Isotopologue Ratios of N - American Chemical Society

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Isotopologue Ratios of N2O and N2 Measurements Underpin the Importance of Denitrification in Differently N‑Loaded Riparian Alder Forests Ü lo Mander,*,†,‡ Reinhard Well,§ Daniel Weymann,∥ Kaido Soosaar,† Martin Maddison,† Arno Kanal,† Krista Lõhmus,⊥ Jaak Truu,† Jürgen Augustin,# and Julien Tournebize‡ †

Institute of Ecology and Earth Sciences, University of Tartu, 51014 Tartu, Estonia Hydrosystems and Bioprocesses Research Unit, National Research Institute of Science and Technology for Environment and Agriculture (Irstea), 1 rue Pierre-Gilles de Gennes CS 10030, F92761 Antony cedex, France § Thünen Institute of Climate-Smart Agriculture, 38116 Braunschweig, Germany ∥ Forschungszentrum Jülich, Agrosphere Institute IBG-3, Wilhelm-Johnen-Straße, 52428 Jülich, Germany ⊥ Institute of Ecology and Earth Sciences, University of Tartu, 51005 Tartu, Estonia # Institute of Landscape Matter Dynamics, Leibniz-Centre for Agricultural Landscape and Land Use Research (ZALF), D-15374 Müncheberg, Germany ‡

S Supporting Information *

ABSTRACT: Known as biogeochemical hotspots in landscapes, riparian buffer zones exhibit considerable potential concerning mitigation of groundwater contaminants such as nitrate, but may in return enhance the risk for indirect N2O emission. Here we aim to assess and to compare two riparian gray alder forests in terms of gaseous N2O and N2 fluxes and dissolved N2O, N2, and NO3− in the near-surface groundwater. We further determine for the first time isotopologue ratios of N2O dissolved in the riparian groundwater in order to support our assumption that it mainly originated from denitrification. The study sites, both situated in Estonia, northeastern Europe, receive contrasting N loads from adjacent uphill arable land. Whereas N2O emissions were rather small at both sites, average gaseous N2-to-N2O ratios inferred from closed-chamber measurements and He−O laboratory incubations were almost four times smaller for the heavily loaded site. In contrast, groundwater parameters were less variable among sites and between landscape positions. Campaign-based average 15N site preferences of N2O (SP) in riparian groundwater ranged between 11 and 44 ‰. Besides the strong prevalence of N2 emission over N2O fluxes and the correlation pattern between isotopologue and water quality data, this comparatively large range highlights the importance of denitrification and N2O reduction in both riparian gray alder stands.

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gas12). In well-aerated, moist conditions (a water filled pore space of soil at 40−60%), N2O can be emitted during nitrification by ammonia-oxidizing bacteria13,14 and archaea15 during the oxidation of hydroxylamine (NH2OH) to nitrite (NO2−). Under low O2 conditions coupled with low organic C content of soils, NO2− can be reduced to N2O and N2 by nitrifier denitrification.16 Apportioning N2O to these source processes is a challenging task that has been traditionally investigated using isotope tracing.16,17 There are also several studies on N2O isotopomer relations that supply information on the contribution of different processes such as production by nitrification, bacterial

INTRODUCTION Riparian ecosystems are important landscape elements that improve water quality in rivers and other water bodies,1−3 but they are also potential hotspots of nitrous oxide (N2O) emission to the atmosphere.4−9 Nitrous oxide plays an important role in altering stratospheric chemistry, including depletion of the ozone layer.10 The radiative forcing of N2O is 296 times higher than that of the same mass of carbon dioxide (CO2), and is therefore a potent greenhouse gas (GHG). Despite its relatively minor contribution to global warming (6%), a small percentage of increase in emissions can lead to a large accumulation of N2O in the troposphere, a phenomenon resulting from the long residence time of N2O, approximately 120 years.11 Nitrous oxide can be produced through a number of different chemical and biochemical pathways, namely nitrification (stepwise conversion of ammonia to nitrate) and denitrification (stepwise conversion of nitrate to nitrogen © 2014 American Chemical Society

Received: Revised: Accepted: Published: 11910

April 8, 2014 September 23, 2014 September 29, 2014 September 29, 2014 dx.doi.org/10.1021/es501727h | Environ. Sci. Technol. 2014, 48, 11910−11918

Environmental Science & Technology

Article

and fungal denitrification, and N2O reduction.18−26 A better understanding of the processes is, however, required in order to improve mitigation strategies.23,27 Denitrification is considered the most important reaction for NO3− remediation in aquifers. This process occurs in O2 depleted layers with available electron donors.28 Considerable NO3− reduction is possible, especially when both, electron donors and acceptors are abundant, for example, in agricultural areas with high N inputs via fertilizers and reduced sulfur in the aquifer28 or in the shallow groundwater of hydromorphic agricultural soils.29 Dinitrogen (N2), the main gaseous component of Earth’s atmosphere, is the final product of this process, and thus the quantification of groundwater N2 arising from denitrification (excess N2) can facilitate the reconstruction of historical N inputs, because NO3− loss can be obtained from the sum of denitrification products.30,31 It is also very important to consider the excess N2 value when calculating indirect N2O emission from the aquifer resulting from N-leaching.21,31,32 Several researchers suggest that the information obtained from measuring the intramolecular distribution of 15N on the central (α) and the end (β) position of the linear N2O molecule is crucial for a better understanding of the apportioning of N2O between nitrification and denitrification, but also source and sink processes.33−35 The N2O site-specific 15 N signatures from bacterial denitrification and the NH2OHto-N2O pathway of nitrification have been shown to be clearly different, making this signature a potential tool for N2O source identification.21,25,32,33,36−38 The majority of studies have been dedicated to the analysis of δ15N and δ18O isotope and isotopomer (δ 1 5 N α and δ 1 5 N β ) values of emitted N2O,22,24,27,26,35,36,38 while there are only a limited number of studies dedicated to the analysis of dissolved N2O in groundwater.21,23,32,39 N2O reduction to N2 affects both, SP and δ18O of residual N2O19,20,38 and clear correlation between these signatures have been shown to be indicative for denitrification with N 2 as main product. 23 Especially, isotopologue-based data on the relative contribution of the saturated and unsaturated zones to total fluxes and on lateral N2O fluxes with groundwater flow to open water bodies are provided by very few studies.23 The main objective of our study is to compare two differently loaded riparian alder stands in terms of gaseous N fluxes from soil and N turnover in the near-surface groundwater. For that purpose we did (1) measure N2O and N2 emission using closed chamber and He−O incubation techniques, respectively; (2) investigate N2O accumulation in shallow groundwater in order to estimate the riparian forest’s potential to emit N2O into the atmosphere via the lateral convective pathway; (3) compare N2O accumulation in groundwater to N2 from denitrification in order to reveal the dynamics of that process; (4) discuss sources of N2O using stable isotope signatures of N2O.

abandoned activities in Porijõgi, the estimated lateral Total N inflow in Viiratsi is about twice as high as in Porijõgi: 45.2 and 25.6 kg N ha−1 yr−1, respectively.8 In both study areas, sampling was conducted in the upper and lower sampling plots (Figure 1).

Figure 1. Riparian study sites in Estonia. Notice a narrow wet plateau in the Filipendula ulmaria community in Viiratsi.

In the Poriõgi area, a 20-m wide gray alder stand grows on a Thapto-Mollic (Endogleyic) Gleysol with a groundwater table depth of 0−0.8 m and 0−0.1 m in the upper and lower plots, respectively. In Viiratsi, a 12-m-wide wet patch (A. incana − Filipendula ulmaria) on Mollic Gleysol (considered as “upper” plot with groundwater table depth 0−0.05 m) is followed by a 28-m-wide gray alder forest on Thapto-Mollic Endogleyic Umbrisol (considered to be a “lower” plot with groundwater depth of 0−0.5 m; Figure 1). In each study plot of both study sites, water sampling wells (ø 50 mm, 1.5 m deep PVC pipes perforated and sealed in a lower 0.5 m part) and collars for gas sampling chambers were installed. Gas Sampling and Analyses. The static closed chamber method40 was used for the measurement of N2O fluxes, and the He−O method40−42 was used for the measurement of N2 emissions. Gas samplers (nontransparent manual conical chambers with a cover made of PVC, height 40 cm, Ø 40 cm, volume 65 L, sealed with a water-filled collar on the soil surface, painted white to avoid heating during application) were installed in seven or eight replicates at upper and lower plots in the Porijõgi and in three replicates in the Viiratsi study area (Figure 1). During each gas sampling session in each plot, we measured the depth of the groundwater table (cm) in water sampling wells and soil temperature at 3 depths (0−10, 20−30, and 30−40 cm). Gas sampling was carried out once a month in April, May, July, August, October, November, and December 2008. At time points 0, 30, and 60 min, gas samples were taken from the enclosures of samplers using previously evacuated gas bottles (100 mL). The soil temperature, redox potential and water

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MATERIALS AND METHODS Study Sites. We studied N2O and N2 emission from soil and N2O isotopologues in groundwater in two differently loaded riparian gray alder (Alnus incana)-dominated forests in agricultural landscapes of southern Estonia: a 38-year-old stand in Porijõgi (58°12′41″N, 26°46′55′E), in which upslope agricultural activities had been abandoned since the middle of the 1990s, and a 55-year-old stand in Viiratsi (58°20′N, 25°39′20″E), which still receives polluted lateral flow from uphill fields fertilized with pig slurry. Owing to the intensive agricultural practices in upslope field in Viiratsi, and the 11911

dx.doi.org/10.1021/es501727h | Environ. Sci. Technol. 2014, 48, 11910−11918


Article

nine replicates from water sampling wells using a peristaltic pump. Water was pumped through the probes into 115 mL serum bottles. To discard water with atmospheric contamination, an overflow of >115 mL water was allowed before the bottles were immediately sealed without trapping air bubbles using butyl rubber septa (Altmann, Holzkirchen, Germany) and crimp caps.31 The samples were stabilized with 0.1 mL of saturated HgCl2 solution and analyzed for dissolved N2O, N2, Ar, and NO3− as described in Weymann et al.31 N2 from denitrification (XexcessN2) was calculated using the following equation:

depth in the sampling wells were measured simultaneously. The gas concentration in the collected air was determined using the Shimadzu 2014 gas chromatographic system (equipped with an electron capture detector (ECD), a flame ionization detector and an autosampler43) in the lab of the Institute of Technology of the University of Tartu. The procedures used for the determination of the emission rates of gases are described by Mander et al.40 Intact soil cores (diameter 6.8 cm, height 6 cm) for use with the He−O method were taken from the topsoil (0−10 cm) at the gas sampler locations each time gas sampling was completed. Soil samples were weighed, kept at low temperature (4 °C) and transported to the laboratory of the Institute of Landscape Biogeochemistry of the Leibniz Centre for Agricultural Landscape Research (ZALF) in Germany. Three replications per site and date were simultaneously placed in special gastight incubation vessels inside a climate chamber at field temperatures (5−20 °C). Four substitution sequences with moderate evacuation (0.047 bar) followed by flushing the vessels with an artificial He/O2 gas mixture (20.58% O2, 347.8 ppm of CO2, 1.780 ppm of CH4, 0.290 ppm of N2O, 3.04 ppm of N2, rest He) were conducted to remove ambient N2. Subsequently, a continuous He/O2 gas flow rate of 15 mL per minute was adjusted to the vessel headspaces, followed by a 24 h period to establish a new flow equilibrium. From each vessel, we measured the N2O headspace concentration once and the N2 concentration three times at the gas inlet and the gas outlet of the individual vessels. Concentration of N2 was analyzed by a micro-GC (Agilent Technologies, 3000 Micro GC), equipped with a thermal conductivity detector (TCD). Gas chromatograph settings were: TCD temperature 60 °C, sample inlet 60 °C, molsieve capillary column (14 m), oven temperature 60 °C, carrier gas He 6.0 (1 mL min−1). N2O concentration was analyzed by a GC (Shimadzu, Duisburg, Germany, GC−14B) equipped with an ECD. GC settings were: ECD temperature 310 °C, column PoraPack 80/100 mesh, oven temperature 60 °C, carrier gas, N2 6.0 (13 mL min−1). Background N2 concentration varied between 3.5 and 4.5 ppm (ca. 3 ppm originate from the artificial He/O2 gas mixture and 1 ppm from diffusion into the incubation measuring device). Flux rates were calculated from the actual gas concentration of the continuous flow rate from the vessel headspace after subtraction of a blank value from a vessel without a soil core, which is equivalent to concentrations from the artificial He/O2 gas mixture. The lowest detectable flux rates: 0.5 μg N2O−N m−2 h−1 and 0.04 mg N2−N m−2 h−1. As a result of restricted core height the N2O emission rates in the lab were 30−50% less than the rates from the field study sites. In order to meet also the magnitude of the N2 flux rates under field conditions, we multiplied the N2:N2O ratio from the lab incubation with the N2O flux rates from the chamber measurements at the study sites. All the raw data of N2O and N2 measurements are presented in the Supporting Information (SI) Table S1a, b. Water Sampling and Analyses. Shallow groundwater depth in upper aquifer was measured once a month from piezometers installed with two to three replicates on the borders of plant communities (Figure 1). Groundwater discharge was estimated based on Darcy’s law and through gauging with weirs installed in groundwater seeping patches within the alder stand. For the analysis of nitrogen gases, NO3−, and their isotopologue signatures, water samples were taken in four to

XexcessN2 = XN2T − XN2EA − XN2EQ

(1)

where X denotes the molar concentration of the parameters, XN2 T represents the molar concentration of the total dissolved N2 in the groundwater sample, XN2 EA is N2 originating from “excess air”, i.e. dissolved gas components in excess of equilibrium and other known subsurface gas sources entrapped in air bubbles near the groundwater table during recharge, and XN2 EQ is the molar concentration of dissolved N2 in equilibrium with the atmospheric concentration.31 Initial NO3− concentration (c NO3− t0) at a given location on the aquifer surface is defined by the NO3− concentration of the recharging water before alteration by denitrification in the groundwater:31 c NO3−t 0 = excess N2 + c NO3− − N + c N2O − N

(2)

Reaction progress of denitrification (RP) is the ratio between the products and the starting material of the process, and can be used to characterize the extent of NO3− elimination by denitrification.31 RP is calculated as follows: RP = (excess N2 + cN2O − N)/cNO3− − Nt 0

(3)

In this study, all of the concentration values are expressed in mg N L−1. Isotope Analyses. The isotopologue signatures of groundwater N2O, i.e. δ18O (δ18O−N2O), average δ15N (δ15Nbulk− N2O) and δ15N from the central N position (δ15Nα), were analyzed after cryo-focusing using isotope ratio mass spectrometry, as described by Well and Flessa.19 The analysis was conducted using a Delta XP IRMS (Thermo−Finnigan, Bremen, Germany), allowing simultaneous detection of mass to charge ratio (m/c) 30, 31 of N2O fragments and m/c 44, 45, and 46 of intact N2O molecules. The IRMS was connected to a modified Precon (Thermo−Finnigan, Bremen, Germany) equipped with an autosampler (model Combi-PAL CTCAnalytics, Zwingen, Switzerland). Pure N2O (Westfalengas, Münster, Germany; purity >99.995%) was used as the reference gas which was analyzed for isotopologue signatures in the laboratory of the Tokyo Institute of Technology, Tokyo, Japan, using the calibration procedures developed earlier.33 This reference signature was used to correct the raw δ15Nα values determined by our instrumentation. 15 N site preference (SP; ‰) was obtained as SP = 2 × (δ15 N α − δ15 Nbulk − N2O)

(4)

The isotopologue ratios of a sample (Rsample) were expressed as the deviation from the 15N/14N and 18O/16O ratios of the reference standard materials (Rstd), atmospheric N2 and standard mean ocean water (SMOW) respectively: δX = (R sample/R std − 1) 11912

(5)



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where X = 15Nbulk−N2O, 15Nα, 15Nβ, or 18O. Typical analytical precision was 0.6%, 0.9%, and 0.9% for δ15Nbulk, δ15Nα, and δ18O, respectively. The detection limit for N2O−N was 1.5 ppb.12 Statistical Analysis of Data. Most of the data had nonnormal distribution (Kolmogorov−Smirnov, Lilliefors, and Shapiro−Wilk’s tests), thus we used the nonparametric statistics whereas both median and average values are presented in the text. Significance of differences between the concentration of different nitrogen forms and isotopologues values in water samples was studied using the Wilcoxon signed-rank test. The Pearson correlation and Spearman rank-order correlation tests were used to characterize the relationship between the variables. The statistical analysis was carried out using Statistica 7.1 (StatSoft Inc.). For all cases, the significance value of p < 0.05 was accepted. In one correlation analysis (excess N2 vs N2O concentration in water), some outlying data were not used in the analysis (SI Table S2a,b).

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RESULTS Groundwater Dynamics. In the Porijõgi upper plot the groundwater depth was somewhat lower (10−35 cm) than in the lower plot (0−7 cm), whereas the wet plateau in the upper part of Viiratsi alder stand (Figure 1) showed significantly higher groundwater Table (5−10 cm) than the downslope position (60−100 cm) (SI, Figure S1A). The annual average ± standard error groundwater input discharge (GID) into both riparian alder stands was estimated to be rather similar: 8.3 ± 1.0 and 8.3 ± 0.9 m3 ha−1 d−1 in Porijõgi and Viiratsi, respectively. Due to the snow cover and sporadically frozen soil, the monthly average GID in both areas was lowest in winter (December−February) whereas the highest values were measured in May and October (SI, Figure S1B). Gaseous Nitrogen Fluxes. The median value of N2O emission during the measurement sessions varied from 0.9 in April to 13.8 in August in Porijõgi and from 5.9 in April to 15.0 μg N2O−N m−2 h−1 in December in Viiratsi (Figure 2A). In Porijõgi the highest values were recorded during the summer months, whereas in Viiratsi emissions peaked in autumn and at the beginning of winter. The emission of N2 in Porijõgi varied from 1580 in December to 3254 μg N m−2 h−1 in May, while in Viiratsi the range was from 700 in May to 2200 μg N m−2 h−1 in October (Figure 2B). In Porijõgi, the N2:N2O ratio changed from 123 in July to 1280 in May, whereas in Viiratsi it varied from 53 to 724 in October (Figure 2C). The median values of N2O fluxes from both riparian zones did not differ significantly throughout the whole study period, being 3.2 and 5.7 μg N2O−N m−2 h−1 in Porijõgi and Viiratsi, respectively. The N2 fluxes were significantly (p < 0.05) higher in Porijõgi: 2020 and 1168 μg N2−N m−2 h−1, correspondingly. The N2:N2O ratio in Viiratsi (median 185) was significantly lower than in Porijõgi (844) (Figure 2D). Different Nitrogen Forms in Water Samples. The median (average) values of NH4+−N concentration in groundwater for the whole study period in Viiratsi were lower (p < 0.05) than those in Porijõgi: 0.01 (0.07) and 0.20 (3.76) mg L−1 respectively). In both sites the difference in NH4+−N values between the upper and lower plots was not significant (p > 0.05; SI, Figure S2A). The measured NO3−−N and N2O−N concentrations were lower (p < 0.05) in Porijõgi than in Viiratsi groundwater (SI, Figure S2A). In the Porijõgi area, lower plot showed smaller (p < 0.05) NO3−−N (SI, Figure S2A) and N2O−N concentrations

Figure 2. Seasonal dynamics (median, 25 and 75% quartile and minmax values) of N2O (A) and N2 (B) emission, N2: N2O ratio (C), and the same parameters generalized for study period and site (D) in Porijõgi and Viiratsi riparian gray alder forests. *, significantly different values (p < 0.05). Upper numbers show median, and lower italic numbers average values. 11913



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Figure 3. Groundwater N2O vs reaction progress (RP) (A), excess N2 vs N2O (B), δ15NbulkN2O vs δ18O−N2O (C), and δ18O−N2O vs SP−N2O (D) in the riparian gray alder forests of Porijõgi (white diamonds) and Viiratsi (black diamonds) in Estonia. For A and B, average and standard error values (SI, Table S2a,b) are presented, for C and D, each single measurement is shown. Correlation between groundwater N2O and RP (A) is shown for Viiratsi only.

Table 1. Pearson Correlation Coefficients Between N2O and N2 Emission, Water Characteristics and Isotopologue Parametersa N2O emission N2 emission a

N2-excess

NH4+−N

NO3−−N

N2O−N

NO3− t0

RP

δ18O−N2O

δ15Nbulk−N2O

SP−N2O

−0.01 0.04

−0.27 −0.27

0.53* 0.66*

0.66* 0.86**

0.92** 0.48

−0.41 −0.62

−0.94 0.32

−0.34 0.15

0.20 0.20

*, p < 0.05; **, p < 0.01.

24.1‰ respectively in the upper and lower positions). No significant differences in isotopologue values between the upper and lower plots were observed in Viiratsi (SI, Figure S2D). The Relationship between the Emitted Nitrogen Gases, Different Nitrogen Forms in Groundwater and N2O Isotopologues. A significant negative linear correlation was ascertained between the groundwater N2O−N concentration and RP values (Figure 3A), whereas excess N2 and N2O−N concentration (Figure 3B), δ15Nbulk−N2O vs δ18O− N2O (Figure 3C), and δ18O−N2O vs SP−N2O values (Figure 3D) were positively correlated. N2O emission was positively correlated with NO3−−N, N2O−N, and NO3−-t0 concentration in groundwater, and a significant positive correlation was also found to exist between N2 emission and NO3−−N and N2O−N concentration (Table 1).

(SI, Figure S2B) than the upper plot. Comparing the sites, N2O−N is highest in Viiratsi, with concentrations mostly >10 μg L−1 and up to 100 μg L−1. The median (average) values of excess N2 concentration in all of the plots were quite similar, varying from 2.40 (2.43) mg L−1 at the lower Porijõgi plot to 3.21 (3.01) mg L−1 in the upper Porijõgi plot. Also, no significant differences were found between the average values of excess N2 of both sites (SI, Figure S2A). The median (average) value of NO3− t0 was significantly higher in Viiratsi than in Porijõgi (3.91 (4.95) and 2.69 (2.85) mg L−1 respectively), whereas there were no significant differences in the NO3− t0 concentration between the sites (SI, Figure S2B). The RP value varied from 0.64 (0.55) at the lower Viiratsi plot to 1.0 (0.94) at the lower Porijõgi plot, whereas no significant differences were found between the plots in Porijõgi (SI, Figure S2C). Possibly, slightly but not significantly higher NH4+−N, NO3−−N, and NO3− t0 concentrations in the downslope position in Viiratsi (SI, Figure S2A and S2B) were caused by low water table in this plot (SI, Figure 1A). N2O Isotopologues. None of the measured isotopologue values differed significantly between the two study areas. For δ18O−N2O, δ15Nbulk−N2O, and SP−N2O, the median (average) values were 54.7 (61.5); 8.8 (16.2) and 14.7 (18.4) ‰ for Porijõgi, and 48.8 (51.8); −2.76 (3,91) and 22.4, 27.4 ± 5.6‰ respectively for Viiratsi (SI, Figure S2D). The upper plot in Porijõ gi showed slightly but not significantly higher δ18O−N2O and δ15Nbulk−N2O values than those in the lower plot (median 68.4 and 23.6‰ for upper and a single value 6.0 ‰ correspondingly in the lower plot), whereas the SP−N2O value was slightly but not significantly lower in the upper Porijõgi plot (median values 12.6 and

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DISCUSSION Gaseous Nitrogen Fluxes. Alders, known as a common species of symbiotic N2 fixing bacteria (actinobacteria) from the Frankia group, are a typical tree species in riparian zones.44 Due to high rates of N2 fixation, some authors have seen alder forests as sources of water body pollution with excess N.45 Several other studies consider riparian alder stands to be effective N removal ecosystems.6 The fixed atmospheric N2 will be transformed to organic N and accumulates in soil, thus being no additional source of leaching.6,45 This contradiction is mainly due to the position of alder stands in the landscape: in riparian zones the excess N is mainly denitrified, whereas leaching takes place in the more aerated conditions of higher altitude locations.45 Due to intense N cycling in alder forests, gaseous N fluxes in these ecosystems are also intensive. For instance, in a study conducted in the Porijõgi area in 2001− 11914



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2003, emissions of N2O ranged from 8 to 31 μg N2O−N m−2 h−1, whereas the N2:N2O ratio varied between 150 and 700.7 In a long-term study (2001−2009) on gaseous N fluxes from the Porijõgi and Viiratsi areas, the range of N2O flux was from −0.6 to 87 μg N2O−N m−2 h−1 in Porijõgi and 0.5−38 μg N2O−N m−2 h−1 in Viiratsi, showing slightly greater but nonsignificant median values in Viiratsi (1.7 and 2.1 μg N2O−N m−2 h−1 for Porijõgi and Viiratsi respectively). The N2:N2O ratio, however, was significantly higher in Porijõgi, ranging between 10 and 7600 and 40−1200 in Porijõgi and Viiratsi correspondingly.8 Thus, the results from our study period are consistent with those gathered in earlier studies. In a study conducted on a gray alder stand planted on an abandoned agricultural area, the average N2:N2O ratio was 171, ranging from 47 to 261.46 It supports the idea of high N2: N2O ratio in alder forests. In some other riparian alder forests with higher summer temperatures, no N2O emission was found during the study period.47 On the other hand, N2:N2O ratio in riparian alder forests was up to two magnitudes higher than that reported for fertilized fields.36 These high ratios can probably be attributed to relatively long residence time of N2O due to low diffusivity of the wet soils and because NO3− concentrations were relatively low compared to the high denitrification rates. Wang et al.48 have summarized various sources and found that N2:N2O ratios can vary significantly, and the main drivers of this variability are the soil nitrate concentration, the availability of easily degradable C substrates, the redox potential, soil moisture, and soil pH. Different Nitrogen Forms in Water Samples. In Viiratsi, NH4+−N concentrations were always very low (1 mg L−1) has mostly been found in Viiratsi, but not in Porijõgi (SI, Table S2a,b). Relatively low NO3− levels can be explained by lower nitrification capacity in the water-saturated zone and/or intense denitrification. The lower NO3− in Porijõgi may also have been caused by less import of N from the abandoned grassland. On the other hand, N2 fluxes were higher in Porijõgi, indicating higher denitrification rate. Mean excess N2 was between 1.5 and 4.5 mg L−1 at almost all of the plots. The highest values were observed in Viiratsi, which coincided with higher NH4+ and NO3− levels (SI, Table S2a,b). A similar pattern was found for aquifers with high N concentrations.31 N2O Isotopologues. High values of δ18O−N2O (>40‰) are typical for N2O production and reduction by denitrification in aquifers (Well et al., 2005a, 2012). Such values are found in Viiratsi at times of elevated NO3− levels (SI, Table 2a,b). The 15N site preference (SP) values (6.0−45.0‰; SI, Table S2a,b) are in a comparable range to the values observed in other aquifers in agricultural use.21,23,32,39 However, Well et al.21 have also shown that the SP value measured in the shallow groundwater of a hydromorphic soil (29−81 ‰) is distinctly greater than surface emitted N2O. Published data on both emitted and soil N2O and the SP values fluctuate within a wide range. Toyoda et al.18 have determined SP values of −5‰ for bacterial denitrification and about 30‰ for nitrification (NO3− and NH2OH), while Sutka et al.25 stated the SP value of 33− 37‰ for fungal denitrification. In another study, Sutka et al.37 found that SP values of N2O produced by the fungi Fusarium

oxysporum and Cylindrocarpon tonkinense were 37.1 ± 2.5‰ and 36.9 ± 2.8 ‰ respectively. Well et al.22 found that SP close to 30 ‰ and δ18O−N2O < 20 ‰ is typical for autotrophic nitrification in soils following NH4+-fertilization. Since there are no analogous studies from riparian ecosystems, we can only compare our results with those gathered in groundwater,23 sediment research49 or wastewater treatment systems analysis.50,51 Our results, especially the SP−N2O and δ18O−N2O values and relations, are in good accordance with these data. N2O reduction to N2 leads to an increase in SP−N2O and δ18O−N2O in the residual N2O.19,20,38,52 In groundwater this has been shown to result in a wide range in both δ18O−N2O and SP−N2O.21,23,39 Moreover, this leads to a close correlation between both signatures where the slope of SP−N2O vs δ18O− N2O has been shown to vary between 539 and 0.821,23 and the lower values were obtained in groundwater with proven intense denitrification. This pattern had been proven and explained to be typical for denitrifying groundwater since it results from isotopologue values of initially produced N2O by bacterial denitrifiers with SP below 0‰25 and δ18O−N2O below 30‰19 with subsequent increase in δ18O−N2O and SP−N2O in the residual N2O during progressing reduction to N2. Exactly this same pattern is evident for our data where the close correlation of δ18O−N2O and SP−N2O with a slope of 1.16 and large range of these values is evident (Figure 3D). Hence, we conclude that N2O originates almost exclusively from bacterial denitrification (including nitrifier denitrification) and that the isotopologue pattern confirms the high N2:N2O ratio of this system (see above). However, quantifying N2O reduction, for example, using a Rayleigh model of isotope fractionation,48,51 is not possible due to the high variability of overall isotope effects of N2O turnover in groundwater.23 The Relationship between Emitted Nitrogen Gases, Different Nitrogen Forms in Groundwater and N2O Isotopologues. The main gaseous flux from both riparian alder stands was in the form of N2. Based on median values it was 185 (Viiratsi) to 844 times (Porijõgi) higher than the amount of N2O emitted. N2O accumulation in the groundwater was moderate, that is, not higher than typical values in nitratecontaminated denitrifying aquifers.31 Therefore, convective mass fluxes of N2O from both study areas were thus small in comparison to surface fluxes. However, at Viiratsi, practically all NO3− has been removed from the groundwater during passage of the riparian zone, though there is twice as much input, N2O emission is not significantly different, and N2 emission is significantly lower than in Porijõgi. The reason is in differences in internal transformations and atmospheric input of N in these systems. In a previous study, Mander et al.53 showed that in Viiratsi symbiotic N2 fixation from the atmosphere was 5 times lower than in Porijõgi: 37.1 and 184.6 kg N ha−1 yr−1, respectively. In opposite, the net N mineralization in Viiratsi (241.1 kg N ha−1 yr−1) was 3 times higher than in Porijõgi (81.9 kg N ha−1 yr−1). This all resulted in N losses (kg N ha−1 yr−) to the water bodies: 9.0 in Viiratsi and 13.2 in Porijõgi.53 Due to the more fluctuating groundwater depth in Porijõgi, a significant part of N2O may be produced by nitrification of NH4+ from organic matter mineralization under unsaturated conditions, at least temporarily. As observed by Weymann et al.,31 the relationship between N2O and RP is similar in aquifers, that is, low N2O−N concentrations were found at high RP values, and vice versa, there were high dissolved N2O values at the RP of 0.6−0.65 11915


Environmental Science & Technology (SI, Figure S2C). This pattern is also evident in the data sets of the plots with high N2O levels in Viiratsi (SI, Table S2a,b). N2O and N2 emission values were positively correlated with NO3−−N, N2O−N and NO3−−t0 concentration in groundwater (Si, Table S2a,b). As expected, excess N2 in groundwater showed a positive correlation with the value for dissolved N2O−N (Figure 3B). We found a significant negative correlation between N2O−N concentration and RP in Viiratsi site (SI, Figure S2A), which is typical evidence of the domination of denitrification processes in aquifers.23 Another indicative characteristic of denitrification was a positive correlation (r = 0.76; p = 0.02) between δ18O− N2O and SP−N2O values (Figure 3D). Since the denitrification-derived values of δ18O−N2O are often higher than those originated from nitrification, Snider et al.54 propose this as an alternative indicator for apportioning N2O sources. Perspectives for Further Studies. Soosaar et al.8 have found that increasing trends in N2 and N2O emissions in the Viiratsi study area may be a result of the age (>50 years) of the gray alder stand, but may also be caused by the long-term nutrient load of this riparian alder stand. This may indicate that the buffering capacity of continuously loaded riparian buffers will decrease over time, which can be affected by ongoing oxidation of reductants by denitrification, decreasing nutrient uptake, and/or supply of new reductants by vegetation.55 This calls for the careful management of these riparian forests (e.g., selective cutting of older trees). Further studies to clarify the impact of age and environmental stress factor on the riparian buffer ecosystems are recommended. For a better understanding of the relationship between nitrification-denitrification processes and in order to distinguish between N2O sources in riparian zones and wetlands in general, a more detailed and long-term comparison of potential lateral N2O fluxes (groundwater discharge) with isotopologue analysis of both groundwater and gaseous emissions is needed. This can be combined with the metagenomic analysis of the functional genes of denitrification (especially nirK, nirS, and nosZ genes) controlling N cycling in wetland ecosystems.56−59 Further studies should focus on the spatial heterogeneity of sites (e,g., on emission hotspots and microsites of reduction/ oxidation in the groundwater system) because this can have a strong effect on the isotopic ratios.60 Some studies have shown remarkable flux of NO from different soils,48 however, for riparian areas no data are available. Another challenge would be to find evidence on ANAMMOX process as N2 source in riparian zones. The first promising evidence on ANAMMOXbacteria have been found.61 Likewise, the role of DNRA in nitrate reduction of riparian ecosystems serves more detailed analysis62

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ACKNOWLEDGMENTS

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REFERENCES

This study was supported by the IAEA’s Coordinated Research Project (CRP) on “Strategic placement and area-wide evaluation of water conservation zones in agricultural catchments for biomass production, water quality and food security”, the Estonian Research Council (grant IUT2-16); and the EU through the European Regional Development Fund (Centre of Excellence ENVIRON and the project BioAtmos). We thank I. Ostermeyer (University of Göttingen, Germany) for dissolved N2O measurements and the Centre for Stable Isotope Research and Analysis at the University of Göttingen for stable isotope analyses.

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ASSOCIATED CONTENT

S Supporting Information *

Table S1a,b has original N2O and N2 emission data, whereas Table 2a,b presents isotopologue and water quality data. Figures S1 and S2 visualize the text info. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 11916



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Isotopologue Ratios of N - American Chemical Society

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