Stable isotopic analyses of soil-emitted N

Here this approach was tested based on laboratory soil incubations with two
different soil types, applying two reference methods for quantification of

N

To overcome the problems with N

N

However, some open questions still remain: (i) are the isotopic fractionation
factors for denitrification processes determined in laboratory experiments
transferable to field conditions? (ii) How robustly can the N

The list with explanations of all abbreviations and specific terms used in the manuscript can be found in the Supplement (Table S1).

Two soil types were used: a mineral arable soil with silt loam texture
classified as a

The incubation vessels were cooled to 2

The data from two selected samplings of this experiment have already been published, with particular emphasis on the O isotopic fractionation (experiment 2.3–2.6 in Lewicka-Szczebak et al., 2016).

The same soils (Min soil and Org soil) as in Exp 1 were used for parallel
incubations under either an anoxic (N

The soils were air dried and sieved at 4 mm mesh size. Afterwards, the soil
was rewetted to obtain a WFPS of 70 % and fertilized with 80 mg N (added
as NO^{®} (Labco
Limited, Ceredigion, UK) and for NA treatment additional samples were collected in one
120 cm

In Exp 1, online trace gas concentration analysis of N

In Exp 2 the samples for gas concentration analyses were collected in Labco
Exetainer^{®} (Labco Limited, Ceredigion, UK)
vials and were analysed using an Agilent 7890A gas chromatograph (Agilent
Technologies, Santa Clara, CA, USA) equipped with an ECD detector. Precision,
as given by the standard deviation (1

Soil water content was determined by weight loss after 24 h drying in
110

Gas samples were analysed using an isotope ratio mass spectrometer (Delta V,
Thermo Fisher Scientific, Bremen, Germany) coupled to an automatic
preparation system (Precon

All isotopic values are expressed as ‰ deviation from the

Soil water was extracted with the method described by Königer et
al. (2011) and

The gas samples from the

For each of the analysed gas species (N

Based on the determined

Knowing

The mineral N concentrations and

formation of natural abundance NO

formation of

mineralization (

nitrate immobilization (

The N

From the statistically significant logarithmic fits between

For

The interpretations and calculations based on

From the calculated

The precision of the quantification of the N

The calibration of the isotopic fractionation approach was performed by
applying

The validation of the isotopic fractionation approach was performed by
applying

Val1 used

Val2 used

Val3 used the same

Until now, isotopomer “maps”, i.e. plots of

Scheme of the mapping approach to simultaneously estimate
the magnitude of N

The

the N

the N

The detailed results presented as time series are shown in Fig. S1 in the Supplement. In general, the switch from oxic to anoxic conditions resulted in an
increase of gaseous N losses. For both treatments of the Min soil (70 and
80 % WFPS), we observed a gradual decrease in

The detailed results presented as time series are shown in Fig. S2 in the Supplement. For the
anoxic treatments we observe a gradual decrease in N

Under oxic conditions, we observe much higher standard deviations for both
N

The detailed results presented as time series are shown in Fig. S3 in the Supplement. The
determined

From all

In Table 1, calculated rates of N transformations are shown. Initial and
final concentrations for nitrate and ammonium were measured, total gaseous
N-loss ([N

Contribution of

Rates of N transformation processes as calculated from

b.d. –

Based on the non-random distribution of N

Contribution of hybrid N

For Min soil we obtained very consistent correlations between

Examples of the relation between

Calculated

Fractionation factors of N

n/a – not applicable – no statistically significant correlation.

These relations look very different for Org soil. Firstly, there is no
significant correlation between

Theoretical

It could also be assumed that

For Org soil, the time course of

From the correlation tested above (Table 2) we found that only for Min soil can

Calibration of the N

For Val1, i.e. using the

For Val2, i.e. using

Validation of the N

The calculated contribution of N

For Val3, i.e. using a common value of

Summarizing the results of these three validation scenarios, we can conclude
that actual

As qualitative indicators of mixing and fractionation processes, we analysed
relations between pairs of isotopic signatures to determine the slopes for
the measured

With the mapping approach we used dual isotope values, i.e.

Relations between isotopic signatures of emitted N

n/a – not applicable – no statistically significant correlation.

In this study quite a high contribution of non-labelled N

A higher contribution of non-labelled N

To our best knowledge, this is one of the very few studies that document a
significant hybrid N

A precondition for the proper quantification of various process rates based
on the

Results from a laboratory incubation experiment to distinguish
between N

With respect to robust estimation of N

Another question is whether the assumption of isotopic fractionation pattern
of closed systems holds. Logarithmic fits provided best correlations with the
measured data, whereas linear correlations that would be indicative for open
system dynamics (Decock and Six, 2013) yielded worse fits (data not shown).
This indicates that the N

To what extent are the observed

For Org soil, much higher absolute values of

The Org soil data thus demonstrate that a high and variable in-time
contribution of fungal denitrification complicates the application of the
N

The successful calibration shows that

The results of the calibration were very similar if we treated the oxic and
anoxic conditions separately and if we used a mean

The results of validation show very different agreement between measured and
calculated

Whereas finding the true

The emitted N

In theory, the slopes for calculated

The

Interestingly, there is no correlation between isotopic values in oxic Exp 2
for Min soil. A single process or the combination of several processes, which
cause large variations in

The graphical interpretations including

For the relation of

Although the interpretation of the relations between particular isotopic
signatures is not completely clear yet, it seems to have potential to
differentiate between mixing and fractionation processes. Note that by using
the literature ranges of isotopic end-member values, they must be
recalculated according to respective substrate isotopic signatures for the
particular study; hence

The mapping approach had been used before based on

Importantly, for Org soil where

We have shown that the N

The attainable precision of the method, determined as mean absolute
difference between the measured and the calculated N

However, for soils of more complex N dynamics, as shown for the Org soil in
this study, the determination of N

The data used in this paper can be found in the Supplement.

The authors declare that they have no conflict of interest.

This study was supported by German Research Foundation (DFG: We/1904-4, LE
3367/1-1). Many thanks are due to Martina Heuer for help in N