gas-flux method to determine N2 emission and N2O pathways: a comparison of different tracer addition approaches

15 N gas flux method allows for quantification of N2 flux and tracing soil N transformations. An important requirement for this method is a homogeneous distribution of the 15 N tracer added to soil. This is usually achieved by soil homogenization and admixture of the 15 N tracer solution or multipoint injection of tracer solution to intact soil. Both 10 methods may create artefacts. We aimed at comparing the results of the gas flux method using both tracer distribution approaches. Intact soil cores with injected 15 N tracer solution show wider range of the results obtained. Homogenized soil shows better agreement between repetitions, but significant differences in 15 N enrichment measured in soil nitrate and in emitted gases were also observed. For intact soil the wider variability of measured values rather results from natural diversity of non15 homogenized soil cores than from inhomogeneous label distribution. Generally, comparison of the results of intact cores and homogenized soil did not reveal statistically significant differences in N2 flux determination. In both cases, pronounced dominance of N2 flux over N2O flux was noted. It can be concluded that both methods showed close agreement and homogenized soil is not necessarily characterized by more homogenous 15 N label distribution. 20 https://doi.org/10.5194/soil-2019-64 Preprint. Discussion started: 5 November 2019 c © Author(s) 2019. CC BY 4.0 License.


Introduction
Determination of soil nitrogen transformation pathways and quantification of gaseous N emissions often requires soil incubation experiments including significant manipulations of natural soil conditions. Especially, the quantification of soil N 2 flux in field studies is very challenging due to high atmospheric background. The most common method for both detailed tracing of soil N transformations and determination of N 2 emission is the application of 15 N tracer (Aulakh et al., 1991;Baily 25 et al., 2012;Bergsma et al., 2001;Buchen et al., 2016;Deppe et al., 2017;Kulkarni et al., 2013;Morse and Bernhardt, 2013;M . 2 14; M . 2 4; W . 2 19). However, this implies a significant impact for the soil due to additional fertilization and soil disturbance depending on the way of tracer addition. For the tracer addition several different strategies may be applied. The two most common techniques are: soil homogenization where the tracer solution is mixed with the soil, or usage of intact soil cores where tracer solution is added through multiple needle injections. Both methods 30 lead to potential bias. Following soil homogenization, the soil structure is changed through sieving and mixing, roots and stones are removed, but this results in the best achievable homogeneity of soil properties and tracer distribution within the soil column and thus better comparability between the repetitions. For needle injections, the soil structure stays unchanged but the pointwise injection may not ensure the homogenous distribution of the tracer which is crucial for the proper application of 15 N gas flux method. Moreover, incomplete equilibration of water content after injecting aqueous tracer 35 solution could lead to increased wetness near the injection spots and thus to enhanced denitrification.
Here we aimed at comparing the results of these different strategies and test how far the determined 15 N pool derived N 2 and N 2 O fluxes are altered due to a particular soil treatment.

Experimental set-up 40
Three treatments were applied: (1) soil was sieved with 4mm mesh size, the tracer solution was added evenly, soil was homogenized and packed into the incubation column (treatment H+M: homogenized + mixed) ; (2) intact soil cores were directly collected into the incubation columns and the tracer solution was added through the injection needles into 12 homogeneously distributed injection points in 6 depths (in total 72 injection points per column) (treatment I+I: intact + injected); (3) soil was sieved with 4mm mesh size (like in treatment H+M), packed into the incubation column, and the tracer 45 solution was added through the injection needles (like in treatment I+I) (treatment H+I: homogenized + injected). For each treatment the soil columns were 0.3 m high with diameter of 0.15 m. Silt loam soil Albic Luvisol from arable cropland of Merklingsen experimental station (Germany) was used (silt content approx. 87%, 11% clay, 2% sand). The soil density of intact cores was 1.3 g ml -1 and the packed columns were compacted to the same density, which gave 6.89 kg soil per column.
For each soil column, 216 mL of 319 mgN L -1 NaNO 3 solution with 73 at% 15 N was added. This resulted in the following 50 initial experimental settings: 75% water-filled pores space (WFPS), 37 mg N kg -1 NO 3 -, 42.5 at% 15 N measured in the subsamples of the homogenized soil. The incubation lasted 8 days. The columns were continuously flushed with a gas mixture with reduced N 2 content to increase the measurements sensitivity (2% N 2 and 21% O 2 in He, (Lewicka-Szczebak et al., 2017)) with a flow of 10 mL min -1 . The gas samples were collected daily in the first 4 days and every second day in the last 4 days into two 12 mL septum-capped Ex ® (L L m Ceredigion, UK) connected to the vents of the 55 incubation columns.

Gas analyses
The gas samples were analysed with a modified GasBench II preparation system coupled to MAT 253 isotope ratio mass spectrometer (Thermo Scientific, Bremen, Germany) according to Lewicka-Szczebak et al. (2013). In this set-up, N 2 O is converted to N 2 prior to analysis, which allows simultaneous measurement of stable isotope ratios 29 R ( 29 N 2 / 28 N 2 ) and 30 R 60

Soil analyses 70
At the end of incubation soil samples were collected from each column using a Goettinger boring rod with diameter of 18 mm (N m H Qu k k m ). Three cores were taken from each column, separated in top (0 to 15 cm) and bottom (15 to 30 cm) layer. For injected treatments ((H+I) and (M+I)) these sample cores were taken between injection point and additional cores were collected from injection points . All soil samples were homogenised and analysed for water ( w 24 11 º ) ion (by extraction in 2M KCl 1:4) and 15N enrichment 75 in nitrate (by bacterial denitrification method (Sigman et al., 2001)).

Statistics
For testing the statistical significance of the differences between treatments ANOVA and Tukey HSD Post-hoc test were applied using R 3.4.2 (R Core Team, 2013).

Gas fluxes and denitrification product ratio
In order to compare the treatments, the time course of the results has to be taken into account because the gas production differed largely between the sampling dates ( Fig.1). Therefore, we checked for statistically significant differences between 85 the treatments individually for each sampling date. The results show well comparable trends and no statistically significant differences between treatments (Fig.1). Notably, r N2O shows very good agreement at the beginning of the experiment, when the large gas concentrations were measured, and start to differentiate when the fluxes drop from the 3 rd day (Fig. 1D), but these differences are not statistically significant. However, if the experiment is evaluated for the cumulative values, significant differences between treatments appear ( Table 1). The cumulated gas fluxes of N 2 O and N 2 are significantly 90 different between the treatments I+I and H+I, whereas the H+M treatment does not differ significantly from the others.
However, comparison of the entire denitrification gas flux (joint N 2 + N 2 O flux) reveal no statistically significant difference between treatments (Table 1). Product ratios are compared as cumulated r N2O (calculated with the cumulated fluxes) and mean r N2O (average value of all sampling points). Cumulated r N2O shows identical pattern of significant differences as the cumulated N 2 and N 2 O fluxes. For mean r N2O values H+M and H+I treatment are significantly different, whereas the I+I 95 treatment does not differ significantly from the others.
There results show that the different tracer application strategies tested had no impact on the total denitrification (N 2 + N 2 O), but the product ratio may be slightly shifted, which results in differences by comparing separately N 2 or N 2 O flux. This presumably results from the differences in distribution of moisture and nitrate between treatments (see Sect. 3.2). Anyway, all determined r N2O values, although partially different, indicate pronounced dominance of N 2 over N 2 O emission. 100 Importantly, no significant differences were noted between the H+M and I+I treatment, only H+I treatment shows higher N 2 O flux, lower N 2 flux and higher r N2O . In this treatment we may deal with joint artefacts associated with soil homogenization and needle injection technique.
The homogenized treatments show better comparability between the repetitionsthey show lower standard deviations for gas emissions and for r N2O (Table 1) and also smaller error bars for the daily measurements (Fig.1). The H+I treatment shows 105 the lowest standard deviations for the cumulative gas emission measurements (Table 1). This indicates that the observed heterogeneity for I+I treatment is not due to needle injection procedure but rather due to intact structure of soil cores, which naturally represent the typical soil heterogeneity.

Soil parameters
A good insight into columns heterogeneity is also provided by the soil analyses performed at the end of experiment (Table  110 2). Clearly, I+I treatment shows the largest standard deviations between repetitions. Also the most pronounced differences between top and bottom soil layer can be noted for this treatment, but only soil moisture is significantly lower for the bottom layer. Since this is not the case for H+I treatment it indicates the natural heterogeneity of intact cores rather than a result of label injection procedure. The values from injection points are never significantly different from samples between injection points (within one treatment) which indicates a good distribution of the tracer solution. 115 Very significant differences between treatments were observed. I+I treatment shows significantly lower nitrate concentration compared to homogenized treatments. This must be due to initial soil nitrate concentration. The soil was stored for two weeks before experiment. Storing of mixed soil or sieving and homogenization procedures probably intensified N mineralization and formation of additional nitrate. Moreover, H+M treatment shows significantly higher 15 N enrichment of NO 3 -(a 15 N NO3 ) than injected treatments. This may be due to injection procedure where the needles might get partially 120 clogged with soil and then addition of tracer solution was lower than planned. The assumption that the injected volume was lower than the target and thus also lower than the addition of tracer solution to H+M treatment, can also be supported by the slightly lower soil moisture and nitrate concentration of the injected treatments.

15 N abundance in soil active pools
Despite the pronounced difference in 15 N content between treatments, the results can be still compared because the 15 N 125 abundance of actively denitrifying pool (a P value) for each sample is individually calculated based on the distribution of N 2 and/or N 2 O isotopologues. We checked how well these calculated a P values for N 2 and N 2 O correspond with the respective 15 N enrichment measured in soil nitrate (a NO3 ) and between each other (Table 3). This comparison gives additional information about the distribution of the 15 N label. We calculated the cumulative relative difference (Table 3: cum diff, calculated as a sum of differences in 15 N enrichment of different pools for all 24 samples) which represents the overall 130 deviation between the analyzed pools. Very high difference was noted between a P values of both gases and a NO3 in H+M treatment. This is mostly due to the first two sampling days, where a P values were significantly lower than a NO3 (mean difference of ca. 15 at% 15 N, Fig.2), whereas for the next samplings they corresponded very well (mean difference of ca. 1 at% 15 N, Fig.2). This shows that initially the gases were produced in soil microsites depleted in 15 N compared to the mean soil value. This is the case for all three treatments, however the largest difference is observed for H+M treatment due to 135 highest a NO3 values. The absolute mean difference (Table 3: mean abs diff, calculated as a mean of modulus of differences in 15 N enrichment of different pools) represent the average variation range of the compared values. Here it is clear that for the comparison between a P_N2 and a P_N2O we obtained quite a good agreement, much better than for comparisons with a NO3 (Table 3). This shows that both gases originate mostly from the same soil pool. Importantly, even in H+M treatment where large difference between a NO3 and a P values was noted, the difference between a P_N2 and a P_N2O is very low. The fact that 140 a P_N2O shows much closer agreement with a P_N2 than a NO3 suggests that, in case of missing data on a P_N2 , which is often the case due to high N 2 detection limit of the gas-flux method, rather the a P_N2O should be used than a NO3 or a theoretical value on 15 N abundance, as it has been also proposed in previous studies (Bergsma et al., 2001;Stevens and Laughlin, 2001).
Interestingly, for I+I treatment lower differences between a NO3 and a P_N2O or a P_N2 values were obtained, but larger difference between a P_N2 and a P_N2O when compared to homogenized treatments (Table 3, Fig. 2). This shows that the multiple injection 145 technique reduced the formation of isolated soil microsites of distinct 15 N enrichment than the a NO3 value measured for total https://doi.org/10.5194/soil-2019-64 Preprint. Discussion started: 5 November 2019 c Author(s) 2019. CC BY 4.0 License.
soil. However, the slightly higher difference between a P values for N 2 and N 2 O suggests not identical origin for both gases, i.e., probable slight admixture of hybrid N 2 (Spott et al., 2011) since the 15 N enrichment of N 2 shows lower values than N 2 O. This could explain the higher cumulated N 2 flux for I+I treatment (Table 1).

Homogenity of 15 N tracer distribution and accuracy of results 150
Surprisingly, the inconsistency in 15 N abundance in total and actively denitrifying nitrate soil pools (Fig. 2) indicates largest inhomogeneity at the beginning of the incubation for the homogenized soil, which is then equilibrated after 2 days of incubation. This resulted most probably from the imperfect mixing of the relatively wet (gravimetric water content of 29.3%) silt loam soil and could be due to delayed equilibration of added 15 N solution into the centre of soil aggregates where denitrification rates are probably highest (Sextone et al., 1985). But, importantly, these first two days are also the ones with 155 the highest gas production and close agreement of results between all three treatments (see Fig. 1). This suggests that practically even non-homogeneous distribution of 15 N label and thus heterogeneity in concentration and 15 N enrichment of nitrate in soil does not lead to severe bias in determining denitrification and its product ratio.
This study allows only for comparison of these different treatments but not for checking with the true emission values, since we have not used any independent method for fluxes determination. However, what can be observed here is the fact that 160 pronounced differences were observed for a 15 N values of different treatments and different pools, but the calculated results for gas fluxes and product ratios were mostly not significantly different between the treatments. This supports the assumption that in real soil situation even the imperfect label distribution allows for obtaining accurate results (Arah, 1997;Deppe et al., 2017). But, importantly, this is possible only if we measure and use a P values representing the 15 N values of the pools actively producing N 2 and N 2 O. The fluxes would be significantly underestimated if the a NO3 value was applied for 165 calculations, e.g., for the first sampling point this would result in about 20% underestimation of the N 2 flux when the measured final a NO3 value was applied, and about 30% underestimation when the initial a NO3 value was applied. Significant differences in 15 N enrichment of total and active nitrate pool has been also found in our previous laboratory and field studies (Buchen et al., 2016;Deppe et al., 2017). It was shown that in such cases the 15 N enrichment of N pool undergoing denitrification is well represented by a P values, but not by a NO3 values. 170 The homogeneity of 15 N label distribution depends not only on the fertilizer application technique but even more on the soil type, water content, initial nitrate and ammonium content. In our previous laboratory experiments quite a good agreement between a NO3 values and a P values was achieved indicating a homogenous denitrifying pool (Lewicka-Szczebak et al., 2017).
In that study similar soil texture was used (silt loam), but initial amount of nitrate and ammonium was very low, and soil samples were prepared at soil moisture of 70% WFPS and rest water was added on top, and soil was incubated in high 175 moisture conditions. But notably, the anoxic conditions showed perfect agreement in a NO3 and a P values whereas for oxic conditions slight differences have also been noted (Lewicka-Szczebak et al., 2017). Oxic conditions can be expected to yield greater disagreement between a NO3 and a P due to absence of nitrification in anoxic microsites and thus less dilution of the 15 N label by soil-derived N sources (Deppe et al., 2017). In the H+M treatment of the actual experiment, inhomogeneity was probably the result of soil homogenization by too high soil moisture (75% WFPS) due to formation of larger aggregates. But 180 this problem can be overcome if the 15 N label is incorporated at low soil moisture and target moisture is established by adding water afterwards (Lewicka-Szczebak et al., 2017, Well et al., 2019.

Conclusions
Soil homogenisation reduced the variability within soil column and between repetitions but not necessarily improved the 15 N label distribution. Wet homogenisation has lead to uneven label and process distribution. Multiple needle injection of 15 N 185 solution resulted in better agreement between 15 N enrichment of soil and emitted gases, indicating even more homogeneous 15 N label distribution than homogenised treatments.
Larger heterogeneity of intact soil cores, noted as larger deviations of all measured values, reflects the natural soil conditions rather than inhomogeneous 15 N label distribution. Importantly, the results obtained with homogenised soil and with intact soil cores do not differ significantly in the determined N 2 flux and denitrification product ratio. Hence, when applying each 190 of these treatments very similar general conclusions will be driven, i.e., the dominance of the N 2 flux over the N 2 O flux. This is thanks to calculation method applying a P values determined individually for each sample which assures the adequate results for flux calculation, even by existence of multiple N pools. It was found that a NO3 values can pronouncedly differ from the a P value of produced gases and its application for N 2 flux determination may result in large bias.

195
Data availability. Original data are available upon request. Material necessary for this study findings is presented in the paper.
Author contribution. DLS and RW designed the experiment and DLS carried it out. Both authors interpreted the results.
DLS prepared the manuscript with significant contribution of RW. 200 Competing interests. The authors declare that they have no conflict of interest.
Acknowledgements. This study was financed by German Research Foundation (DFG: LE 3367/1-1). Many thanks are due to   Table 1: Comparison of cumulated fluxes, cumulated product ratio (cum r N2O ) and mean product ratios (mean r N2O ) in three treatments: homogenized and mixed (H+M), intact and injected (I+I), homogenized and injected (H+I). Statistically significant differences are indicated (*p<0.05, **p<0.01, ***p<0.001). Table 3: Differences between the measured 15 N abundance in soil nitrate (a NO3 ) and determined 15 N abundance of 15 N-pool derived N 2 (a P_N2 ) and N 2 O (a P_N2O ) expressed as the cumulative relative difference for all samples (n=24) (cum diff = ), mean absolute difference (mean abs diff = ). In the above equations a 1 and a 2 represent the 15 N enrichment of two compared pools (a NO3 or a P_N2 or a P_N2O ).