<p>Natural and engineered nitrogen (N) removal processes in aqueous systems represent important sources of nitrogenous gas emissions, including the potent greenhouse gas nitrous dioxide (N<sub>2</sub>O). The relevance of microbial and abiotic formation pathways can be assessed using <sup>15</sup>N tracing techniques. While <sup>15</sup>N-N<sub>2</sub>O analysis using optical analyzers is straightforward, quantification of <sup>15</sup>N fractions in inorganic N compounds, ammonium (NH<sub>4</sub><sup>+</sup>), nitrite (NO<sub>2</sub><sup>-</sup>), and nitrate (NO<sub>3</sub><sup>-</sup>), is typically time-consuming and labor-intensive.</p> <p>In this study, we developed an <strong>a</strong>utomated <strong>s</strong>ample-<strong>p</strong>reparation unit coupled to a <strong>m</strong>embrane-<strong>i</strong>nlet quadrupole <strong>m</strong>ass <strong>s</strong>pectrometer (3n-ASSP-MIMS) for the online quasi-simultaneous analysis of <sup>15</sup>N fractions in NH<sub>4</sub><sup>+</sup>, NO<sub>2</sub><sup>-</sup>, and NO<sub>3</sub><sup>-</sup>. The technique was designed and validated for applications at moderate (100 - 200 μmol L<sup>-1</sup>) to high (2 – 3 mmol L<sup>-1</sup>) N, as found in sewer systems, wastewater in treatment plants, or eutrophic surface waters, and <sup>15</sup>N spiking (f<sub>15</sub>) between 1 and 33%.</p> <p>The potential of 3n-ASSP-MIMS was demonstrated in a feasibility study, where the technique, in conjunction with <sup>15</sup>N-N<sub>2</sub>O analyses by FTIR spectroscopy, was applied to pinpoint nitrifier denitrification as the primary N<sub>2</sub>O formation pathway during partial NH<sub>4</sub><sup>+</sup> oxidation to NO<sub>2</sub><sup>-</sup> in a lab-scale sequencing batch reactor.</p>

Engineered nitrogen (N) removal processes in water treatment plants and N-transformation reactions in polluted environments represent prominent sources of the potent greenhouse gas, nitrous oxide (N2O). The relevance of microbial and abiotic formation pathways can be assessed by using N-15 tracer techniques. While N-15-N2O analysis with optical analyzers is straightforward, the quantification of atom % N-15 of inorganic N compounds, such as ammonium (NH4+), nitrite (NO2-), and nitrate (NO3-), requires discrete sample analyses that are time-consuming and labor-intensive. In this study, we developed an automated sample preparation unit, coupled to a membrane inlet quadrupole mass spectrometer, for the online, quasi-simultaneous analysis of atom % N-15 in NH4+, NO2-, and NO3-. This technique was designed and validated for N-15-spiking applications at moderate (100-200 mu mol L-1, 1 atom % N-15) to high (2-3 mmol L-1, 33 atom % N-15) dissolved inorganic N concentrations typically encountered in sewer systems or contaminated watersheds. The high potential of the developed system, in combination with N-15-N2O analysis by Fourier-transform infrared spectroscopy, to constrain N transformations and sources of N2O was demonstrated in a feasibility study, where nitrifier denitrification was identified as the primary N2O formation pathway during the partial NH4+ oxidation to NO2- in a lab-scale sequencing batch reactor.

Denitrification in groundwater of aquifers is an important process that helps to maintain environmental standards, yet there is a limited number of studies that determine the spatial variation of denitrification conditions in aquifers on a regional scale. This paper presents a procedure for the regionally differentiated derivation of denitrification conditions in groundwater based on measured values of the redox-sensitive parameters oxygen, iron, manganese, DOC and nitrate as well as information on aquifer typologies. We applied this procedure to Germany, using measured values from more than 24,000 groundwater monitoring sites from 2007-2016. Annual concentrations of the five parameters at the monitoring sites were regionalized using an optimized, iterative inverse distance weighting procedure, using 15 aquifer typologies for spatial delineation. The annual grids (2007-2016) of each parameter were then overlaid and a median over time was calculated. Discrete ranks were then assigned to the concentrations of each parameter depending on their redox class, and ultimately, after overlaying the five parameters, a mean value was calculated describing the nitrate degradation capacity in groundwater.To assess the plausibility of the derived denitrification conditions in groundwater, the latter were linked to reaction constants of denitrification in groundwater that assume a dependence of the extend of denitrification on the travel time in groundwater. A comparison of the modeled quantities of denitrified nitrate with the proportion of denitrified nitrate determined with the N 2 /Ar method at 820 groundwater monitoring wells in three German Federal States showed good agreement. Accordingly, the method presented here is suitable to be used consistently for larger regions for the regionally differentiated derivation of denitrification conditions in groundwater. For regions with denitrifying groundwater conditions, the results provide an explanation for the frequently observed discrepancy between high nitrate emissions from soil and low nitrate concentrations in the groundwater of intensively used agricultural areas.

Denitrification in groundwater is an important process that helps to maintain environmental standards, yet there are very few studies that determine the spatial variation of denitrification conditions in aquifers on a regional scale. We introduce a procedure to derive spatially continuous estimates of denitrification conditions in groundwater based on the interpolation of measurements of the redox-sensitive parameters oxygen, nitrate, iron, manganese and DOC, combined with the quantification of denitrification using a 2D-hydrodynamic model based on first-order reaction kinetics. We applied this procedure to Germany, using measured values from more than 24,000 groundwater monitoring sites from 2007 to 2016. Annual concentrations of the five parameters at the monitoring sites were regionalized using an optimized, iterative inverse distance weighting procedure within 15 aquifer typologies for spatial delineation. The annual grids (2007–2016) of each parameter were then overlaid and a median over time was calculated. Discrete ranks were then assigned to the concentrations of each parameter based on their redox class, and ultimately, after overlaying the five parameters, a mean value was calculated describing the nitrate degradation conditions in groundwater. After assigning half-life times and reaction constants to those denitrification conditions, we quantified denitrification in groundwater using the hydrodynamic model WEKU.To assess the plausibility of the derived denitrification in groundwater, we compared our results with the proportion of denitrified nitrate determined with the N2/Ar method at 820 groundwater monitoring wells in three German Federal States, which showed an overall good agreement. Accordingly, the method presented here is suitable to be used for the regionally differentiated derivation of denitrification conditions in groundwater. For regions with denitrifying groundwater conditions, the results provide an explanation for frequently observed discrepancies between high nitrate emissions from the soil and low nitrate concentrations in the groundwater of intensively used agricultural areas.

RationaleExisting methods for the measurement of the 15N/14N isotopic composition of ammonium and nitrate are either only suitable for labelled samples or require considerable sample preparation efforts (or both). Our goal was to modify an existing analytical approach to allow for natural abundance precision levels.MethodsPublished reaction protocols were used to convert ammonium into N2 by NaOBr and nitrate into N2O by TiCl3. A membrane inlet system was developed and coupled to an isotope ratio mass spectrometer to allow precise determination of the analytes.ResultsConcentrations of ≥35 μmol/L N for both ammonium or nitrate could be analysed for δ15N values with precisions of better than 0.9 mUr. While ammonium analyses exhibited a small concentration dependency and an offset of 2.7 mUr at high ammonium concentrations irrespective of the standard isotopic composition, nitrate analysis showed no offset but a blank contribution visible at very low concentrations.ConclusionsThe presented method is capable of fast measurement of δ15N values in ammonium and nitrate from aqueous samples with reasonable accuracy at natural abundance levels. It will thus facilitate the application of isotopic methods to studies of nitrogen cycling in ecosystems.

Valide Modelle zur flächenhaften Abschätzung von Nitratausträgen aus der Landwirtschaft ins Grundwasser sind ein unerlässliches Prognosewerkzeug. Eine wesentliche Herausforderung für die Validierung solcher Modelle ist die räumliche und zeitliche Inkongruenz zwischen Daten aus Grundwassermessstellen und modellierten Nitrateinträgen ins Grundwasser und der Umstand, dass viele der bestehenden Grundwassermessstellen bisher nicht zur Validierung genutzt werden können.

We evaluated the abiotic formation of dissolved organic nitrogen (DON) by the fast reaction of iron (Fe) with nitrate (NO3-) in the dissolved organic matter (DOM) of volcanic soils in a temperate rainforest (>5000 mm precipitation per year). During five days, the educts and products of abiotic reactions under anoxic conditions were measured in a microcosm experiment depending on the Fe and NO3- concentrations. A control zero-Fe was not used because there was no chemical reaction with nitrate addition. Using a novel technique of automated sample preparation for inorganic N (SPIN) attached to a membrane inlet quadrupole mass spectrometry (MIMS), the N-15 abundances and inorganic N concentrations were determined directly in aqueous solutions. The results were explained in the context of the Ferrous Wheel Hypothesis which states that Fe(II) is utilized to reduce NO3- to nitrite (NO2-) that is incorporated into DOM. Fe(II) is regenerated from Fe(III) in anaerobic soil microsites. Here we tested one part of this hypothesis, the processes occurring in DOM (instead of soil organic matter). Using the SPIN-MIMS technique, we could overcome Ferrous Wheel Hypothesis criticism regarding possible Fe interference during NO3- analysis. The total recovery of N-15 added as NO3- fluctuated between 63 and 101%, and the remaining N-15 was measured as gaseous N2O. The N-15-labelled NO3- added decreased immediately after 15 min of incubation. After five days of incubation, approximately 25% of the labelled NO3- (e(- )acceptors) added was transformed to DON in the presence of a high amount of Fe(II) (e(-) donors). Small amounts of N2O and CO2 provided further evidence of NO3- reduction and DOM oxidation, respectively. From these results, we propose a new theoretical model that includes the Ferrous Wheel Hypothesis, where only the transformation of NO3- to DON was proven. The present results explain the high retention of NO3- in DOM from volcanic soils in ecosystems with high precipitation. (C) 2018 Elsevier Ltd. All rights reserved.

Zusammenfassung Valide Modelle zur flächenhaften Abschätzung von Nitratausträgen aus der Landwirtschaft ins Grundwasser sind ein unerlässliches Prognosewerkzeug. Eine wesentliche Herausforderung für die Validierung solcher Modelle ist die räumliche und zeitliche Inkongruenz zwischen Daten aus Grundwassermessstellen und modellierten Nitrateinträgen ins Grundwasser und der Umstand, dass viele der bestehenden Grundwassermessstellen bisher nicht zur Validierung genutzt werden können. Mithilfe der N 2 /Ar-Methode können erstmals auch Grundwassermessstellen, die reduziertes Grundwasser fördern, zur Modellvalidierung verwendet werden. Dazu wurden niedersachsenweit über 484 Grundwassermessstellen beprobt und jeweils mit dem DENUZ-Modell modellierte potenzielle Nitratkonzentrationen im neugebildeten Grundwasser mit Nitrateintragskonzentrationen, die mit der N 2 /Ar-Methode berechnet wurden, verglichen. Die Ergebnisse der Modellvalidierung zeigen eine gute Übereinstimmung beider Methoden im Bereich der niedersächsischen Geest. In grundwassernahen Niederungsregionen, in denen Nitratabbauprozesse im Boden und Grundwasser ineinander übergehen, modelliert das DENUZ-Modell ca. 27 % höhere Nitratemissionen ins Grundwasser als die N 2 /Ar-Methode. Die hohe räumliche und zeitliche Variabilität der Nitrateinträge ins Grundwasser bedingt die Einbeziehung einer großen Anzahl von Grundwassermessstellen bei der Modellvalidierung.

Earlier an automated sample preparation unit for inorganic nitrogen (SPIN) coupled to a membrane inlet quadrupole mass spectrometer (MIMS) was developed for automated and sensitive determination of the N-15 abundances and concentrations of nitrate, nitrite and ammonium of aqueous solutions without any sample preparation. Here we describe an alternative analytical protocol to convert NO3- to N2O instead of NO before measurement. This is advantageous because NO strongly interacts with surfaces, requires long purge times, and still shows considerable carryover between samples, all of which is avoided when N2O is used as analyte. The sensitivity of the measurement of NO3- as N2O is comparable to the earlier measurements with NO as analyte.

N2O is a potent greenhouse gas with an atmospheric lifetime of 114 years which also contributes to ozone layer destruction. Mitigating N2O emissions is especially challenging to the agricultural sector that is responsible for the majority of anthropogenic N2O release. In order to develop effective mitigation strategies, a detailed understanding of drivers for N2O production and reduction in agriculturally managed soils is needed. Denitrification is recognized as one of the most important source processes for N2O emissions from soils. However, the last step in denitrification, the reduction of N2O to N-2 is the only known sink for N2O in soil. Although the impact of single parameters on denitrification is quite well documented, there is still a knowledge gap when it comes to the impact of complex farming systems on N2O production and reduction. In this experiment, we incubated soil samples from the DOK long term field trial in Therwil/Switzerland comparing organic (BIOORG) and conventional (CONMIN) farming systems with an a non-fertilized control (NOFERT). Soil samples were incubated under 90% WFPS after fertilization with (NH4NO3)-N-15 equivalent to a moderate fertilization event in the field with 40 kg N ha(-1). In order to assess soil's potential for N2O production and reduction, we combined direct measurements of denitrification end products N2O and N-2 with molecular analysis of functional denitrifying communities involved in NO and N2O reduction on DNA and mRNA levels. In order to monitor N cycling processes under the chosen conditions, stable isotope tracing was employed to quantify nitrification and NO3- consumption rates. Results revealed increased NO3- consumption and greatest potential for N2O emissions in BIOORG as a result of increased soil organic carbon contents. Production of N-2 was similar in BIOORG and CONMIN and significantly lower in NOFERT, most likely due to significantly decreased pH inhibiting N2O reduction. This caused the greatest N20/(inhibiting N2O reduction. This caused the greatest N2O/(N2O + N-2) ratios in NOFERT (0.88 +/- 0.02) followed by BIOORG (0.79 +/- 0.01) and CONMIN (0.68 +/- 0.02) (p < 0.001). Lowest N2O/(N2O + N-2) ratios in CONMIN were reflected by lowest N2O emissions and coincided with elevated nosZ transcript copies in the beginning of incubation. Although highest N2O emissions in BIOORG were detected, the incubation setup cannot directly be translated to field conditions. Nevertheless, our results emphasize that farming system induced changes on soil geochemical parameters like soil pH and soil organic carbon affect microbial N2O production and reduction processes during denitrification. (C) 2017 Elsevier Ltd. All rights reserved.

An automated sample preparation unit for inorganic nitrogen (SPIN) coupled to a membrane inlet quadrupole mass spectrometer (MIMS) was developed for automated and sensitive determination of the 15N abundances and concentrations of nitrate, nitrite, and ammonium in aqueous solutions without any sample preparation. The minimum N concentration for an accurate determination of the 15N abundance is 7 μmol/L for nitrite and nitrate, with a relative standard deviation (RSD) of repeated measurements of <1%, and 70 μmol/L with an RSD < 0.4% in the case of ammonium. The SPIN-MIMS system provides a wide dynamic range (up to 3500 μmol/L) for all three N species for both isotope abundance and concentration measurements. The comparison of parallel measurements of 15N-labeled NH4+ and NO3- from soil extracts with the denitrifier method and the SPIN-MIMS system shows a good agreement between both methods.

Knowledge about the spatial variability of in situ denitrification rates (Dr(in situ)) and their relation to the denitrification capacity in nitrate-contaminated aquifers is crucial to predict the development of groundwater quality. Therefore, 28 push–pull 15N tracer tests for the measurement of in situ denitrification rates were conducted in two sandy Pleistocene aquifers in northern Germany. The 15N analysis of denitrification-derived 15N-labelled N2 and N2O dissolved in water samples collected during the push–pull 15N tracer tests was performed using isotope ratio mass spectrometry (IRMS) in the lab and additionally for some tracer tests online in the field with a quadrupole membrane inlet mass spectrometer (MIMS) in order to test the feasibility of on-site real-time 15N analysis. Aquifer material from the same locations and depths as the push–pull injection points was incubated, and the initial and cumulative denitrification after 1 year of incubation (Dcum(365)) as well as the stock of reduced compounds (SRC) was compared with in situ measurements of denitrification. This was done to derive transfer functions suitable to predict Dcum(365) and SRC from Dr(in situ). Dr(in situ) ranged from 0 to 51.5 μg N kg−1 d−1. Denitrification rates derived from on-site isotope analysis using MIMS satisfactorily coincided with laboratory analysis by conventional IRMS, thus proving the feasibility of in situ analysis. Dr(in situ) was significantly higher in the sulfidic zone of both aquifers compared to the zone of non-sulfidic aquifer material. Overall, regressions between the Dcum(365) and SRC of the tested aquifer material with Dr(in situ) exhibited only a modest linear correlation for the full data set. However, the predictability of Dcum(365) and SRC from Dr(in situ) data clearly increased for aquifer samples from the zone of NO3−-bearing groundwater. In the NO3−-free aquifer zone, a lag phase of denitrification after NO3− injections was observed, which confounded the relationship between reactive compounds and in situ denitrification activity. This finding was attributed to adaptation processes in the microbial community after NO3− injections. It was also demonstrated that the microbial community in the NO3−-free zone just below the NO3−-bearing zone can be adapted to denitrification by NO3− injections into wells for an extended period. In situ denitrification rates were 30 to 65 times higher after pre-conditioning with NO3−. Results from this study suggest that such pre-conditioning is crucial for the measurement of Dr(in situ) in deeper aquifer material from the NO3−-free groundwater zone and thus for the prediction of Dcum(365) and SRC from Dr(in situ).

Knowledge about the spatial variability of denitrification rates and the lifetime of denitrification in nitrate-contaminated aquifers is crucial to predict the development of groundwater quality. Therefore, regression models were derived to estimate the measured cumulative denitrification of aquifer sediments after one year of incubation from initial denitrification rates and several sediment parameters, namely total sulphur, total organic carbon, extractable sulphate, extractable dissolved organic carbon, hot water soluble organic carbon and potassium permanganate labile organic carbon.For this purpose, we incubated aquifer material from two sandy Pleistocene aquifers in Northern Germany under anaerobic conditions in the laboratory using the 15N tracer technique. The measured amount of denitrification ranged from 0.19 to 56.2 mg N kg−1 yr−1. The laboratory incubations exhibited high differences between non-sulphidic and sulphidic aquifer material in both aquifers with respect to all investigated sediment parameters. Denitrification rates and the estimated lifetime of denitrification were higher in the sulphidic samples. For these samples, the cumulative denitrification measured during one year of incubation (Dcum(365)) exhibited distinct linear regressions with the stock of reduced compounds in the investigated aquifer samples. Dcum(365) was predictable from sediment variables within a range of uncertainty of 0.5 to 2 (calculated Dcum(365)/measured Dcum(365)) for aquifer material with a Dcum(365) > 20 mg N kg−1 yr−1. Predictions were poor for samples with lower Dcum(365), such as samples from the NO3− bearing groundwater zone, which includes the non-sulphidic samples, from the upper part of both aquifers where denitrification is not sufficient to protect groundwater from anthropogenic NO3− input. Calculation of Dcum(365) from initial denitrification rates was only successful for samples from the NO3−-bearing zone, whereas a lag-phase of denitrification in samples from deeper zones of NO3− free groundwater caused imprecise predictions.In our study, Dcum(365) of two sandy Pleistocene aquifers was predictable using a combination of short-term incubations and analysis of sediment parameters. Moreover, the protective lifetime of denitrification sufficient to remove NO3− from groundwater in the investigated aquifers is limited, which demonstrates the need to minimise anthropogenic NO3− input.

Denitrifying aquifers are sources of the greenhouse gas N2O. Isotopic signatures reflect processes of production and reduction of N2O, but it is not clear to which extent these can be used to quantify those processes. We investigated the spatial distribution of isotopologue values of N2O (delta O-18, average delta N-15, and N-15 site preference, SP) in two denitrifying sandy aquifers to study N2O production and reduction and associated isotope effects in groundwater. For the first time, we combined this approach with direct estimation of N2O reduction from excess-N-2 analysis. Groundwater samples were collected from 15 monitoring wells and four multilevel sampling wells and analysed for NO3-, dissolved N2O, dissolved O-2, excess N-2 from denitrification and isotopic signatures of NO3- and N2O. Both aquifers exhibited high NO3- concentrations with average concentrations of 22 and 15 mg N L-1, respectively. Evidence of intense denitrification with associated N2O formation was obtained from mean excess-N-2 of 3.5 and 4.3 mg N L-1, respectively. Isotopic signatures of N2O were highly variable with ranges of 17.6-113.2 parts per thousand (delta O-18), 55.4 to 89.4 parts per thousand (delta N-15(bulk)) and 1.8-97.9 parts per thousand (SP). delta N-15 and delta O-18 of NO3- ranged from 2.1 parts per thousand to 65.5 parts per thousand and from -5 parts per thousand to 33.5 parts per thousand, respectively.The relationships between delta N-15 of NO3- , delta N-15(bulk) and SP were not in good agreement with the distribution predicted by a Rayleigh-model of isotope fractionation. The large ranges of delta O-18 and SP of N2O as well as the close correlation between these values could be explained by the fact that N2O reduction to N-2 was strongly progressed but variable.We confirm and explain that a large range in SP and delta O-18 is typical for N2O from denitrifying aquifers, showing that this source signature can be distinguished from the isotopic fingerprint of N2O emitted from soils without water-logging. We conclude that isotopologue values of N2O in our sites were not suitable to quantify production or reduction of N2O or the contribution of different processes to the total N2O flux, apparently because these values were not only governed by individual pathways but eventually also by the spatial distribution of substrates and activity within the aquifers. These observations could be explained by the dynamics of N2O production, reduction and transport in water-saturated systems with heterogenic distribution of microbial activity and by a combination of diffusive and enzymatic isotope effects. (C) 2012 Elsevier Ltd. All rights reserved.