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A modified method for trapping and analysing 15N in NO released from soils Ronghua Kang, Jan Mulder, and Peter Doersch Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b05096 • Publication Date (Web): 03 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017
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Analytical Chemistry
A modified method for trapping and analysing leased from soils
15
N in NO re-
Ronghua Kang, Jan Mulder, and Peter Dörsch* Faculty of Environmental Sciences and Natural Resource Management, Norwegian University of Life Sciences, N-1432, Aas, Norway. Corresponding author: Peter Dörsch, Email:
[email protected], Tel.: +47 67231836 ABSTRACT: 15N isotope tracing is an effective and direct approach to investigate sources of nitric oxide (NO) formed in soils. However, NO is highly reactive and rapidly converted to nitrogen dioxide (NO2) in the presence of ozone, making it impossible to directly measure 15N in NO. Various wet-chemical methods for conversion of NO to nitrite (NO2-) and nitrate (NO3-) have been proposed for 15N analysis in high-concentration NO sources, such as combustion processes. In contrast, NO concentrations in soil surface-near air are usually small (ppbv-range), posing major challenges to conversion efficiency and blank correction. Here we present a modified method in which NO is oxidized quantitatively to NO2 by chromium trioxide (CrO3), before conversion to NO2and NO3- in an alkaline hydrogen peroxide (H2O2) solution. A denitrifier method was used to reduce NO2- and NO3- in the trapping solution quantitatively to nitrous oxide (N2O) for subsequent 15N analysis. NO trapping efficiencies of > 85% were obtained with 50 ppb NO in a 0.5 L min-1 air stream bubbling through a solution of 1.2 M H2O2 and 0.5 M NaOH. In a laboratory test with distinct 15 NO abundances, the overall precision was 0.29‰ (δ-values) for natural abundance NO and 0.13 atom% for labeled NO, suggesting that our method can be used for both natural abundance studies and 15N labeling experiments. In a soil incubation experiment with 15NH4NO3, NH415NO3 or Na15NO2 amendments, we found distinct 15N abundances in NO, indicating that our method is well suited to investigate NO sources in soils.
INTRODUCTION
NO have to fulfil two criteria: i) NO has to be converted to a stable N species and ii) resulting NO2-/NO3- concentrations should be large enough allowing for 15N analysis by Isotope Ratio Mass Spectrometry (IRMS). Both processes should be efficient, precise, and reproducible and proceed with as little 15 N fractionation as possible. Various chemical oxidizers have been proposed for the effective conversion of NO and NO2 to NO2- and NO3-, such as acidic or alkaline hydrogen peroxide (H2O2)15, 16, mixtures of potassium permanganate and sodium hydroxide (KMnO4/NaOH)17, Fenton reagent18, sodium persulfate in H2O219, or a combination of ultra violet light (UV) and H2O220. However, most procedures were developed and tested for high NOx concentrations (e.g. in exhaust gases) and not for NO at low concentrations (as in dynamically sampled soil emissions). Heaton (1990)16 trapped NOx in alkaline H2O2 and found significantly different ranges of δ15N values for the two main sources of anthropogenic NOx, vehicle exhaust (-13 to 2‰) and exhaust from coal-fired boilers (+6 to +13‰). Recently, Walters et al. (2015)21 collected NOx from vehicles, including gasoline and diesel-powered engines, using sulfuric acid (H2SO4) and H2O2 as trapping agents, and obtained δ15N values ranging from -19.1 to 9.8‰. These studies considered isotopic fractionation during the NOx emission process, but not during NOx collection and conversion. If the efficiency for NOx conversion to NO3- and NO2- is small, the faster reaction of the lighter 14NO with the trapping agent may cause isotopic
Nitrogen oxides (NOx) consisting predominately of nitric oxide (NO) and nitrogen dioxide (NO2) are important precursors of acid rain1 and play a central role in photochemical pollution2. More than 50% of atmospheric NOx derives from combustion processes (fossil fuel, biomass). Microbial nitrogen (N) transformations in soils contribute an estimated 16 30% to the global NOx budget3, 4. In soils, NO is formed as a by-product of microbial nitrification5 or as an intermediate of denitrification6, through nitrosylation between nitrite (NO2-) and organic N7, 8 or through dismutation of nitrous acid (HNO2), the protonated form of NO2- 9. The complexity of NO sources in soil makes it difficult to study source processes, thus stable isotope approaches are needed to identify precursors and processes. 15 N labeling of soil N pools is commonly used to apportion N2O or N2 to biogenic processes in soils 10-12 and could in principal also be used to apportion NO to its source processes. However, the determination of 15N in NO is not straightforward, as NO is highly reactive and rapidly converted to NO2 in the presence of atmospheric ozone. In addition, background NO concentrations in the surface-near air are usually small (ppbv) and soil emitted NO becomes strongly diluted when employing dynamic chamber approaches with typically high through-flow rates13, 14. Therefore, methods for NO trapping allowing for the subsequent analysis of 15N abundance in
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fractionation, resulting in an underestimation of the 15N abundance in the trapped NOx. Recently, Fibiger et al. (2014)17 presented a robust method of collecting and converting NOx from diesel engines to NO3- with KMnO4 and NaOH reporting 100% conversion efficiency and thus no fractionation. However, the study of Fibiger et al. (2014)17 also demonstrated that methods based on wet-chemical conversion are fraught by potentially high NO3- backgrounds in the trapping reagent, which are difficult to remove. So far, few studies have used 15NO for source separation in soil. Li et al. (2008)22 reported δ15N values in NO released from soil after fertilizer application, by converting the NO to NO2 on a solid oxidizer (CrO3/H3PO4), followed by entrapment in KOH/guaiacol as NO2-. Most other studies on NO turnover in soils23, 24 based on 15N techniques have used Ogawa filters to assess microbial25, 26 or abiotic NO-producing mechanisms27. However, the trapping efficiency and isotopic fractionation associated with these techniques are unknown. Myers and Overcamp (2002)28 found that the removal efficiency of alkaline H2O2 solution for NO2 was significantly greater than for NO. Thus, the oxidation of NO to NO2 prior to trapping increases the overall NO conversion efficiency in alkaline H2O2 solution. Based on the studies of Heaton (1990)16 and Myers and Overcamp (2002)28, we designed a trapping method in which NO is oxidized to NO2 by chromium trioxide (CrO3) prior to conversion to NO2- and NO3- in a H2O2-NaOH solution. We further tested whether the resulting NO2- and NO3- in the trapping solution could be converted to N2O by a modified denitrifier method11, 29 for convenient 15N analysis by IRMS.
detection limit (~ 1.0 µM). The concentration of NO2 at the outlet of the trapping bubbler was measured with a NOx analyzer (LMA-3D, Drummond Technology, Canada). To find the optimum conditions for NO trapping (H2O2 concentration, NaOH concentration, and gas flow rate), we conducted experiments in which only one variable was changed at a time. For instance, we kept the NaOH concentration, gas flow rate and inlet NO concentration constant, and increased H2O2 concentration from zero until obtaining maximum trapping efficiency.
EXPERIMENTAL SECTION
To test the applicability of biological methods to convert the resulting NO3- to gaseous N for IRMS analysis, we added manganese dioxide (MnO2) as a catalyst to remove excess H2O2. Excess H2O2 had to be removed, as it would inhibit growth of Pseudomonas aureofaciens, a denitrifying bacterial strain that quantitatively converts NO3- to N2O under anoxic conditions (“denitrifier method”31, 32). A single-use syringe filter (0.45 µm, VWR International, Norway) was used to remove MnO2. After filtration and neutralizing the solution to pH ~7 (by adding 5 M HCl; Supporting Information Ch.1), the solution was inoculated with the growing P. aureofaciens culture. The resulting N2O was analyzed by isotope ratio mass spectrometry (IRMS) coupled to a pre-concentration unit (PreCon-GC-IRMS, Thermo Finnigan MAT, Bremen, Germany). In order to obtain ion beams of constant size, the sample volume was adjusted corresponding to 100 nmol N per bottle. International standards, IAEA N3 (δ15N = 4.7 ‰ air N2) and USGS 32 (δ15N = 180.0 ‰ air N2) were included in each sample series for data correction. Yu et al. (2016)33 and Zhu et al. (2013)11 showed that a modified denitrifier method can be used to convert NO3- in soil water samples, as well as in 0.25 M KCl soil extracts, obtained by dilution from 2M KCl soil extracts. Neutralization of NaOH in the trapping process with HCl results in Na+ and Cl- concentrations in the range of 0.2 to 1.0 M. We tested the efficiency to convert NO3- to N2O by P. aureofaciens with a sample solution containing 0.5 M NaCl in addition to NaNO3 (13.5 µM) and for a standard solution containing KNO3 only.
Figure 1. Schematic diagram of the experimental setup: 1. Gas cylinder with 10 ppm NO in N2; 2. Gas cylinder with synthetic air; 3. Gas mixer; 4. 20 L Tedlar gas sampling bag; 5. Valve for adjusting the gas flow rate; 6. Pump; 7. Three-way valve; 8. CrO3 oxidizer; 9. Trapping bubbler with H2O2-NaOH solution (diameter: 1.0 cm, height: 30 cm).
Experimental reagents. To oxidize NO, we used chromium oxide (CrO3) prepared according to Levaggi et al. (1974)30. A 17% CrO3 solution was absorbed on the surface of molecular sieves, 1/16 inch pellets (Linde 5Å). After drying at 105oC and hydration with ambient air, a red oxidizer was obtained. Analytically pure 30% H2O2 (9.0 M) (VWR International, Norway) was diluted with Milli-Q water to 0.6, 1.2, 1.8, 2.4 and 2.7 M, respectively. Solid NaOH (Merck, Germany) was dissolved in Milli-Q water to prepare NaOH solution at concentrations of 0.4, 0.8, 1.0, 1.6 and 2.0 M, respectively. Equal volumes of diluted H2O2 and NaOH were mixed to yield an alkaline H2O2 solution with H2O2 concentrations ranging from 0.3 to 1.35 M and NaOH concentrations ranging from 0.2 to 1.0 M. Concentrated HCl (37% HCl (12 M), VWR International, Norway) was diluted to 5 M HCl with Milli-Q water to neutralize NaOH after trapping. Experimental setup. The NO trapping system is shown in figure 1. Different concentrations of NO were prepared from a certified 10 ppm NO standard (in N2) and synthetic air using a gas mixer (Beijing Sevenstar Electronics Co. China). The gas containing NO of known concentration was pumped through the oxidizer tube (containing CrO3) to convert NO to NO2. Next, the gas including the produced NO2 (and remaining NO, Supporting Information Ch. 1), was passed through a trapping bubbler (Diameter: 1 cm, Height: 30 cm, containing the H2O2NaOH mixture), where NO2 and NO were trapped in the solution as NO3-. The concentration of NO2- was always below
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Analytical Chemistry
tion limit: ~ 1.0 µM). The δ15N value of the blank trapping solution was -3.43 ± 0.22‰ and this value, together with the amount of blank NO3- was used to correct δ15N of sampled NO. Applying the method to soil incubations. To apply the method to a range of realistic NO fluxes, a laboratory soil experiment with labeled NH4+, NO3- and NO2- was carried out, involving 15N measurements in NO released at different soil moistures. Each 20 g (15.6 g dry weight equivalent) of disturbed acidic surface soil (0 - 5 cm; pH 4.3; bulk density 1.2 g cm-3), characterized as an Orthic Acrisol36, from a subtropical forested catchment at Tie Shan Ping, Chongqing, SW China (for details see37) was loosely packed into twelve 250 ml wideneck glass bottles (40.3 cm2 surface area). The caps of the bottle were equipped with inlet and outlet tubing, through which the headspace could be flushed continuously with synthetic air at a flow rate of 0.5 L min-1. In order to minimize gas diffusion limitation, the incubated soil was not compacted and the soil layer was only 0.7 cm thick. Three labeling treatments were applied while one treatment remained unlabeled, all of them in three replicates. For labeling, 2 ml solution of either 15 NH4NO3 (98 atom%, 17.2 mM 15N), NH415NO3 (98 atom%, 17.2 mM 15N), Na15NO2 (98 atom%, 5 mM 15N), or distilled water were injected into the soil followed by thorough mixing. The total N addition for both NH4NO3 treatments was 68.8 µmol, which roughly equals 0.25 g N m-2. For the NaNO2 treatment, the N addition amounted to 10 µmol N. After N addition and mixing, synthetic air was pumped through the headspace of the bottles to flush NO released from the soil into the trapping solution. To increase the amount of trapped NO relative to background NO3-, the trapping time for each sample was set to 60 min. Before trapping, the NO concentration in the gas stream was measured by a NOx analyzer (LMA3D, Drummond Technology, Canada) to calculate the NO flux according to equation (2):
Sample volumes of 1 to 5 ml were He-washed in 120 ml serum bottles sealed with permeable septa. Thereafter, 2 ml of the P. aureofaciens culture was injected and the bottles were incubated at room temperature. To quantify the conversion efficiency, the accumulation of N2O in the headspace of the bottles was monitored by a robotized GC setup34. The conversion efficiency of NO3- to N2O as a function of time was expressed as the percentage of N2O-N in headspace relative to the amount of NO3--N added. Testing the method. During our optimization experiments, we found that trapping efficiency slightly decreased with decreasing NO concentration. To check whether incomplete trapping would induce fractionation, a standard gas mixture containing 10 ppm non-labeled NO in N2 was diluted with synthetic air, producing gas with 50, 100 and 200 ppb NO. The trapping time for each sample was 20 min, and the volume of the trapping solution was 10 ml, resulting in 12.7 cm high trapping column. After the NO was trapped, the NO3- and NO2- were converted and analyzed for δ15N as described above. Griess reaction with or without VCl335 was used to determine NO3- and NO2- concentrations in blank and samples spectrophotometrically. The concentrations were used to calculate the blank-to-sample ratio of N needed to correct the measured δ15N. Since no certified 15NO standard is available for testing the reproducibility and accuracy of the 15N abundance in NO, we produced NO with different atom% 15N by reducing a mixture of labeled (98 atom%) Na15NO2 (Sigma Aldrich, USA) and unlabeled NaNO2 with acidic NaI (eq. 1, Supporting Information Ch. 2). The resulting enrichments in NO2- were 1.25 atom% and 2.28 atom% 15N, as analyzed by IRMS after direct conversion of NO2- by the denitrifier method. The chemical conversion of NO2- to NO was carried out in a He-washed 120 ml serum bottle, resulting in concentrated NO (Supporting Information Ch. 2). In order to perform the trapping test at realistically low NO concentration (~ 100 ppb), the labeled NO was transferred to a He-washed 20 L Tedlar bag using a gas-tight syringe, where the NO was diluted with synthetic air (Purity: 99.9995%). We also determined the conversion efficiency of NaNO2 to NO by measuring NO concentration by an NO analyzer (M200E, Advanced Pollution Instrumentation, Inc, USA) and pressure increase by a digital manometer (Keller Leo 2, Switzerland) in the conversion bottle. This was done to evaluate the possibility for 15N fractionation due to incomplete conversion of the enriched NO2-. 15NO, diluted with synthetic air in the gas bag, was trapped in H2O2-NaOH solution under conditions previously determined to yield optimum conversion efficiency.
F =
∗ ∗
∗
(2)
where F is the NO flux (µg N m-2 h-1), Cs the NO concentration in the outlet gas stream (ppb), Cr the NO concentration in the inlet gas stream (ppb) (Cr = 0 in the present experiment because synthetic air was used), Q the gas flow rate (L min-1), A the cross-section area of the soil surface (m2), N the atomic weight of nitrogen (14.007 g mol-1) and Vm the molar volume of gas at room temperature (L mol-1). Nitric oxide emissions from soils strongly depend on soil moisture content25, 38. At large soil moisture levels (> 60% water filled pore space; WFPS), NO production is mainly driven by microbial denitrification, whereas nitrification becomes an important NO source at intermediate and smaller soil moisture content39, 40, 25. To provoke NO formation from different soil N pools, we let the initially moist soil microcosms dry out during the experiment by flushing the headspace continuously with dry synthetic air. Moisture loss was recorded as weight loss. We selected five time points during dry out to measure NO fluxes and trapped released NO. The percent recovery of applied 15N in emitted NO was calculated according to Nadelhoffer et al. (2004)41, as
2NaNO2 + 2NaI + 4CH3COOH ⟶ 2NO + I2 + 4CH3COONa + 2H2O (1) Industrially produced, 30% H2O2 solution (9.0 M) contains considerable amounts of NO3- (in our case ~20 µM) which could result in significant errors when only a small amount of NO is trapped. Since trapping efficiency of H2O2-NaOH for NO2 is larger than for NO, we could dilute the H2O2 solution to 1.2 M, thus lowering the NO3- background to 5.0 ± 1.0 µM. No detectable NO3- was found in the NaOH solution (Detec-
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% 15Nrec_NO = m NO, pool * (atom% om% 15NNO, ref) / m tracer * 100
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rate increased from 0.1 of 0.5 L min-1 (Fig. 2c). Under optimum conditions (1.2 M H2O2, 0.5 M NaOH, and 0.5 L min-1 gas flow rate), we found that the efficiency increased with increasing inlet NO concentration (Fig. 2d). However, even with low inlet NO concentration (> 50 ppb NO), a trapping efficiency of > 85% could be obtained. The quantitative conversion of trapped NO3- to N2O by the denitrifier method required about 55 h for a standard solution with 100 nmol KNO3 in Milli-Q water. With a background concentration of 0.5 M NaCl stemming from the neutralization of the 0.5 M NaOH trapping solution with HCl, about 72 h were required until 98% of the NO3- was converted to N2O (Fig. 3). This finding is in agreement with known toxicities of salts on soil microbial processes45. The quantitative conversion of sampled NO3- to N2O indicated that the denitrifier method is suitable for our samples.
NNO, pool – at(3)
where % 15Nrec_NO is the mass percentage of 15N recovered in NO, m NO, pool the cumulative N mass of emitted NO (g N m-2), atom% 15NNO, pool and atom% 15NNO, ref the 15N atom% of NO released from labeled and non-labeled pools, respectively, and m tracer the total 15N input (15N dose of labeled fertilizer, g N m-2).
RESULTS and DISCUSSION Optimizing the H2O2 – NaOH trapping system. Increasing the H2O2 concentration in the trapping solution from 0.3 to 0.6 M (the NaOH concentration and gas flow rate were constant at 0.5 M and 0.5 L min-1, respectively) caused an increase of the trapping efficiency from 90% to 96% (Fig. 2a). This likely reflects the increase in free radicals (•OH and •OOH) in the tested concentration range leading to faster and more efficient oxidation of NO2 to NO3- 42, 43. Increasing the H2O2 concentration above 0.6 M did not improve the trapping efficiency appreciably. A similar pattern of increased trapping efficiency with increasing H2O2 concentration was observed in other scrubbing systems20, where UV/H2O2 was used to remove NO from flue gas.
Figure 3. Conversion of a KNO3 standard dissolved in Milli-Q water and NO trapped as NO3- in a final matrix of 0.5 M NaCl to N2O by Pseudomonas aureofaciens. Both sources of NO3- were at natural abundance.
Fractionation and reproducibility of 15N in NO. Since the trapping efficiency depended on the NO concentration in the inlet stream (Fig. 2d), we used this relationship to test whether the 15NO value would be affected by incomplete trapping. We used a commercially available gas cylinder (NO in N2) and diluted with different amounts of synthetic air. After trapping and conversion to N2O (denitrifier method), the δ15N of NO was -23.4 ± 0.29‰, irrespective of the NO concentrations entering the trapping solution (50, 100, to 200 ppb NO; Tab. 1). Hence, trapping efficiency had no effect on δ15N of NO. The relative standard deviations averaged 1.2%, which is similar to findings in other 15N tracer studies46, 47. The consistent δ15N in trapped NO indicates that there was no fractionation during trapping and conversion. Furthermore, even though the background N in blanks relative to the sample was large (> 0.3), especially for samples with NO trapped at low concentration, our experimental results suggest that the blank correction can be carried out with high precision, as long as the NO3- background is constant due to the addition of a fixed amount of 1.2 M H2O2.
Figure 2. Effect of trapping conditions on NO trapping efficiency (Trapping efficiency = (C gas mixer – C outlet) / C gas mixer): (a) H2O2 concentration; (b) NaOH concentration; (c) gas flow rate; (d) inlet NO concentration. Each figure shows the effect of one variable while other variables were held constant at values noted in the panels.
The trapping efficiency was strongly dependent on alkalinity and increased from 30% without NaOH addition to 90% with 0.2 M NaOH (Fig. 2b), most likely due to the greater solubility of acidic NO2 in alkaline solution. Alternatively, H2O2 in alkaline solution produces more free radicals44, leading to more efficient oxidation of NO2 to NO3-. Increasing the concentration of NaOH to > 0.5 M did not further enhance the trapping efficiency. The trapping efficiency depended weakly on the gas flow rates and inlet NO concentrations (Fig. 2c and 2d). The trapping efficiency increased from 76% to 96% as the gas flow
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Analytical Chemistry
Table 1. Reproducibility of δ15N-NO for sampling from the same source with different NO concentrations NO concentrations (ppb)
δ15N (‰)
Ratio of blank N* to total N
50
-23.36
0.62
50
-23.31
0.73
100
-23.22
0.52
100
-23.03
0.52
100
-23.35
0.57
200
-23.95
0.37
200
-23.24
0.38
200
-23.58
0.39
200
-23.78
indicates that, except for the NaNO2 treatment, the amount of added N was small relative to the mineral N pool in the soil37. In contrast, significant differences in the 15N atom% of NO were observed (Fig. 4b), with larger values for the NH415NO3 treatment as compared with the 15NH4NO3 treatment, indicating that NO3--dependent denitrification was an important source of NO over a wide range of soil moistures (90 - 20% WFPS). The 15N atom% of NO in the 15NH4NO3 treatment was initially small and increased slowly, reaching a peak at 16% WFPS, most probably reflecting increased nitrification activity at smaller soil moisture48. The significantly smaller 15N recovery in the 15NH4NO3 treatment (Tab. 3) was likely due to the dilution of 15NH4+ with unlabeled NH4+ from the relatively large native soil pool37, and/or fast immobilization of added NH4+ in microbial biomass29 and on clay surfaces. Adding Na15NO2 to this acidic soil resulted in instantaneous NO release, with large 15N atom% values (Fig. 4). Cumulative recovery of 15N in NO (~ 84%) was largest in the Na15NO2 treatment (Tab. 3). A similar immediate recovery of 15NO2- in NO has been observed in other studies24, 49, indicating that chemical decomposition of NO2- is an important source of NO emitted from soil.
0.42 -
*The blank N comes from the NO3 contamination of the H2O2 solution. The trapping time was 20 min, and the volume of the trapping solution 10 ml
Measurements of 15N abundance were highly reproducible, both for NO at natural 15N abundance (Tab. 1) and for NO enriched in 15N (Tab. 2). The 15N atom% of trapped NO, produced from 1.25 and 2.28 atom% enriched NaNO2, averaged 0.86% and 1.91% with standard deviation of 0.13% (n = 4) and 0.15% (n = 6), respectively (Tab. 2). Measured atom% 15N in NO was systematically smaller than that in Na15NO2 determined directly by the denitrifier method. This deviation was probably due to incomplete conversion (68.6 ± 5.6%, n = 6) of NO2- to NO (eq. 1), which may result in NO depleted in 15N relative to its precursor (NO2-). Table 2. Reproducibility of 15N atom% in trapped NO produced from 15N enriched NaNO2 Atom% of 15N in Na15NO2 (%)
Atom% of 15N in sampled NO (%)
Mean ± SD
0.83 1.25
0.80 0.77
0.86 ± 0.13
1.06 1.79 2.09 2.28
1.97 2.06
1.91 ± 0.15
1.82 1.72 The trapping time was 20 min, and the volume of trapping solution 10 ml. SD: standard deviation
Soil incubation experiments. In laboratory incubations, we found no significant difference in NO net-fluxes between the treatments with labeled N and the unamended control throughout the dry-out period, with the exception of the Na15NO2 treatment. The latter showed instantaneous high release rates, which levelled off after 15 hour (Fig. 4a). This
Figure 4. (a) NO fluxes and (b) atom% of 15N in NO during dryout. Error bars are standard deviations. The trapping time was 60 min, and the volume of trapping solution 10 ml
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Table 3. Mean cumulative recovery (± standard deviation; n=3) of added 15N in NO (%) 0.5 h
6h
24 h
48 h
72 h
NH4NO3
0.01± 0.002
0.08± 0.02
0.82± 0.31
2.53± 0.88
3.53± 1.02
NH415NO3
0.15± 0.04
1.85± 0.40
13.18± 2.76
28.64± 8.38
33.25± 9.30
Na15NO2
18.01± 0.02
40.91± 12.01
74.02± 16.22
81.93± 27.91
83.16± 17.13
15
Absolute and relative standard deviations of the mean 15NO atom% values obtained in the soil experiment (n = 3) are shown in table 4 together with WFPS values. The relative standard deviation of 15NO atom% increased during the dryout, probably because the variability in soil moisture within treatments increased, inducing variable portions of NO produced from labeled and unlabeled N species. Similar standard deviations have been reported from other soil experiments using Ogawa filter24 or GC-QMS48.
Table 4. Mean atom% 15N in NO (n=3) released from soil amended with differently labeled N-substrates at different mean WFPS (n=3) during dry-out 15
15
NH4NO3
NH415NO3
Na15NO2
Control
Mean ± SD
N atom%
WFPS%
0.5 h
6h
24 h
48 h
72 h
0.5 h
6h
24 h
48 h
72 h
0.57 ± 0.07
0.68 ± 0.07
0.95 ± 0.23
1.44 ± 0.21
0.95 ± 0.38
88.2 ± 1.4
74.6 ± 1.6
45.5 ± 1.6
16.4 ± 2.8
3.3 ± 1.6
RSD, %
12.3
10.3
24.2
14.6
40.0
1.6
2.1
3.5
17.1
48.5
Mean ± SD
5.35 ± 1.35
5.73 ± 1.63
8.10 ± 1.38
6.91 ± 0.29
1.89 ± 0.50
87.2 ± 2.1
75.5 ± 2.8
47.4 ± 2.8
16.4 ± 0.1
4.2 ± 1.6
RSD, %
25.2
28.4
17.0
4.2
26.5
2.4
3.7
5.9
0.6
38.1
Mean ± SD
24.22 ± 0.79
6.99 ± 0.84
2.94 ± 0.37
2.30 ± 0.37
0.56 ± 0.09
89.6 ± 2.8
77.4 ± 4.3
49.3 ± 4.3
20.2 ± 5.9
5.2 ± 2.8
RSD, %
3.3
12.0
12.6
16.1
16.1
3.1
5.6
8.7
29.2
53.8
n = 1*
0.38
0.38
0.38
0.37
0.37
87.7 ± 3.2
76.5 ± 3.2
49.3 ± 4.3
21.1 ± 4.3
5.2 ± 2.8
3.6
4.2
8.7
20.4
53.8
SD: standard deviation; RSD: relative standard deviation. * Two of three replicate samples for the control treatment had to be omitted 15 because of N carry-over from samples with highly enriched NO during IRMS analysis.
CONCLUSIONS
Thus, the method has potential for atmospheric NO source tracing. In a soil incubation experiment with labeled NH4+, NO3- and NO2-, distinct 15NO atom% values were found, demonstrating that the method is well suited to investigate different NO sources in soil.
Trapping of NO as NO2 (after CrO3 oxidation) in a solution with 1.2 M H2O2 and 0.5 M NaOH was shown to be a robust method to collect and concentrate NO from air at concentrations as low as 50 ppb with satisfactory efficiency (~ 85%) and trapping time (~ 20 min). The precision of the 15N analysis in the resulting NO3- (including the conversion of NO3- to N2O) was 0.29‰ for NO at natural abundance and 0.13 atom% for labeled NO. This makes the method a feasible approach to determine 15N in NO released from soils at typically small mixing ratios, both at natural abundance and from experimentally enriched sources. Interference with background NO3contained in H2O2 is a problem, especially when the amount of trapped NO is small, but could be reduced to acceptable levels by diluting the H2O2 to 1.2 M (5.0 µM NO3-). Moreover, the ratio of blank to total N can be decreased by prolonging the trapping time, so that the method should also work for low ambient NO concentrations. Consistent δ15N values in NO obtained from repeated trapping experiments (with the same source of NO) suggested that blank correction could be carried out successfully as long as the same batch of H2O2 is used.
Corresponding Author *Email:
[email protected] Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT Ronghua Kang thanks Norwegian Quota program for supporting her PhD study. This study is supported by the Norwegian Research Council (project 209696/E10) ‘Forest in South China: an important sink for reactive nitrogen and a regional hotspot for N2O?’ We thank Prof. Duan Lei from Tsinghua University for providing the gas mixer. We are grateful to Trygve Fredriksen for laboratory assistance.
REFERENCES
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(25) Roelle, P. A.; Aneja, V. P.; Gay, B.; Geron, C.; Pierce, T. Atmos. Environ. 2001, 35, 115-124. (26) Schindlbacher, A.; Zechmeister-Boltenstern, S.; ButterbachBahl, K. J. Geophys. Res-Atmos. 2004, 109. (27) Venterea, R. T.; Rolston, D. E. J. Geophys. Res-Atmos. 2000, 105, 15117-15129. (28) Myers, E. B.; Overcamp, T. J. Environ. Eng. Sci. 2002, 19, 321-327. (29) Yu, L. F. thesis. NMBU, 2016. (30) Levaggi, D.; Kothny, E. L.; Belsky, T.; Vera, E. D.; Mueller, P. K. Environ. Sci. Technol. 1974, 8, 348-350. (31) Sigman, D. M.; Casciotti, K. L.; Andreani, M.; Barford, C.; Galanter, M.; Bohlke, J. K. Anal. Chem. 2001, 73, 4145-4153. (32) Casciotti, K. L.; Sigman, D. M.; Hastings, M. G.; Bohlke, J. K.; Hilkert, A. Anal. Chem. 2002, 74, 4905-4912. (33) Yu, L. F.; Zhu, J.; Mulder, J.; Dörsch, P. Global. Change. Biol. 2016, 22, 3662-3674. (34) Molstad, L.; Dörsch, P.; Bakken, L. R. J. Microbiol. Meth. 2007, 71, 202-211. (35) Doane, T. A.; Horwath, W. R. Anal. Lett. 2003, 36, 27132722. (36) IUSS Working Group WRB. World Soil Resources Reports No. 106 2014, FAO, Rome. (37) Zhu, J.; Mulder, J.; Wu, L. P.; Meng, X. X.; Wang, Y. H.; Dörsch, P. Biogeosciences. 2013, 10, 1309-1321. (38) Homyak, P. M.; Sickman, J. O. J. Arid. Environ. 2014, 103, 46-52. (39) Cardenas, L.; Rondon, A.; Johansson, C.; Sanhueza, E. J. Geophys. Res-Atmos. 1993, 98, 14783-14790. (40) Ormeci, B.; Sanin, S. L.; Peirce, J. J. J. Geophys. Res-Atmos. 1999, 104, 1621-1629. (41) Nadelhoffer, K. J.; Colman, B. P.; Currie, W. S.; Magill, A.; Aber, J. D. Forest. Ecol. Manag. 2004, 196, 89-107. (42) Kuropka, J.; Mieczyslaw, A.; Gostomczyk, M.A. Environ. Protect. Eng. 1990, 16, 86–97. (43) Muruganandham, M.; Yang, J. S.; Wu, J. J. Ind. Eng. Chem. Res. 2007, 46, 691-698. (44) Brooks, R. E.; Moore, S. B. Cellulose. 2000, 7, 263-286. (45) Rath, K. M.; Maheshwari, A.; Bengtson, P.; Rousk, J. Appl. Environ. Microb. 2016, 82, 2012-2020. (46) Zhang, L.; Altabet, M. A.; Wu, T. X.; Hadas, O. Anal. Chem. 2007, 79, 5297-5303. (47) Liu, D. W.; Fang, Y. T.; Tu, Y.; Pan, Y. P. Anal. Chem. 2014, 86, 3787-3792. (48) Russow, R.; Sich, I.; Neue, H. U. Chemosphere. 2000, 2, 359366. (49) Sich, I.; Russow, R. Rapid. Commun. Mass. Sp. 1999, 13, 1325-1328.
(1) Larssen, T.; Lydersen, E.; Tang, D. G.; He, Y.; Gao, J. X.; Liu, H. Y.; Duan, L.; Seip, H. M.; Vogt, R. D.; Mulder, J.; Shao, M.; Wang, Y. H.; Shang, H.; Zhang, X. S.; Solberg, S.; Aas, W.; Okland, T.; Eilertsen, O.; Angell, V.; Liu, Q. R.; Zhao, D. W.; Xiang, R. J.; Xiao, J. S.; Luo, J. H. Environ. Sci. Technol. 2006, 40, 418-425. (2) Altshull, A. P.; Bufalini, J. J. Environ. Sci. Technol. 1971, 5, 39-&. (3) Davidson, E. A.; Kingerlee, W. Nutr. Cycl. Agroecosys. 1997, 48, 37-50. (4) Jaegle, L.; Steinberger, L.: Martin, R. V.; Chance, K. Faraday. Discuss. 2005, 130, 407-423. (5) Wrage, N.; Velthof, G. L.; van Beusichem, M. L.; Oenema, O. Soil. Biol. Biochem. 2001, 33, 1723-1732. (6) Firestone, M. K.; Davidson, E. A. Life. Sci. R. 1989, 47, 7-
21. (7) Shoun, H.; Kim, D. H.; Uchiyama, H.; Sugiyama, J. Fems. Microbiol. Lett. 1992, 94, 277-281. (8) Spott, O.; Russow, R.; Stange, C. F. Soil. Biol. Biochem. 2011, 43, 1995-2011. (9) Venterea, R. T.; Rolston, D. E.; Cardon, Z. G. Nutr. Cycl. Agroecosys. 2005, 72, 27-40. (10) Panek, J. A.; Matson, P. A.; Ortiz-Monasterio, I.; Brooks, P. Ecol. Appl. 2000, 10, 506-514. (11) Zhu, J.; Mulder, J.; Bakken, L.; Dorsch, P. Biogeochemistry. 2013, 116, 103-117. (12) Morse, J. L.; Bernhardt, E. S. Soil. Biol. Biochem. 2013, 57, 635-643. (13) Butterbach-Bahl, K.; Kock, M.; Willibald, G.; Hewett, B.; Buhagiar, S.; Papen, H.; Kiese, R. Global. Biogeochem. Cy. 2004, 18. (14) Pilegaard, K.; Skiba, U.; Ambus, P.; Beier, C.; Bruggemann, N.; Butterbach-Bahl, K.; Dick, J.; Dorsey, J.; Duyzer, J.; Gallagher, M.; Gasche, R.; Horvath, L.; Kitzler, B.; Leip, A.; Pihlatie, M. K.; Rosenkranz, P.; Seufert, G.; Vesala, T.; Westrate, H.; ZechmeisterBoltenstern, S. Biogeosciences. 2006, 3, 651-661. (15) Snape, C. E.; Sun, C. G.; Fallick, A. E.; Irons, R.; Haskell, J. Abstr. Pap. Am. Chem. S. 2003, 225, U843-U843. (16) Heaton, T. P. E. Tellus. 1990, 42, 304-307. (17) Fibiger, D. L.; Hastings, M. G.; Lew, A. F.; Peltier, R. E. Anal. Chem. 2014, 86, 12115-12121. (18) Guo, R. T.; Pan, W. G.; Zhang, X. B.; Ren, J. X.; Jin, Q.; Xu, H. J.; Wu, J. Fuel. 2011, 90, 3295-3298. (19) Wang, Z. P.; Wang, Z. W.; Ye, Y.; Chen, N.; Li, H. W. Chem. Eng. Sci. 2016, 145, 133-140. (20) Liu, Y. X.; Zhang, J. Ind. Eng. Chem. Res. 2011, 50, 38363841. (21) Walters, W. W.; Goodwin, S. R.; Michalski, G. Environ. Sci. Technol. 2015, 49, 2278-2285. (22) Li, D. J.; Wang, X. M. Atmos. Environ. 2008, 42, 4747-4754. (23) Felix, J. D.; Elliott, E. M. Atmos. Environ.2014, 92, 359-366. (24) Homyak, P. M.; Blankinship, J. C.; Marchus, K.; Lucero, D. M.; Sickman, J. O.; Schimel, J. P. P. Natl. Acad. Sci. USA. 2016, 113, E2608-E2616.
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