Natural Nitrogen Isotope Ratios as a Potential Indicator of N 2 O Production Pathways in a Floodplain Fen

: Nitrous oxide (N 2 O), a major greenhouse gas and ozone depleter, is emitted from drained organic soils typically developed in ﬂoodplains. We investigated the e ﬀ ect of the water table depth and soil oxygen (O 2 ) content on N 2 O ﬂuxes and their nitrogen isotope composition in a drained ﬂoodplain fen in Estonia. Measurements were done at natural water table depth, and we created a temporary anoxic environment by experimental ﬂooding. From the suboxic peat (0.5–6 mg O 2 / L) N 2 O emissions peaked at 6 mg O 2 / L and afterwards decreased with decreasing O 2 . From the anoxic and oxic peat (0 and > 6 mg O 2 / L, respectively) N 2 O emissions were low. Under anoxic conditions the δ 15 N / δ 14 N ratio of the top 10 cm peat layer was low, gradually decreasing to 30 cm. In the suboxic peat, δ 15 N / δ 14 N ratios increased with depth. In samples of peat ﬂuctuating between suboxic and anoxic, the elevated 15 N / 14 N ratios ( δ 15 N = 7–9% (cid:24) ambient N 2 ) indicated intensive microbial processing of nitrogen. Low values of site preference (SP; di ﬀ erence between the central and peripheral 15 N atoms) and δ 18 O-N 2 O in the captured gas samples indicate nitriﬁer denitriﬁcation in the ﬂoodplain fen.


Introduction
Floodplains and other riparian ecosystems are important buffers controlling water quality and providing several other ecosystem services [1]. However, beside their water purification and habitat provision roles, riparian zones can be significant sources of greenhouse gas (GHG) emission [2]. Periodically flooded floodplains are a significant source of nitrous oxide (N 2 O), a powerful GHG and a major ozone-depleting gas [3][4][5]. N 2 O is produced in soil via nitrification under aerobic conditions, where ammonia is oxidized, and by denitrification, which occurs under anaerobic conditions, where nitrate is sequentially reduced to nitrite, NO, N 2 O, and pure molecular nitrogen (N 2 ) [6]. The gaseous nitrogen losses directly depend on soil moisture, which affects oxygen availability in the soil. Hence, understanding the relationship between soil moisture, oxygen content, and N 2 O emissions is highly important to understand N 2 O production and consumption mechanisms in floodplains.
The effect of draining or flooding on nitrous oxide emissions has been studied in several studies. N 2 O emissions follow a bell-shaped distribution with the peak at intermediate soil moisture [7]. Experiments show that variation in soil oxygen content induces high N 2 O emissions [8]. Lower soil nitrate levels have been observed during the flooding periods, whereas a peak in N 2 O emissions followed by a sudden drop has been observed as an after-effect of flooding [9]. These short-lived peaks, which were recorded when the water table was below soil surface, are also found to be a major source Water 2020, 12, 409 2 of 12 of global N 2 O emissions. When the soil was continuously and completely submerged, N 2 O emissions dropped significantly [6,7,10,11].
For understanding the N 2 O production, analysis of isotopic signatures has been developed in the past decades [12]. The N 2 O molecule has an asymmetric structure (N-N-O) and the two N atoms are distinct referred to as beta (β) and alpha (α)-N β N α O [12]. It has also been shown that there is a preference for enrichment of 15 N at the central (α) position of the N 2 O molecule in atmosphere [13]. The ground state zero-point vibrational energy of 14 N 15 N 16 O is less than 15 N 14 N 16 O, which is why formation of the former is favoured over the latter under equilibrium conditions [14]. Also, the 14 N- 16 O is reduced more easily as compared to 15 N- 16 O, due to its low bond strength [15]. The enrichment of 15 N at the central (α position) of the N 2 O molecule has been observed during nitrification and denitrification [16,17]. Moreover, studies conducted with pure cultures of microbes producing N 2 O have observed high site preferences (expressed as SP = δ 15 N αδ 15 N β ) for nitrification and low site preferences of~0% for bacterial denitrification respectively [18][19][20][21][22]. High site preference has also been observed in fertilized arable soils indicating autotrophic nitrification [23]. Contrary to these results, there have also have been studies that have shown non-uniform site preference during denitrification conditions, which creates a dilemma on answering the question of whether site preference can be used as a tool to differentiate between nitrification and denitrification [24][25][26][27][28].
The majority of 15 N analyses related to N 2 O fluxes have been made in mineral soils, and only a limited number of studies consider peat soils. For instance, Rückauf et al. (2004) [29] performed 15 N tracer experiments in drained and reflooded microcosms filled with fen peat and found that denitrification was the main N transformation process, whereas N 2 O emission from reflooded (anoxic) conditions was significantly lower than that from drained microcosms. Similar results were gained in field studies by Tauchnitz et al. (2015) [30] with 15 N tracer studies on nitrogen gases released from a transition bog and found high N 2 O and low N 2 from drained conditions and the reversed situation in rewetted cases. Likewise, Yang et al. (2011) [31] found significantly higher emission from drained soils with oxic conditions, using field-based 15 N-N 2 O pool dilution technique to measure gross N 2 O production in soil. However, the natural isotope composition of N 2 O to differentiate between nitrification and denitrification source processes has not been studied before in floodplain peats.
The objective of this study is to analyze the impact of water table depth and oxygen content on N 2 O production pathways in a drained nitrogen-rich floodplain fen using experimental flooding and the natural isotope composition of N 2 O.

Site Description
We collected gas samples from three positions in a drained fen in the floodplain of the Emajõgi River, Estonia (58 • Figure 1). We collected the gas samples from chambers to glass vials of 50 ml for gas analysis and 100ml for gas-isotope analysis. The chambers were organized in equilateral triangles. The side of the equilateral triangle was 1.6 meters. White 65-litre chambers were used on top of the collars to trap the gas emitted from the soil. Observation wells were placed to read the water table. The three positions were labelled as A, B, and C depending on their distance from the river. Position C was closest to the river and A was farthest away with B at the centre. During one-hour sessions, samples were collected after every 20 min and ten such sessions were conducted for this study. Position C was flooded with ditch water for 2 h before collecting samples using a garden pump to achieve anoxic conditions. Oxygen sensors (Fibox 4 by PreSens, Regensburg, Germany) were placed at depths of 5 cm, 25 cm, and 50 cm for a vertical oxygen profile at all three sampling positions. Soil temperature was monitored at all positions in each session at depths of 10 cm, 20 cm, 30 cm, and 40 cm. Soil samples were collected from at different depths. Water 2020, 12, x FOR PEER REVIEW 3 of 12

Soil and Gas Isotope Analysis
Soil samples were collected at various depths. For bulk nitrogen isotope analysis, samples were dried to remove moisture and 1 milligram of each was packed into a tin capsule. Soil samples were then analyzed using a Delta V Plus mass spectrometer coupled with a Flash HT element analyser (Thermo Scientific, Bremen, Germany) in the Isotope Ratio Mass Spectrometer laboratory at University of Tartu. Nitrogen isotopes were calibrated against IAEA-N1 and IAEA-N2 international standards. Analytical precision was better than ±0.2‰. Soil chemical analysis was done at the Estonian University of Life Sciences. 100 mL of NH4 + -acetate solution was paired with titanium-yellow reagent. A flow injection analyser was used to determine plant-available magnesium (Mg 2+ ). To analyse available calcium (Ca 2+ ), the flame photometrical method was used with the same solution. Soil pH was calculated on a 1N KCl solution and flow-injection analysis was used on a 2M KCl extract of soil to determine soil NH4-N and NO3-N. Oven-dried samples were used to determine total nitrogen via dry combustion method on a varioMAX CNS elemental analyzer (manufacturer: elementar, Langenselbold, Germany). Organic matter of oven-dry soil was calculated by loss on ignition at 360 °C. Soil bulk density was calculated considering that peat consists of organic matter, mineral matter, and water. Individual bulk densities of 0.23, 2.65, and 1 g/cm 3 for mineral matter, organic matter, and water, respectively, were used to calculate the bulk density of each peat sample [32,33].
Gas-phase N2O concentrations were measured using a gas chromatograph equipped with an electron detector (GC-2014, Shimadzu, Kyoto, Japan). Soil gas N2O isotopomer ratios (bulk nitrogen δ 15 Ngas), and 15 N site preference (SP) were concentrated and purified on a modified PreCon [34] and GasBench II and analyzed on a Delta V mass spectrometer (Thermo Scientific: Waltham, MA, USA) ( Figure 2). We replaced the viton seals with rubber rings in PreCon and also bypassed the oven. We removed the sample loop in the GasBench II and used the same ports to connect PreCon, and used 100 ml sample bottles. For mass 31 measurements we used higher amplification (3.00 × 10 11 ) according to Potter et al. [35]. The isotope and site preference values were calculated according to Toyoda and Yoshida [12] and calibrated against standard reference gas.

Soil and Gas Isotope Analysis
Soil samples were collected at various depths. For bulk nitrogen isotope analysis, samples were dried to remove moisture and 1 milligram of each was packed into a tin capsule. Soil samples were then analyzed using a Delta V Plus mass spectrometer coupled with a Flash HT element analyser (Thermo Scientific, Bremen, Germany) in the Isotope Ratio Mass Spectrometer laboratory at University of Tartu. Nitrogen isotopes were calibrated against IAEA-N1 and IAEA-N2 international standards. Analytical precision was better than ±0.2% . Soil chemical analysis was done at the Estonian University of Life Sciences. 100 mL of NH 4 + -acetate solution was paired with titanium-yellow reagent. A flow injection analyser was used to determine plant-available magnesium (Mg 2+ ). To analyse available calcium (Ca 2+ ), the flame photometrical method was used with the same solution. Soil pH was calculated on a 1N KCl solution and flow-injection analysis was used on a 2M KCl extract of soil to determine soil NH 4 -N and NO 3 -N. Oven-dried samples were used to determine total nitrogen via dry combustion method on a varioMAX CNS elemental analyzer (manufacturer: elementar, Langenselbold, Germany). Organic matter of oven-dry soil was calculated by loss on ignition at 360 • C. Soil bulk density was calculated considering that peat consists of organic matter, mineral matter, and water. Individual bulk densities of 0.23, 2.65, and 1 g/cm 3 for mineral matter, organic matter, and water, respectively, were used to calculate the bulk density of each peat sample [32,33]. Gas-phase N 2 O concentrations were measured using a gas chromatograph equipped with an electron detector (GC-2014, Shimadzu, Kyoto, Japan). Soil gas N 2 O isotopomer ratios (bulk nitrogen δ 15 N gas ), and 15 N site preference (SP) were concentrated and purified on a modified PreCon [34] and GasBench II and analyzed on a Delta V mass spectrometer (Thermo Scientific: Waltham, MA, USA) ( Figure 2). We replaced the viton seals with rubber rings in PreCon and also bypassed the oven. We removed the sample loop in the GasBench II and used the same ports to connect PreCon, and used 100 ml sample bottles. For mass 31 measurements we used higher amplification (3.00 × 10 11 ) according to Potter et al. [35]. The isotope and site preference values were calculated according to Toyoda and Yoshida [12] and calibrated against standard reference gas.
Isotopomer ratios were noted as δ values defined as:  Isotopomer ratios were noted as δ values defined as: The 15 R α and 15 R β are the 15 N/ 14 N ratios at central (α) and terminal (β) nitrogen position in the linear N2O molecule, respectively. 15 Rbulk denotes the average value of 15 N/ 14 N ratios. Standard is an international standard of atmospheric N2 for N. The 15 N site preference was calculated from isotopomer ratios as SP = δ 15 N α − δ 15 N β . The δ values and SP value are expressed in per mil (‰). The standard deviations of the measurements were 0.3‰ for δ 15 Nbulk and 0.9‰ for δ 15 N α .
Conversion of measured ratios into isotopomers ratios was done using the following equations [12] 45 R = 15

N2O Emissions Varying with Soil Chemistry
Soil properties of all the three positions at different depths are reported in Table 1. Our results for the C/N ratio show similarly low values among the three positions, ranging from 9% to 11%. Hence at our experimental site the variability of N2O emissions must depend significantly on other factors such as water table depth and oxygen concentration, and not only on C/N ratio. Klemedtsson et al. (2005) [36] have shown that at C/N ratios > 20, N2O emissions are low irrespective of other physical factors such as pH, water table, and soil oxygen content but when C/N ratios are low they affect N2O emissions. Our results showed a negative trend N2O emissions and bulk density. Leifeld (2018) [37] found similar results. Liu et al. (2019) [38] found a positive correlation between bulk density and N2O emissions, which is in contradiction with our results. The concentrations of ammonium and nitrate increased from position A to C, without a trend with N2O emissions. This indicated that inorganic nitrogen was not a driving factor responsible for the N2O emissions. Organic matter content increased from position A to B and then decreased from position B to C, but due to similar C/N ratios, we expect it would not affect the N2O emissions. No strong trends were Conversion of measured ratios into isotopomers ratios was done using the following equations [12] Here 45 R and 46 R represent the isotopomers of N 2 O that contribute to mass 45 and 46, 17 R represents the heavy oxygen and 31 R and 32 R correspond to isotopomers of NO.

N 2 O Emissions Varying with Soil Chemistry
Soil properties of all the three positions at different depths are reported in Table 1. Our results for the C/N ratio show similarly low values among the three positions, ranging from 9% to 11%. Hence at our experimental site the variability of N 2 O emissions must depend significantly on other factors such as water table depth and oxygen concentration, and not only on C/N ratio. Klemedtsson et al. (2005) [36] have shown that at C/N ratios > 20, N 2 O emissions are low irrespective of other physical factors such as pH, water table, and soil oxygen content but when C/N ratios are low they affect N 2 O emissions. Our results showed a negative trend N 2 O emissions and bulk density. Leifeld (2018) [37] found similar results. Liu et al. (2019) [38] found a positive correlation between bulk density and N 2 O emissions, which is in contradiction with our results. The concentrations of ammonium and nitrate increased from position A to C, without a trend with N 2 O emissions. This indicated that inorganic nitrogen was not a driving factor responsible for the N 2 O emissions. Organic matter content increased from position A to B and then decreased from position B to C, but due to similar C/N ratios, we expect it would not affect the N 2 O emissions. No strong trends were observed between site preference and soil chemistry factors such as bulk density and C/N ratio. Also, according to the World Reference Base for Soil Resources (2007), soils containing 12% or more carbon are classified as peat. In our experimental site, all three positions had more than 12% of carbon and hence could be classified as peat [39].

N 2 O Emissions Varying with Water Table
In response to changing water table depth, the N 2 O emissions showed two maxima at water levels between −40 and −50 cm, and when the soil was submerged (Figure 3). Dobbie et al. (2001 and [9,40] have also observed similar trends in their experiment where they have shown an increase in N 2 O emissions with increasing water-filled pore space (WFPS) [9,40,41]. Furthermore, Goldberg et al. (2009) [42] have observed that N 2 O in fen peat is mostly produced at depths between 0.3 and 0.5 m. Also, it has been shown that the emissions peak at one optimum point and are at the lowest in dry or saturated soil [43,44]. This is similar to the soil-moisture optimum observed worldwide [7]. We also observed temporal variation of emissions from each position. High spatial and temporal variability of fluxes seems to be typical for N 2 O and has been observed in both mineral and organic soils [7][8][9]27,30,37,38,40]. Characteristically large temporal variations of N 2 O emissions have also reported in a review paper by Henault et al. (2012) [45].
Water 2020, 12, x FOR PEER REVIEW 5 of 12 observed between site preference and soil chemistry factors such as bulk density and C/N ratio. Also, according to the World Reference Base for Soil Resources (2007), soils containing 12% or more carbon are classified as peat. In our experimental site, all three positions had more than 12% of carbon and hence could be classified as peat [39].

N2O Emissions Varying with Water Table
In response to changing water table depth, the N2O emissions showed two maxima at water levels between −40 and −50 cm, and when the soil was submerged (Figure 3). Dobbie et al. (2001 and [9,40] have also observed similar trends in their experiment where they have shown an increase in N2O emissions with increasing water-filled pore space (WFPS) [9,40,41]. Furthermore, Goldberg et al. (2009) [42] have observed that N2O in fen peat is mostly produced at depths between 0.3 and 0.5 m. Also, it has been shown that the emissions peak at one optimum point and are at the lowest in dry or saturated soil [43,44]. This is similar to the soil-moisture optimum observed worldwide [7]. We also observed temporal variation of emissions from each position. High spatial and temporal variability of fluxes seems to be typical for N2O and has been observed in both mineral and organic soils [7][8][9]27,30,37,38,40]. Characteristically large temporal variations of N2O emissions have also reported in a review paper by Henault et al. (2012) [45].

N 2 O Emissions Varying with Flooding Time.
The N 2 O emissions were observed to increase after the flooding over 80 minutes, after which a sudden drop was observed in N 2 O emissions and emissions remained low at longer flooding times ( Figure 4A). This also explains the high N 2 O emissions observed during the positive water table as when the flooding process would begin, it would take some time to achieve anoxic conditions and during this time some N 2 O bursts or peaks were observed. Hansen et al. (2014) [46] have observed a similar trend in agricultural soils where emissions increased after flooding until a maximum at a certain flooding time and after that decreased with a steep fall [46]. Similar trends have also been observed in rice paddies in Indonesia and China [47,48].
Water 2020, 12, x FOR PEER REVIEW 6 of 12

N2O Emissions Varying with Flooding Time.
The N2O emissions were observed to increase after the flooding over 80 minutes, after which a sudden drop was observed in N2O emissions and emissions remained low at longer flooding times ( Figure 4A). This also explains the high N2O emissions observed during the positive water table as when the flooding process would begin, it would take some time to achieve anoxic conditions and during this time some N2O bursts or peaks were observed. Hansen et al. (2014) [46] have observed a similar trend in agricultural soils where emissions increased after flooding until a maximum at a certain flooding time and after that decreased with a steep fall [46]. Similar trends have also been observed in rice paddies in Indonesia and China [47,48].

N2O Emissions Varying with Oxygen Content.
Under anoxic conditions (0 mg O2/L), most of the N2O emissions were low ( Figure 4B). This could indicate reduction of N2O to N2 due to a lack of oxygen. From the suboxic peat (0.5-6 mg O2/L) emissions were high and showed a peak at oxygen content of 6 mg/L. From the oxic peat (6-12 mg O2/L), N2O emissions were lower than from the suboxic peat but higher than from the anoxic peat. Vor et al. (2003) [40] and Rubol et al. (2012) [8] show similar trends in N2O emissions where suboxic or intermediate oxygen content results in the highest N2O emissions from the soil [10,49,50]. Furthermore, Zhua et al. (2013) [51] have also shown that as conditions approach an anoxic nature, heterotrophic denitrification is the major active process [51]. This coheres with our results and hypothesis and is further confirmed by the isotope results below.

Variation of Soil δ 15 Nbulk soil
The δ 15 N values of total soil nitrogen showed low δ 15 N (‰ air N2) values with little variation with depth at position B, possibly due to its suboxic nature ( Figure 5). However, in flooded position C there is a decreasing δ 15 N bulk soil trend with peat depth in the upper part of the section, which starts to increase again from the soil depth of 40 cm. This might be due to the flooding effect. In contrast, at position A, the δ 15 Nsoil values increased with soil depth to 30 cm, after which the δ 15 Nsoil decreased.

N 2 O Emissions Varying with Oxygen Content
Under anoxic conditions (0 mg O 2 /L), most of the N 2 O emissions were low ( Figure 4B). This could indicate reduction of N 2 O to N 2 due to a lack of oxygen. From the suboxic peat (0.5-6 mg O 2 /L) emissions were high and showed a peak at oxygen content of 6 mg/L. From the oxic peat (6-12 mg O 2 /L), N 2 O emissions were lower than from the suboxic peat but higher than from the anoxic peat.  [51] have also shown that as conditions approach an anoxic nature, heterotrophic denitrification is the major active process [51]. This coheres with our results and hypothesis and is further confirmed by the isotope results below.

Variation of Soil δ 15 N bulk soil
The δ 15 N values of total soil nitrogen showed low δ 15 N (% air N 2 ) values with little variation with depth at position B, possibly due to its suboxic nature ( Figure 5). However, in flooded position C there is a decreasing δ 15 N bulk soil trend with peat depth in the upper part of the section, which starts to increase again from the soil depth of 40 cm. This might be due to the flooding effect. In contrast, at position A, the δ 15 N soil values increased with soil depth to 30 cm, after which the δ 15 N soil decreased. The increasing trend of δ 15 N soil has also been observed by Brenner et al. (2001) [52] during their study in grasslands in California. They indicated soil inputs such as soil roots, as the cause for this trend [52]. However, as the flooding process was conducted for a time span of over a month, during which ten sessions were conducted in our experiment, the microbial activity due to fluctuating water table depth and oxygen content could have been responsible for the enrichment of heavy nitrogen isotope during this time. Increased δ 15 N values have also been observed in forest soil by Snider et al. (2009) [53].
Water 2020, 12, x FOR PEER REVIEW 7 of 12 The increasing trend of δ 15 Nsoil has also been observed by Brenner et al. (2001) [52] during their study in grasslands in California. They indicated soil inputs such as soil roots, as the cause for this trend [52]. However, as the flooding process was conducted for a time span of over a month, during which ten sessions were conducted in our experiment, the microbial activity due to fluctuating water table depth and oxygen content could have been responsible for the enrichment of heavy nitrogen isotope during this time. Increased δ 15 N values have also been observed in forest soil by Snider et al. (2009) [53].  Figure 4B).

δ 15 Ngas and Site Preference in N2O
Both total N2O emissions and δ 15 N values of the gas samples were highest at the water table depth of −40 to −50 cm (Figure 3). Schmidt et al. [16] have explained a decrease in heavy isotope at the central position of the N2O molecule with increase of water table depth. The 15 N α enrichment trend in our data was similar to the total N2O emissions showing an optimum around the −40 cm water table (Figure 3). The increasing enrichment ( Figure 3) is in coherence with the results found by Sutka et al. [21] who observed an increase of δ 15 N α produced by denitrifying bacteria with depletion of soil oxygen. SP values were negative ( Figure 6A). This, with negligible N2O emission ( Figure 4B) and high soil 15 N abundance ( Figure 5) indicated denitrification. In the suboxic peat the negative SP, high N2O emissions and low soil 15 N abundance indicated incomplete denitrification.
Most of the earlier studies on N2O site preference observed an increasing trend for site preference vs. N2O oxygen isotopic composition [54][55][56][57] though no clear trend was observed between the oxygen isotope composition of N2O and site preference under heterotrophic denitrification in pure bacterial cultures [21]. Interestingly in our suboxic peat (Pos B) site preference increased with δ 18 O ( Figure 6B). The positive relationship between site preference and δ 18 O has been observed by a few studies focused on denitrification in soils [53,58]. Under the varying soil oxygen status, heavy oxygen enrichment was higher than heavy nitrogen enrichment, showing significant correlation ( Figure 6C). This can be considered as one of the indicators of denitrification as the dominant producer of N2O in the floodplain fen. Similar trends have been observed by Menyailo et al. (2006) [59] in their study of Siberian soils under denitrifying conditions. In our study, low values of site preference (SP; difference between the central and peripheral 15 [21] who observed an increase of δ 15 N α produced by denitrifying bacteria with depletion of soil oxygen. SP values were negative ( Figure 6A). This, with negligible N 2 O emission ( Figure 4B) and high soil 15 N abundance ( Figure 5) indicated denitrification. In the suboxic peat the negative SP, high N 2 O emissions and low soil 15 N abundance indicated incomplete denitrification.
Most of the earlier studies on N 2 O site preference observed an increasing trend for site preference vs. N 2 O oxygen isotopic composition [54][55][56][57] though no clear trend was observed between the oxygen isotope composition of N 2 O and site preference under heterotrophic denitrification in pure bacterial cultures [21]. Interestingly in our suboxic peat (Pos B) site preference increased with δ 18 O ( Figure 6B). The positive relationship between site preference and δ 18 O has been observed by a few studies focused on denitrification in soils [53,58]. Under the varying soil oxygen status, heavy oxygen enrichment was higher than heavy nitrogen enrichment, showing significant correlation ( Figure 6C). This can be considered as one of the indicators of denitrification as the dominant producer of N 2 O in the floodplain fen. Similar trends have been observed by Menyailo et al. (2006) [59] in their study of Siberian soils under denitrifying conditions. In our study, low values of site preference (SP; difference between the central and peripheral 15 N atoms) and δ 18 O-N 2 O in the captured gas samples indicate nitrifier denitrification in the floodplain fen ( Figure 6B,C). This is also supported by the findings in relevant publications on N 2 O production pathways [60][61][62]. captured gas samples indicate nitrifier denitrification in the floodplain fen ( Figures 6B,C). This is also supported by the findings in relevant publications on N2O production pathways [60][61][62]. Fluctuating water table in riparian zones [56] and floodplain fens [63] is a natural phenomenon that is increasing N2O emissions. Agricultural use and climate change in floodplain fens will intensify N2O losses and therefore, mitigation measures are necessary. Poyda et al. (2016) [64] propose to avoid arable use of floodplain fens. A productive three-cut system grassland would yield the lowest emission rate from high groundwater (long-term mean < 20 cm below the surface) [64]. For northern Europe, climate change models forecast increasing precipitation sum and more frequent climate events (floods and droughts; IPCC, 2014) [4], wherefore the range of water-table fluctuation will be most likely increasing in floodplains. Therefore, adaptation strategies and mitigation measures are becoming increasingly important. Using nuclear techniques will be helpful to understand processes and identify N2O sources. That is necessary for the further development of mitigation measures [65].

Conclusions
In the anoxic floodplain fen peat, N2O emission was low. Accumulation of the heavy nitrogen isotope in the soil, low values of site preference, and δ 18 O-N2O in the captured gas samples indicate nitrifier denitrification in the anoxic floodplain peat. In the suboxic peat the isotopic signals were Fluctuating water table in riparian zones [56] and floodplain fens [63] is a natural phenomenon that is increasing N 2 O emissions. Agricultural use and climate change in floodplain fens will intensify N 2 O losses and therefore, mitigation measures are necessary. Poyda et al. (2016) [64] propose to avoid arable use of floodplain fens. A productive three-cut system grassland would yield the lowest emission rate from high groundwater (long-term mean < 20 cm below the surface) [64]. For northern Europe, climate change models forecast increasing precipitation sum and more frequent climate events (floods and droughts; IPCC, 2014) [4], wherefore the range of water-table fluctuation will be most likely increasing in floodplains. Therefore, adaptation strategies and mitigation measures are becoming increasingly important. Using nuclear techniques will be helpful to understand processes and identify N 2 O sources. That is necessary for the further development of mitigation measures [65].

Conclusions
In the anoxic floodplain fen peat, N 2 O emission was low. Accumulation of the heavy nitrogen isotope in the soil, low values of site preference, and δ 18 O-N 2 O in the captured gas samples indicate nitrifier denitrification in the anoxic floodplain peat. In the suboxic peat the isotopic signals were similar but N 2 O emissions were high, indicating that the denitrification was incomplete. Further investigation should focus on distinguishing N 2 O production pathways using microbial analysis (metagenomic and qPCR approaches) and labelled 15 N techniques. This is important for better regulation of land use in floodplains to mitigate N 2 O emissions.