Pierre Chevallier
email: pierre.chevallier@ird.fr

Jean-Luc Seidel

Jean-Denis Taupin

Ornella Puschiasis

Headwater Flow Geochemistry of Mount Everest (Upper Dudh Koshi River, Nepal)

Keywords: major ions, trace elements, stable isotopes, precipitation, river flow, water use, Central Himalaya

The aim of this work, conducted in the upper valley of the Khumbu on the southern part of Mount Everest, is to approach in parallel three topics: (i) the dynamics of the water geochemistry, major ions and trace elements; (ii) the stable water isotopes of precipitation and rivers; and (iii) the water uses by the inhabitants. As in most mountain environments, the Khumbu area is threatened by climate change, which impacts the cryosphere and consequently the people and the landscapes. Moreover, changes in water use are also related to new needs stemming from tourism, which strongly affect local livelihood. For the first two topics, new results are presented. They provide details on the global chemical quality of the river water and show how certain elements are seasonally influenced and how other elements allow us to distinguish the water origins within the study zone. Beside the use of stable isotopes to determine mainly the origin of the water flow in the rivers, the isotopic patterns confirm the double climatic influence of the westerly fluxes in the winter season and of the Asian monsoon in the summer season. Regarding water use, the study confirms that the chemical quality of the water course is good, but does not conclude on the biological quality, which has not been investigated. In conclusion, we attempt to predict the future of the geochemistry patterns submitted to the double pressure of climate change and the surge in tourism.

Introduction

The future of the cryosphere (glacier and snow cover) in the Hindu Kush Himalaya high mountains is a major concern [START_REF] Pörtner | IPCC Special Report on the Ocean and Cryosphere in a Changing Climate[END_REF]Wester et al. 2019); it is threatened not only by global warming due to greenhouse gas emissions, but also by local air pollution due to the atmospheric brown cloud, and especially the transportation and deposition of black carbon aerosol [START_REF] Bonasoni | Atmospheric Brown Clouds in the Himalayas: First Two Years of Continuous Observations at the Nepal Climate Observatory-Pyramid (5079 m)[END_REF][START_REF] Kaspari | Recent Increase in Black Carbon Concentrations from a Mt. Everest Ice Core Spanning 1860-2000 AD[END_REF][START_REF] Jacobi | Black carbon in snow in the upper Himalayan Khumbu Valley, Nepal: observations and modeling of the impact on snow albedo, melting, and radiative forcing[END_REF] from human activities in the densely inhabited regions of the south and southwest of the central Himalaya mountain range as well as the rapid increase of tourist activity [START_REF] Jacquemet | The Sherpa Community in the 'Yak Donald's' Era : Locational Struggles for Access to Resources in Mount Everest Touristic Region (Nepal)[END_REF].

In this context, the Paprika and Preshine projects (see Funding section) joined the efforts of Nepalese, French, and Italian research teams (see Acknowledgments section) to explore the impact of these anthropogenic constraints on the water cycle dynamics in the Dudh Koshi River Basin (Figure 1) on the south side of Mount Everest (8847 m). Several reasons justified the choice of this study zone that is characterized by two confluent watercourses, the Imja River and the Khumbu River, respectively, originating from the southern and western faces of the main range of Mount Everest and its satellite summits.

Among these reasons, the following are particularly important in the context of this article:

• Relative facility of access with the Everest Base Camp Trail;

• Settlements at high altitude (above 3500 m) where inhabitants are facing a wide range of changes beside climatic one as a more tourism-centered economy involving integration to global markets, agropastoral and lifestyles changes, and new migration patterns [START_REF] Aubriot | Reconfiguration of the Water-Energy-Food Nexus in the Everest Tourist Region of Solukhumbu, Nepal[END_REF]); • Presence of a high-altitude scientific laboratory, the Pyramid of Lobuche (5050 m), administrated by the Italian Ev-K2/CNR association in agreement with the Nepalese Academy of Science and Technology (NAST).

In this framework, a working task of the Paprika and Preshine projects has been devoted to the identification of water resources and the perception of this held by the local inhabitants. In the preliminary exchanges with the scientists of the Paprika and Preshine projects, the Khumbu valley inhabitants manifested a deep concern regarding their water resource and its future. They were acutely aware that climate and economic changes will have a direct impact on the environment in which they live. As a consequence, it appears as a necessity to also aboard the climate processes and the water balance through the geochemical angle, even though this was not the primary route chosen for these projects.

The village of Pangboche, which includes several settlements, was chosen as the most convenient. The main settlement is located at an elevation of approx. 3950 m on the right bank of the Imja River, 4 km after the confluence of its two main branches: the Khumbu River flowing from the north and the Upper Imja River flowing from the west. An important concern of the local population is the future of the water resources, not only in quantity, but also in quality. Considering the latter point, it was also stated that the water quality could be a substantial indicator of the water flow processes, especially regarding the different origins of these flows: glacier melt, snowmelt, groundwater, or direct surface flow.

During the past decade, geochemistry studies have been conducted of the Himalaya water flows (e.g., [START_REF] Jeelani | Geochemical Characterization of Surface Water and Spring Water in SE Kashmir Valley, Western Himalaya: Implications to Water-Rock Interaction[END_REF][START_REF] Ghezzi | Findings on Water Quality in Upper Mustang (Nepal) from a Preliminary Geochemical and Geological Survey[END_REF]), but no study has been undertaken in the context of very high altitudes covering dynamically an entire year. This issue underlines the exploratory character of the current study, which, however, does not aim to address all the questions raised in this exceptional framework.

Numerous isotopic precipitation and river studies have been carried out in Himalaya at the local or regional scale in the past few years [START_REF] Garzione | Predicting Paleoelevation of Tibet and the Himalaya from Δ18O vs Altitude Gradients in Meteoric Water across the Nepal Himalaya[END_REF][START_REF] Wen | The Altitude Effect of Δ18O in Precipitation and River Water in the Southern Himalayas[END_REF][START_REF] Jeelani | Variation of Δ18O and ΔD in Precipitation and Stream Waters across the Kashmir Himalaya (India) to Distinguish and Estimate the Seasonal Sources of Stream Flow[END_REF][START_REF] Balestrini | Chemistry and Isotopic Composition of Precipitation and Surface Waters in Khumbu Valley (Nepal Himalaya): N Dynamics of High Elevation Basins[END_REF][START_REF] Balestrini | Wet Deposition at the Base of Mt Everest: Seasonal Evolution of the Chemistry and Isotopic Composition[END_REF][START_REF] He | Stable Isotopes in Monsoon Precipitation and Water Vapour in Nagqu, Tibet, and Their Implications for Monsoon Moisture[END_REF][START_REF] Ren | Controls on the Stable Isotopes in Precipitation and Surface Waters across the Southeastern Tibetan Plateau[END_REF][START_REF] Florea | Stable Isotopes of River Water and Groundwater along Altitudinal Gradients in the High Himalayas and the Eastern Nyainqentanghla Mountains[END_REF]Jeelani and Deshpande 2017;Jeelani et al. 2017;[START_REF] Guo | Controls of Precipitation Δ18O on the Northwestern Tibetan Plateau: A Case Study at Ngari Station[END_REF][START_REF] Li | Spatial Distribution and Controlling Factors of Stable Isotopes in Meteoric Waters on the Tibetan Plateau: Implications for Paleoelevation Reconstruction[END_REF][START_REF] Kumar | Tracing Isotopic Signatures (ΔD and Δ18O) in Precipitation and Glacier Melt over Chorabari Glacier-Hydroclimatic Inferences for the Upper Ganga Basin (UGB), Garhwal Himalaya[END_REF][START_REF] Verma | Hydroclimatic Significance of Stable Isotopes in Precipitation from Glaciers of Garhwal Himalaya, Upper Ganga Basin (UGB), India[END_REF][START_REF] Shen | Precipitation δ 18 O on the Himalaya-Tibet Orogeny and Its Relationship to Surface Elevation[END_REF][START_REF] Singh | Moisture Sources for Precipitation and Hydrograph Components of the Sutri Dhaka Glacier Basin, Western Himalayas[END_REF]. The main aim of these studies was to link the isotopic variability recorded in precipitation to climate parameters and air mass circulation and the transfer of this isotopic signal through the global and complex altitudinal hydrosystem from glaciers to tropical valleys. Regional precipitation studies of the southern external border of Himalaya (Jeelani and Deshpande 2017) from Kashmir (western Himalaya) to Assam (eastern Himalaya), or of the whole Tibetan Plateau [START_REF] Li | Spatial Distribution and Controlling Factors of Stable Isotopes in Meteoric Waters on the Tibetan Plateau: Implications for Paleoelevation Reconstruction[END_REF], showed that the isotopic variation observed in precipitation across the Himalayas conforms to the regional repartition of the two main moisture sources: the westerly fluxes and Asian monsoon. Local studies of precipitation isotopes were carried out in central Nepal, Kathmandu, and the north of Kathmandu [START_REF] Wen | The Altitude Effect of Δ18O in Precipitation and River Water in the Southern Himalayas[END_REF] as well as in the Khumbu Valley at the Pyramid Laboratory [START_REF] Balestrini | Chemistry and Isotopic Composition of Precipitation and Surface Waters in Khumbu Valley (Nepal Himalaya): N Dynamics of High Elevation Basins[END_REF][START_REF] Balestrini | Wet Deposition at the Base of Mt Everest: Seasonal Evolution of the Chemistry and Isotopic Composition[END_REF].

The current paper aims to present, analyze and discuss, on the one hand, the geochemical behavior of the water flows used by the inhabitants of Pangboche for their activities, during the year 2011, exploring the conductivity, pH, major ions, and trace elements dissolved in the water; and, on the other hand, the water-stable isotopes in the precipitation and river flows of the Khumbu area, during the period from November 2014 to May 2017. The paper aims to link these data to the water origins and seasonal variability in the context of global change.

2 Study area and methods

Study area

The climate of the study area is dominated from June to September by monsoon dynamics [START_REF] Wang | The Asian Monsoon[END_REF][START_REF] Bookhagen | Toward a Complete Himalayan Hydrological Budget: Spatiotemporal Distribution of Snowmelt and Rainfall and Their Impact on River Discharge[END_REF][START_REF] Immerzeel | Climate Change Will Affect the Asian Water Towers[END_REF][START_REF] Turner | Climate Change and the South Asian Summer Monsoon[END_REF], but winter and pre-monsoon precipitations can occur from December to April due to the Western Disturbances, which are part of the westerlies entry, originating from the Mediterranean region [START_REF] Pisharoty | Western Disturbances and Indian Weather[END_REF][START_REF] Madhura | Changes in Western Disturbances over the Western Himalayas in a Warming Environment[END_REF].

Above 5000 m in general, and lower during the winter, satellite imagery shows that the snow cover can be wide; however, the difficulty of monitoring snowfall in high mountains [START_REF] Sevruk | Reliability of Precipitation Measurements[END_REF]Tahir et al. 2011) does not allow us to quantify precisely the volume of snowfall in the study area. Recent studies have explored the spatial distribution of precipitation in this area [START_REF] Savéan | Water Budget on the Dudh Koshi River (Nepal): Uncertainties on Precipitation[END_REF][START_REF] Gonga-Saholiariliva | Geostatistical Estimation of Daily Monsoon Precipitation at Fine Spatial Scale: Koshi River Basin[END_REF][START_REF] Eeckman | Providing a Non-Deterministic Representation of Spatial Variability of Precipitation in the Everest Region[END_REF][START_REF] Mimeau | Evaluation of Gridded and In Situ Precipitation Datasets on Modeled Glacio-Hydrologic Response of a Small Glacierized Himalayan Catchment[END_REF]. They roughly show, beside a large local heterogeneity mainly due to the steep relief, the valley orientation and the slope aspect, a positive gradient with the altitude until a peak of annual precipitation between 2500 and 3200 m and a negative one above.

The geology of the southern area of Mount Everest has been detailed by [START_REF] Bortolami | Limnology of High Altitude Lakes in the Mt Everest Region, Nepal[END_REF] and by [START_REF] Searle | The Structural Geometry, Metamorphic and Magmatic Evolution of the Everest Massif, High Himalaya of Nepal-South Tibet[END_REF]. It is dominated by Precambrian-Early Paleozoic sillimanite gneisses in the high faces, which alternate in some places with intrusive Miocene leucogranites. The highest zone (Everest, Lhotse), above 6500 m, presents Ordovician shale series and limestone layers. Except for those slopes covered by glaciers or rock glaciers, the valley slopes and valley bottoms are mainly composed of fluvio-glacial deposits and debris, with the presence of moraines of different levels and ages. According to [START_REF] Bortolami | Limnology of High Altitude Lakes in the Mt Everest Region, Nepal[END_REF], the composition of the rocks leads to a low weathering and a low impact on the chemical composition of the flows. The same author notes that the aquifers located in the debris material have a high porosity. Their thicknesses are largely unknown. By contrast, the fissured rocks, which constitute the bed rock, are generally impermeable. A few other studies are devoted to groundwater storage in the Himalayas. Although their findings are not very helpful with regard to the geological characteristics [START_REF] Dongol | Shallow Groundwater in a Middle Mountain Catchment of Nepal: Quantity and Quality Issues[END_REF][START_REF] Jeelani | Aquifer Response to Regional Climate Variability in a Part of Kashmir Himalaya in India[END_REF] or the scale of the approach used [START_REF] Andermann | Impact of Transient Groundwater Storage on the Discharge of Himalayan Rivers[END_REF], these studies highlight a notable contribution of snow and glacier melt to groundwater. [START_REF] Andermann | Impact of Transient Groundwater Storage on the Discharge of Himalayan Rivers[END_REF] assess the storing capacity of the whole Dudh Koshi basin (3700 km²) to be approx. 300 mm, i.e., less than 20% of the average annual discharge, which represents a low impact of the groundwater in our study area, considering that most of the reservoirs are very likely concentrated in the bottom material of the middle and low elevations. Other authors, including [START_REF] Nepal | Understanding the Hydrological System Dynamics of a Glaciated Alpine Catchment in the Himalayan Region Using the J2000 Hydrological Model[END_REF], [START_REF] Savéan | Water Budget on the Dudh Koshi River (Nepal): Uncertainties on Precipitation[END_REF], and [START_REF] Eeckman | Comparing the ISBA and J2000 Approaches for Surface Flows Modelling at the Local Scale in the Everest Region[END_REF], consider in their modeling approaches that the volume stored over a long period in groundwater reservoirs is negligible.

Water resources for local population

The Khumbu zone (Figure 1) encompasses an area of approximately 1100 km² along the border between Nepal and the Tibet Autonomous Region of China. All these territorial and economic mutations have led to profound changes in water resources, increasing needs previously limited to domestic (drinking water, cooking, personal hygiene), agricultural (irrigation of barley), and religious purposes (water-driven prayer wheels, water spirit shrine) [START_REF] Aubriot | Reconfiguration of the Water-Energy-Food Nexus in the Everest Tourist Region of Solukhumbu, Nepal[END_REF]. Water is taken directly from springs or small streams flowing through the settlements or channeled by pipes to houses, since large rivers are not the primary source of water for villagers [START_REF] Mcdowell | Climate-Related Hydrological Change and Human Vulnerability in Remote Mountain Regions: A Case Study from Khumbu, Nepal[END_REF]. In Pangboche village, changes appeared some 20 years ago with the installation of running water supplying guest houses, a bottled-water manufacturing plant in 2003, and a micro-hydroelectric plant in 2004 [START_REF] Puschiasis | Des enjeux planétaires aux perceptions locales du changement climatique : pratiques et discours au fil de l'eau chez les Sherpa de la vallée du Khumbu (Everest, Népal)[END_REF]. Water has become a "commodity" (André-Lamat 2017) with a proliferation of uses for tourism (shower, flushing toilets, bottled water) and for electrification. Khumbu inhabitants have become highly dependent on reliable water supply systems to respond to the new types of usages, which is key to local development. Nevertheless, there is a lack of a proper management system at a regional level to reduce the pressure on water resources.

Methods of sampling and analysis

Labels A to Y refer to the sampling points shown in Figure 1 and described in Table 1. They are ordered by altitude from bottom to top.

Conductivity, major and trace elements

Temperature, pH, and electrical conductivity (Tref =25°C) were measured in the field, using a portable pH meter and conductivity meter (WTW 3210i®). Water samples (125 mL) were filtered in the field with a PP syringe and Durapore® membrane (0.22 µm) and stored in acid-washed HDPE bottles. Aliquots for major cations and trace elements were acidified with ultrapure HNO3 (1‰ v/v). Samples were stored at 4°C before reaching Montpellier for analysis.

Five sites in the surroundings of Pangboche village were selected for the water sampling (G, H, I, J, L):

• The G point on the Imja River represents the reference site for the main river after the confluence of the Khumbu branch and the Imja branch. These catchments include, on the one hand, the large glaciers of the south side of the Everest Range and, on the other hand, the Imja moraine lake, which concentrates the melting runoff from the majority of the glaciers of the upper Imja Valley. • The Tauche point (J) on the east stream of the southern slope of the Tauche Peak (6542 m).

This watercourse collects the melt flow from the very small glacier located on the peak summit. • The Teouma (L) and Kisang (I) points on the central stream flow do not receive water of glacial origin. Because this watercourse flows through the village of Pangboche, the first point was chosen upstream and the second downstream from the village in order to analyze how the water quality of the stream is influenced by the village. • The Chomar (H) source on the west side of Pangboche was also sampled, because its water was drawn and bottled in 2011 by a small company to be sold to tourists under the brand name of "Namaste Sabina Tabuche Beiu." After 2013, the company no longer produced bottled water for unknown reasons.

The water flows were sampled 18 times between February and November 2011 following the complete annual cycle (Figure 2). A total of 12 samplings benefitted from a complete protocol (64 samples) and 6 more samplings, in winter and autumn, from measurements of electrical conductivity and pH only.

Two complementary water samples were collected in June 2012 in Imja Lake (Y) and a rainfall reference was taken in the settlement of Pheriche (P). In addition, several measurements of electrical conductivity were carried out in different watercourses within the Imja River basin.

Chemical analyses were performed at the HydroSciences water chemistry laboratory in Montpellier (France). Total alkalinity was measured by acid titration with HCl 0.01 N (Gran method). Major ions (Cl -, NO3 -, SO4 2-, Ca 2+ , Mg 2+ , Na + , and K + ) were analyzed by ionic chromatography (Dionex ICS 1000®). The precision error was < ± 5%. Trace elements (Li, B, Al, Si, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Rb, Sr, Mo, Cd, Cs, Ba, Pb, and U) were analyzed with Q-ICPMS (X series2 Thermo Scientific®) on the AETE (Analyse des Elements en Trace dans l'Environnement) technical platform of Montpellier University. The precision error was < ± 8%.

Stable isotopes

Six sampling campaigns in rivers located between 1985 m (Kharikola, label A) and 5000 m (foot of glaciers, X, Y) were carried out (November to December 2014, November 2015, November 2016, March 2015, May 2016, May 2017).

In addition, during the study interval from November 2014 to December 2016, monthly rainfall was collected at Pangom (2890 m, E) using a homemade rain gauge with an 80-cm² cross-section in a 5-L plastic tank inside an isotherm box, which was linked to the gauge with a flexible pipe and hermetically sealed to avoid direct evaporation.

The rainfall and river samples were stored in amber glass bottles (25 mL) with conical plugs and transported in shaded conditions to the laboratory in Montpellier.

Water-stable isotopes were measured with an Isoprime® mass spectrometer on the LAMA platform of HydroSciences Montpellier (LAboratoire Mutualisé d'Analyse des isotopes stables de l'eau). The oxygen isotopic composition was measured after equilibration of 200 µL of water with CO2 via the dual-inlet technique, with an overall precision of ± 0.06‰. Deuterium was measured by continuousflow using a Eurovector Pyr-OH ® elemental analyzer converting 0.5-µL injections of water to H2 on Cr powder at 1070°C, with an overall precision of ±0.6‰.

Water isotopic compositions are reported as δ 18 O and δ 2 H on the V-SMOW scale.

Results

Electrical conductivity and pH

Figure 2 shows the dynamics of the electrical conductivity and pH during the year 2011 at the five measurement points (G, H, I, J, L) of the Pangboche area.

Regarding electrical conductivity, the absolute values are below 60 µS/cm. These very low values indicate the slight level of mineralization of the flows. Specifically, the five sites present two main behaviors: (i) Kisang and Teouma located on the same watercourse have an almost identical and constant extremely low conductivity during the year (approx. 20 µS/cm), (ii) for Tauche and Imja the values are 2-3 times higher, with a slight decrease during and shortly after the monsoon (July-October), meaning that the increasing runoff generates a dilution effect. The Chomar site fits between the two, with a decrease during the monsoon season.

The pH varies between 7 and 8, except for Pangboche-Chomar bottled drinking water (8.6) and for the rain sample (6.8). The different sites present a short-term variability from date to date. However, two main behaviors can be observed: (i) for the slope water courses (Tauche, Kisang, Teouma, and Chomar), the amplitude of the short-term variability reaches 0.5 and a relative dilution effect appears during the monsoon; (ii) the valley river (Imja) has an almost stable pH value (≈ 7.8) during the course of the year, but higher than the pH of the slope water courses.

Major ions

The following ranges in concentration were shown by the major cations and anions: Ca

2+ (1.1-11.1 mg/L), Mg 2+ (0.1-0.7 mg/L), Na + (0.4-1.8 mg/L), K + (0.2-1.3 mg/L), HCO3 -(5.1-32.3 mg/L), SO4 2- (0.4-14.5 mg/L), NO3 -(0.1-1.5 mg/L), Cl -(0.1-0.6 mg/L).
Silica ranges from 1.3 to 20.8 mg/L. Average concentrations of major ions are reported in Table 2. The Piper diagram for major cations and anions (Figure 3 and Table 2) shows variations in the chemical composition of the surface waters, which is dominated by Ca 2+ and HCO3 -. The waters are mainly of Ca + -Mg + -HCO3 -type. Waters influenced by glacier melt (Tauche and Imja sites) exhibit an enrichment in SO4 2-, particularly for the Tauche site during the monsoon season. Ca 2+ is the dominant cation contributing more than 70% to the cation budget, followed by Na + (< 20%) and Mg 2+ (<10%). In Kisang waters and to a less extent in Teouma waters, before the monsoon season, Na + is the dominant cation. Cl -and NO3 -concentrations are very low, <0.6 mg/L and <1.5 mg/L, respectively. The HCO3 -contribution to the anion budget ranges between 60 and 90%, except for Tauche water during the monsoon season, which evolves to a Ca + -Mg + -SO4 2-type. The origin of sulfates is found in sulfide oxidation via glacier runoff, as suggested by [START_REF] Hodson | Chemical Denudation and Silicate Weathering in Himalayan Glacier Basins: Batura Glacier, Pakistan[END_REF], because no anhydrite or gypsum have been identified in the region. The plot of Ca + +Mg + +Na + versus HCO 3 -+SO 4 2-from all the waters shows that most of the samples lie close to the 1:1 line, indicating the dissolution of calcite, dolomite, silicates, and sulfides (Figure 4) as suggested by [START_REF] Crespo | Ionic and stable isotope chemistry as indicators of water sources to the Upper Mendoza River basin, Central Andes of Argentina[END_REF].

For all major elements, including SiO2, the temporal evolution of the concentrations displays a dilution effect during the monsoon season, except for the Imja site where an increase in the concentration of all the major elements occurs during the same season, especially in August 2011, which corresponds to the maximum glacier melt. This enrichment demonstrates that the glaciated catchment undergoes more intense chemical weathering taking place beneath the glacier than catchments that do not have a glacier because the CO2 dissolved in the proglacial zone with the aerated flow conditions characteristic of the meltwater environments may promote chemical weathering by maintaining the acid potential of the water (Reynold & Johnson 1972) in [START_REF] Singh | Major Ion Chemistry and Weathering Control in a High Altitude Basin: Alaknanda River, Garhwal Himalaya, India[END_REF].

Trace elements

Average concentrations for measured trace elements are presented in Table 3. Dissolved trace elements such as Li, B, Ni, Zn, Cu, Rb, Sr, Ba, and U show a dilution effect during the monsoon season at the Teouma, Kisang, Tauche, and Chomar sites whereas the Imja site in the same season exhibits, except for boron, a high concentration increase, by a factor of 10 to 100, as shown, for example, for rubidium (Figure 5). The origin of this increase can be found in the glacier melt enriched in subglacial material leached by the heavy rain during the monsoon. This phenomenon underlines the integrating power of the Imja Khola River. On the other hand, Al, Ti, Fe, and Mn to a lesser extent increase in all the sites during the monsoon season. In comparison with the Upper Mustang rivers in the western region of Nepal Himalaya [START_REF] Ghezzi | Findings on Water Quality in Upper Mustang (Nepal) from a Preliminary Geochemical and Geological Survey[END_REF], the concentrations are elevated, from 5 to 6600 µg/L for Al, from 0.5 to 485 µg/L for Ti, from 5 to 7130 µg/L for Fe, and from 2 to 200 µg/L for Mn. These elements are mobilized by the surface runoff during the monsoon season and their common origin is to be found in the weathered bedrock.

In regards of the WHO drinking water guidelines, no major or trace elements exceed the recommended values, even if the concentrations for some major or trace elements of the Imja River are elevated.

Stable isotopes

Three complete years (2014-2016) of meteorological data (P, T) are available at the Pangom station (2885 m, label D) [START_REF] Chevallier | Paprika -Preshine Hydrology Data Sets in the Everest Region (Nepal). 2010-18[END_REF], and a monthly sampling of rainfall for water isotope analysis was carried out between November 2014 and December 2016. The comparison of all the results is only qualitative owing to the small temporal record at the Pangom weather station.

The mean annual temperature measured at Pangom in 2015 was 6.7°C (7.6°C in 2014 and 8.0°C in 2016) with lower temperatures in the dry season (min. in December: -1.1°C) and higher temperatures during the monsoon season (Figure 6) (max. in June: 12°C). In the higher part of the Khumbu valley at the Pyramid Laboratory station, monthly temperatures follow the same pattern ranging between -12 and 4°C for the 2012-2014 interval [START_REF] Balestrini | Wet Deposition at the Base of Mt Everest: Seasonal Evolution of the Chemistry and Isotopic Composition[END_REF].

Precipitations

Annual rainfall in Pangom was 3046 mm in 2015 (3683 mm in 2014 and 3947 mm in 2016). The South Asia monsoon (JJAS) accounts for more than 80% of the precipitation amount (Figure 6) (84.7 to 86.1% from 2014 to 2016) with no specific rainier month in this season (JAS between 888 and 1075 mm).

During the sampling interval (November 2014 to December 2016, with a gap in May 2016), a large isotopic variation is observed (Figure 6): 3.25 to -15.26‰ V-SMOW for δ 18 O and 43.3 to -109.5‰ V-SMOW for δ 2 H. Figure 6 shows the isotopic composition of all precipitations giving a local water line following the equation: δ 2 H = 8.57 δ 18 O + 20.5 (R 2 = 0.997; n = 25)

The slope is slightly higher than the slope of the global meteoric water line (GMWL, δ 2 H = 8.13 δ 18 O + 10.13) defined by [START_REF] Rozanski | Isotopic Patterns in Modern Global Precipitation[END_REF] and also shows an intercept d = 20.5 higher, close to the meteoric line of the precipitation in the southern Tibetan plateau [START_REF] Yao | A Review of Climatic Controls on Δ18O in Precipitation over the Tibetan Plateau: Observations and Simulations[END_REF] The relationship between isotope values and temperature (temperature effect) shows an inverse correlation with a low coefficient (R = -0.336; n = 25) as well as with rainfall (amount effect) with R = -0.392, which are not significant using a t-distribution with n-2 degrees of freedom at a significance level a = 0.05; the critical values associated with df = 23 are ±0.396 (Bravais-Pearson table ). The monsoon season corresponds to a more depleted rainfall [START_REF] Dansgaard | Stable Isotopes in Precipitation[END_REF]. In fact, the main effect on isotope variability in our site is the origin of air masses, as shown in previous regional studies cited in this article or in the local Khumbu Valley at the Pyramid Laboratory [START_REF] Balestrini | Wet Deposition at the Base of Mt Everest: Seasonal Evolution of the Chemistry and Isotopic Composition[END_REF]).

The year 2015, which is complete, gives a weighted annual value (Σ(Pi δi) / ΣPi) of -9.24‰ V-SMOW for δ 18 O and -60.1‰ V-SMOW for δ 2 H (2016: 95.9‰ of rainfall, -8.20‰ and -50.6‰), which is in good agreement with the altitude effect in the region, regarding the results obtained in Kathmandu (1320 m, -8.7‰) and Nyalang (3811 m, -11.3‰) [START_REF] Wen | The Altitude Effect of Δ18O in Precipitation and River Water in the Southern Himalayas[END_REF][START_REF] Balestrini | Wet Deposition at the Base of Mt Everest: Seasonal Evolution of the Chemistry and Isotopic Composition[END_REF]; at the Pyramid Laboratory station (5050 m), the isotope content during the monsoon season in 2012 and 2013 does not show a large variation (-17.74 to -17.81‰ in δ 18 O). The gradient is -0,23‰ / 100 m, in good agreement with the range of the isotope altitudinal gradient in Himalaya (-0.15, -0.33‰) [START_REF] Wen | The Altitude Effect of Δ18O in Precipitation and River Water in the Southern Himalayas[END_REF].

The d-excess is a good indicator for evaluating the contribution of different water vapor sources [START_REF] Clark | Environmental Isotopes in Hydrogeology[END_REF] We note in the total observed interval a good correlation between oxygen-18 and d-excess values (R 2 = 0.62; n=25), which reaches R 2 = 0.72 if the more enriched point is removed (April 2016); this may indicate a slight alteration in isotope air mass signal by the evaporation process.

To conclude, in Pangom as in other Himalayan sites, the change in air circulation patterns, marine vapor from the Indian Ocean and the Bay of Bengal, on the one hand, and continental vapor from the Western Disturbances, on the other, modify the isotope composition and d-excess of precipitation.

Rivers

For the river isotope sampling, in the general spatial pattern, depleted values are observed in the headwaters of the streams and enriched values at lower elevations of the catchments. The water isotope variability of stream water shows a lower variability with respect to local precipitation at Pangom, in spite of altitudinal sampling (1981-4935 m) and six campaigns between November 2014 and May 2017 (local sampling between one and six campaigns) that ranged from -10.10 to -18.39‰ for δ 18 O and from -64.5 to -135.8‰ for δ 2 H (Figure 7, Table 4). The sampling was carried out at the beginning of the extra-monsoon season, with possibly the influence of the end of the monsoon season, and at the end of the extra-monsoon season. However, the variability registered is mainly very low, lower than 0.6‰ for δ 18 O for 89% of local stations. Two stations with at least five samplings (D -Pangom and J -Tauche) show a large difference; both stations in March 2015 have a high depleted value (-16.65 and -16.53‰ in δ 18 O, respectively); not considering this result, Pangom has a mean value of -10.47‰ with a range interval of 0.45 (n = 4) and Tauche has a mean value of -15.31‰ with a range interval of 1.22 (n = 5). Both sampling points are located in non-glaciated catchment for Pangom and small glaciated catchment (less than 1% of the basin area) for Tauche. The river in this extra-monsoon season with few precipitations (at Pangom in 2015, precipitation amounts are 19 mm in January, 19 mm in February and 66 mm in March) is supplied mainly by superficial aquifer whose main characteristic is an isotopic composition relatively constant over the year at a local altitudinal point [START_REF] Jeelani | Application of Water Isotopes to Identify the Sources of Groundwater Recharge in a Karstified Landscape of Western Himalaya[END_REF].

The main hypothesis to explain this important change is the presence of snow cover at higher altitude with more depleted isotopic content during this season, which was melting and supplied by direct runoff from the river.

Although there were no measurements during the monsoon season in this study, the works of [START_REF] Balestrini | Chemistry and Isotopic Composition of Precipitation and Surface Waters in Khumbu Valley (Nepal Himalaya): N Dynamics of High Elevation Basins[END_REF] in the high part of the Khumbu Valley (> 4200 m) have shown a higher variability (2‰) close to glacier and morainic lake inflow and low variability (0.39‰ in δ 18 O) at Pheriche (label P in Figure 1) during monthly sampling between July and October 2008. The authors suggest that the area extension of the hydrological basin buffers the isotopic signal. Another fact is the difficulty to link directly the isotope content of monthly precipitation and isotopic content of river water at the same time.

The isotopic composition of the stream water in the whole study interval is close to the local precipitation regression line (Figure 7) following the equation: δ 2 H = 8. Contrary to the results of another study in southern Himalaya by [START_REF] Wen | The Altitude Effect of Δ18O in Precipitation and River Water in the Southern Himalayas[END_REF], who found a very good correlation between the isotope content in the Boqu River and altitude (n = 39, R 2 = 0.90, Sept. 2011) between 1845 and 5060 m, the relationship between the altitude of the sampling sites and isotope content is not well correlated (R 2 = 0.399; n=18) very likely due to the heterogeneity of the catchments and their glacier cover varying between 0% and 70%. The river in catchments with glacier cover at all altitudes is mainly controlled by the ice melting and by higher depleted value with respect to no glacier catchments. This is the case of the Phakding samples, located at a low altitude (2620 m), which show an isotope content in δ 18 O of -15.89‰, with a 20.9% glacier cover (Everest catchment). By comparison, the weighted mean annual rainfall in Pangom (2890 m) yields -9.24‰, in better accordance with the isotope values collected in the Pangom River (watershed without glacier) and reflecting the isotope composition of rainfall in the whole study area (-11.71‰).

By contrast, rivers in catchments without any or with small glacier cover (Kharikola, Pangom, Tauche, Chomar, Teouma), spring (Lobuche spring), or surface flow not connected to glacier melt such as wet saturated pastures (kharka in Nepali; e.g., Phulung Kharka) show (Figure 8) a better correlation with altitude (R 2 = 0.809; n = 8). Focusing only on the river sampling points that depending on glacier melt, the correlation with altitude is significantly improved (R 2 = 0.525; n = 10). Indirectly, the lower the altitude, the more the glacier component in the river decreases and the more the aquifer component increases.

The isotopic altitude gradient for river sampling in no or in a lightly glaciated catchments shows for δ 18 O a value of -0.20‰/100m; this is in the range (-0.11/-0.36‰/100 m) of other studies referenced by [START_REF] Wen | The Altitude Effect of Δ18O in Precipitation and River Water in the Southern Himalayas[END_REF] and [START_REF] Ren | Controls on the Stable Isotopes in Precipitation and Surface Waters across the Southeastern Tibetan Plateau[END_REF] in Himalaya and is close to results found by [START_REF] Florea | Stable Isotopes of River Water and Groundwater along Altitudinal Gradients in the High Himalayas and the Eastern Nyainqentanghla Mountains[END_REF], in the same zone, i.e., -0.28‰/100 m. By comparison with the study of [START_REF] Florea | Stable Isotopes of River Water and Groundwater along Altitudinal Gradients in the High Himalayas and the Eastern Nyainqentanghla Mountains[END_REF], in the Dudh Koshi River with a sampling set located between Gorak Shek (5180 m), upstream of Lobuche, and downstream of Phakding (2550 m) in May 2011, values in δ 18 O are between -17.9 and -9.7‰. The relationship between δ 18 O and δ 2 H demonstrates a lower slope and d-excess value than our global study, with the equation: δ 2 H = 7.8 δ 18 O + 4.0 (R 2 = 0.94; n = 32) Individually, tributary streams and direct sampling in the Dudh Koshi River show a d-excess ranging between 4.4‰ and 12.7‰ (72% of samples lower than 10‰), which is globally lower than the d-excess measured in this study. The slope is lower than 8, but overall the low d-excess values imply that an evaporation process during the study interval [START_REF] Florea | Stable Isotopes of River Water and Groundwater along Altitudinal Gradients in the High Himalayas and the Eastern Nyainqentanghla Mountains[END_REF]) may be due to the sampling time (end of extra-monsoon season), with warmer conditions than during the sampling times of this study.

Discussion

Water origins

Not surprisingly, the isotope properties of the precipitation highlight the double climatic influence from the arrival of the westerlies in winter and from the Asian monsoon in summer.

The isotope response to ground ice melt has been investigated since the mid-1970s (Stuiver et al., 1976;[START_REF] Fujino | Determination ofoxygen isotopic concentration in the ground ice of a tundra area[END_REF] who relies on isotope fractionation that occurs during phase changes (i.e. freezing, condensation, adsorption) and the resulting difference in the slope of the regression of δD on δ 18 O with a slope between 3 and 7 [START_REF] Jouzel | Melting andrefreezing at the glacier sole and the isotopic composition of the ice[END_REF][START_REF] Lacelle | On the d18O, dD and D-excess relations in meteoric precipitation and during equilibrium freezing: theoretical approach and field examples[END_REF]. [START_REF] Ala-Aho | Using stable isotopes to assess surface water source dynamics and hydrological connectivity in a high-latitude wetland and permafrost influenced landscape[END_REF] show in western Siberia the possible discrimination of water origin between precipitation, river, lakes and thawing permafrost. The slope of the regression of δD on δ 18 O was lower than the precipitation (7.6) with soils/permafrost (4.64) < lakes (5.54) < rivers (6.08) and a strong variability of median isotope content in precipitation (-15.6‰ in δ 18 O), rivers (-15.3‰), soils/permafrost (-13.0‰) and lakes (-11.1‰) > rivers (-15.1‰). In our study case the river points do not show an obvious influence of thawing permafrost during the sampling period which correspond before or after monsoon when temperatures are lower, may-be during monsoon period of higher temperature, an important thawing permafrost could be detected in some rivers.

Locally, the results of the current study confirm that few chemical patterns can be used to distinguish the waterflow origins during the different seasons. Rb, as Li, Cu, Sr, Ba and SiO2, originating from the minerals of the bedrock, characterizes substantially the water originating from glacier melt, as shown in Figure 5, especially during the monsoon season. Nevertheless, the isotopic results appear to be less useful for that task, even if differences are observed: The isotopic climate signal in the water courses is very likely mixed with the signal emitted by the storage in groundwater temporary reservoirs, which limits a clear interpretation. Factually, it depends on the sampling location and of the ratio of glacierized area. In the high altitudes (> 4000 m) the river reflects the isotope content mainly of the ice and snow melt because the climatic conditions do not allow an important weathering of the rocks and a strong development of an aquifer structure; the consequence is a reduced groundwater capacity, and a fast groundwater circulation reflecting isotope content of ice and snow. At lower altitude, the weathering is higher and the aquifer can develop itself with a higher storage capacity involving local recharge by rainfall; the isotope content is enriched with respect to ice and snow melt (altitude effect): the lower the altitude, the richer is the isotope content of total flow. It is a consequence of the higher base flow in the dry season and of the higher contribution of the surface runoff in the monsoon season, this last being enriched in isotope with respect to ice and snow melt more depleted.

However, the meltwater marking (glacier as snow cover melt) by the isotopes can be more visible than by the chemical signature in downstream sampling sites, because it is less dissolved, with the inconvenience of a smoothed seasonal effect. The result at the Phakding (20.9‰ glacier covered) and Pangom (no glacier) stations, detailed in the previous section, is, therefore, significant.

The pH remains in a relatively narrow range in the different sampling points and does not seem useful for characterizing the water paths or the seasons, while electrical conductivity shows slightly higher values for flows originating from glaciers and lower values in the monsoon runoffs of streams not fed by glacier melt (Figure 2).

In addition to the previous considerations, complementary observations on electrical conductivity were made in the main rivers of this area and in the Imja Lake during a 3-day interval in the second half of June 2012, in the early monsoon season. They are summarized in Table 5, which shows that the flows arising from the Imja Lake are approximately twofold less mineralized than those of the Khumbu upper valley. Both basins have similar areas and ratios of glaciated surfaces. Because the flows are from the same geological and glaciological origin, the conductivity difference observed can only be explained by the presence of the lake. The higher value observed in the lateral Imja River, which does not flow from the lake, confirms this hypothesis. This phenomenon could be attributed to the sedimentation only within the lake of the solid load generated by the glacier abrasion, due to quieter hydraulic conditions. This hypothesis is reinforced by the visual observations at the confluence between the Imja and the Khumbu rivers, which showed that the turbulence of the runoff was higher in the Khumbu branch than in the Imja branch (Figure 9).

Water uses

Before commenting, it must be stressed that in the framework of this study no analysis was performed regarding the bacterial quality of the water used by the inhabitants and by the visitors. The reason is that, at the time of the study, the only laboratory for analyzing samples, poorly equipped and without dedicated manpower, was located in the Khumjung Hospital, close to the village of Namche Bazar, several hours' walking distance from the sampling points. This is unfortunate, because the sources of bacterial pollution are numerous, especially high-altitude pastures, perfunctory toilet installations, uncontrolled waste disposal, etc. [START_REF] Manfredi | Solid Waste and Water Quality Management Models for Sagarmatha National Park and Buffer Zone, Nepal Implementation of a Participatory Modeling Framework[END_REF]). These sources of pollution are increasing with the rapid development of tourist activities. Furthermore, inhabitants in Pangboche expressed more concerns about the future of the water quality than about water quantity since no proper sewage system exists and they are witnessing a dissemination of plastic waste in streams.

The quality of the natural water can be chemically considered good with a very low mineralization degree. It appears that the reticence to consider the water of the large valley river is mainly justified by the danger in accessing and harvesting it and also by its white color, due to the fine particles mentioned in the previous section. This particular property gave its name to the Dudh Koshi River, which means Milk River in Nepali. Nevertheless, these statements cannot conclude on the drinkability of the river water, since, as written above, organic and microbiological analysis have not been done in the current study framework.

The sampling points L (Pangboche-Teouma) and I (Pangboche-Kisang) were chosen for their location, upstream and downstream of the village, respectively, in order to examine an eventual degradation due to the human activities. No notable differences can be observed in terms of the major ions (Table 2). Nevertheless, in Figure 4, the trace elements collected throughout the year in Teouma (upstream) appear grouped, when a dispersion is noted for those collected downstream in Kisang. This dispersion is very likely due to the limited pollution of the water used in the village. Shortly after Kisang, the slope torrent is intercepted by the large Dudh Koshi River, where the low chemical pollution is dissolved.

Finally, the issue of bottled water consumption by tourists is of some importance to the local economy, because it is a non-negligible income for the communities [START_REF] Puschiasis | Des enjeux planétaires aux perceptions locales du changement climatique : pratiques et discours au fil de l'eau chez les Sherpa de la vallée du Khumbu (Everest, Népal)[END_REF][START_REF] Jacquemet | The Sherpa Community in the 'Yak Donald's' Era : Locational Struggles for Access to Resources in Mount Everest Touristic Region (Nepal)[END_REF]. Several small companies have been established in the region to exploit this niche. One of them collected water at the Pangboche-Chomar point (labeled H in Figure 1) and, after a basic filtration process, bottled it. The bottled water was also analyzed with the same procedure as the other samples, except that it was transported to France after several months in the original PET bottle and not in a standard analysis recipient (see Section 2.3.1). As shown in Figure 4, no difference is observed between the bottle and the other samples from the Chomar site. In Table 2, a few differences are notable, especially regarding pH, Cl -, NO3 -, and Na + . They can be explained by the very long storage in a bad-quality recipient, but this is the reality for most of the water bottles available in the study zone. Nevertheless, in terms of chemical quality, the bottled water and the river water are similar. It seems that for some reason the water company did not pursue the exploitation of the Chomar site, which no longer functioned after 2013. However, water bottles from other places, particularly those collected in the surroundings of Namche Bazar, were sold in the Pangboche shops and lodges.

Conclusion

Between many studies, the recent IPCC special report on the cryosphere future [START_REF] Pörtner | IPCC Special Report on the Ocean and Cryosphere in a Changing Climate[END_REF] confirms the loss of cryospheric mass and the rapid permafrost thaw. It is also verified in the Central Himalaya, which presents threats on physical entities (water resources, flood, landslide, avalanche) and on ecosystems (forest, tundra). The Khumbu inhabitants are strongly preoccupied by this situation, which directly impacts their livelihood. In addition, they face difficulties in terms of inequalities in the social water management system, failing to regulate proper access of water in villages [START_REF] Puschiasis | Des enjeux planétaires aux perceptions locales du changement climatique : pratiques et discours au fil de l'eau chez les Sherpa de la vallée du Khumbu (Everest, Népal)[END_REF][START_REF] Faulon | Tourisme, gestion sociale de l'eau et changement climatique dans un territoire de haute altitude : le massif de l'Everest au Népal[END_REF]. More than climatic variations, changes in water use over the past decades are due to a growing need for tourism and for domestic purposes. Regarding the future of accessible water resources, this does not seem to be threatened in terms of quantity, even if seasonal pressures should lead to a better water management, especially during the high season of tourism activities [START_REF] Aubriot | Reconfiguration of the Water-Energy-Food Nexus in the Everest Tourist Region of Solukhumbu, Nepal[END_REF]). However, water quality could evolve in a worrisome direction.

Apart from the bacteriological issue, which is not considered here, the points addressed could change as follows:

• The isotopic signature of precipitation and rivers is a good indicator of climate change and flow paths. It should be periodically analyzed. On the one hand, it could follow changes observed in the seasonal precipitation patterns in the study region [START_REF] Shea | A Comparative High-Altitude Meteorological Analysis from Three Catchments in the Nepalese Himalaya[END_REF]. On the other hand, it facilitates the recognition of the transitory storage of surface water (glacier, snow cover, or underground reservoirs).

• In this study the chemical properties of the water used for domestic activities do not seem to be hazardous to human and animal health at present. However, with changes in precipitation and river regimes, in land use and land cover due to economic income, in cropping strategies, in the very low level of waste and toilet water management, the current fragile balance could be seriously jeopardized. The project of road construction in the valley, for instance, validated until Surkhe, close to Lukla ("The Rising Nepal: Everest Region Closer with Bridge over Sunkoshi" 2020), will lead to the importation of chemical products and molecules that have been thus far unknown.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions

Pierre Chevallier coordinated the whole study, including the field collection, wrote the introduction, discussion, and conclusion sections, and assembled the different parts of the paper. Jean-Luc Seidel contributed to the geochemistry analysis of the major ions and trace elements and Jean-Denis Taupin of the stable isotopes. Ornella Puschiasis lived in Pangboche during the whole year in 2011 at an elevation of 4000 m, collecting the water samples and she wrote the parts regarding water use by the local inhabitants.

Funding

The global research action, the sample analysis, and the PhD grant, followed by a post-doctoral grant, of Ornella Puschiasis were supported by Agence Nationale de la Recherche -France (references: ANR-09-CEP-005-04/PAPRIKA and ANR-13-SENV-005-03/PRESHINE). Some monitoring equipment was acquired through the Glacioclim SNO (French National Observation Service).

Tables 851

  . In Himalaya, low d-excess values characterize moisture coming from the Indian Ocean and the Bay of Bengal, and high values continental moisture carried by the Western Disturbance (Jeelani and Deshpande 2017). At Pangom in 2015 and 2016, d-excess values during the monsoon season were between 11.3 and 18.7‰ (n = 8, mean = 13.6‰), while in the two extra-monsoon seasons (2015 and 2016) d-excess values were between 14.3 and 23.9‰ (n = 14, mean = 19.1‰). At the Pyramid Laboratory, we have the same pattern for isotope content and d-excess during the monsoon (depleted values up to -30‰ for δ 18 O and d-excess < 15‰) and the extra-monsoon seasons (more enriched values and high d-excess until 26‰). The difference observed with the local meteoric line (LML) in d-excess is due to the different repartition of measurements in the monsoon season (n = 38) and the extra-monsoon season (n = 8).

  Figure 1. Bottom left: location of the Dudh Koshi River basin (red line). Left: Dudh Koshi River basin and sub-

  Figure 1. Bottom left: location of the Dudh Koshi River basin (red line). Left: Dudh Koshi River basin and subbasins, southern Mount Everest. Right: details of the Upper Khumbu and Imja area. The red dots represent the sampling points. The labels refer to Table 1. Sources: for relief, digital elevation model based on Spot-HRS images with a 40-m resolution (Gardelle et al. 2013); for glacier extension, Randolf Glacier Inventory (RGI Consortium 2017).

Figure 2 .

 2 Figure 2. Year 2011: Daily precipitation (PR) and temperature (T) data at Pangboche weather station vs. electrical conductivity (EC) and pH at the five sampling points around Pangboche (~4000 m). The monsoon season (JJAS) is highlighted in green.

Figure 3 .Figure 5 .

 35 Figure 3. Piper diagram of the major ions in the collected samples.

Figure 6 :

 6 Figure 6: Monthly precipitation data in Pangom (E): precipitation (PR), air temperature (T), and stable isotopes (δ18O, δD, D-excess, ‰ VSMOW). The monsoon seasons (JJAS) are highlighted in green.

Figure 7 :Figure 8 :

 78 Figure 7: δ 18 O vs δ 2 H for precipitation samples in Pangom (monsoon and extra-monsoon seasons in red and green, respectively) and for river samples in blue, compared with the Global Meteoric Water Line (GMWL)

Figure 9 :

 9 Figure 9: Confluence of the Khumbu River (down left) and the Imja River branch (upper right). The image has been saturated for a better depiction of the difference in turbidity between both branches. Picture by P. Chevallier, 19 June 2012.

  It is included in the Nepalese administrative division of the Solu-Khumbu district. The area is divided into three major distinct valleys-Imja Valley, Dudh Koshi Valley, and Bhote Khosi Valley-forming a U-shape, a testimonial to the glacial erosion and draining of the main rivers in the region. The Dudh Koshi first meets the Imja Khola on the eastern side of the region, and it then meets the Bhote Koshi before running out of

	Khumbu toward the south into a deep gorge. Khumbu settlements span elevations from 2805 m
	(Jorsalle) to 5170 m (Gorak Shep). The villages are located extensively on the rare alluvial terraces,
	hanging valleys, and amphitheater slumps and comprise mostly south-and north-facing slopes.
	Khumbu corresponds to the former Village Development Committees (VDCs, before administrative
	restructuring in 2017) of Khumjung and Namche hosting approximately 3500 residents belonging
	mostly to the Sherpa ethnic group with a growing number of non-Sherpa residents (Rais, Tamangs,
	Magar, and Bahun-Chhetri). With the establishment of the Sagarmatha National Park (SNP) in 1976
	and its designation as a UNESCO World Heritage Site in 1979, the economy of the region has shifted
	to a more tourism-centered form (Spoon 2011). The number of visitors to the SNP increased to 45,000
	in 2017 (Jacquemet 2018) leading to an important trekking and expedition tourism hub, including the
	popular trekking route to Mount Everest Base Camp.

  In Pangom, if one distinguishes between the seasons, the equations are: These values belong to the extra-monsoon season and are linked to notable continental recycling mainly from non-fractional processes such as transpiration or soil evaporation. In the regional study byJeelani and Deshpande (2017), stations in Nepal and Assam also showed a high d-excess (> 20‰) associated with a high δ 18 O > -1‰ value, suggesting a dominant influence of transpiration, increasing the δ 18 O of vapor over the forest floor[START_REF] Lai | Deuterium Excess Reveals Diurnal Sources of Water Vapor in Forest Air[END_REF].The isotopic values recorded during the 2 years, including monsoon and extra-monsoon seasons, display different patterns, globally more depleted in the monsoon season (2015, -10.05‰; 2016, -8.48‰, weighted mean for δ 18 O) and more enriched in the extra-monsoon season (2015, -4.48‰; 2016, -0.81‰, weighted mean for δ 18 O). The much more enriched values in the extramonsoon season in 2016 could be an effect on isotope composition and deuterium excess in the beginning of the monsoon season with a possibly late isotope re-equilibration of the air mass, perhaps due to higher mixing with the recycled continental vapor. Indeed, in June and July 2016, isotope values were more enriched than in June and July 2015, also with a greater d-excess. Then, it is only in August 2016, in the middle of the monsoon season, that isotope values and d-excess seem to be consistent with isotope values in the monsoon season, and this continues until the beginning of the extra-monsoon season (October 2016) with a dephasing of 2 months.

	ONDJFMAM (non-monsoon)	δ 2 H = 8.46 δ 18 O + 20.6	(R 2 = 0.997; n = 17)
	JJAS (monsoon),	δ 2 H = 8.52 δ 18 O + 18.4	(R 2 = 0.999; n = 8).
	The more enriched monthly values above -1‰ in δ 18 O (n=5) do not show an evaporation mark; a high
	deuterium excess is observed (between 17.4 and 21.6‰) for low to medium rainfall amount (21 to 142
	mm).
			):
	δ 2 H = 8.89 δ 18 O + 23.0	(R 2 = 0.980; n = 374).

At the Pyramid Laboratory station at the weekly scale between June 2012 and December 2013,

[START_REF] Balestrini | Wet Deposition at the Base of Mt Everest: Seasonal Evolution of the Chemistry and Isotopic Composition[END_REF]

found: δ 2 H = 8.17 δ 18 O + 16.6

  For each season the slope and the d-excess value can differ greatly, but the number of samples is small (except for May 2017, which is close to the global river equation) and these variations are not representative of a particular process:

	November 2014	δ 2 H = 8.11 δ 18 O + 4,1	(R 2 = 0.972; n = 5)
	March 2015	δ 2 H = 7.56 δ 18 O + 22.2	(R 2 = 0.999; n = 9)
	November 2015	δ 2 H = 6.71 δ 18 O + 9.3	(R 2 = 0.934; n = 11)
	May 2016	δ 2 H = 8.24 δ 18 O + 16.3	(R 2 = 0.994; n = 10)
	November 2016	δ 2 H = 8.08 δ 18 O + 13.3	(R 2 = 0.992; n = 9)
	May 2017	δ 2 H = 8.47 δ 18 O + 19.1	(R 2 = 0.997; n = 17)
	The mean d-excess value at each sampling point is between 5.6 and 14.4‰. For the whole dataset,
	d-excess values vary between 3.1 and 15.6‰ with 19% of samples lower than 10‰, which are altered
	by an evaporation process. The samples with d-excess between 10 and 14‰ do not show a special
	trend, likely linked to glacier melt and aquifer discharge where the main accumulation and recharge
	are during monsoon season when the d-excess value of the monthly precipitation is between 11 and
	14‰. For d-excess values higher than 14‰, there is possibly a partial contribution of rainfall or melted
	snow during the extra-monsoon season where d-excess values of monthly precipitation can reach 24‰
	during the 2014-2016 interval.
	23 δ 18 O + 15.0	(R 2 = 0.987; n = 58)

Table 1 :

 1 Main characteristics of the sampling points (ordered by altitude)

	852
	Name (+)	Label		Coordinates		Data type	Sample	Glaciers
			(++)	Longitude	Latitude	Altitude	(*)	(**)	(***)
						(m)
	Kharikhola	A	86°43′16″	27°36′22″	1981	R	IS	N
	Phakding	B	86°42′47″	27°44′53″	2620	R	IS	Y
	Jorsalle		C	86°43′19″	27°46′48″	2850	R	IS	Y
	Pangom river	D	86°44′38″	27°35′24″	2880	R	IS	N
	Pangom village	E	86°44′35″	27°35′17″	2890	P	IS	-
	Phuki Tenga	F	86°44′35″	27°49′37″	3200	R	IS	Y
	Pangboche-Imja	G	86°47′53″	27°51′40″	3917	R	MT IS	Y
	Pangboche-Chomar	H	86°46′59″	27°51′14″	3930	R & bottle	MT	N
	Pangboche-Kisang	I	86°47′31″	27°51′18″	3971	R	MT	N
	Pangboche-Tauche	J	86°47′35″	27°51′32″	4005	R	MT	S
	Shomare	K	86°48′40″	27°52′5″	4021	R	IS	N
	Pangboche-Teouma	L	86°47′17″	27°51′32″	4148	R	MT IS	N
	Imja confluence	M	86°49′08″	27°52′52″	4172	R	CP	Y
	Khumbu confluence	N	86°49′05″	27°52′59″	4172	R	CP	Y
	Pheriche hydro	O	86°49'08″	27°53′13″	4216	R	IS	Y
	Pheriche village	P	86°49′16″	27°53′46″	4260	P R	MT IS	Y
	Dingboche village	Q	86°50′06″	27°53′35″	4370	R	IS	Y
	Dingboche hydro	R	86°50′28″	27°53′46″	4372	R	IS	Y
	Tauche Kharka	S	86°47′17″	27°52′23″	4405	R	IS	S
	Phulung Kharka	T	86°49′01″	27°54′36″	4504	R	IS	S
	Tukla		U	86°48′54″	27°55′59″	4700	R	IS	Y
	Chukung	V	86°52′41″	27°54′04″	4752	R	CP	Y
	Lobuche river	W	86°48′50″	27°57′25″	4840	R	IS	Y
	Lobuche spring	X	86°48′43″	27°57′18″	4935	R	IS	S
	Imja Lake	Y	86°54′29″	27°54′00″	5001	R	MT	Y
	(+) precipitation samples are in gray; the others are river samples.
	(++) see location in Figure 1
	(*)	Data type:	P=precipitation; R=river
	(**)	Sample:	MT=major/trace + conductivity/pH; IS=isotopes; CP= only conductivity/pH
	(***)	Glacier:	N=no; Y=yes; S=not significant

Table 5 :

 5 Additional observations of electrical conductivity, performed on 19, 21, and 22 June 2012.

	860
	Name	Label	Electrical conductivity
		(+)	(µS/cm)
	Pangboche-Imja	G	53.0
	Khumbu confluence N	67.6
	Pheriche hydro	O	62,6
	Imja confluence	M	35.4
	Dingboche hydro	R	32,9
	Chukung	V	41.5
			29,7
	Imja Lake (*)	Y	30,7
			29,6
	(+) see location in Figure 1
	(*) 3 different sampling points in the outlet narrows.

Table2. Main characteristics of the geochemical samples: water temperature, pH, electrical conductivity, and major ions.

Acknowledgments

The analyses of major ions and trace elements were done by Sandra Van Exter and Jean-Luc Seidel, and the analysis of stable isotopes by Nicolas Patris at the Laboratoire HydroSciences Montpellier (CNRS, IRD, University of Montpellier). Dawa Nuru Sherpa translated the dialogs with Pangboche's inhabitants and facilitated the measurements. Ang Jangmu Sherpa collected and stored the precipitation samples in Pangom. Yves Arnaud, Olivia Aubriot, Anneke De Rouw, François Delclaux, Judith Eeckman, Michel Esteves, Frédéric Hernandez, Devesh Koirala, Luc Neppel, Rémi Muller, Marie Savéan, Joëlle Smadja, and Patrick Wagnon participated in the field operations, as well as extraordinary, indispensable and friendly local porters.

Yves Arnaud and Isabelle Sacareau were, respectively, the coordinators of the Paprika and Preshine projects, which benefited from a partnership with the following institutions: Nepalese Academy of Science and Technology (Kathmandu, Nepal), EvK2-CNR Association (Bergamo, Italy), International Center of Integrated Mountain Development (Kathmandu, Nepal), Tribhuvan University (Kathmandu, Nepal), and Department of Hydrology and Meteorology (Kathmandu, Nepal).

Finally, the authors thank the two anonymous reviewers who allow to substantially improve the definitive version of the paper.


MD. Krystel Nyangoh Timoh

David Moszkowicz

Maud Creze

Mazen Zaitouna

Margaux Felber

Cédric Lebacle

Djibril Diallo

Jelena Martinovic

Ashutosh Tewari

Vincent Lavoué

Gevorg Ghukasyan

Gerard Benoit

Thomas Bessede

Affiliations

The male external urethral sphincter is autonomically innervated

Keywords:

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Radical prostatectomy is responsible for erectile dysfunction in around 10 to 46% of patients and for urinary incontinence in around 16% (Ficarra et al., 2012a;Ficarra et al., 2012b) .With a view to improving postoperative erectile function Walsh described a nerve-sparing radical prostatectomy procedure aimed at preserving the neurovascular bundles (NVB) [START_REF] Walsh | Impotence Following Radical Prostatectomy: Insight into Etiology and Prevention[END_REF]. Furthermore, several recent clinical studies suggest that preservation of the NVB is associated with improved postoperative urinary continence after radical prostatectomy [START_REF] Reeves | Preservation of the neurovascular bundles is associated with improved time to continence after radical prostatectomy but not long-term continence rates: results of a systematic review and meta-analysis[END_REF] [START_REF] Srivastava | Effect of a risk-stratified grade of nerve-sparing technique on early return of continence after robot-assisted laparoscopic radical prostatectomy[END_REF][START_REF] Steineck | Degree of preservation of the neurovascular bundles during radical prostatectomy and urinary continence 1 year after surgery[END_REF].

Postoperative urinary continence is multifactorial, but the urethral sphincter (US), and in particular the external US (EUS), are major actors of urinary continence (Bessede et al., 2017). It has previously been suggested that the autonomic supply for the US is similar to the neural pathway of the erectile bodies (Hinata et al., 2014), however anatomical evidence based on specific neuronal markers is lacking. Classically, autonomic nerves are described to run above the levator ani muscle while somatic nerves run below. In a previous study using immunohistochemistry and three-dimensional reconstruction, our team described the innervation of the medial part of the levator ani muscle by autonomic nerves (Nyangoh Timoh et al., 2017).

Therefore, we hypothesized that the EUS innervation, is supplied by autonomic innervation using the same pathway that NVB.

The aim of the present study was to describe autonomic US innervation using specific muscular and neuronal antibody markers and 3D reconstruction.

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Material and Methods

Human fetuses

The fetal specimens were obtained from late miscarriages. All parents gave written consent and authorization for the scientific use of the cadaver. Only specimens without maceration and without morphological or neurological macroscopic abnormalities on pathology examination were used.

The study was approved by the French Biomedicine Agency (PFS15-011).

Three human male fetuses had a gestational age of between 18 and 40 weeks.

Macroscopic dissection

The entire pelvis was removed en-bloc with the pelvic organ and pelvic bone, and was fixed in formalin (10% formaldehyde) for 48 h. Tissues were then cut into transverse or sagittal slices at 4-mm intervals. The tissue slices were placed in baskets, processed, and embedded in cardboard molds filled with paraffin. Slices were kept warm in water at 37°C and then mounted whole on Superfrost glass slides. They were then dried at 37°C overnight.

Series of 5 μm-thick sections were subsequently created without intervals.

Staining and immunolabeling (Supporting information 1: Table 1).

The first section of each level was taken as the reference and was stained with Masson's trichrome or eosin and hematoxylin to provide information about the topography of anatomical structures. Smooth muscles were detected with polyclonal antibodies against alpha-smooth muscle actin (SMA).

Neuronal markers were detected with polyclonal antibodies against protein S100 (PS100) for labeling all nerves, peripheral myelin protein (PMP 22) for the somatic peripheral nervous system, tyrosine hydroxylase (TH) for adrenergic nerves, the neural isoform of nitric oxide synthase (nNOS) for the pro-erectile nerve bundles, and the calcitonin gene-related peptide (CGRP) for primary sensory neuropeptide immunolabeling (Moszkowicz et al., 2011) .

The avidin-biotin-peroxidase detection procedure was carried out with a Vectastain ABC kit (ref. PK6100; Vector Laboratories, Burlingame, CA, USA). Chromogenic detection was performed with a DAB detection kit (DAB,Vector Laboratories). Non-immune serum or IgG at an equivalent dilution was used as a control for all the immunohistochemical analyses.

Histological examination

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Serial stained and immunolabeled two-dimensional (2D) sections were used for threedimensional (3D) reconstruction. Using high magnification (4× to 40×) analyses of the Masson's trichrome and eosin and hematoxylin-stained sections, it was possible to identify the various anatomical structures. Subsequent sections, treated with an antibody against S100, were used to identify pelviperineal nerves. By comparing the Masson's trichrome-stained sections with sections stained with specific antibodies against SMA, S100, TH, PMP22, CGRP and nNOS, it was possible to determine the structure of muscle (smooth (SMA)) and the nature of the nerve fibers identified: i.e., somatic (PMP22), autonomic (TH), sensory (CGRP) or nitrergic (nNOS).

The sections were taken at almost the same level with a negligible interval between sections (5 µm). The computer system comprised a personal laptop computer (Windows XP) equipped with an Epson Perfection V750 digitization system, Silverfast AI digitization software, Adobe Photoshop image-processing software, and Surfdriver software for Windows (Winsurf image reconstruction software, version 4.3). All sections were digitized at a resolution of 4800 dots per inch, and the images were then stacked and aligned. The pelvic anatomical structures and nerve fibers were outlined manually on all histological sections. A 3D analysis of the location, course, and distribution of the nerve fibers and muscle structure was then carried out. An animated reconstruction was generated (Supporting information 2).

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Results

Global morphology of the urethral sphincter (US)

The US is composed of a deep internal smooth muscle layer and a superficial external striated muscle layer.

External US (EUS)

The EUS contains striated muscle cells only. It is circular, completely surrounds the urethra (Fig. 1B1, 1B2, 1B3 and supporting information 3), and is in continuation with the musculus vesicoprostaticus above (Fig. 1A1, 1A2, 1A3 and supporting information 3).

Cranially, the anterior wall of the EUS is thicker than its posterior wall with transversal sections showing a signet-ring appearance (Fig. 1B1, 1B2, and1B3).

Caudally, and as it approaches the bulb of the penis, the posterior wall of the EUS thins and fades along the midline whereas it thickens into two pillars or wings laterally (Fig. 1C1, 1C2, 1C3 and supporting information 3). At this posterior opening of the EUS, glands, vessels and nerves penetrate into the US. This opening is also where the smooth muscle central compartment attaches to the IUS (Fig. 1C1, 1C2, 1C3 and Fig. 4).

Internal US (IUS)

The IUS contains smooth muscle cells only. It is composed of three layers: a thin longitudinal superficial layer, a dense circular layer and a thin longitudinal deep layer (Fig. 1B1, 1B2, and1B3).

Cranially, the IUS is in continuation with smooth muscle (anti-SMA positive) areas within the prostate that correspond to McNeal's zones: ventrally with the anterior fibromuscular stroma of the prostate, and dorsally with the peripheral zone of the prostate (Fig. 1B1, 1B2, 1B3, Fig. 3, Fig. 3B2 and 3B3).

Caudally, the posterior aspect of the IUS is attached to the smooth muscle central compartment (Fig. 2B1, and2B2) and also receives nerves and vessels that reach the urethral wall or the cavernous and spongiosis corpus (Fig. 3D and Fig. 4).

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Innervation of the US

Autonomic system

Smooth muscle areas are innervated by nerves arising from the inferior hypogastric plexus (IHP).

The IHP consists of hypogastric and pelvic splanchnic nerves. It is possible to distinguish retrorectal, laterorectal, interprostatorectal, lateroprostatic, and anteroprostatic fibers (Fig. 3A).

The distal part of the IHP is localized in a triangular zone between the levator ani muscle laterally, the urethra anteriorly and the rectum posteriorly. The nerves (S100 positive) are: sympathetic (anti-TH positive), sensory (anti-CGRP positive), nitrergic (anti-NOS positive) (Fig. 3A, 3B, 3C, 3D, 3E, 3F and 3G). There are no somatic (anti-PMP22 negative) nerves (Fig. 4G).

Urethral innervation is supplied by lateroprostatic and prerectal nerve fibers all of which pass through the rectourethral muscle between the rectum and the urethra (Fig. 5), and then through the middle of the US opening. Some fibers then penetrate the urethra along the midline in the ventral direction, while others curve around the bulb and penetrate the urethral wall at 3 and 9 o'clock (Fig. 4 and Fig. 5).

Nerve fibers spread into the EUS, the IUS and the urethral submucosa (Fig. 4). The autonomic system therefore innervates both the striated EUS and the smooth IUS.

The nitrergic fibers originate from the NVB and innervate the EUS and IUS. These fibers also supply innervation for the erectile bodies. (Fig. 4).

Somatic system

After branching off to form the dorsal nerve of the penis, the pudendal nerve (PN) gives nerve fibers that curve medially to the EUS at the level of the wings. These nerves are thus perineal and they penetrate the EUS at its posterior aspect. The nerves are somatic (anti-PMP22 positive). No somatic branches coming from pelvic nerves are observed for the EUS (Supporting information 4).

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Discussion

We demonstrate that both the EUS and IUS are innervated by autonomic and somatic nerves. The autonomic nerves supplying the urethra: 1) run within the NVB, and 2) arise from the IHP to run at the posteromedial aspect of the prostate apex and through the rectourethral muscle.

Human fetal material is a good model to study pelvic anatomy (Nyangoh Timoh et al., 2017;Nyangoh Timoh et al., 2018). First, the small size of fetus pelvis enables en-bloc removal and processing with mega-cassette embedding which limits data loss and structure displacement compared to surgical dissection. Therefore, the whole neuroanatomy and muscular pelvic floor anatomy is preserved. Second, the small size of the fetus pelvis also makes 3D reconstruction easier. Third, as the nerves are relatively larger in the fetus as opposed to the adult, the neuroanatomy is more easily visualized. Finally, the use of multiple specific muscular and neuronal markers is more feasible with fresh materials.

Numerous discrepancies exists about the involvement of autonomic nerves from the IHP to the EUS.

Classically, the striated muscle EUS is described as being innervated by somatic nerve fibers, and the smooth muscle IUS innervated by autonomic nerves [START_REF] Srivastava | Effect of a risk-stratified grade of nerve-sparing technique on early return of continence after robot-assisted laparoscopic radical prostatectomy[END_REF][START_REF] Strasser | Anatomic and functional studies of the male and female urethral sphincter[END_REF]. However, our team has demonstrated: 1) the existence of autonomic (IHP) and somatic (PN) communications within the levator ani muscle (Alsaid et al., 2011), and 2) that the medial part of the levator ani muscle is innervated by nerves arising from the IHP (Nyangoh Timoh et al., 2017).

In 1987, Kumagai already observed unmyelinated axons in the EUS (Kumagai et al., 1987). Some authors then went on to provide evidence of the involvement of pelvic branches for US innervation using conventional dissection. These pelvic branches were described as somatic nerves coming from the PN or from the nerve to the levator ani [START_REF] Akita | Origins and courses of the nervous branches to the male urethral sphincter[END_REF], or as autonomic nerves from the IHP (Hinata et al., 2014;Hollabaugh et al., 1997;[START_REF] Takenaka | Variation in course of cavernous nerve with special reference to details of topographic relationships near prostatic apex: histologic study using male cadavers[END_REF]. Such controversy highlights the limitation of gross anatomy dissection studies in the field of nerve characterization (Bessede et al., 2017).

In the present study using specific neuronal markers, we observed autonomic nerves arising from Another controversy remains about the exact location of fibers that supply the US. In the present study, we observed that autonomic nerves supplying the US are located posterolaterally at midprostate, then shift to a posteromedial position to join the prostate apex, pass through the smooth rectourethral muscle, and innervate the US before ultimately continuing towards the erectile bodies.

In past we have shown that peri prostatic nerves rather form a hammock and it converges in the Accepted Article midline near the apex [START_REF] Tewari | Anatomical grades of nerve sparing: a risk-stratified approach to neural-hammock sparing during robot-assisted radical prostatectomy (RARP)[END_REF]. Ganzer also observed pelvic nerves running towards the dorsal region of the EUS (Ganzer et al., 2015). This posteromedial position of autonomic nerves supplying the EUS represents a functional risk during apical dissection and the retrourethral detachment of rectourethral muscle fibers.

The classic anatomical description of the NVB indicates that they contain cavernous nerves which are located in the posterolateral aspect of the prostate. NVB are part of IHP. Recent studies have suggested that the cavernous nerves do not strictly follow the course of the NVB, do not form a fascicle and have a fan-like distribution at several levels [START_REF] Alsaid | Tridimensional computer-assisted anatomic dissection of posterolateral prostatic neurovascular bundles[END_REF]Costello et al., 2004;[START_REF] Takenaka | Variation in course of cavernous nerve with special reference to details of topographic relationships near prostatic apex: histologic study using male cadavers[END_REF]. In the present study, we demonstrated that autonomic fibers from the NVB supply innervation to the EUS (Fig. 6A and6B). This anatomical evidence underpins clinical observations that preserving the NVB may limit urinary incontinence [START_REF] Reeves | Preservation of the neurovascular bundles is associated with improved time to continence after radical prostatectomy but not long-term continence rates: results of a systematic review and meta-analysis[END_REF][START_REF] Srivastava | Effect of a risk-stratified grade of nerve-sparing technique on early return of continence after robot-assisted laparoscopic radical prostatectomy[END_REF][START_REF] Steineck | Degree of preservation of the neurovascular bundles during radical prostatectomy and urinary continence 1 year after surgery[END_REF].

The origin of the somatic control of the EUS is also a matter of debate. It has been reported that the pelvic somatic nerves supplying innervation to the EUS come from the intrapelvic PN and/or from the nerve to the levator ani (Hollabaugh et al., 1997). However, luxol fast blue is not as effective as neuronal markers. In the present study using antimyelin markers, we did not observe any somatic fibers within the IHP that would supply nerves to the EUS. However, we did observe an infralevator perineal branch coming from the PN and supplying the EUS. These findings are consistent with Strasser, Narayan, and Jueneman (Juenemann et al., 1988;Narayan et al., 1995;[START_REF] Strasser | Anatomic and functional studies of the male and female urethral sphincter[END_REF]. Moreover, we located this perineal branch below the rectourethral muscle, which suggests that it is not in the surgical field of retropubic or laparoscopic radical prostatectomy. On the other hand, this somatic pathway is exposed during perineal prostatectomy.

From a physiopathological point of view, autonomic nerves in the EUS may help early return of urinary continence (Hinata et al., 2014). These branches also innervate the medial part of the levator ani muscle and the rectourethral muscle which may indicate a coordinating role. Moreover, the high density of sensory nerves is of major importance for the reflex activity of the EUS. Similarly, the high density of NOS fibers may help regulate striated muscle performance (Hinata et al., 2014).

Autonomic nerves could also target the vessels rather than muscles and regulate blood flow.

From a clinical point of view, we can systematize three nerve pathways to the EUS: 1) autonomic nerves running within the NVB, 2) autonomic nerves running from the IHP to the posteromedial aspect of the prostate apex and remaining above and through the rectourethral muscle and, 3) somatic nerves arising from the PN, joining the midline but remaining below the rectourethral muscle (Fig. 6). As mentioned above, recent clinical studies suggest that preserving the NVB

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during surgery improves postoperative continence after radical prostatectomy [START_REF] Reeves | Preservation of the neurovascular bundles is associated with improved time to continence after radical prostatectomy but not long-term continence rates: results of a systematic review and meta-analysis[END_REF][START_REF] Srivastava | Effect of a risk-stratified grade of nerve-sparing technique on early return of continence after robot-assisted laparoscopic radical prostatectomy[END_REF][START_REF] Steineck | Degree of preservation of the neurovascular bundles during radical prostatectomy and urinary continence 1 year after surgery[END_REF]. The present study provides anatomical evidence for this hypothesis. Using neuronal markers, we observed autonomic nerves, sympathetic, sensory and highly nitrergic coming from the IHP and supplying the urethra. These nerves also innervate and pass through the rectourethral muscle which should be considered as a structure to be preserved by a cautious peeling during the retroapical dissection. Ultimately, our results demonstrate that autonomic nerve fibers of the US are more exposed than its somatic fibers during retropubic or laparoscopic radical prostatectomy.

Despite these results, our study has some limitations.

Although the fetal model is a mature and established model for neuroanatomical study, its use can be criticized. However, at the end of the embryonic period, the pelvic anatomy is comparable to that in adults (Fritsch, 1989). Second, the small sample size could lead to bias. However, the same anatomic observations were established for the three fetuses. Moreover, the axis sections were different (axial and sagittal) so as to provide several views. Third, it may remain unclear if nerve anatomical localization have functional relevance. However, in the present study, the use of neuronal antibodies helps to understand nerve function. Finally, the absence of a somatic intrapelvic branch may be related to the fact that myelinization is a late event during fetal development. However, we were able to identify the somatic control of the EUS coming from perineal branch of PN. Moreover, as the total pelvis was removed en-bloc and we took continuous serial sections at 4 μm intervals, we were able to strictly follow the course of the nerves.

Conclusion

This study provides anatomical evidence of an autonomic component in the innervation of the external US that travels in the neurovascular bundle. During radical prostatectomy, the rectourethral muscle and the neurovascular bundles are to be preserved, particularly during apical dissection. The prostate apex is sectioned with the anterior fibromuscular stroma of the prostate (anti-SMA positive, dark green area), transitional zone (dark blue) and the musculus vesicoprostaticus above (anti-SMA negative, dark red area).

Cranial membranous urethra : (B,B1) Transverse section stained with Hematoxylin's eosin. (B2) (B3) Transverse section stained with anti-SMA antibody. (B3) is the same section than (B2) with legends.

The EUS (light purple, anti-SMA negative) is circular, completely surrounds the urethra and is in continuation with the musculus vesicoprostaticus above.

The internal US (dark purple, anti-SMA positive) is composed of three layers: from superficial to deep: a thin longitudinal layer, a dense circular layer and another thin longitudinal layer. It continues with the anterior fibromuscular stroma of the prostate ventrally, and dorsally with the peripheral zone of the prostate. Caudally the posterior wall of the external US thickens into 2 pillars posterolaterally. This opening allows attachment of the smooth muscle central compartment to the internal US.

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Color code: Urethra (Ur, yellow), prostate with the anterior fibromuscular stroma of the prostate (AFMS, dark green area) transitional zone (TZ, dark blue) and the musculus vesicoprostaticus above (MVP, dark red area). internal urethral sphincter (IUS, dark purple), external urethral sphincter (EUS, light purple), levator ani muscle (LAM, orange), rectum (beige), smooth muscle cells of the middle compartment (SMMC) with the core (or rectourethral muscle, light green), the endolevator area (or medial part of the levator ani, endo, pink). (A2) is the same section than (A1) with legends.

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External US continues the musculus vesicoprostaticus (anti-SMA negative, dark red area) above.

Internal US continues with smooth muscle (ant-SMA positive) areas within the prostate.

Parasagittal: Sections are stained with Masson's trichrome (B), anti-SMA antibody (B1 and B2).

(B2) is the same section than (B1) with legends.

The posterior aspect of the internal US (dark purple) receives smooth muscle cells from the smooth muscle of the middle compartment (infralevator area, light blue, anti-SMA positive).

Color code: prostate with the anterior fibromuscular stroma of the prostate (AFMS, dark green area) transitional zone (TZ, dark blue), central zone (ZC, light red) and the musculus vesicoprostaticus above (MVP, dark red area), external urethral sphincter (EUS, light purple), internal urethral sphincter (IUS, dark purple), levator ani muscle (LAM, orange), rectum (beige), smooth muscle cells of the middle compartment (infra levator area, light blue), obturator internal muscle (OIM, brown).

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  the IHP and supplying innervation to the EUS. These findings are consistent with the study of Hinata et al. reporting the presence of NOS+ nerve fibers within the EUS (Hinata et al., 2014). Hinata concluded that the external US is innervated from the IHP (Hinata et al., 2014).

  Using luxol fast blue, Karam observed intrapelvic supralevator somatic nerve fibers supplying the urethra (Karam et al., 2005).

Figure 1 :

 1 Figure 1: Serial transverse sections of a 21-week-old male fetus scanned at an optical

  Caudal membranous urethra: (C,C1) Transverse section stained with Masson's Trichrome. (C2) (C3) Transverse section stained with anti-SMA antibody. (C3) is the same section than (C2) with legends .

Figure 2 :

 2 Figure 2: Sagittal (A, B, C) and parasagittal (D, E, F) sections of a 21-week-old male fetus scanned at an optical resolution of 4800 dpi. Sagittal: Sections are stained with hematoxylin and eosin (A), anti-SMA antibody (A1 and A2).

Figure 3 :

 3 Figure 3: Sagittal sections of a 21-week-old male fetus scanned at an optical resolution of 4800

Figure 4 :

 4 Figure 4: Transverse sections of a 21-week-old male fetus scanned at an optical resolution of

Figure 5 :

 5 Figure 5: Computer-assisted anatomical dissection (CAAD) of the pelvic structure in a 21-

Figure 6 :

 6 Figure 6: The three nerve pathways of the External Urethral Sphincter innervation.

Acknowledgement section

The authors sincerely thank those who donated their bodies to science so that anatomical research could be performed. Results from such research can potentially increase mankind's overall knowledge that can then improve patient care. Therefore, these donors and their families deserve our highest gratitude.

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The study was approved by the French Biomedicine Agency (PFS15-011).

Alsaid B, Moszkowicz D, Peschaud F, Bessede T, Zaitouna M, Karam I, Droupy S &Benoit G.

(2011). Autonomic-somatic communications in the human pelvis: computer-assisted anatomic dissection in male and female fetuses. Journal of anatomy, 219:565-573. Bessede T, Sooriakumaran P, Takenaka A &Tewari A. (2017). Neural supply of the male urethral sphincter: comprehensive anatomical review and implications for continence recovery after radical prostatectomy. World journal of urology, 35:549-565. Costello AJ, Brooks M &Cole OJ. (2004). Anatomical studies of the neurovascular bundle and cavernosal nerves. BJU international, 94:1071-1076. Ficarra V, Novara G, Ahlering

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Supporting information 1:

Supporting information 2 Supporting information: Movie File of the pelvic floor of fetus B.

Code: the centro-levator area (centro) is in light green; the infra-levator (infra) in light blue; the levator ani muscle in orange; the external anal sphincter in dark blue; the external urethral sphincter in light purple; the prostate in dark red; the rectum in beige; and the internal urethral sphincter in dark purple; the bulbospongiosus muscle in pink; nerve to levator ani in green; the pudendal nerve in blue; the inferior hypogastric plexus in yellow.

Supporting information 3:

Computer-assisted anatomical dissection (CAAD) of the pelvic structure in a 21-week-old male fetus.

A to C: Structures are revealed on right lateral views.

A:

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The internal US (in dark purple) surrounds the urethra (yellow) down to the urethral bulb

The external US (in semi-transparent light purple) surrounds the internal US.

Appearance of the bulbospongiosus muscle (BSM, pink)

Appearance of the smooth muscle middle compartment (SMMC) with the core (or rectourethral muscle, light green), the endolevator area (or medial part of the levator ani muscle dark pink), the infralevator area (or deep transverse perineal muscle, light blue).

B:

The rectum is surrounded by the SMMC C:

Levator Sections (A) is stained with Masson's trichrome.

Sections (B) and (B1) are stained with anti-S100 antibody.

Sections (B2) is stained with anti-PMP22 antibody.

(B1, B2) are magnified views from rectangle in (B) with anti-S100 and anti-PMP22 staining.

The pudendal nerve is shown with green arrowhead.

The pudendal nerve gives perineal nerve fibers that curve medially to the EUS at the level of the wings. These nerves are somatic (positive anti-PMP22) (black arrowhead in (B2).

Color code : External urethral sphincter (EUS, light purple), internal urethral sphincter (IUS, dark purple), levator ani muscle (LAM, orange), rectum (beige), smooth muscle cells of the middle