; in leaves of the C3 plants were found to be affected by plant species, ranging from a minimum of?28.67 ‰; to a maximum of?23 ‰. However, they were relatively similar within each plant species although they were grown at different sites. In the second study area (salt affected soil), a higher carbon discrimination value (Δ¹³C ‰) was exhibited by P. juliflora, indicating that the latter is a salt tolerant species; however, its δ¹⁵N was highly positive (+7.03 ‰) suggesting a negligible contribution of the fixed N₂. Hence, it was concluded that the enhancement of N₂ fixation might be achieved by selection of salt-tolerant Rhizobium strains.     

15N Distribution in Wheat and Chemical Fractionation of Root-Borne 15N in the Soil

Abstract

Spring wheat plants were grown in split-root containers and labelled with ¹⁵N by fertilizing one compartment of the container with ¹⁵NH₄ ¹⁵NO₃ (95 at.-% ¹⁵N exc.). After the harvest, approx. 90% of the ¹⁵N incorporated by the plants were found in the shoots and 3% in the roots; approx. 7% had been released into the soil of the unlabelled compartment. The ¹⁵N in the soil of the unlabelled compartment was extracted with KCl and hydrolysed with HCl. Approx. 60% of the ¹⁵N was found in the hydrolysable organic N pool of the soil and 9% in the fraction of the soluble and exchangeable inorganic nitrogen.     

Transfer of Symbiotically Fixed Nitrogen in an Alfalfa-Grass Mixture Studied Through Isotope Dilution in a Pot Experiment

Abstract

The nitrogen transfer between alfalfa and ryegrass was studied through isotope dilution at three different levels of N fertilization (20 mg N/pot, 200 mg N/pot, 400 mg N/pot) in a pot experiment using quartz sand as a substrate. An isogenic, nodulating, but non nitrogen fixing alfalfa line was used as a reference crop. Fixed N was transferred to the grass in the 20 mg N treatment and contributed markedly to the N nutrition of the grass (about 50% of the N in the plants). No transfer of fixed N could be detected in the higher fertilized treatments, although nitrogen fixation was only slightly inhibited by the presence of the fertilized mineral N. It is concluded that N transfer is strongly influenced by the N concentration in the substrate and transferred N contributes only slightly to the productivity of the legume/grass mixture under the given experimental conditions.     

Amino Acid 15N/14N Analysis at Natural Abundances: A New Tool for Soil Organic Matter Studies in Agricultural Systems

Abstract

The effects of landuse, fertilizer history and soil type on the quantity and isotopic quality of hydrolysable soil amino acids were examined in 3 grassland and 2 arable soils. Results showed, (i) that overall concentrations of individual amino acids were highest in the grassland soils, (ii) that ‰δ¹⁵N values of the individual amino acids differed considerably between the five soils, and (iii) that the combination of amino acid ‰δ¹⁵N values and concentrations could be used to distinguish between landuse, crop type and fertilizer history. This preliminary study indicates that the pathways of transformation of soil amino acid N are influenced by long term N inputs and that associated biological processes are reflected in differences in concentrations and ‰δ¹⁵N values of individual soil amino acids.     

Using the natural 15N abundance to assess the main nitrogen inputs into the sand dune area of the North-Western Negev desert (ISRAEL)

The variation of the natural ¹⁵N abundance is often used to evaluate the origin of nitrogen or the pathways of N input into ecosystems. We tried to use this approach to assess the main input pathways of nitrogen into the sand dune area of the north-western Negev Desert (Israel). The following two pathways are the main sources for nitrogen input into the system:

1. Biological fixation of atmospheric nitrogen by cyanobacteria present in biological crusts and by N₂-fixing vascular plants (e.g. the shrub Retama raetam);

2. Atmospheric input of nitrogen by wet deposition with rainfall, dry deposition of dust containing N compounds, and gaseous deposition.

Samples were taken from selected environmental compartments such as biological crusts, sand underneath these crusts (down to a depth of 90?cm), N₂-fixing and non-N₂-fixing plants, atmospheric bulk deposition as well as soil from arable land north of the sandy area in three field campaigns in March 1998, 1999 and 2000. The δ¹⁵N values measured were in the following ranges: grass ?2.5‰ to +1.5‰; R. reatam: +0.5‰ to +4.5‰; non-N₂-fixing shrubs +1‰ to +7‰; sand beneath the biological crusts +4‰ to +20‰ (soil depth 2–90?cm); and arable land to the north up to 10‰. Thus, the natural ¹⁵N abundance of the different N pools varies significantly. Accordingly, it should be feasible to assess different input pathways from the various ¹⁵N abundances of nitrogen. For example, the biological N fixation rates of the Fabaceae shrub R. reatam from the ¹⁵N abundances measured were calculated to be 46–86% of biomass N derived from the atmosphere. The biological crusts themselves generally show slight negative ¹⁵N values (?3‰ to ?0.5‰), which can be explained by biological N fixation. However, areas with a high share of lichens, which are unable to fix atmospheric nitrogen, show very negative values down to ?10‰. The atmospheric N bulk deposition, which amounts to 1.9–3.8?kg?N/ha?yr, has a ¹⁵N abundance between 4.4‰ and 11.6‰ and is likely to be caused by dust from the arable land to the north. Thus, it cannot be responsible for the very negative values of lichens measured either. There must be an additional N input from the atmosphere with negative δ¹⁵N values, e.g. gaseous N forms (NO _x , NH₃). To explain these conflicting findings, detailed information is still needed on the wet, particulate and gaseous atmospheric deposition of nitrogen.     

Course of Uptake of Weed-Borne Nitrogen by Maize,Tested with 15N

Abstract

The course of uptake of weed-borne nitrogen by maize was tested with ¹⁵N in a pot experiment with silty loam after common growth of maize and Chenopodium album L., and mulching the weed in the 5-leaf stage of maize. Harvests 4,8 and 12 weeks after mulching show that the maize took up 35, 63 and 70% of the weed-borne nitrogen, resp., in consequence of a rapid and almost complete mineralization. The portion of weed-borne nitrogen in total N of the maize was 16% at all harvest dates. The differences in yield between weeded and unweeded maize were not significant neither at 5-leaf stage nor at corn maturity.     

Analysis of the coexisting pathways for NO and N2O formation in Chernozem using the 15N-tracer SimKIM-Advanced model

The nitrogen (N) cycle consists of a variety of microbial processes. These processes often occur simultaneously in soils, but respond differently to local environmental conditions due to process-specific biochemical restrictions (e.g. oxygen levels). Hence, soil nitrogen cycling (e.g. soil N gas production through nitrification and denitrification) is individually affected through these processes, resulting in the complex and highly dynamic behaviour of total soil N turnover. The development and application of methods that facilitate the quantification of individual contributions of coexisting processes is a fundamental prerequisite for (i) understanding the dynamics of soil N turnover and (ii) implementing these processes in ecosystem models. To explain the unexpected results of the triplet tracer experiment (TTE) of Russow et al. (Role of nitrite and nitric oxide in the processes of nitrification and denitrification in soil: results from ¹⁵N tracer experiments. Soil Biol Biochem. 2009;41:785–795) the existing SimKIM model was extended to the SimKIM-Advanced model through the addition of three separate nitrite subpools associated with ammonia oxidation, oxidation of organic nitrogen (N_org), and denitrification, respectively. For the TTE, individual treatments with ¹⁵N ammonium, ¹⁵N nitrate, and ¹⁵N nitrite were conducted under oxic, hypoxic, and anoxic conditions, respectively, to clarify the role of nitric oxide as a denitrification intermediate during N₂O formation. Using a split nitrite pool, this analysis model explains the observed differences in the ¹⁵N enrichments in nitric oxide (NO) and nitrous oxide (N₂O) which occurred in dependence on different oxygen concentrations. The change from oxic over hypoxic to anoxic conditions only marginally increased the NO and N₂O release rates (1.3-fold). The analysis using the model revealed that, under oxic and hypoxic conditions, N_org-based N₂O production was the dominant pathway, contributing to 90 and 50 % of the total soil N₂O release. Under anoxic conditions, denitrification was the dominant process for soil N₂O release. The relative contribution of N_org to the total soil NO release was small. Ammonia oxidation served as the major pathway of soil NO release under oxic and hypoxic conditions, while denitrification was dominant under anoxic conditions. The model parameters for soil with moderate soil organic matter (SOM) content were not scalable to an additional data set for soil with higher SOM content, indicating a strong influence of SOM content on microbial N turnover. Thus, parameter estimation had to be re-calculated for these conditions, highlighting the necessity of individual soil-dependent parameter estimations.     

Distribution and Utilization of 15N in Cowpeas Injected into the Stem Under Influence of Water Deficit

Abstract

Investigations were carried out on Vigna unguiculata L. Walp. to estimate the distribution and utilization of ¹⁵N in different organs after stem injection during vegetative, flowering and pod filling stage. During flowering effects of water deficit were also examined. In well watered plants, within 4 days after injection, 65% of ¹⁵N accumulated in leaves. This was drastically reduced to 42% by water deficit. ¹⁵N accumulation in stems increased under water deficit. The translocation of ¹⁵N from the stem base to roots were not altered by water deficit. During pod filling 62% of recovered ¹⁵N in the plants had accumulated in seeds, 24% in leaves and 11% in stems within 4 days, whereas the uptake of nitrogen in pod walls and roots remained low (2%). These results demonstrate that the method of injecting very small quantities (1 mg/plant) of ¹⁵N into the stem base allows an exact and detailed quantitative assessment of N translocation/distribution with regard to different organs, different growth stages and different treatments.     

N2O concentration and isotope signature along profiles provide deeper insight into the fate of N2O in soils†

Nitrous oxide is an important greenhouse gas and its origin and fate are thus of broad interest. Most studies on emissions of nitrous oxide from soils focused on fluxes between soil and atmosphere and hence represent an integration of physical and biological processes at different depths of a soil profile. Analysis of N₂O concentration and isotope signature along soil profiles was suggested to improve the localisation of sources and sinks in soils as well as underlying processes and could therefore extend our knowledge on processes affecting surface N₂O fluxes. Such a mechanistic understanding would be desirable to improve N₂O mitigation strategies and global N₂O budgets. To investigate N₂O dynamics within soil profiles of two contrasting (semi)natural ecosystem types (a temperate acidic fen and a Norway spruce forest), soil gas samplers were constructed to meet the different requirements of a water-saturated and an unsaturated soil, respectively. The samplers were installed in three replicates and allowed soil gas sampling from six different soil depths. We analysed soil air for N₂O concentration and isotope composition and calculated N₂O net turnover using a mass balance approach and considering diffusive fluxes. At the fen site, N₂O was mainly produced in 30–50 cm soil depth. Diffusion to adjacent layers above and below indicated N₂O consumption. Values of δ¹⁵N and δ¹⁸O of N₂O in the fen soil were always linearly correlated and their qualitative changes within the profile corresponded with the calculated turnover processes, suggesting further reduction of N₂O. In the spruce forest, highest N₂O production occurred in the topsoil, but there was also notable production occurring in the subsoil at a depth of 70 cm. Changes in N₂O isotope composition as to be expected from local production and consumption processes within the soil profile did hardly occur, though. This was presumably caused by high diffusive fluxes and comparatively low net turnover, as isotope signatures approached values measured for ambient N₂O towards the topsoil. Our results demonstrate a highly variable influence of diffusive versus production/consumption processes on N₂O concentration and isotope composition, depending on the type of ecosystem. This finding indicates the necessity of further N₂O concentration and isotope profile investigations in different types of natural and anthropogenic ecosystems in order to generalise our mechanistic understanding of N₂O exchange between soil and atmosphere.     

Accuracy and Precision for Measurements of the Mass Ratio 30/28 in Dinitrogen from Air Samples and its Application to the Investigation of N Losses from Soil by Denitrification

Abstract

In the 1950s Hauck introduced a special version of the ¹⁵N dilution technique (¹⁵N flux method) for the determination of N losses from the soil by denitrification. Although this method is very useful and reliable its application has been rather infrequent up to now. This is mainly due to the need to measure the m/z 30 in addition to the usually measured m/z 28 and 29 for dinitrogen, because the ¹⁵N in the enriched air sample taken from an enclosure (cover box) at the soil surface is nonrandom. The signal from the m/z 30 is very low and difficult to measure with sufficient precision because other species (e.g. NO) also having the m/z 30 often interfere with its measurement. In this study the accuracy and precision of an easy to use CF-IRMS with sample batch operation to measure the ratio 30/28 was investigated. The relative standard deviation (RSD = precision) from natural abundance up to 2 at.% was always <1%. After correction of the mass ratio 30/28 (R30), by means of a formula obtained by linear regression of theoretical R30 against measured R30, the accuracy of the abundance calculated from this corrected R30 was very high. From the achieved precision and assuming a cover box height of 10 cm (headspace volume of 7 1), and a collection time of 2 h, a limit of detection for N₂ losses by denitrification equivalent to 16 g N/ha*d or 6 kg N/ha*a can be estimated. The performance of the ¹⁵N dilution method using the equipment and procedure described is demonstrated by means of results from an incubation experiment with [¹⁵N]nitrate-amended soils.     

Deposition of 15N into Soil Layers of Different Proximity to Roots by Wheat Plants

Abstract

The translocation of root borne N compounds to different distances from the roots was studied by use of rectangular pots with three separated soil zones. Wheat plants were grown for 28 days (4 leaf stage) and subsequently pulse labelled by exposure to 15 ppm ¹⁵NH₃ (generated from (¹⁵NH₄)₂SO₄ with 95 at.-% ¹⁵N exc.) every other day with the rooting medium sealed from the atmosphere. Six pulses were applied in total.

The plants assimilated 65% of the label offered. The final ¹⁵N enrichment in the shoots was approx. 13 at.-% exc. and in the roots approx. 5 at.-% exc. These abundances were high enough to detect traces of ¹⁵N in soil approximately 1 cm distant from the roots. Most of the ¹⁵N recovered was retained in the shoots (about 90%), 5% were present in the roots and another 5% had been released into the rhizosphere. Considering the ¹⁵N released, 62% were found in the central root zone, 26% in the adjacent layer and 12% in the outer zone.     

Equilibrating of 15N-Gases by Electrodeless Discharge: A Method of Indirect Mass Spectrometric Analysis of 30N2 for Denitrification Studies in Soils

Abstract

The estimation of denitrification in soil by the ¹⁵N tracer technique includes isotope analysis of gas samples with a nonrandom distribution of the N₂ mole masses of 28, 29 and 30. In that case the emission of total ¹⁵N is underestimated by calculating ¹⁵N atom fractions from the ²⁹N₂/²⁸N₂ ratio if ³⁰N₂ is not considered. ³⁰N₂ can be measured indirectly in N₂ enriched with ¹⁵N with nonrandom distribution of mole masses by mass spectrometric analysis. The nitrogen fraction of gas samples was transferred to discharge tubes. Microwaves (60 sec) generated an electrodeless discharge of the gas which caused a temporary split-up of N₂ molecules and thus established an equilibrium distribution of the mole masses. The ²⁹N₂/²⁸N₂ ratio was measured in equilibrated and in untreated samples to calculate the real emission of ¹⁵N. The measurements of ¹⁵N standard gases by this method satisfactorily coincided with calculated values for ¹⁵N atom fraction above a concentration of 50 δ‰.