Ilann Bourgeois
email: ilann.bourgeois@univ-grenoble-alpes.fr

Joel Savarino

Nicolas Caillon

Hélène Angot

Albane Barbero

Franck Delbart

Didier Voisin

Jean-Christophe Clément

Tracing the fate of atmospheric nitrate in a subalpine watershed using ∆ 17 O

Keywords: atmospheric deposition, nitrate isotopes, subalpine streams, Lautaret Pass

Nitrogen is an essential nutrient for life on Earth, but in excess, it can lead to environmental issues (e.g., N saturation, loss of biodiversity, acidification of lakes, etc.). Understanding the nitrogen budget (i.e., inputs and outputs) is essential to evaluate the prospective decay of the ecosystem services (e.g., freshwater quality, erosion control, loss of high patrimonial-value plant species, etc.) that subalpine headwater catchments provide, especially as these ecosystems experience high atmospheric nitrogen deposition. Here, we use a multi-isotopic tracer (∆ 17 O, δ 15 N and δ 18 O) of nitrate in aerosols, snow and streams to assess the fate of atmospherically deposited nitrate in the subalpine watershed of the Lautaret Pass (French Alps). We show that atmospheric deposition contributes significantly to stream nitrate pool yearround, either by direct inputs (up to 35 %) or by in situ nitrification of atmospheric ammonium (up to 35 %). Snowmelt in particular leads to high exports of atmospheric nitrate, most likely fast enough to impede assimilation by surrounding ecosystems.

Yet, in a context of climate change, with shorter snow seasons, and increasing nitrogen emissions, our results hint at possibly stronger ecological consequences of nitrogen atmospheric deposition in the close future.

Introduction

Reactive Nitrogen (Nr) has long been known for its duality. At low to moderate concentrations, it is an essential nutrient for natural ecosystems in general, and plant nutrition in particular ( 1 ). In excess, Nr becomes an environmental issue for endemic terrestrial plant species and aquatic ecosystems ( [START_REF] Aber | Nitrogen Saturation in Northern Forest Ecosystems[END_REF][START_REF] Vitousek | SOURCES AND CONSEQUENCES[END_REF][START_REF] Galloway | The Nitrogen Cascade[END_REF] ), and may pose threats to human health ( 5 ). Human activities have increased N input to the environment by 150% over the last century ( 6), with a rising contribution -among other distribution processes -of atmospheric deposition [START_REF] Matson | The Globalization of Nitrogen Deposition: Consequences for Terrestrial Ecosystems[END_REF][START_REF] Galloway | Transformation of the nitrogen cycle: recent trends, questions, and potential solutions[END_REF] ). Fossil-fuel combustion, agriculture and food production are responsible for direct or indirect emissions of atmospheric Nspecies with the capacity to reach remote ecosystems such as mountainous regions [START_REF] Baron | High Elevation Ecosystem Responses to Atmospheric Deposition of Nitrogen in the Colorado Rocky Mountains, USA[END_REF][START_REF] Preunkert | A seasonally resolved alpine ice core record of nitrate: Comparison with anthropogenic inventories and estimation of preindustrial emissions of NO in Europe[END_REF] ) where Nr equilibrium is very sensitive to small perturbations ( [START_REF] Baron | Ecosystem Responses to Nitrogen Deposition in the Colorado Front Range[END_REF][START_REF] Kirchner | Nitrogen deposition along differently exposed slopes in the Bavarian Alps[END_REF] ). Alpine and subalpine watersheds are particularly vulnerable to this increasing Nr-supply ( [START_REF] Clark | Estimated losses of plant biodiversity in the United States from historical N deposition (1985-2010)[END_REF][START_REF] Burns | The effects of atmospheric nitrogen deposition in the Rocky Mountains of Colorado and southern Wyoming, USA-a critical review[END_REF] ), as they are usually N-limited ecosystems [START_REF] Kaye | Competition for nitrogen between plants and soil microorganisms[END_REF]. The critical load for these ecosystems has been estimated to 1-4 kg-N.ha -1 .yr -1 , making alpine and subalpine grasslands the most sensitive to N deposition among grasslands and tall forb habitats ( [START_REF] Bobbink | Review and revision of empirical critical loads and dose-response relationships. Review and revision of empirical critical loads and doseresponse relationships: proceedings of an expert workshop[END_REF][START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF][START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] ). In some regions of the globe, such as in the Colorado Front Range, USA, shifts from Nlimitation to N-saturation state have been observed leading to regional and national policies to regulate this phenomenon ( [START_REF] Baron | Hindcasting nitrogen deposition to determine an ecological critical load[END_REF][START_REF] Wolfe | Recent ecological and biogeochemical changes in alpine lakes of Rocky Mountain National Park (Colorado, USA): a response to anthropogenic nitrogen deposition[END_REF][START_REF] Williams | Critical loads for inorganic nitrogen deposition in the Colorado Front Range[END_REF] ). As a consequence of increasing nitrogen deposition, a slow but persistent change of alpine and subalpine landscape has been noticed in several regions ( [START_REF] Bassin | Species Composition of Subalpine Grassland is Sensitive to Nitrogen Deposition, but Not to Ozone, After Seven Years of Treatment[END_REF][START_REF] Mcdonnell | Modeled subalpine plant community response to climate change and atmospheric nitrogen deposition in Rocky Mountain National Park, USA[END_REF] ). Species more competitive for nitrogen uptake tend to develop in abundance at the expense of usually oligotrophic native plants, leading to plant diversity loss and changes in ecosystem functioning and services.

Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395

Freshwater quality and erosion control are, per instance, threatened by this cascade of changes ( [START_REF] Bowman | Nitrogen Critical Loads For Alpine Vegetation And Terrestrial Ecosystem Response: Are We There Yet?[END_REF][START_REF] Chapin | Biotic control over the functioning of ecosystems[END_REF] ). Yet, and mainly because of the paucity of monitoring facilities in isolated areas and of the logistical difficulties in accessing these regions, key biogeochemical processes regulating N cycling in mountains are still not well understood [START_REF] Mast | Links between N Deposition and Nitrate Export from a High-Elevation Watershed in the Colorado Front Range[END_REF].

More specifically, atmospheric nitrate (NO 3 -) and ammonium (NH 4 +

), deriving principally from anthropogenic NO x (NO and NO 2 ) and agricultural NH 3 emissions, are transferred to the land surface through wet and dry deposition. Dissolved inorganic nitrogen (DIN) species (i.e., NO 3 -and NH 4 + ) are of capital importance in ecological studies given their high solubility in water; the strong hydrological connectivity and typical topography of alpine and subalpine watersheds can lead to a fast transport and a potentially long reach of the above-mentioned nutrients in surrounding ecosystems ( [START_REF] Campbell | Nitrogen deposition and release in alpine watersheds[END_REF][START_REF] Clow | Relations between basin characteristics and stream water chemistry in alpine/subalpine basins in Rocky Mountain National Park, Colorado[END_REF]. Traditionally, environmental studies on NO 3 -sources partitioning are essentially based on δ [START_REF] Kaye | Competition for nitrogen between plants and soil microorganisms[END_REF] N and δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O-NO 3 -values ( [START_REF] Kendall | Tracing sources of nitrate in snowmelt runoff using the oxygen and nitrogen isotopic compositions of nitrate[END_REF][START_REF] Ohte | Nitrogen and oxygen isotope measurements of nitrate to survey the sources and transformation of nitrogen loads in rivers[END_REF][START_REF] Mayer | Sources of nitrate in rivers draining sixteen watersheds in the northeastern US: Isotopic constraints[END_REF] ), to constrain the biogeochemical processes affecting the various N forms in mountains. Yet, the wide range of observed values for δ [START_REF] Kaye | Competition for nitrogen between plants and soil microorganisms[END_REF] N and δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O-NO 3 -from atmospheric and other sources limits accurate quantification of contributing sources [START_REF] Michalski | Tracing Atmospheric Nitrate Deposition in a Complex Semiarid Ecosystem Using Δ17[END_REF][START_REF] Liu | Using 15 N, 17 O, and 18 O To Determine Nitrate Sources in the Yellow River, China[END_REF] ).

In this study we focus -for the first time (to the best of our knowledge) -on yearround NO 3 -dynamics in a mountainous watershed in the French Alps, using a multiisotopic (∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O, δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O, δ 15 N) tracer of NO 3 -in subalpine surface waters. This relatively new method has been increasingly used, as it enables a more refined understanding of atmospheric NO 3 -(NO 3 atm ) contribution to ecosystems N pool [START_REF] Michalski | Tracing Atmospheric Nitrate Deposition in a Complex Semiarid Ecosystem Using Δ17[END_REF][START_REF] Rose | Triple Nitrate Isotopes Indicate Differing Nitrate Source Contributions to Streams Across a Nitrogen Saturation Gradient[END_REF][START_REF] Tsunogai | Accurate and precise quantification of atmospheric nitrate in streams draining land of various uses by using triple oxygen isotopes as tracers[END_REF] ). We hypothesized that (i) the triple isotopes approach will enable a clear delineation of NO 3 -sources and the quantification of their respective contribution to small and medium-scale watersheds N budget and (ii) seasonal dynamics of NO 3 atm exports in stream will provide additional insights on N cycling controls in high altitude catchments.

Material and Methods

Study Site

The studied catchments are located in the upper Romanche valley, near the Lautaret Pass (central French Alps, SI Figure S1). The experimental site is part of the national research infrastructure for the study of continental ecosystems and their biodiversity February 2015 and 13.9 °C in July 2015. More detailed information about the sampling site can be found elsewhere ( [START_REF] Legay | Comparison of inorganic nitrogen uptake dynamics following snowmelt and at peak biomass in subalpine grasslands[END_REF][START_REF] Clement | The effects of snow-N deposition and snowmelt dynamics on soil-N cycling in marginal terraced grasslands in the French Alps[END_REF] ).

The first studied sub-watershed (hereafter called Lautaret) is mainly constituted of calcareous substrate and extends over 3.4 km². At the outlet of this sub-watershed runs a stream called Tufiere. Samples were collected at an altitude of ca. 2015 m a.s.l, just before the stream merges with the outlet of the second sampled watershed (Figure S1). Altitude on this watershed spans from 1665 to 2725 m a.s.l. Mean slope is 25%, and the vegetation cover Normalized Difference Vegetation Index (NDVI) is 0.38.

The second sub-watershed (hereafter called Laurichard) is constituted of granitic rocks on an ancient volcanic layer and extends over 5.3 km². It is part of the North exposed side of the Combeynot mountain range, which highest peak culminates at 3155 m. The Laurichard stream, fed in part by glacier melt, runs down this mountainous area and is sampled downhill, just before it joins the Tufiere stream.

Altitude on this watershed ranges from 1665 to 3155 m a.s.l. Mean slope is 30%, and the vegetation cover NDVI is 0.33.

The last river is Romanche, which integrates the response of the whole Romanche Valley, and is fueled by smaller tributaries such as Tufiere and Laurichard from the Lautaret Pass and the Ecrins National Park down to the artificial Chambon Lake. Our sampling point for this river is about 1000 m lower in altitude and approximately 12 km away from the first two tributaries (contributing area ≈ 220 km²).

Field sampling

Aerosols were collected on the Lautaret watershed from November 2014 to September 2015 using a homemade apparatus (similar to a Digitel ® DA77) equipped with a Digitel ® PM2.5 cutting head. We used QMA Whatman quartz filters for aerosols collection, burnt at 800 °C for 1 h to lower the blank signal ( 38 ). Filters were changed on a weekly basis, thus accumulating enough material for isotopic analysis.

Airflow was fixed at 0.5 m 3 per hour.

From December 2014 to April 2015 a snow pit (SP) was dug every month on the Lautaret watershed to provide profiles of NO 3 atm concentration and isotopic values in the snowpack. For each pit, snow was dug until the soil was reached, then after cleaning the vertical profile, samples were collected every 3 cm starting from surface snow.

Water samples were collected in the three streams (SI Figure S1, in red) from November 2014 to December 2015 to obtain a full one-year hydrological cycle and stream N chemistry. The samples were taken according to the Niwot Ridge LTER protocol ( 39 ). Samples were taken on a weekly basis when possible, and at most 2 weeks separated two consecutive samples.

More details regarding aerosols, snow and water sampling can be found in the SI, Appendix 1. Unfortunately, neither rivers discharge measurements nor wet deposition collection could be done at the time of the study because of the lack of equipment.

Appreciation of snowmelt timeframe, peak and length was based on visual observation of snow cover and rivers flow. The Laurichard stream could not be sampled from mid-January to mid-April as the riverbed was completely covered by the snowpack.

Samples pre-treatment and NO 3 -concentration analysis

Aerosols samples were analyzed by extracting NO 3 -from filters, immersed in 40 mL of ultrapure water in a centrifugal ultrafiltration unit (Milipore Centricon plus-70, 100

Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395 000 Daltons), shaken a few times, and then centrifuged at 4500 RPM for 20 minutes.

The extract was then collected in a 50 mL Corning ® centrifuge tube and kept frozen until further analysis.

Water and snow samples underwent the same pre-treatment for isotopic analysis.

Frozen samples were melted at room temperature overnight before being filtered using 0.45 µm Whatman GD/X syringe filters linked to a peristaltic pump.

Ultimately, aerosols, snow and stream samples were concentrated using an anionic resin (0.6 mL AG 1-X8 resin, Cl --form, Bio-Rad) with recovery efficiency over 98.5% ( 40 ) and eluted with 10 mL of a 1 M NaCl solution for isotopic analysis ( 41 ). Sample aliquots were taken before the resin concentration step for [NO 3 -] determination using a Continuous Flow Analysis spectrophotometer (SEAL Analyzer QuAAtro) based on cadmium-reduction of NO 3 -to nitrite ( 42 ). [NO 3 -] values are given with an uncertainty of 0.02 mg.L -1 , calculated as the mean standard deviation of ten successive measurements of the calibrating standards.

Isotopic analysis

Aerosols, snow and river samples were analyzed for ∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O, δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O and δ 15 N of NO 3 using the bacterial denitrifier technique in combination with the N 2 O gold decomposition method ( [START_REF] Casciotti | Measurement of the Oxygen Isotopic Composition of Nitrate in Seawater and Freshwater Using the Denitrifier Method[END_REF][START_REF] Kaiser | Triple Oxygen Isotope Analysis of Nitrate Using the Denitrifier Method and Thermal Decomposition of N 2 O[END_REF][START_REF] Morin | Tracing the Origin and Fate of NOx in the Arctic Atmosphere Using Stable Isotopes in Nitrate[END_REF] ). More details about the analytical procedure can be found elsewhere ( 46) and in the SI, Appendix 1.

All samples isotopic values were corrected for any isotopic effect possibly occurring during the procedure by processing simultaneously international reference materials standards, with a mean uncertainty of 0.2, 1.4 and 0.4 ‰ respectively.

End-member mixing analysis

The triple stable isotope approach using [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O/ [START_REF] Bobbink | Review and revision of empirical critical loads and dose-response relationships. Review and revision of empirical critical loads and doseresponse relationships: proceedings of an expert workshop[END_REF] O and [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O/ [START_REF] Bobbink | Review and revision of empirical critical loads and dose-response relationships. Review and revision of empirical critical loads and doseresponse relationships: proceedings of an expert workshop[END_REF] O enables exclusive quantification of unprocessed NO 3 atm . Because chemical pathways leading to the production of atmospheric NO 3 -involve NO x oxidation by ozone, which is the bearer of [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O-excess in the first place, it shows a significant positive deviation from the terrestrial fractionation line (TFL: δ 17 O ≈ 0.52 * δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O) ( [START_REF] Thiemens | History and applications of mass-independent isotope effects[END_REF][START_REF] Kaiser | Technical note: Consistent calculation of aquatic gross production from oxygen triple isotope measurements[END_REF] ). ∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O is a quantification of this deviation from the TFL, calculated as ∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] Post-deposition processes such as denitrification or assimilation should enrich δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O and δ 15 N along a 2:1 to 1:1 line ( [START_REF] Kendall | Tracing anthropogenic inputs of nitrogen to ecosystems[END_REF][START_REF] Granger | N and O isotope effects during nitrate assimilation by unicellular prokaryotic and eukaryotic plankton cultures[END_REF] ), but co-occurring soil production of NO 3 -by nitrification can lead to lower enrichment proportions ( 53). The ∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O value of residual NO 3 -are unaffected by post-deposition processes leading to NO 3 -loss because it is independent of the absolute δ 18 O values ( 32 ). It therefore provides a robust quantification of unprocessed NO 3 atm , and subsequently, of NO 3 bio as described in Eq. 2.

Statistical Analysis

We used two non-parametric statistical tests in our study. The choice for nonparametric tests lies in the small population size of the aerosols (n=26), snow (n=30, 22, 29, 36 and 26 for December, January, February, March and April SP, respectively), and stream water samples (n=42, 29 and 28 for Tufiere, Laurichard and Romanche, respectively).

A Mann-Whitney test was applied on river samples to determine whether mean concentrations and isotopic values were significantly different between streams. A Spearman test was applied to evaluate the correlation between stream water parameters (typically ∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O, δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O, δ [START_REF] Kaye | Competition for nitrogen between plants and soil microorganisms[END_REF] N and [NO 3 -]). Differences and correlations were held significant when the p value reached a 0.01 credible interval.

Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395

All statistical analyses were conducted using R software (v3.2.3).

Results

Nitrate input to ecosystems: NO 3 atm

Evolution of mean concentration, ∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O, δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O and δ 15 N of NO 3 -in snow pits throughout the winter and in aerosols throughout year 2015 can be found in Table 1 and Figure S3. This peak was also measured in April (orange line in Figure S3), but at a lower depth (40-60 cm) and to a lesser extent (2.7 mg.L -1 ), due to elution of the snowpack NO 3 by snowmelt water. ∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O-NO 3 -in snow varied between 20 and 35 ‰ (SI, Figure S3b), well within the atmospheric range of NO 3 -isotopic values reported in the literature ( [START_REF] Morin | Comprehensive isotopic composition of atmospheric nitrate in the Atlantic Ocean boundary layer from 65°S to 79[END_REF][START_REF] Savarino | Isotopic composition of atmospheric nitrate in a tropical marine boundary layer[END_REF] ). δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O and δ 15 N values were between 60 and 90 ‰ and -10 and 0 ‰, respectively (SI, Figure S3c andd), consistent with a reservoir exclusively made of

Nitrate output in rivers

All streams [NO 3 -] data can be found in the SI, Table S1. Laurichard showed significantly higher year-round [NO ] in summer (Figure 1a).

Tufiere had the widest range in ∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] 1b). Mean ∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O-NO 3 -was significantly higher in Romanche than in Tufiere and Laurichard, whereas there was no significant difference between Tufiere and Laurichard mean ∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O-NO 3 -(p-value = 0.5).

All streams showed similar range for δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O-NO 3 -and δ 15 N-NO 3 -, between -5 and 25 ‰ for δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O-NO 3 -and between -6 and 13 ‰ for δ 15 N-NO 3 -. Our findings were in the same range of previously measured NO 3 -isotopic values in alpine streams ( [START_REF] Mayer | Sources of nitrate in rivers draining sixteen watersheds in the northeastern US: Isotopic constraints[END_REF][START_REF] Campbell | Pathways for nitrate release from an alpine watershed: Determination using δ 15 N and δ 18 O: ALPINE WATERSHED NITRATE δ 15[END_REF][START_REF] Burns | Analysis of δ15N and δ18O to differentiate NO3-sources in runoff at two watersheds in the Catskill Mountains of New York[END_REF] ). Our choice to use snow-NO 3 -as the atmospheric end-member laid on simple but robust assumptions. First, snow is a good conservator of atmospheric nitrate in high accumulation sites (> 30 cm of snow accumulation) and preserves its isotopic composition ( [START_REF] Savarino | Nitrogen and oxygen isotopic constraints on the origin of atmospheric nitrate in coastal Antarctica[END_REF][START_REF] Hastings | Seasonal variations in N and O isotopes of nitrate in snow at Summit, Greenland: Implications for the study of nitrate in snow and ice cores[END_REF] ). Second, the snowpack is representative of winter bulk deposition, as it aggregates both wet and dry deposition ( [START_REF] Williams | Storage and release of solutes from a subalpine seasonal snowpack: soil and stream water response[END_REF]61 ). Third, most of NO 3 atm measured in streams was assumed to come from snowmelt, as previously observed in other seasonally snow-covered catchments ( [START_REF] Williams | Storage and release of solutes from a subalpine seasonal snowpack: soil and stream water response[END_REF]62 ). Little temporal variation of NO 3 concentration and isotopic values in snow pits profiles, along with no isotopic evidence of post-deposition processes further supported this assumption (SI, Appendix 2, Figures S3 andS4).

Discussion

By inputting mean snow ∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O-NO 3 -in Eq.2, we obtained: (δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O-H 2 O) ( [START_REF] Kendall | Tracing anthropogenic inputs of nitrogen to ecosystems[END_REF][START_REF] Rose | Drivers of atmospheric nitrate processing and export in forested catchments[END_REF] ). Note that our approach (i.e., ∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O vs δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O) might yield lower uncertainty compared to the traditional one (i.e., δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O calculation) which comes with a number of assumptions and caveats that are thoroughly dissected elsewhere ( [START_REF] Rose | Drivers of atmospheric nitrate processing and export in forested catchments[END_REF]65 ).

(Eq.
Conversely, we found a significant negative correlation between ∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] permafrost-related landform, which increased surface velocities over the past twenty years hint at increased permafrost temperatures, possibly to the point of thawing ( 76 ).

Considerably higher year-round [NO 3

-] in Laurichard compared to snow-fed Tufiereby 1-2 orders of magnitude -suggested that Laurichard NO 3 -exports were mainly driven by increasing soil exposure ( 77) more than any other geomorphic or biogeographic feature ( 78). Sources of such elevated levels of NO 3 -, while still debated ( [START_REF] Slemmons | The influence of glacial meltwater on alpine aquatic ecosystems: a review[END_REF][START_REF] Williams | Effects of glaciers on nutrient concentrations and phytoplankton in lakes within the Northern Cascades Mountains (USA)[END_REF] ), were likely linked to glaciers retreat and permafrost thawing leading to enhanced stream solutes fluxes ( [START_REF] Hood | Riverine organic matter and nutrients in southeast Alaska affected by glacial coverage[END_REF][START_REF] Barnes | Thawing glacial and permafrost features contribute to nitrogen export from Green Lakes Valley, Colorado Front Range, USA[END_REF] ), increased nitrification and/or mobilization of stored N from disturbed soils ( 83). Steeper slopes (SI, Figure S1), and potentially increased deposition loading because of increased orographic precipitations ( 21)relative to the Lautaret watershed -were certainly responsible for the occasional higher ∆ [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O-NO 3 -peaks, concomitant with rain events (Figure 1b). or uptake by the riparian vegetation ( 85). Denitrification and assimilation should discriminate δ [START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O against δ 15 N of residual NO 3 -following a line with a positive slope (i.e., enrich the residual nitrate with heavier isotopes), which was not observed in our study (SI, Figure S5). However, frequent recharge of NO 3 -by smaller tributaries such as Laurichard may have overprinted any biological isotopic signature ( 53).

On all basins, the strong controls seemingly exerted either by snowmelt (Tufiere and assimilation in an ecosystem and its remediation capacity, and can lead to intensive exports of N in streams [START_REF] Lovett | A New Conceptual Model of Nitrogen Saturation Based on Experimental Nitrogen Addition to an Oak Forest[END_REF]. Given the highest percentage of unprocessed NO 3 atm during hydrological events, our results provided additional evidence that catchment hydrology is certainly responsible for the kinetic N saturation in subalpine watersheds. Anyhow, assessment of N deposition load could help us estimate whether the critical threshold has been crossed, as evidenced in other alpine environments [START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF][START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF][START_REF] Boutin | Subalpine Pyrenees received higher nitrogen deposition than predicted by EMEP and CHIMERE chemistry-transport models[END_REF] ), and will be the object of future studies. S2). In a 21 st century with a shorter snow season, a higher fraction of the yearly deposited NO 3

-will be brought to subalpine ecosystems at a slower rate, during the growth season, instead of abruptly at snowmelt. If NO 3 -saturation is indeed kinetic -as our study suggested -then this climate driven change in NO 3 -influx timing could result in higher NO 3 -absorption in the environment, and lead to capacity saturation. This would come with a wide number of changes in the ecosystem, which have been extensively detailed elsewhere at a global scale ( 2,3,8 ), and more specifically in high altitude catchments [START_REF] Baron | Ecosystem Responses to Nitrogen Deposition in the Colorado Front Range[END_REF][START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF][START_REF] Elser | Shifts in lake N:P stoichiometry and nutrient limitation driven by atmospheric nitrogen deposition[END_REF] ).

These findings emphasize the need for more joint monitoring of subalpine soils and plant communities, with the objective being a better understanding of atmospheric N deposition consequences on biogeochemical cycling in these semi-isolated ecosystems. In particular, prospective studies should continue focusing on potential synergetic effects of land use management coupled with atmospheric deposition on ecosystems alteration, to better orient policy makers in N emissions mitigation and adaptation efforts.

Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero Red, blue and green colors stand for Tufiere, Laurichard and Romanche, respectively. Outliers (surrounded by stars) were not considered in the linear regression model, as we assume they result from biological processes (e.g., denitrification) and would blur determination of NO 3 -sources.

3 -

 3 With the aim to clearly delineate the fate of atmospheric Nr in a subalpine watershed, we first characterized the triple isotopic composition of NO 3 -in aerosols and snow deposited in high altitude catchments (> 1000m a.s.l.) of the central French Alps (i.e., the input Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395 flux). Second, we evaluated the fate of the deposited NO atm by monitoring NO 3 riverine exports from the watersheds (i.e., the output flux).

(

  International Atomic Energy Agency USGS 32, USGS 34 and USGS 35) through the same analytical batch. The isotopic standards were prepared in the same background matrix as samples (i.e., 1M NaCl). Standard deviation of the residuals Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395from the linear regression between the measured reference standards (n = 20) and their expected values served as indicator of the accuracy of the method. In our study, the ∆[START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O, δ[START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O and δ 15 N values of aerosols, snow and streams NO 3 -are given, with respect to atmospheric N 2 (AIR) and Vienna Standard Mean Ocean Water (VSMOW)

3 -(Eq. 1 ) ∆ 17 O-NO 3 -sample = ƒ atm * ∆ 17 O-NO 3 -atm + ( 1 -ƒ atm ) * ∆ 17 O-NO 3 -With ƒ atm (= %NO 3 - 3 - 3 - 3 - 3 -

 31173173117333333 O = δ[START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O -0.52 * δ[START_REF] Nanus | Spatial variation of atmospheric nitrogen deposition and critical loads for aquatic ecosystems in the Greater Yellowstone Area[END_REF] O in the present work. ∆[START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O value of atmospheric NO3 -generally ranges between 20 and 35 ‰ in temperate latitudes ([START_REF] Morin | Comprehensive isotopic composition of atmospheric nitrate in the Atlantic Ocean boundary layer from 65°S to 79[END_REF][START_REF] Savarino | Nitrogen and oxygen isotopic constraints on the origin of atmospheric nitrate in coastal Antarctica[END_REF] ) whereas ∆[START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O value of NO 3 -from all other sources (industrial fertilizers, nitrification) or biologically processed NO 3 atm , is 0 ([START_REF] Michalski | Tracing Atmospheric Nitrate Deposition in a Complex Semiarid Ecosystem Using Δ17[END_REF][START_REF] Michalski | Oxygen and nitrogen isotopic composition of nitrate in commercial fertilizers, nitric acid, and reagent salts[END_REF] ). In this study, we used these two distinct ∆[START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O values as end-members in a simple mixing model to quantify unprocessed NO atm proportion in streams. Calculation of atmospheric contribution, noted ƒ atm , was derived from the following equation: bio atm ), the mole fraction of the atmospheric contribution ( 32 ), ∆ 17 O-NO sample the measured value of ∆ 17 O-NO 3 -in a sample and ∆ 17 O-NO bio the value of 17 O-excess of biologically-derived NO 3 -(NO 3 bio ). Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395 As ∆ 17 O-NO Choice for the atmospheric end-member values of ∆ 17 O-NO atm is further discussed in section 4.1.

3 -]

 3 First snow falls occurred mid-November 2014 with several freezethaw events before it started to accumulate. On days of snow pits sample collection, snowpack depths were 66, 64, 139, 142 and 101 cm in December 2014, January, February, March and April 2015, respectively. Maximum snow accumulation (ca. 1.6 m) occurred early March 2015 (SI, Appendix 1). By the end of April 2015, the snowpack had completely melted. In December and January, low [NO 3-] were measured in all snow pits layers (SI, FigureS3a). In February and March, a peak of [NO up to 5 mg.L -1 between 70 and 90 cm of snow accumulation was recorded.

NO 3 -

 3 atm[START_REF] Kendall | Tracing anthropogenic inputs of nitrogen to ecosystems[END_REF]

4. 1 NO 3 -

 13 atm end-member Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395

3 - 3 - 3 - 3 -

 3333 The error range on ƒ atm was inferred from snow ∆[START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O-NO 3 -standard deviation, and calculated as 2 %.It must be underlined that snow as atmospheric end-member might not account correctly for wet or dry deposition of NO 3 -in summer. Wet N deposition has de facto been shown to contribute significantly to freshwaters NO 3 -contents, either through direct inputs due to flashy hydrology ( 34,56 ) or through indirect contribution via enhanced nitrification of atmospheric NH 4 + ( 63 ). We show that summer deposition of NO 3 -(wet or dry) as atmospheric end-member results in higher calculated NO 3 atm fraction in streams, up to 6% in this study (SI, Appendix 3). For that reason, all calculated ƒ atm values in the following sections must be considered as a lower estimate of NO 3 atm inputs in this system. Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395No correlation was found between ∆ 17 O-NO 3 -and [NO 3 -] in Tufiere, Laurichard and Romanche (Figure 2a, b and c), indicating that NO atm is not the main contributor to streams nitrate load on a yearly basis. For all streams, ∆ 17 O-NO 3 -was significantly correlated to δ 18 O-NO 3 -(Figure 2d, e and f) with the best correlation found in Romanche. Deviations from linear regressions are attributed to occasionally intense biological processes affecting δ 18 O signal but leaving ∆ 17 O intact. Otherwise, the strong linear correlations observed here suggest that mixing of sources more than biological processes could explain the observed seasonal variations of NO 3 -isotopic values. As a consequence, by extrapolating this linear relationship to ∆ 17 O-NO 3 -= 0 (i.e., 100 % biologically derived NO 3 -), we can determine the mean δ 18 O value of NO bio produced in the three watersheds: -6.0, -4.6 and -6.6 ‰ in Tufiere, Laurichard and Romanche, respectively. These results for NO bio were consistent with other studies estimation of δ 18 O-NO bio in streams, either calculated via the same method ( 32,64 ) or deduced from measured δ 18 O values of soil-water and O 2 , assuming they respectively contributed to NO 3 --O accordingly to δ 18 O-NO 3 -= 1/3 (δ 18 O-O 2 ) + 2/3

3 -

 3 2g, h and i) in the three streams, with once again the best fit for Romanche. With the same method as cited above, we can infer the mean δ 15 N-NO 3 bio : 4.4, 5.0 and 6.1 ‰ in Tufiere, Laurichard and Romanche, respectively. These values characterizing the NO bio end-member are typical of NO

4. 3 Seasonal variations of NO 3 - 3 -

 333 atm exportsAll stream water samples in this study had positive ∆[START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O-NO 3 -values, showing unquestionable inputs of NO atm in all streams year-round (Figure 1b). However, Tufiere and Laurichard, although sampled at the same altitude, showed different NO 3 -export patterns, certainly due to different hydrological behaviors and respective basins characteristics ( 58 ). Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395

NO 3 - 3 - atm deposits and contributed up to 35 % 3 - 3 - 3 - 3 -Oppositely, NO 3 -

 333533333 dynamic in Tufiere were most likely governed by snowmelt. Brief but high peaks of[START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O-excess of NO 3 -in March 2015, coincident with higher [NO 3 -], were attributed to direct runoff of snowmelt water to the stream ( 56,64 ). Rapid melting of the lower watershed snowpack washed away labile NO of the total stream NO 3 -pool. Leached NO bio from snow-free soils by subsurface melt or rain water accounted for both higher [NO 3 -] and lower ∆ 17 O-NO 3 -in the stream later in spring ( 74 ). Gentle hillslopes and the calcareous substrate of the Lautaret sub-watershed were also likely to facilitate snowmelt water infiltration and mixing with groundwater, a common feature in subalpine catchments ( 75 ). Starting early May, a slow but persistent increase in ∆ 17 O-NO 3 -suggested a resurgence of NO atm loaded water entering the stream, increasing NOatm proportion up to 25 % again at the end of August. Significantly higher precipitation in May and in August, compared to the previous months (SI, Figure S2a) could have triggered this resurgence by filling the aquifer with rain water, thus flushing stored water. The wide temporal range of this ∆ 17 O increase, encompassing six months from May to October, is in line with a temporally spread transit time of infiltrated melt water along an altitude gradient. Direct contribution of rain NO atm was unlikely to explain such pattern, given the low response of Tufiere [NO 3 -] and ∆ 17 O-NO 3 -to the storm in June (Figure 1a and b). Considering the basin topography and geology (i.e., gentler slopes and higher vegetation cover), most rain N inputs were certainly retained in soils or assimilated before either infiltration or direct runoff of rainwater in Tufiere ( 63 ). atm contributed only between 4 and 15 % of total NO 3 -in Laurichard stream from April to May (Figure 1b), coincident with the snowmelt period for this northern exposed watershed. Laurichard rock glacier is an actively monitored Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395

Lower vegetation cover -and therefore reduced plant NO 3 - 3 - 3 - 3 - 3 - 3 - 3 -

 3333333 atm uptake-and the poor buffering capacity of Laurichard watershed granitic bedrock are additional factors that could have fostered punctually enhanced exports of NO atm from wet deposition ([START_REF] Williams | Nitrate content and potential microbial signature of rock glacier outflow, Colorado Front Range[END_REF][START_REF] Rose | Drivers of atmospheric nitrate processing and export in forested catchments[END_REF] ). The storm in June 2015 (FigureS2) led to the strongest input of NO atm in this stream, accounting for 29 % of Laurichard NO 3 -pool (Figure1b). Simultaneous ∆[START_REF] Baron | Empirical Critical Loads of Atmospheric Nitrogen Deposition for Nutrient Enrichment and Acidification of Sensitive US Lakes[END_REF] O-NO 3 peaks and [NO 3 -] minima in July and September were further evidence of this flashy hydrology and illustrated stream glacial melt water dilution by rain. Conversely, we did not observe an early snowmelt runoff-linked peak in Romanche stream water ∆ 17 O-NO 3 -in spring. Instead, NO atm contribution increased steadily from April to July (34 % of NO atm on July 9 th ) then declined until reaching a baseflow value of about 5 % of NO atm during the dormant season. Romanche is the main stream of the valley, fueled by myriad smaller tributaries like Tufiere and Laurichard. Lower altitude catchments contributed most in spring, but as air temperature and Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395 solar radiation increased in summer, melting snowpack from higher watersheds of the Ecrins National Park may have led to enhanced NO atm export in Romanche stream leading to the wide ∆ 17 O-NO 3 -temporal pattern observed in this stream. Why [NO 3 -] decreased from Laurichard to Romanche is uncertain, but dilution by snow-fed streams like Tufiere between sampling locations is a possibility. Other possible sinks include denitrification, in-stream algal and phytoplankton assimilation

3 - 3 - 4 + 3 - 4 . 4

 334344 Romanche) or by glacier melt water (Laurichard) on stream NO 3 atm exports precluded the quantification of the direct NO atm summer deposition contribution. Summer deposition loads of NO atm to streams were either buffered by vegetation uptake and/or soil immobilization on Lautaret watershed, subdued by high NO 3 bio except on intense rain events on Laurichard watershed, or combined with the wide temporal response of the Romanche to snowmelt from uplands. Nevertheless, summer deposition of NH atm contributed significantly to subalpine soils N pool and was partly responsible for the NO bio exports in streams. Simultaneous monitoring of ∆ 17 O-NO 3 -, streams discharge, water isotopes and chemical tracers of hydrological paths remain needed to further constrain water sources and their respective contribution to NO 3 -exports in subalpine streams. Longer monitoring of streams exports is also warranted to better evaluate annual variability in this subalpine watershed, as year 2015 was significantly drier than Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395 previous years at the Lautaret Pass. Hydrological responses to rain events, and resulting NO 3 -export dynamics under different precipitation conditions, need to be assessed. Nitrogen saturation and ecological implication Despite presenting different N export dynamics, all streams shared uninterrupted presence of NO 3 -year-round, with a significant contribution from atmospheric deposition. In a supposedly N-limited ecosystem, such a pattern was unexpected as demand for dissolved inorganic nitrogen (DIN) from plants and microbial communities in spring and summer should have exceeded mobile NO 3 -reservoir in soils, precluding leaching to the streams. Nitrogen saturation has first been evaluated in an ecosystem subjected to increasing N deposition ( 2 ). Several stages of saturation have been distinguished, ranging from 0 (N-limited ecosystem) to 3 (fully saturated ecosystem characterized by N-leaching and higher streams [NO 3 -]). [NO 3 -] in Tufiere and Romanche was within the range of reported alpine and mountain stream [NO 3 -] ( 84 ), but [NO 3 -] in Laurichard was higher than in streams pinned as indicators of stage 2 or 3 ( 34 ). However, similar seasonal variations of [NO 3 -] in Tufiere, Laurichard and Romanche -high in spring after snowmelt, decreasing in summer, then increasing again as air temperature starts falling down (Figure 1) -were symptomatic of plants and microbial biomasses working as N sinks during the growing season. Evaluation of N saturation according to the conceptual model of biological demand exceeded by N supply does not take into account other drivers of N exports ( 58 ), and may not be adapted to describe the N saturation state of subalpine watersheds. Another approach to N saturation has been described as kinetic N saturation, where the rate of N inputs exceeds the rate of N Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395

N 3 - 3 -

 33 inputs at the watershed scale, through NO atm direct contribution or NH 4 + atm nitrification, are likely to substantially impact surrounding ecosystems, especially during the growing season following snowmelt. For instance, the lake Chambon, a freshwater reservoir fueled by the Romanche stream, which supplies downhill villages and cities -including Grenoble (163 000 inhabitants) -with drinking water, showed high NO atm contribution from May to November (up to 25 %, Table

While [NO 3 - 3 - 3 -

 333 ] in the lake averaged 2.0 ± 0.8 mg.L -1 during the growing season, far below the limit of 50 mg.L -1 (guideline value from The World Health Organization translated in the EU Water Framework Directive 2000/60/EC), the presence of NO atm in this elevated freshwater reservoir is yet another warning signal of potential threats due to atmospherically deposited pollutants. Evidence of lakes fertilization by atmospheric deposition in high altitude watersheds has been demonstrated before[START_REF] Hundey | Agriculture causes nitrate fertilization of remote alpine lakes[END_REF]. Fast cycling dynamics of high atmospheric Nr in lakes (89) can lead to welldocumented ecological consequences such as acidification (17), shift of algal and phytoplankton communities ([START_REF] Hobbs | Nitrogen deposition to lakes in national parks of the western Great Lakes region: Isotopic signatures, watershed retention, and algal shifts: NITROGEN DEPOSITION TO NORTHERN LAKES[END_REF][START_REF] Spaulding | Paleolimnological Records of Nitrogen Deposition in Shallow, High-Elevation Lakes of Grand Teton National Park, Wyoming[END_REF] ) and disturbance of lakes food web (92), and highlight the necessity to protect critical water resources. In winter, limited hydrological connectivity between soils and streams cannot explain the year-round export presence of unprocessed NO 3 atm in subalpine streams, highlighting the Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395 potential contribution of groundwater contamination to streams exports. Previous studies also showed that most alpine and subalpine plants acquire N during snowmelt ( 93 ). In traditionally N-poor subalpine ecosystems, increasing inputs of NO atm together with climate change could accelerate diversity and composition losses of plant communities.

  (69) Jusselme, M.-D.; Saccone, P.; Zinger, L.; Faure, M.; Le Roux, X.; Guillaumaud, N.; Bernard, L.; Clement, J.-C.; Poly, F. Variations in snow depth modify N-related soil microbial abundances and functioning during winter in subalpine grassland. Soil Biol. Biochem. 2016, 92, 27-37. (70) Hiltbrunner, E.; Schwikowski, M.; Körner, C. Inorganic nitrogen storage in alpine snow pack in the Central Alps (Switzerland). Atmos. Environ. 2005, 39 (12), 2249-2259. (71) Schmidt, S. K.; Lipson, D. A. Microbial growth under the snow: implications for nutrient and allelochemical availability in temperate soils. Plant Soil 2004, 259 (1), 1-7. (72) Williams, M. W.; Brooks, P. D.; Mosier, A.; Tonnessen, K. A. Mineral nitrogen transformations in and under seasonal snow in a high-elevation catchment in the Rocky Mountains, United States. Water Resour. Res. 1996, 32 (10), 3161-3171. (73) Saccone, P.; Morin, S.; Baptist, F.; Bonneville, J.-M.; Colace, M.-P.; Domine, F.; Faure, M.; Geremia, R.; Lochet, J.; Poly, F.; et al. The effects of snowpack properties and plant strategies on litter decomposition during winter in subalpine meadows. Plant Soil 2013, 363 (1-2), 215-229. (74) Liu, F.; Williams, M. W.; Caine, N. Source waters and flow paths in an alpine catchment, Colorado Front Range, United States: SOURCE WATERS AND FLOW PATHS IN ALPINE CATCHMENTS. Water Resour. Res. 2004, 40 (9). (75) Cowie, R. M.; Knowles, J. F.; Dailey, K. R.; Williams, M. W.; Mills, T. J.; Molotch, N. P. Sources of streamflow along a headwater catchment elevational gradient. J. Hydrol. 2017, 549, 163-178. (76) Bodin, X.; Thibert, E.; Fabre, D.; Ribolini, A.; Schoeneich, P.; Francou, B.; Reynaud, Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395

Figure 2

 2 Figure 2 Correlation between nitrate ∆ 17 O (‰) and a), b), c) log([NO 3 -]); d), e), f)

Figure 3 4 + 3 - atm and NO 3 -from nitrified NH 4 + 3 -Figure 1

 3433431 Figure 3 Dual isotopes plot (∆ 17 O vs. δ 15 N) illustrating the mixing between three sources of NO 3 -in our study sites. The plain triangles feature the mixing range between snow NO 3 atm and NO 3 -from nitrified NH 4 + atm in grey, NO 3 -from nitrified

Figure 2

 2 Figure 2

  Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52(10), 5561-5570. , DOI : 10.1021/acs.est.7b02395

  , A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395Biogeochemistry 2009, 95 (1), 77-94. (61) Campbell, J. L.; Mitchell, M. J.; Mayer, B.; Groffman, P. M.; Christenson, L. M. Mobility of Nitrogen-15-Labeled Nitrate and Sulfur-34-Labeled Sulfate during Snowmelt. Soil Sci. Soc. Am. J. 2007, 71 (6), 1934. (62) Brooks, P. D.; Williams, M. W. Snowpack controls on nitrogen cycling and export in seasonally snow-covered catchments. Hydrol. Process. 1999, 13 (14), 2177-2190. (63) Nanus, L.; Williams, M. W.; Campbell, D. H.; Elliott, E. M.; Kendall, C. Evaluating Regional Patterns in Nitrate Sources to Watersheds in National Parks of the Rocky Mountains using Nitrate Isotopes. Environ. Sci. Technol. 2008, 42 (17), 6487-6493. (64) Darrouzet-Nardi, A.; Erbland, J.; Bowman, W. D.; Savarino, J.; Williams, M. W. Landscape-level nitrogen import and export in an ecosystem with complex terrain, Colorado Front Range. Biogeochemistry 2012, 109 (1-3), 271-285. (65) Snider, D. M.; Spoelstra, J.; Schiff, S. L.; Venkiteswaran, J. J. Stable Oxygen Isotope Ratios of Nitrate Produced from Nitrification: 18O-Labeled Water Incubations of Agricultural and Temperate Forest Soils. Environ. Sci. Technol. 2010, 44 (14), 5358-5364. (66) Xue, D.; Botte, J.; De Baets, B.; Accoe, F.; Nestler, A.; Taylor, P.; Van Cleemput, O.; Berglund, M.; Boeckx, P. Present limitations and future prospects of stable isotope methods for nitrate source identification in surface-and groundwater. Water Res. 2009, 43 (5), 1159-1170. (67) Mara, P.; Mihalopoulos, N.; Gogou, A.; Daehnke, K.; Schlarbaum, T.; Emeis, K.-C.; Krom, M. Isotopic composition of nitrate in wet and dry atmospheric deposition on Crete in the eastern Mediterranean Sea: ISOTOPIC COMPOSITION OF NITRATE IN DEPOSITION. Glob. Biogeochem. Cycles 2009, 23 (4), n/a-n/a. (68) Guha, T.; Lin, C. T.; Bhattacharya, S. K.; Mahajan, A. S.; Ou-Yang, C.-F.; Lan, Y.-P.; Hsu, S. C.; Liang, M.-C. Isotopic ratios of nitrate in aerosol samples from Mt. Lulin, a highaltitude station in Central Taiwan. Atmos. Environ. 2017, 154, 53-69.

Acknowledgments

This study was supported by grants from the Labex OSUG@2020 ("Investissements d'avenir" -ANR10 LABX56), the ARC -Environnement Région Rhone-Alpes and the ACS Paragon Plus Environment Environmental Science & Technology Version postprint Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395 Grenoble-Chambéry DIPEE CNRS. This work also benefited from the National Research Agency support ("Investissements d'avenir" -ANR11 INBS-0001AnaEE-Services) and from the SAJF research station (UMR 3370, UGA/CNRS) infrastructures and competences. The study took place on the long-term ecological research site Zone Atelier Alpes, a member of the ILTER-Europe. We would like to thank J.-L. Jaffrezo, F. Masson, V. Lucaire, E. Vince, C. Arnoldi and S. Albertin for help with either laboratory or field work. We acknowledge J. Renaud for help with SIG. ACS Paragon Plus Environment Environmental Science & Technology Version postprint Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10), 5561-5570. , DOI : 10.1021/acs.est.7b02395

Supporting Information

Appendix 1 includes complementary information on sampling and analytic protocols.

List of Figures

Version postprint

Comment citer ce document : Bourgeois, I., Savarino, J., Caillon, N., Angot, H., Barbero, A., Delbart, F., Voisin, Clement, J.-C. (2018). Tracing the fate of atmospheric nitrate in a subalpine watershed using Delta O-17. Environmental Science and Technology, 52 (10)