Novel Method for Nitrogen Isotopic Analysis of Soil-Emitted Nitric

Article

A novel method for nitrogen isotopic analysis of soil-emitted nitric oxide (NO) Zhongjie Yu, and Emily M. Elliott Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 03 May 2017 Downloaded from http://pubs.acs.org on May 8, 2017

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Environmental Science & Technology

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A novel method for nitrogen isotopic analysis of soil-

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emitted nitric oxide (NO)

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Zhongjie Yu* and Emily M. Elliott

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University of Pittsburgh, Department of Geology and Environmental Science, Pittsburgh,

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PA 15260, USA

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* Corresponding author. Phone: (973) 718 0238; Fax: (412) 624 8780; Email: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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The global inventory of NOx (NOx=NO+NO2) emissions is poorly constrained with a

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large portion of the uncertainty attributed to soil NO emissions that result from soil

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abiotic and microbial processes. While natural abundance stable N isotopes (δ15N) in

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various soil N-containing compounds have proven to be a robust tracer of soil N cycling,

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soil δ15N-NO is rarely quantified due to the measurement difficulties. Here, we present a

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new method that collects soil-emitted NO through NO conversion to NO2 in excess ozone

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(O3) and subsequent NO2 collection in a 20% triethanolamine (TEA) solution as nitrite

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and nitrate for δ15N analysis using the denitrifier method. The precision and accuracy of

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the method were quantified through repeated collection of an analytical NO tank and

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inter-calibration with a modified EPA NOx collection method. The results show that the

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efficiency of NO conversion to NO2 and subsequent NO2 collection in the TEA solution

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is >98% under a variety of controlled conditions. The method precision (1σ) and

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accuracy across the entire analytical procedure are ±1.1‰. We report the first analyses of

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soil δ15N-NO (-59.8‰ to -23.4‰) from wetting-induced NO pulses at both laboratory

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and field scales that have important implications for understanding soil NO dynamics.

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TOC Art

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INTRODUCTION

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Emissions of nitrogen oxides (NOx = NO + NO2) degrade air quality and affect global

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tropospheric chemistry,1, 2 posing a significant danger to ecosystem and human health.3-6

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Although fossil fuel combustion is currently the largest source of atmospheric NOx,7, 8

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NO is also produced in and emitted from natural and fertilized soils.9-11 Due to the spatial

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segregation of different NOx sources and the short boundary layer lifetime of NOx, there

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are substantial areas of the world (e.g., tropical and agricultural regions) where the local

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NOx budget is controlled exclusively by soil NO emissions.3, 7, 12-15 In these regions, soil

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NO emissions govern the formation and lifetime of tropospheric ozone (O3) and hydroxyl

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radical, driving reaction chains that produce environmentally important trace gases (e.g.,

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nitric acid and peroxyacetyl nitrate) and biogenic secondary aerosols.4, 13, 16

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Various processes, both microbial10, 17-19 and abiotic20-22, are capable of producing

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NO in soils. Although the strong dependence of soil NO emission on edaphic and

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climatic factors has long been demonstrated by laboratory and field studies,23-27 a

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process-based understanding of soil NO dynamics is lacking.14, 28 More importantly, soil

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NO emission often exhibits an episodic nature (e.g., time scale of minutes), with pulse-

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like emission events being often triggered by rewetting of dry soils.12, 13, 29-33 In dry

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agricultural soils, massive NO pulses triggered by coupled fertilization and precipitation

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during warm seasons can result in daily O3 enhancement up to 16 ppbv.13 Unfortunately,

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the sources of and processes controlling the pulsed soil NO emission are still

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mysterious,13, 14 making it difficult to model and up-scale field-observed NO fluxes.

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While empirical bottom-up models estimate that soil NO emission accounts for about

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15% of the global NOx inventory,1 inversion of satellite-based NO2 observations have



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indicated significant underestimates in soil NO emission (e.g., up to a factor of 3) at

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various spatiotemporal scales.7, 12-16, 29, 34 Indeed, with the substantial reductions in NOx

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emissions from combustion sources in many countries,2, 35 soils as a source of

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atmospheric NOx may be more important than we thought, and there is a pressing need to

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elucidate mechanisms underlying soil NO dynamics.13, 14

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Stable nitrogen (N) isotope compositions at natural abundances (notated as δ15N)

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in various soil N-containing compounds are a robust tracer of soil N cycling.36-39

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Incorporation of δ15N-NO measurements into the soil N isotope systematics is expected

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to provide a new process-level information of key mechanisms regulating NO production

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and consumption in soil. Moreover, there is a growing interest in using δ15N of

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atmospheric N oxides (e.g., NO2 and nitrate (NO3-)) as a tracer to partition NOx emission

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sources over large spatial and temporal scales.40-43 This interest stems from the

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observations that NOx emitted from different sources has distinct δ15N values44-49 and that

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soil-emitted NO is presumably lower in δ15N than NOx from other natural and

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anthropogenic sources.50-52

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Despite its promising potential, soil δ15N-NO is rarely measured due to the

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intermittent nature and low magnitudes of soil NO emission. A summary of published

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NOx collection methods is provided in Table S1 in the supporting information (SI)),

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highlighting that none of existing methods have been rigorously verified for their

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suitability for soil-emitted NO. In pioneering work, Li and Wang50 fertilized a soil

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monolith in the laboratory and collected NO by first converting NO to NO2 using a

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chromium trioxide (CrO3)-impregnated solid oxidizer and then trapping the converted

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NO2 in an annular denuder as nitrite (NO2-) for δ15N analysis. However, it is well


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documented that NO oxidation efficiency of the CrO3 oxidizer varies dramatically with

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sample relative humidity (RH) (e.g., 60%).53, 54 Due to this overlooked

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humidity interference, it is unclear whether N isotopic fractionation can occur during the

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NO oxidation under varying soil conditions. Recently, Fibiger et al.55 and Wojtal et al.56

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presented a NOx collection method that utilizes a KMnO4+NaOH solution to actively

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collect NOx as NO3- for δ15N-NOx determination. Sample δ15N-NOx must be calculated

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using an isotope mass balance due to a high reagent blank in the solution (5-7 µM NO3-,

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δ15N=~2‰)49, 55. While the precision of this approach is ±1.5‰, this method is

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incompatible for δ15N-NO measurement of low and diffuse soil NO emissions, because

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larger error is propagated from the isotope mass balance calculation if concentration and

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δ15N value of collected soil NO are significantly lower than the blank.

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Here, we present a new method for soil δ15N-NO determination (hereafter, “DFC-

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TEA method”). This method collects NO through NO conversion to NO2 in excess O3

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and subsequent NO2 collection in a triethanolamine (TEA) solution as NO2- and NO3- for

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δ15N analysis. The NO collection approach is coupled to a soil dynamic flux chamber

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(DFC) system for simultaneous NO flux and δ15N-NO measurements. Both laboratory

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and field method verifications have been conducted to demonstrate suitability of the

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DFC-TEA method for accurate and precise soil δ15N-NO determination.

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EXPERIMENTAL SECTION

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DFC system setup

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The DFC is a technique that has been developed to continuously measure soil-atmosphere

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fluxes of various compounds including NO.18, 19 A schematic of the developed DFC



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system is shown in Figure 1. The system consists of five components: air purification unit,

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gas dilution unit, flux chamber, NO-NOx-NH3 analyzer, and NO collection train. Zero air

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free of NOx and O3 is produced in the air purification unit for purging the flux chamber

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and providing air to a O3 generator (Model 146i, Thermo Fisher Scientific) in the NO

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collection train. NO, NO2, and ammonia (NH3) concentrations in the chamber headspace

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are measured alternately by a chemiluminescent analyzer (Model 17i, Thermo Fisher

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Scientific) at 10 s intervals for flux calculations. For method development, reference NO,

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NO2, and NH3 from three analytical tanks were diluted into the purging flow to simulate

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soil gas emissions inside the chamber. Two versions of the DFC system were developed

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for laboratory and field experiments. In the laboratory DFC system, a 1 L Teflon flow-

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through jar is used as the flux chamber. For the field DFC system, we fabricated a

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cylindrical flow-through chamber (39 cm I.D. and 30 L inner volume; Figure 1b),

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following considerations57, 58 for minimizing pressure differentials in chamber headspace.

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Control tests indicate that NO transmission from the chamber is greater than 98.3%.

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Details about the flux measurement, the chamber tests and the specifications for each

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DFC component are provided in the SI.

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NO collection train

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To collect NO for δ15N-NO analysis, a Teflon-coated diaphragm pump is used to sample

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chamber air passing through the NO collection train (Figure 1). The sample flow rate (1.6

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standard liter per minute (slpm)) is controlled by a mass flow controller. For the NO

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conversion in excess O3, a length of Teflon tubing (9.5 mm I.D., ca. 240 cm length)

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serves as the reaction tube. A O3 flow of 0.4 slpm, produced from photolysis of O2 in

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zero air at 185 nm by the O3 generator, is mixed with the sample flow at the starting point


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of the reaction tube (Figure 1). To prevent generation of HOx radicals during the

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photolysis, water vapor is removed from the zero air using two drying columns and a

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Teflon filter is attached before the O3 addition point to decompose remaining HOx

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radicals.59 Long-term (5 months) average O3 concentration after the mixing of the sample

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and O3 flows was 2911±32 ppbv as measured by an O3 monitor (Model 202, 2B

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Technologies). The flow leaving the reaction tube is forced to pass through a 500 mL gas

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washing bottle with a fritted cylinder containing a solution of TEA (Fisher Scientific,

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Certified Grade) in water (20% (v/v), 70 mL). The stopper of the gas washing bottle was

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lengthened so that 70 mL of the solution just covered the frit.

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Determination of reaction time

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Reaction of NO with excess O3 forms NO2 (R1 in Table S3 in the SI). In a dark

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environment, the efficiency of NO to NO2 conversion is limited by the formation of

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higher nitrogen oxide species (i.e. nitrate radical (NO3) and dinitrogen pentoxide (N2O5);

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R2-R5 in Table S3 in the SI).59-63 In order to model the NO conversion in the reaction

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tube, the reaction time is needed. Following Fuchs et al.,61 the reaction time in the

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reaction tube was experimentally determined by sampling zero air that contained a

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constant NO concentration (27 ppbv) using the NO collection train and varying the

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excess O3 concentrations (266-2890 ppbv). The ending point of the reaction tube was

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attached to the sampling inlet of the chemiluminescent analyzer for NO concentration

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determination. The NO concentration decay was then fitted to a single exponential

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function assuming pseudo-first order loss of NO in excess O3 (details are described in the

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SI). Due to the inner tubing of the chemiluminescent analyzer, the estimated reaction

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time essentially includes the reaction tube plus the analyzer inner tubing. To correct this



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overestimate, the reaction time of the inner tubing was estimated by repeating the

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experiment with the mixing point of the sample and O3 flow directly attached to the

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analyzer inlet for NO concentration determination.

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Preparation of TEA solution

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Triethanolamine is a tertiary amine and has long been used to scrub acidic gases in fuel

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gas treating processes and to coat passive filters for ambient NO2 monitoring.52, 64-66 We

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used a 20% TEA solution for NO2 collection. Its reagent N blank was determined to be

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0.12±0.04 µM (details are given in the SI).

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It is reported that aging of the TEA solution can cause significant efficiency

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decrease in collecting NO2.55 This aging problem may occur to a greater degree with

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more diluted TEA solutions.55 Therefore, to minimize alteration of TEA from its original

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state, we sub-sampled new TEA (i.e., freshly opened bottle) into 15 mL glass vials in a

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glovebox with a 95% N2 + 5% H2 atmosphere to avoid contact with ambient air. Vials

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were then capped, tightly wrapped with Parafilm, sealed in Ziploc bags, and stored under

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dark at 4 °C until further use. One glass vial was opened to make a fresh 20% TEA

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solution immediately prior to each sample collection. The storage time of TEA used in

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this study was up to approximately 4 months since sub-sampling.

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Measurement of NO2- and NO3- in TEA solution

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Both NO2- and NO3- can be produced from the reaction between NO2 and TEA.64-66 NO2-

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+NO3- concentration in the TEA collection samples was measured using a modified

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spongy cadmium method.67 Detailed measurement protocol is given in the SI. Control

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tests using 10 µM NO2- or NO3- in 20% TEA solution indicate that the precision (1 σ,

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n=8) of the method is ±0.09 µM and ±0.36 µM for NO2- and NO3- measurements,


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respectively. Due to the multiple reduction and neutralization steps during the

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measurements and the N blank inherent to the 20% TEA solution (~0.12 µM), standards

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were always prepared in 20% TEA solution for concentration calibration.

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Isotopic analysis

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The isotopic composition of collected NO2- and NO3- in the TEA solution was measured

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using the bacterial denitrifier method.68-69 In brief, denitrifying bacteria lacking the N2O

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reductase enzyme (Pseudomonas aureofaciens) are used to convert 5-20 nmol of NO2-

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and NO3- into gaseous N2O. Using He as a carrier gas, the N2O is then purified in a series

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of chemical traps, cryo-focused, and finally analyzed on a GV Instruments Isoprime

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Continuous Flow Isotope Ratio Mass Spectrometer at m/z 44, 45, and 46 at the University

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of Pittsburgh Regional Stable Isotope Lab for Earth and Environmental Science

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Research.

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Special considerations were taken during the isotopic analysis to ensure precise

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and accurate measurement of the δ15N of the TEA collection samples. First, the TEA

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collection samples were neutralized using 12 N HCl to pH ~8 before sample injection to

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avoid overwhelming the buffering capacity of the bacterial medium.70 Second, in light of

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the expected low δ15N of soil-emitted NO50-52 and the presence of NO2- as the dominant

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collection product (see below), a NO2- isotopic standard with low δ15N value (KNO2,

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RSIL20, USGS Reston; δ15N = -79.6‰, δ18O = 4.5‰)70 is used together with other

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international NO3- reference standards (IAEA-N3, USGS34, and USGS35) to calibrate

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δ15N and δ18O measurements. Third, following the IT principle (i.e., identical treatment

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of sample and reference material), a blank-matching strategy is used to make the isotopic

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standards in the same matrix (i.e., 20% TEA) as collection samples and to match both the



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molar N amount and injection volume (±5%) between the collection samples and the

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standards (Figure 2). This ensures that the isotopic interference of any blank N associated

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with the bacterial medium68, 71 and the TEA solution is minimized. The percentage

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difference (Pdiff) in the major N2O (m/z 44) peak area between each collection sample and

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RSIL20 measured within the same batch is calculated to quantify how precisely the

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blank-matching strategy is implemented (Figure 2). Finally, the ∆17O (∆17O=δ17O -

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0.52×δ18O)72 of the analyte N2O is independently measured for collected samples with

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sufficient concentration for 50 nmol injection using the N2O thermal decomposition

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method.73 The resolved ∆17O is then used to correct the isobaric interference on the δ15N

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analysis resulting from the NO oxidation by O3 according to Kaiser et al.73

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Quantification of method precision and accuracy

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The precision of the DFC-TEA method was quantified through repeated NO collection

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using the reference NO tank (50.4 ppmv). The collection was conducted under a variety

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of conditions, including differing NO concentrations (12-749 ppbv), chamber

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temperatures (11.5-30.8 °C), RH (27.1-92.0%), purging flow rates in the field chamber

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(5-20 slpm), and coexistence of NH3 in high concentrations (500 ppbv). In light of the

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high temporal variability of soil NO emission, we limited the collection time for each

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sample to be less than 2 h. Given that soils can produce and emit nitrous acid (HONO)74,

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the δ15N-NO analysis by soil HONO emission was minimized by forcing the sample flow

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to pass through a HONO scrubber (250 mL fritted gas washing bottle containing 50 mL

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of 1 mM phosphate buffer solution at pH 7.0)76 before entering the NO collection train.

and that HONO positively interferes NO2 collection in TEA solution,65 interference of


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While there is no certified isotopic standard for gaseous NO, the accuracy of the

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DFC-TEA method was evaluated through inter-calibration with a modified EPA NOx

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collection method. A detailed description of the modified EPA method has been provided

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by Felix et al.44 and Walters et al.47 In brief, gas samples from the NO and NO2 tanks

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were collected directly into evacuated 1 L borosilicate gas sampling bulb containing 10

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mL of a NO2 absorbing solution (H2SO4+H2O2) on a vacuum line. The absorbing solution

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oxidizes NO2 into NO3-. For the NO collection, the collection was terminated with a

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small vacuum remaining in the bottle. The bottle was then quickly vented to the

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laboratory atmosphere to allow introduction of O2 into the bottle for the conversion of

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NO to NO2.77 After the collection, the bottles were allowed to stand for 1 week with

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occasional shaking to facilitate the conversion of NOx to NO3-. The residual NOx

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headspace concentration was measured after a 1 week period and indicates that the

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collection was 100%. The absorbing solution was then collected and neutralized for δ15N

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analysis using the denitrifier method. The results show that the NO and NO2 tanks had

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δ15N values of -71.4±0.5‰ (n=4) and -39.8±0.2‰ (n=3), respectively.

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Laboratory soil δ15N-NO measurements

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To test the DFC-TEA method using real soil samples, approximately 4 kg of soil was

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collected from the upper 10 cm of an urban forest soil in Pittsburgh, PA. Before use, soil

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samples were sieved by passing through a 2-mm sieve and air-dried for 14 days. To

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trigger NO pulses, 35 g of air-dried soil samples was added to the Teflon jar, mixed

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thoroughly, and wetted by deionized water to achieve 100% water holding capacity. With

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the continuous purging of the jar headspace, the soil samples were subject to drying-out

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over the next 48 h, and NO was collected periodically for the δ15N-NO analysis.



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Field soil δ15N-NO measurements

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To verify the DFC-TEA method under varying field conditions, the field DFC system

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was deployed using the University of Pittsburgh Mobile Air Quality Laboratory to

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measure δ15N-NO in a field soil rewetting experiment (see Figure S10 in the SI for the

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field setup). A waterproof tarp (300 cm×240 cm) was erected over a fallow, urban plot in

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Pittsburgh, PA for 2 weeks (8/15/2016 - 8/29/2016) to exclude precipitation inputs. After

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the drying period, four soil plots were respectively wetted on four consecutive days using

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500 mL of MilliQ water, 20 mM KNO3 (δ15N=46.5±0.3‰), 10 mM NaNO2

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(δ15N=1.0±0.4‰), and 20 mM NH4Cl (δ15N=1.7±0.1‰) solutions. These N amendment

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solutions were chosen because they are common precursors of NO in major NO-

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producing processes.31, 36 Previous studies have reported that common δ15N values of N

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fertilizers are not very different from 0‰ (e.g., -4.4‰ to 0.3‰),78 while δ15N values of

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atmospherically deposited NO2, NO3- and NH4+ could vary over a wider range (e.g., -

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10‰ to 15‰), depending on source contributions.42, 79 Hence, the δ15N values of the

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amended NO2- and NH4+ are within the environmentally relevant range, whereas the δ15N

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of the added NO3- is significant higher. The NO, NO2, and NH3 fluxes were continuously

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measured before and after the soil rewetting, and NO was collected periodically for the

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δ15N-NO analysis.

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RESULTS AND DISCUSSION

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We evaluate each step in the NO collection and report isotopic results that document the

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overall precision and accuracy of the δ15N-NO analysis. We then present δ15N-NO


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measurements from laboratory and field soil rewetting experiments that demonstrate

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utility of the DFC-TEA method for resolving soil NO dynamics.

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NO conversion in excess O3

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The reaction time of the inner tubing of the chemiluminescent analyzer and the reaction

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tube plus the inner tubing were estimated to be 1.4 s and 6.4 s, respectively, resulting in a

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reaction time of the reaction tube of 5 s at the measured flow temperature (22 °C) (Figure

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S7 in the SI). This estimated reaction time is consistent with the residence time calculated

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from the assumption of plug flow in the reaction tube. Based on this reaction time and the

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average O3 concentration of 2911 ppbv, numerical model calculations including reactions

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R1-R580 and NO3 loss on the interior tubing wall (R6 in Table S2 in the SI)81 indicate that

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NO is quantitatively converted in the reaction tube and that the specific conversion of NO

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to NO2 is between 98.7% and 99.0% over a wide range of NO concentrations (0-1000

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ppbv) at 22 °C (Figure S8a in the SI). Notably, the remaining NO from the conversion

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exists primarily as N2O5 (Figure S8b in the SI).

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Deviations from controlled laboratory condition in the field may result in

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variations in the modeled NO conversion efficiency.61 We therefore modeled the effects

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of temperature variation and soil emission of biogenic volatile organic carbon (BVOC)82

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on the NO conversion (reactions R7 and R8 in Table S2 in the SI)83. The results indicate

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that the conversion of NO to NO2 is not likely to fall below 98% over a temperature

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range of 0-40°C in conjunction with high BVOC emissions (e.g., 100 ppbv isoprene in

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the chamber) (details on the extended modeling are given in the SI). In addition, slight

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variations in the reaction time may result from changes in temperature and pressure of the

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sample flow (e.g., pressure increase induced by the attachment of the gas washing bottle).



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While the effect of these variations on the NO conversion is difficult to empirically

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quantify, any uncertainty in converting NO under the tested conditions is reflected in the

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over method precision and accuracy for the δ15N-NO measurement.

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NO2 collection in TEA solution

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The 20% TEA solution was 100% efficient at collecting NO2. This was confirmed

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by collecting a flow of reference NO2 at 1 ppmv using the laboratory DFC system (Table

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1). Importantly, because the 20% TEA solution foams rigorously upon sparging, the

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applied total flow rate (1.6 slpm of the sample flow plus 0.4 slpm of the O3 flow) was

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chosen to avoid solution spill. We have also tested TEA solution from another brand

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(BioUltra) that foams much less rigorously (coarse bubbles). However, consistent low

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collection efficiency (0.05). The deviations from 100% NO recovery likely reflect inefficiencies in the NO

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conversion (see above), the high uncertainty in the NO3- concentration determination

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(e.g., for the 12 ppbv and 25 ppbv NO collection samples), and/or NO loss within the

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system (e.g., NO loss in the HONO scrubbing solution and on the interior wall of the

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field chamber). Importantly, the high and consistent NO recovery is a direct evidence that


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the sub-sampling was effective to minimize the TEA aging problem, if any, for a storage

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time of at least 4 months.

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For all the tank collection samples (n=52), about 90% of the collected NO or NO2

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was in the form of NO2-, and the remainder as NO3- (Table 1). A 90% NO2-+10% NO3-

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stoichiometry has been previously reported for active NO2 sampling using TEA-coated

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cartridges.65 While a satisfactory explanation for the NO3- production cannot be given at

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this time,65 the observed stoichiometry is best approximated by the redox reaction

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between NO2 and TEA in the presence of water that gives a theoretical 1:1 conversion of

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NO2 to NO2-.64, 65, 84 Although it is well known that N2O5 hydrolyzes in water as HNO3,85

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which is preserved as NO3- in an alkaline TEA solution, whether N2O5 produced in the

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NO conversion can be collected in the TEA solution as NO3- is not possible to quantify in

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this case due to the high uncertainty in the NO3- concentration determination (i.e., ±0.36

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µM) but will be the subject of future research.

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It is worth noting that collection efficiency of the 20% TEA solution may be

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subject to decrease over longer collection periods due to presence of O2, O3, and CO2 in

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the sample flow that can compete with NO2 for TEA oxidation and decrease solution pH.

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Given that the DFC-TEA method described here is developed to characterize transient

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variations of soil NO emissions, use of 20% TEA solution for prolonged collection (i.e.,

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>2 h) should be further investigated to ensure high and consistent collection efficiency.

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Analytical uncertainty of the denitrifier method and the total N blank

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The pooled standard deviation for each of the isotopic standards made in 20% TEA

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solution and measured along with individual sample sets was: 0.3‰, 0.3‰, and 0.8‰ for

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δ15N of IAEA-N3, USGS34, and RSIL20, respectively; 0.7‰ and 0.7‰ for δ18O of



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IAEA-N3 and USGS34, respectively; and 1.2‰ for ∆17O of USGS35. The lower

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precision of the δ15N analysis of RSIL20 (0.8‰ relative to 0.3‰ for other standards) is

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due to the larger uncertainty in measuring diluted RSIL20 solutions that require large

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injection volumes (Figure 3a). To further understand this volume dependence, we

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estimated the total N blank associated with the δ15N analysis of the TEA samples (i.e.,

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TEA N blank + blank N associated with the denitrifier medium68, 71) by quantifying

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shrinkage of the N isotope-ratio scale between USGS34 and RSIL20 measured in each

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run of the TEA collection samples86 (more details are described in the SI). The results

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show that the fractional blank size (fB) ranged between 0.04 and 0.18 across different runs

345

and was significantly, positively correlated with the sample volume and the measured

346

δ15N of RSIL20 (δ15NRSIL20-m) (Figure 3a). Fitting a linear equation to the molar amount

347

of the total N blank and the sample volume indicates that the N blank likely consisted of

348

a constant component of 0.46±0.12 nmol and a sample volume-dependent component of

349

0.23±0.06 nmol·mL-1 (Figure S5 in the SI; Figure 2); this is consistent with the blank size

350

estimated by injecting blank 20% TEA solution (details are provided in the SI). From the

351

linear relationship between fB and δ15NRSIL20-m, the δ15N of the blank N appears to be

352

~10‰ across different runs (Figure 3a). These consistent and predictable behaviors of the

353

total N blank indicate with a high degree of confidence that its isotope effect is implicitly

354

corrected during the δ15N analysis using the blank-matching strategy.

355

Isobaric interference from mass-independent oxygen isotopic composition

356

The δ18O of RSIL20 calibrated against IAEA-N3 and USGS34 ranged from -

357

25.8±0.9‰ to -22.7±1.5‰ across different runs, with an average of -23.7±1.1‰. This

358

results in an isotopic offset of about 28‰ between the measured apparent δ18O and the


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359

“true” δ18O (4.5‰) of RSIL20, in line with the branching fractionation between NO3- and

360

NO2- during denitrification (25-30‰).70 This implies that the oxygen isotopic exchange

361

between NO2- and water is limited in the alkaline TEA solution. Not surprisingly,

362

positive ∆17O values were observed in the N2O generated from collected samples. To

363

understand the transfer of the ∆17O anomaly from O3 during the NO conversion, a

364

theoretical ∆17O of the NO2 produced from the NO+O3 reaction (R1 in Table S2 in the

365

SI) was calculated to be 22.5±1.8‰ (details are provided in the SI). This theoretical ∆17O

366

value is not very different from the measured ∆17O values, which had an average of

367

19.7±0.8‰ across different runs (Table 1), indicating that the NO+O3 reaction essentially

368

dominated during the NO conversion. The measured ∆17O values led to a 1.0-1.2‰

369

correction of the measured δ15N values. For samples without sufficient concentrations for

370

∆17O measurement, the average ∆17O value (19.7‰) was used for the correction. This is

371

not a complete correction, in that the expression of the isobaric interference depends on fB

372

relative to each sample. Nevertheless, the resultant overcorrection on the δ15N of the low

373

concentration samples is 0.05; Figure S6b in the SI). The DFC-TEA method and the EPA NOx

383

collection method generally agree within 0.3‰, although discrepancies within individual

384

sets of collected samples ranged from -1.3‰ to 1.6‰. The largest discrepancies between

385

the two methods occurred with the lowest sample concentrations (i.e., 12 ppbv NO

386

collection samples) (Figure 3b). For these low concentration samples, isotopic analyses

387

were conducted on 5 nmol of NO2-+NO3- (achieved with a 3.8 mL injection of the

388

collection samples) and of a fB of 0.18. Therefore, although Pdiff did not correlate with the

389

sample concentration (Figure S6a in the SI), the collection samples with lower

390

concentrations were more prone to random error in matching blank between the standards

391

and samples due to their higher fB. Consequently, for accurate δ15N-NO analyses, soil NO

392

should be collected to achieve >3 µM NO2-+NO3- in the solution within 2 h (equivalent to

393

collecting a flow of >26 ppbv NO over a 2 hour period). Blank 20% TEA solution should

394

then be used to dilute both soil NO collection samples and isotopic standards within a

395

batch to a common concentration for injection of 10 nmol of N using the denitrifier

396

method. Control tests using a soil NO sample collected from the laboratory rewetting

397

experiment (δ15N = -37.1‰, [NO2-]+[NO3-] = 9.2 µM) indicate that dilution-induced

398

uncertainty in the δ15N was 3 µM

399

NO2-+NO3- in the solution (data not shown). This uncertainty is within the analytical

400

uncertainty (i.e., ±0.8‰ for RSIL20). Therefore, we always group samples with similar

401

concentrations such that the dilution factor does not exceed 3.

402

Overall, our inter-calibration effort demonstrates that although the NO recovery

403

was slightly less than 100%, fractionation during chamber mixing and NO conversion

404

and collection is effectively minimized under the tested conditions. The derived standard


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405

deviation of ±1.1‰ based on all the collection samples with an average Pdiff of ±7% (i.e.,

406

sample peak area is within 100±7% of that of RSIL20) represents the overall accuracy

407

and precision across the entire method, accounting for propagated errors from the total N

408

blank and its mismatch between the standards and samples. While the method precision is

409

lower than that of the modified EPA method (Table S1 in the SI), our integrated method

410

featuring simultaneous NO flux measurement and collection is the first to show its

411

suitability for unbiased soil δ15N-NO determination under realistic, varying soil

412

conditions. Furthermore, the method is more convenient than previous methods and does

413

not require time-consuming pretreatments for δ15N analysis (Table S1 in the SI). Given

414

the good result from the inter-calibration, the tank NO can be utilized as a secondary

415

standard for correcting the isobaric interference. For instance, the tank NO can be

416

collected before and after soil NO collection; ∆17O of the tank collection samples can

417

then be estimated using an empirical relationship scaling a 1‰ increase from accepted

418

δ15N value (-71.4‰ in this case) to every 18.8‰ increase in ∆17O to correct the soil

419

collection samples.86

420

Application to pulsed soil NO emissions

421

Pulsed NO emission was triggered by soil rewetting under both the laboratory and field

422

conditions (Figure 4). In the laboratory, the pulsed NO emission had evident temporal

423

variations with a rapid initial NO pulse being triggered upon the rewetting (Figure 4a).

424

While the initial NO pulse was absent under the field conditions, possibly due to the

425

relatively high pre-wetting soil water content (0.17 cm3·cm-3), the rewetting and N

426

amendments caused significantly increased NO emission as compared to the pre-wetting



427

emission (47±16 nmol·m-2·min-1). Particularly, a dramatic increase in NO emission was

428

triggered by the NO2- addition (Figure 4b).

429

Twenty and 15 samples were collected for the δ15N-NO analysis from the

430

laboratory and field experiments, respectively (Table S5 and S6 in the SI). The average

431

NO recovery was 102.2±5.6% and 108.6±11.0% for the laboratory and field collection

432

samples, respectively. The >100% recovery was detected mostly in samples collected

433

under the NO2- addition (NO recovery = 117.5±11.6%; Table S6 in the SI). We suspect

434

that the >100% NO recovery might result from our underestimation of the soil NO

435

emission due to the slow response time of the chemiluminescent analyzer (>30 s),

436

especially given that transient fluctuations in the NO flux were likely triggered by the

437

NO2- addition (Figure 4b). Alternatively, the >100% recovery could result from soil

438

emission of NOy (NOy = NO2 + HONO + HNO3 + other non-NO reactive N oxides),87

439

that can potentially be collected in the TEA solution as NO2- and/or NO3-.65 If soil NOy

440

emission was significant during measurement, it would be detected as NO2 by our

441

chemiluminescent analyzer with a molybdenum convertor.88 Because the NO2 flux never

442

exceed 2% of the simultaneous NO flux (Figure S11 and Figure S12 in the SI),

443

contributions of NOy emission to the NO recovery and the measured δ15N-NO are

444

considered negligible. Future application of the DFC-TEA method can be coupled to a

445

faster NO measurement system and existing denuder and wet chemistry methods76, 89 that

446

quantitatively scrub NOy without significant loss of NO.

447

The measured soil δ15N-NO exhibited intriguing patterns that are indicative of

448

mechanisms underlying the soil NO emissions (Figure 4). In the laboratory rewetting of

449

the air-dried soil samples, the initial NO pulse had higher δ15N values (-36.7~-39.9‰)


Page 20 of 44

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450

than NO emission after 12 h post-wetting (-52.0~-53.6‰; Figure 4a). Recent work by

451

Homyak et al.33 provided evidence that in arid soils, abiotic reactions govern the rapid

452

initial NO pulse, whereas microbial processes control later emissions as microbes recover

453

from drought stress. Therefore, the higher δ15N-NO values associated with the initial NO

454

pulse may suggest that abiotic reactions likely bear a smaller isotopic fractionation on

455

NO production than microbial processes. However, the temporal variation of δ15N-NO

456

could also result from changing rates of microbial NO production90 or sequential

457

resuscitation of different microbial groups91 during the rewetting. Further constraints on

458

the relevant isotope effects are needed to tease apart the relative importance of abiotic

459

and microbial pathways sustaining pulsed NO emissions in soils. In the field rewetting

460

experiment where an initial NO pulse was lacking, the measured soil δ15N-NO responded

461

differently to the added N precursors (Figure 4b). First, the δ15N-NO values that evolved

462

from the NH4+ (-59.8‰ to -56.0‰) and NO2- (-34.4‰ to -23.4‰) amendments were

463

significantly lower and higher relative to the control (MilliQ water addition; -44.3‰ to -

464

41.3‰), respectively, in spite of the almost equal δ15N of the added NH4+ and NO2-.

465

Secondly, despite the high δ15N of the added NO3- (i.e., 46.5‰), the δ15N-NO values

466

measured from the NO3- amendment (-40.7‰ to -39.4‰) were not significantly different

467

from those in the control. The measured soil δ15N-NO and its differential responses to the

468

amended N sources indicate that various soil NO-producing processes (e.g., nitrification,

469

denitrification, and chemodenitrification), stimulated by different N amendments, likely

470

bear distinguishable isotopic imprints on NO production, similar to what has been

471

observed in soil nitrous oxide (N2O) studies.37-39 For example, N2O production in soil

472

was found to be associated with a larger isotope effect for nitrification of NH4+ (e.g., -



473

45‰ to -67‰) than denitrification of NO2- (e.g., -35‰ to -22‰).37-39 Thus, soil δ15N-NO

474

measurement could potentially provide important implications for understanding

475

couplings between soil NO and N2O emissions, in that NO is the precursor of N2O in

476

most abiotic and microbial processes.10, 17-20 Finally, the measured soil δ15N-NO values

477

are significantly lower than other measured NOx emissions sources,44, 45, 47-49 confirming

478

use of soil δ15N-NO as a robust tracer of regional N deposition.41, 42 Quantification of

479

isotope effects associated with NO dynamics in soils therefore represents an important

480

avenue for future research on the soil-atmosphere cycling of reactive N.

481 482

ACKNOWLEDGEMENT

483

This material is based upon work supported by a National Science Foundation CAREER

484

award (Grant No. 1253000) to EME. We thank Kathrine Redling, Vivian Feng, Madeline

485

Ellgass, and Madeline Gray for assistance with isotopic measurements.

486 487

SUPPORTING INFORMATION AVAILABLE

488

Supporting information includes a description of: (1) comparison between the DFC-TEA

489

method and previously published NOx collection methods; (2) the dynamic flux chamber

490

system; (3) the protocol for NO2- and NO3- concentration measurement using the

491

modified spongy cadmium reduction method; (4) the total N blank and the blank-

492

matching strategy; (5) extended modeling of the NO conversion in excess O3; (6)

493

calculation of the theoretical ∆17O value of the NO2 produced from the NO+O3 reaction;

494

(7) experimental setup of the field rewetting experiment; (8) soil NOy emissions in the


Page 22 of 44

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495

laboratory and field soil rewetting experiments; and (9) complete datasets of the

496

laboratory and field collection samples.

497 498

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873 874 875

Page 40 of 44

Table 1. Summary of the reference NO and NO2 tank collection using the DFC-TEA method under varying environmental conditions. The complete dataset is given in Table S4 in the SI.a RH (%)

NO2-+NO3(µM)

Recoveryb (%)

NO2- percent (%)

Pdiff (%)

δ15Nc (‰)

25.3

132.5 4.7

101.4 3.6

87.4 0.3

3.3 5.1

-40.1 0.8

44.6 27.1 34.2

1.4 4.1 11.9

95.0 100.7 98.2

97.0 93.1 94.0

-0.7 -1.2 0.7

-73.0 (-71.7) -70.3 (-69.2) -71.0 (-69.9)

18.8

749 ppbv NO (n=4) 120 22.8 47.5 14.2 NO collection – laboratory DFC system – temperature effect 34 ppbv NO (n=4) 120 11.5 92.0 4.0 101 ppbv NO (n=4) 120 30.8 28.8 11.8 NO collection – laboratory DFC system – interference 34 ppbv NO 120 23.0 33.1 4.0 + 500 ppbv NH3 (n=3) 101 ppbv NO 120 23.1 46.5 11.7 + 500 ppbv NH3 (n=4) 101 ppbv NO 120 22.3 89.7d 11.6 + HONO (n=4) NO collection – field DFC systeme 25 ppbv NO (n=4) 120 21.4 40.8 3.1 34 ppbv NO (n=4) 120 21.9 50.4 3.9 56 ppbv NO (n=3) 120 21.2 44.8 6.2 101 ppbv NO (n=4) 120 21.7 36.6 11.7

99.3

90.7

3.5

-70.6 (-69.4)

20.6

99.8 98.7

89.3 89.5

2.4 3.6

-71.1 (-70.0) -70.8 (-69.7)

20.0

99.5

88.2

-3.8

-70.1 (-69.0)

97.5

91.5

2.6

-71.2 (-70.2)

19.5

96.7

89.8

2.8

-71.0 (-69.9)

19.6

103.9 97.6 96.0 97.1

94.3 92.8 92.9 89.5

4.3 -1.8 -2.0 1.0

-72.9 (-71.7) -70.7 (-69.6) -71.5 (-70.4) -71.0 (-69.9)

19.5

Mean Standard error (1 σ)

98.5 3.5

91.7 3.4

1.1 5.1

-71.1 (-70.0) 1.1 (1.1)

19.7 0.8

Time T (min) (°C) NO2 collection – laboratory DFC system 1002 ppbv NO2 (n=4) 135 23.7 Standard error (1 σ) Sample

NO collection – laboratory DFC system 12 ppbv NO (n=3) 120 23.0 34 ppbv NO (n=4) 120 24.8 101 ppbv NO (n=4) 120 23.1

876 877 878 879 880 881 882 883 884 885 886 887 888

a: Out of 56 NO and NO2 tank collection samples, 52 samples yielded consistent results

wherein 4 samples were detected as outliers on the basis of erroneous concentrations. These outliers were not included in this table. b: NO (NO2) recovery was calculated by dividing measured NO2-+NO3- concentration by the theoretical concentration calculated using the collection time, sample flow rate (1.6 slpm), NO (NO2) concentration, and the TEA solution volume. The TEA solution volume was corrected for evaporative loss by weighing the gas washing bottle containing the solution before and after each sample collection. c: Relative to N2 in the air. δ15N values before the isobaric correction are shown in the brackets. d: RH was measured after the HONO scrubber instead of in the Teflon chamber. e: The chamber purging flow rates were 20 slpm, 15 slpm, 9 slpm, and 5 slpm for the 25 ppbv NO, 34 ppbv NO, 56 ppbv NO, and 101 ppbv NO collection, respectively.


∆17O (‰)

Page 41 of 44


889 890

Figure 1. (a) Schematic of the DFC system (not to scale) consisting of the following: (1)

891

diaphragm pump, (2) air purification columns, (3) drying columns, (4) humidifier, (5)-(7)

892

NO, NO2, and NH3 reference tanks, (8) mass flow controller, (9) flux chamber, (10)

893

temperature and relative humidity sensor, (11) in-line PTFE particulate filter assembly,

894

(12) HONO scrubber, (13) moisture exchanger, (14) reaction tube, (15) gas washing

895

bottle containing TEA solution, (16) O3 generator, (17) NO-NOx-NH3 analyzer; (b)

896

picture showing the field chamber. Specifications of each component of the DFC system

897

are given in the Table S2 in the SI.



898 899

Figure 2. Illustration of the blank-matching strategy for correcting N blanks associated with the

900

TEA solution and denitrifier method.


Page 42 of 44

Page 43 of 44


901 902

Figure 3. (a) The measured δ15N of RSIL20 (δ15NRSIL20-m) as a function of the fraction of

903

analyte N2O-N derived from the total N blank (fB). Sample injection volume, standard

904

deviation of δ15NRSIL20-m, and number of replicates for the individual runs are given in the

905

brackets. The dot in red (56 ppbv collection sample) was not included in the linear

906

regression. (b) The measured δ15N of the NO collection sample as a function of the

907

sample NO2-+NO3- concentration. The dash line and the shaded area represent the mean ±

908

(1 σ) of the δ15N of the NO tank measured using the modified EPA NOx collection

909

method.

910 911 912 913 914 915



916 917

Figure 4. NO emission (lines) and δ15N-NO (dots) results from the laboratory (a) and

918

field (b) rewetting experiments. The error bar on the x-axis denotes the time span of each

919

collection sample. In the field rewetting experiment, four soil plots were respectively

920

wetted on four consecutive days using 500 mL of MilliQ water (black), 20 mM KNO3

921

(red; δ15N=46.5±0.3‰), 10 mM NaNO2 (dark blue; δ15N=1.0±0.4‰), and 20 mM NH4Cl

922

(light blue; δ15N=1.7±0.1‰) solutions.


Page 44 of 44

Novel Method for Nitrogen Isotopic Analysis of Soil-Emitted Nitric

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