Alfonso Mucci

Maurice Levasseur

Yves Gratton

Chloe Martias

Michael Scarratt

Denis Gilbert

Jean-Eric Tremblay

Gustavo Ferreyra

Bruno Lansard

Chloé Martias

Jean-Éric Tremblay

Tidally induced variations of pH at the head of the Laurentian Channel

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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the intrusion of waters from the Upper St. Lawrence Estuary and the Saguenay Fjord. The presence of a cold intermediate layer impedes the upwelling of the deeper, hypoxic, lower pH and aragoniteundersaturated waters of the Lower St. Lawrence Estuary to depths shallower than 50 meters.

RÉSUMÉ

La tête du chenal Laurentien est une région très dynamique d'une richesse biologique exceptionnelle. Afin d'évaluer l'impact des apports en eaux douces, du mélange tidal, et de l'activité biologique sur le pH des eaux de surface dans cette région, une suite de variables physiques et chimiques a été mesurée dans la colonne d'eau sur deux cycles de marée. La contribution relative des quatre sources d'eau-type qui convergent dans cette région a été évaluée à l'aide d'un algorithme d'optimisation multi-paramétrique (OMP). Les résultats de l'OMP ont été utilisés pour reconstruire les propriétés de la colonne d'eau en assumant un mélange conservateur et la différence entre les propriétés issues du modèle et des mesures effectuées sur le terrain ont servies à identifier les facteurs qui contrôlent le pH des eaux de surface. Les eaux de surface sont généralement sous-saturées par rapport à l'aragonite, surtout à cause de l'intrusion d'eau provenant de l'estuaire fluvial du Saint-Laurent et du fjord du Saguenay. La présence de la couche intermédiaire froide (CIF) durant les marées de morte-eau tamponne l'acidification des eaux de surface dans cette région de l'estuaire. La présence d'une couche intermédiaire froide limite la remontée des eaux profondes, hypoxiques, de faible pH et sous-saturées par rapport à l'aragonite de l'estuaire maritime du Saint-Laurent à des profondeurs de moins de 50 mètres.

INTRODUCTION

The oceans have absorbed approximately 30% of the anthropogenic CO2 released to the atmosphere since the beginning of the industrial revolution [START_REF] Sabine | The oceanic sink for anthropogenic CO2[END_REF]). Consequently, over the last century, the pH of the global ocean surface has decreased by an estimated 0.1 unit, equivalent to a 30% increase in the proton concentration [START_REF] Caldeira | Ocean model predictions of chemistry changes from carbon dioxide emissions to the atmosphere and ocean[END_REF]. The increased acidity has lowered the saturation state of ocean waters with respect to calcite and aragonite (the two most common CaCO3 polymorphs that constitute the shells and skeleton of many marine organisms) and, in combination with other stresses such as global warming, likely affected the ecology of carbonate-secreting organisms [START_REF] Fabry | Impacts of ocean acidification on marine fauna and ecosystem processes[END_REF][START_REF] Miller | Shellfish face uncertain future in high CO2 world: influence of acidification on oyster larvae calcification and growth in estuaries[END_REF][START_REF] Ries | Marine calcifiers exhibit mixed responses to CO2-induced ocean acidification[END_REF][START_REF] Kroeker | Impacts of ocean acidification on marine organisms: quantifying sensitivities and interaction with warming[END_REF] as well as non-calcifiers because pH plays a critical role in mediating many physiological processes [START_REF] Fabry | Impacts of ocean acidification on marine fauna and ecosystem processes[END_REF][START_REF] Kroeker | Impacts of ocean acidification on marine organisms: quantifying sensitivities and interaction with warming[END_REF].

The pH of surface waters in coastal systems is controlled by more dynamic processes than in the open ocean. In these systems, the pH of surface waters may exhibit important short (hours) and long (season) term variations in response to freshwater inputs and vertical mixing (Abril et al. 2000;[START_REF] Feely | The combined effects of ocean acidification, mixing, and respiration on pH and carbonate saturation in an urbanized estuary[END_REF]. Freshwater rivers and inner estuaries are typically supersaturated in CO2 with respect to the atmosphere [START_REF] Meybeck | Riverine transport of atmospheric carbon: sources, global typology and budget[END_REF][START_REF] Raymond | Carbon dioxide concentration and atmospheric flux in the Hudson River[END_REF][START_REF] Barth | Carbon cycle in St Lawrence aquatic ecosystems at Cornwall (Ontario), Canada: seasonal and spatial variations[END_REF][START_REF] Barwell-Clarke | Institute of Ocean Sciences Nutrient Methods and Analysis[END_REF][START_REF] Dinauer | Spatial variability of surface-water pCO2 and gas exchange in the world's largest semi-enclosed estuarine system: St. Lawrence Estuary (Canada)[END_REF] as a result of inputs from their drainage basin and the activity of heterotrophic organisms sustained by natural or anthropogenic terrestrial and riverine organic carbon inputs (Frankinouille et al. 1996(Frankinouille et al. , 1998;;[START_REF] Feely | The combined effects of ocean acidification, mixing, and respiration on pH and carbonate saturation in an urbanized estuary[END_REF]. Consequently, waters are often characterized by circum-neutral to slightly acidic pHs [START_REF] Wallace | Coastal ocean acidification: the other eutrophication problem[END_REF] and are a net source of CO2 to the atmosphere (see compilation in [START_REF] Chen | Reconciling opposing views on carbon cycling in the coastal ocean: Continental shelves as sinks and near-shore ecosystems as sources of atmospheric CO2[END_REF]. The high primary productivity characterizing estuarine systems, in combination with their deep-water circulation, favor the accumulation of respiratory CO2 at depth in stratified estuaries and a further decrease in pH [START_REF] Taguchi | Carbon dioxide stored and acidified low oxygen bottom waters in coastal seas, Japan[END_REF][START_REF] Cai | Acidification of subsurface coastal waters enhanced by eutrophication[END_REF][START_REF] Mucci | Acidification of Lower St. Lawrence Estuary bottom waters[END_REF][START_REF] Wallace | Coastal ocean acidification: the other eutrophication problem[END_REF]Hagens et al. 2015). In urbanized areas, this phenomenon can be exacerbated by eutrophication (Borges and Gypens 2010;[START_REF] Sunda | Eutrophication induced CO2-acidification of subsurface coastal waters: interactive effects of temperature, salinity, and atmospheric pCO2[END_REF][START_REF] Melzner | Future ocean acidification will be amplified by hypoxia in coastal habitats[END_REF]. Since variations in pH in coastal waters are typically recurrent and of greater amplitude than those observed in the open ocean, one could surmise that the estuarine biota will be more resilient to pH fluctuations than open ocean biota. In order to evaluate the risk posed by ocean acidification on marine organisms in coastal systems, it is thus essential to know the chemical properties of source waters, the relative contribution of each to the coastal waters and their temporal variations. This knowledge is also required in order to develop realistic experimental protocols to assess the sensitivity of estuarine organisms to ocean acidification.

The head of the Laurentian Channel at the western limit of the Lower St. Lawrence Estuary (LSLE, Figure 1) is one of the most dynamic regions of the St. Lawrence Estuary. Here, complex tidal phenomena due to rapid shoaling (tidal movements, including internal tides and strong flows over the steep entrance sill) generate significant mixing of near-surface waters with the deeper saline waters, resulting in a nutrient-rich surface layer that sustains the feeding habitat of several large marine mammals. Four different water masses converge at this location: the brackish surface waters of the Upper St. Lawrence Estuary (USLE), the brackish surface waters discharged at the mouth of the Saguenay Fjord, the cold intermediate layer (CIL) waters of the LSLE and the hypoxic bottom waters of the LSLE [START_REF] Mucci | Acidification of Lower St. Lawrence Estuary bottom waters[END_REF]. With the exception of the Saguenay River waters, the other water masses are supersaturated in CO2 with respect to the atmosphere (Table 1), the supersaturation having been acquired from their tributaries and/or the accumulation of metabolic CO2 from microbial respiration of autochtonous or allochthonous organic matter [START_REF] Yang | Chemical dynamics of the "St. Lawrence" riverine system: δDH2O, δ 18 OH2O, δ 13 CDIC, δ 34 Ssulfate, and dissolved 87 Sr/ 86[END_REF][START_REF] Barth | Carbon cycle in St Lawrence aquatic ecosystems at Cornwall (Ontario), Canada: seasonal and spatial variations[END_REF]Wang and Veizer 2000). The objective of this study was to assess how the confluence and mixing of these different water masses affect the chemical and biological properties of the surface waters over two tidal cycles at a fixed station at the head of the Laurentian Channel. After identifying the properties of the individual end-member water-mass types, we estimated the relative contribution of each water-mass type to the surface waters at the fixed station by solving for a set of linear equations for conservative and non-conservative properties. A reconstruction of the water column properties at the fixed station and a comparison with field measurements are used to identify factors that control the pH of surface waters in this region over the study period.

Study Site:

The St. Lawrence Estuary

The St. Lawrence Estuary (SLE) receives the second largest freshwater discharge (11900 m 3 s -1 ) in North America (El-Sabh and Silverberg 1990). It begins at the landward limit of the salt intrusion near Ile d'Orléans and extends 400 km seaward to Pointe-des-Monts and the Gulf of St.

Lawrence (GSL), a semi-enclosed sea connected to the Atlantic Ocean via Cabot Strait and the Strait of Belle-Isle (Fig. 1). The Upper St. Lawrence Estuary (USLE) is the relatively shallow, well mixed, and fully oxygenated transition zone between the mouth of the St. Lawrence River at Quebec City and the marine region (SP ≈ 25 and greater) beginning at Tadoussac (Fig. 1). At the transition between the Upper and Lower St. Lawrence Estuary, the water column deepens from 50 m to 300 m over less than 20 km, in an area characterized by diverse and complex tidal phenomena [START_REF] Gratton | Satellite observations of tidal upwelling and mixing in the St. Lawrence Estuary[END_REF][START_REF] Saucier | Tidal circulation and buoyancy effects in the St. Lawrence Estuary[END_REF]. The dominant bathymetric feature of the Lower 2. The surface layer displays large seasonal variations in temperature and salinity due to atmospheric and buoyancy (i.e., runoff events) forcing. In winter, the surface layer becomes progressively colder and denser, as tributary flow decreases, air temperatures plummet, and ice forms, until it merges with the intermediate layer [START_REF] Gilbert | Interannual variability (1948-1994) of the CIL core temperature in the Gulf of St. Lawrence[END_REF][START_REF] Galbraith | Effect of ocean acidification on the early life stages of the blue mussel Mytilus edulis[END_REF]. The deep waters are a mixture of Labrador Current and North Atlantic Central waters whose proportions vary on a decadal or secular timescale [START_REF] Bugden | Changes in the temperature-salinity characteristics of the deeper waters of the Gulf of St. Lawrence over the past several decades[END_REF][START_REF] Gilbert | A seventy-two year record of diminishing deep-water oxygen levels in the St. Lawrence estuary: the northwest Atlantic connection[END_REF]. The transit time of a parcel of water between the seaward end of the Laurentian Channel at the edge of the continental shelf and the head of the Channel at Tadoussac has been estimated at 4 to 7 years [START_REF] Bugden | Changes in the temperature-salinity characteristics of the deeper waters of the Gulf of St. Lawrence over the past several decades[END_REF][START_REF] Gilbert | Propagation of temperature signals along the northwest Atlantic continental shelf edge and into the Laurentian Channel[END_REF]) whereas the mean age of the deep waters of the LSLE is believed to be ~20 years [START_REF] Mucci | Acidification of Lower St. Lawrence Estuary bottom waters[END_REF]. Given the properties of the source waters, benthic and pelagic respiration along the Channel, and their mean age, the bottom waters of the LSLE are hypoxic (<20% oxygen saturation; [START_REF] Gilbert | A seventy-two year record of diminishing deep-water oxygen levels in the St. Lawrence estuary: the northwest Atlantic connection[END_REF], charged with metabolic CO2 and therefore acidified [START_REF] Mucci | Acidification of Lower St. Lawrence Estuary bottom waters[END_REF]. The landward end of the Laurentian Channel marks the transition from the Lower to the Upper St.

Lawrence Estuary, the mouth of the Saguenay Fjord, and the marine region (SP ≈ 25 and greater) beginning at Tadoussac (Fig. 1). Typical semi-diurnal (M2) tidal current amplitudes in the LSLE are on the order of 0.2 m s -1 [START_REF] Saucier | Tidal circulation and buoyancy effects in the St. Lawrence Estuary[END_REF], so that tidal excursions are on the order of 3 km.

The Saguenay Fjord

The Saguenay Fjord is a 93 km long, 1-6 km wide and 275 m deep U-shaped submerged valley, bounded by sheer, vertical walls that reach up more than 300 m above the water line.

Situated approximately 150 km northeast of Quebec City on the north shore of the St. Lawrence Estuary, it connects with the estuary at Tadoussac through a 20-m deep sill. Its bathymetry is defined by three basins separated by two sills at 60 and 120 m depth, located approximately 20 km and 30 km, respectively, from the mouth of the fjord. A short account of its geological history and physiographic features can be found in [START_REF] Schafer | The Saguenay Fjord: A major tributary to the St. Lawrence Estuary[END_REF] and [START_REF] Locat | Le Fjord du Saguenay: Une physiographie et un registre exceptionnels[END_REF].

The water column of the Saguenay Fjord is characterized by a sharp pycnocline that separates two distinct water masses. The thick bottom layer is well-mixed and oxygenated, with waters penetrating landward from the St. Lawrence Estuary as they episodically spill over the sills [START_REF] Therriault | Penetration of the deep layer of the Saguenay Fjord by surface waters of the St-Lawrence Estuary[END_REF][START_REF] Siebert | Deepwater exchange processes in the Saguenay Fjord[END_REF][START_REF] Stacey | The energetics and tidally induced reverse renewal in a twosilled fjord[END_REF][START_REF] Bélanger | Observation and Modelling of a Renewal Event in the Saguenay Fjord[END_REF][START_REF] Belzile | Water renewals in the Saguenay Fjord[END_REF]. Bottom-water salinities are approximately 30.5 [START_REF] Mucci | The differential geochemical behavior of arsenic and phosphorus in the water column and sediments of the Saguenay Fjord Estuary, Canada[END_REF], with temperatures ranging from 0.4 to 1.7 o C [START_REF] Fortin | Synthèse des connaissances sur les aspects physiques et chimiques de l'eau et des sédiments du Saguenay[END_REF]. The surface layer consists of brackish waters (SP~0-10) resulting from the turbulent mixing of the outflow from the Saguenay River (and smaller tributaries such as the Valin, Ha! Ha! and Mars Rivers) and the underlying marine waters. The thickness of the surface layer, perhaps best defined by the depth of the 18.00 sigma-tee isopycnal, typically increases towards the mouth of the fjord, but pinches off towards the surface at high tide or when denser waters overspill the entrance sill. The surface water temperatures range from freezing in winter to 16 º C in summer [START_REF] Fortin | Synthèse des connaissances sur les aspects physiques et chimiques de l'eau et des sédiments du Saguenay[END_REF]. Detailed hydrographic characteristics of the fjord can be found in [START_REF] Schafer | The Saguenay Fjord: A major tributary to the St. Lawrence Estuary[END_REF] and [START_REF] Gratton | Océanographie physique du fjord du Saguenay[END_REF].

Figure 3 shows a 3-day average of sea-surface temperatures derived from satellite data (OGSL 2014). A prominent tongue of cold surface waters due to upwelling and tidal mixing extends from the head of the Laurentian Channel (near Tadoussac) to about Rimouski. Our fixed station is located at the northern edge of this cold tongue.

Methods

Water column sampling

On June 10-15, 2013, we sampled the whole water column at six stations and surface waters (3 -5 m below the surface) at four stations along a saw-tooth transect at the head of the Lower St.

Lawrence Estuary (LSLE), the mouth of the Upper St. Lawrence Estuary (USLE) and the mouth of the Saguenay Fjord (Figure 1). In addition, we sampled the whole water column every four hours and surface waters (~3 m) every two hours over 50 hours at a fixed station (48°11.2'N, 69°34.4'W) a few kilometers east of the fjord's mouth in the northern half of the head of the LSLE in order to characterize changes in the properties of these surfaces waters over two tidal cycles.

Finally, the whole water column at four stations (Stations 23,22,20,18) in the LSLE and western Gulf of St. Lawrence was sampled on an earlier cruise (June 3-8, 2013) and these results served to better define the properties of the source-water types encountered at the fixed station (see Water mass analysis section below). Water samples were collected using a 12-Niskin bottle/CTD (SeaBird SBE 911) rosette sampler onboard the R/V Coriolis II. Sampling depths were typically, 3,10,20,40,60,80,100,150 m deep, in addition to 15 m above the bottom. The pH (on the NBS scale -pHNBS), dissolved oxygen (DO), temperature and conductivity probes were calibrated by the manufacturer during the winter months preceding the cruise. Nevertheless, discrete samples were taken from the Niskin bottles for laboratory measurements of pH (total proton concentration scale -pHT), DO and salinity and the CTD records re-calibrated post-cruise. Additional surface water samples were collected, on distinct outings, in Chicoutimi (Saguenay River end-member) and near Quebec City (St. Lawrence River end-member) during or within a week of the research cruise.

Analytical methods

Discrete salinity samples taken throughout the water column were analyzed on a Guildline Autosal 8400 salinometer calibrated with IAPSO standard seawater. The instrument has a theoretical accuracy < ± 0.002. Dissolved oxygen (DO) concentrations were determined by Winkler titration [START_REF] Grasshoff | Methods of seawater analysis[END_REF]) of water samples recovered from the Niskin bottles. The relative standard deviation, based on replicate analyses of samples recovered from the same Niskin bottle, was better than 1%. These measurements further served to calibrate the SBE-43 oxygen probe mounted on the rosette.

Water samples destined for pH and titration alkalinity (TA) measurements were transferred directly from the Niskin bottles to, respectively, 125 mL plastic bottles without headspace and 250 mL glass bottles, as soon as the rosette was secured onboard. In the latter case, a few crystals of HgCl2 were added before the bottle was sealed with a ground-glass stopper and Apiezon ® Type-M high-vacuum grease.

The pH of the 125 mL samples was determined onboard using a Hewlett-Packard UV-Visible diode array spectrophotometer (HP-8453A) and a 5-cm quartz cell after thermal equilibration of the sampling bottles in a constant temperature bath at 25.0±0.1°C. Phenol red (PR; [START_REF] Robert-Baldo | Spectrophotometric determination of seawater pH using phenol red[END_REF] and m-cresol purple (mCP; [START_REF] Clayton | Spectrophotometric seawater pH measurements: total hydrogen ion concentration scale calibration of m-cresol purple and at-sea results[END_REF] were used as indicators. The absorbance at the wavelengths of maximum absorbance of the protonated (HL) and deprotonated (L) indicators were measured and recorded. A similar procedure was carried out several times each day using TRIS buffers prepared at practical salinities (SP) of ~35 and ~25 [START_REF] Millero | The pH of estuarine waters[END_REF]). The pH on the total proton concentration scale (pHT) of the samples and buffer solutions were calculated according the equation of [START_REF] Byrne | Standardization of standard buffers by visible spectrometry[END_REF]. The salinity-dependence of the dissociation constants and molar absorptivities of the indicators were taken from [START_REF] Robert-Baldo | Spectrophotometric determination of seawater pH using phenol red[END_REF] for phenol red and from [START_REF] Clayton | Spectrophotometric seawater pH measurements: total hydrogen ion concentration scale calibration of m-cresol purple and at-sea results[END_REF] for m-cresol purple. The salinitydependence of the phenol red indicator dissociation constant and molar absorptivities was extended (from SP = 5 to 35; Bellis 2002) to encompass the range of salinities encountered in this study, but computed pHTs from the revised fit were not significantly different from those obtained with the relationship provided by [START_REF] Robert-Baldo | Spectrophotometric determination of seawater pH using phenol red[END_REF]. All measurements were converted to the total proton scale using the salinity of each sample and the HSO4 -association constants given by [START_REF] Dickson | Standard potential of the (AgCl + 1/2H2 = Ag + HCl(aq)) cell and the dissociation constant of bisulfate ion in synthetic sea water from 273[END_REF]. Reproducibility and accuracy of our TRIS buffer measurements were on the order of 0.005 pH units or better. The computed pHTs at 25°C and 1 atmosphere total pressure were then used in combination with the measured TA to calculate the pHTs at the in-situ temperature and pressure using the Microsoft Excel version of CO2SYS [START_REF] Pierrot | MS Excel Program Developed for CO2 System Calculations[END_REF], based on the original algorithm of [START_REF] Lewis | Program Developed for CO2 System Calculations[END_REF], and the carbonic acid dissociation constants of [START_REF] Cai | The chemistry, fluxes, and sources of carbon dioxide in the estuarine waters of the Satiala and Altamaha Rivers, Georgia[END_REF], the total boron concentration [B]T value from [START_REF] Uppström | The boron/chlorinity ratio of deep-sea water from the Pacific Ocean[END_REF], and the standard acidity constant of the HSO4 -ion (K(HSO4)) from [START_REF] Dickson | Standard potential of the (AgCl + 1/2H2 = Ag + HCl(aq)) cell and the dissociation constant of bisulfate ion in synthetic sea water from 273[END_REF].

The titration alkalinity (TA) was determined at the land-based laboratory within one week of sampling by open-cell automated potentiometric titration (Titrilab 865, Radiometer ® ) with a pH combination electrode (pHC2001, Red Rod ® ) and a dilute (~0.025N) HCl titrant. The latter was calibrated against Certified Reference Materials (CRM Batch#94, provided by A. G. Dickson, Scripps Institute of Oceanography, La Jolla, USA). Samples were drawn from the 250 mL sample bottle and weighed on an analytical balance to ± 0.1 mg. The average relative error, based on the average relative standard deviation on replicate standard and sample analyses, was better than 0.15%.

Nutrients

Subsamples for nutrient determinations were filtered through a 0.45 µm disposable syringe filter to remove particles. Dissolved NH4 + concentrations were determined immediately onboard with the fluorometric method described by [START_REF] Holmes | A simple and precise method for measuring ammonium in marine and freshwater ecosystems[END_REF], with a detection limit of 0.01 µM.

For nitrate, nitrite, soluble reactive phosphorus (SRP) and soluble reactive silicon (SRS), filtered samples were collected into acid-washed 15-ml polyethylene tubes and quickly frozen and stored at -20°C. Back at home laboratory, frozen samples were rapidly thawed and concentrations of inorganic nutrients were determined using the routine colorimetric method adapted from Hansen and Koroleff (2007) with a Bran and Luebbe Autoanalyzer III. The analytical detection limit was 0.02 µM for nitrite, 0.04 µM for nitrate+nitrite (hereafter dissolved inorganic nitrogen, DIN), 0.05 µM for SRP and 0.1 µM for silicate.

Isotopic composition

Water samples for isotopic analysis were collected in 13 mL screw-top plastic test tubes.

The stable oxygen isotopic composition (δ 18 O) of the water samples was analyzed at the GEOTOP-UQAM stable isotope laboratory. 200-μL aliquots of the water samples and two laboratory internal reference waters were transferred into 3-mL vials stoppered with a septum cap. The vials were then placed in a heated rack maintained at 40°C. Commercially available CO2 gas was introduced in all the vials using a Micromass AquaPrep and allowed to equilibrate for 7 hours. The headspace CO2 was then sampled by the Micromass AquaPrep, dried on a -80°C water trap, and analyzed on a Micromass Isoprime universal triple collector isotope ratio mass spectrometer in dual inlet mode.

Data were normalized against the two internal reference waters, both calibrated against V-SMOW and V-SLAP. Data are expressed in ‰ with respect to V-SMOW, and the average relative standard deviation on replicate measurements of sample waters is better than 0.05‰.

Water mass analysis

The water mass analysis was performed using the optimum multi-parameter algorithm (OMP) of [START_REF] Karsteen | OMP Analysis Package for Matlab[END_REF], originally developed by [START_REF] Tomczak | A multiparameter extension of temperature/salinity diagram techniques for the analysis of non-isopycnal mixing[END_REF]. OMP analysis is based on a simple model of linear mixing. It assumes that all water mass properties undergo the same mixing process, i.e. their mixing coefficients are identical, as is the case in turbulent mixing.

The spatial water mass distribution can therefore be determined through a linear set of mixing equations. OMP determines the contributions of pre-defined end-member water masses (so called source-water types or SWT) to a sample. The SWT contributions or fractions (Xi) for each data point are obtained by finding the best linear mixing combination in parameter space defined by temperature, salinity, oxygen, total alkalinity and δ 18 O which minimizes the residuals in a non-negative least squares analysis. The solution includes two physically realistic constraints: (i) the contributions from all sources must add up to 100%, and (ii) all contributions have to be nonnegative.

Based on the description of the water circulation in the LSLE, four different source-water types contribute to the water column properties at the head of the Laurentian Channel: the freshwaters of the St. Lawrence and Saguenay Rivers, the saline and cold intermediate layer water and the upwelling saline hypoxic bottom water of the LSLE. Even though the properties of the source-water types are variable in both space and time, we use the dataset acquired during this study as well as data acquired a week before on another cruise along a seaward transect through the Lower Estuary and Gulf of St. Lawrence to define the most representative properties for each source-water type (Table 1). This allows the direct comparison of the water mass fractions and their vertical distribution at the time of sampling. Whereas OMP analysis was initially used to distinguish and calculate the relative contributions of each source-water type in a parcel of water using temperature, practical salinity (SP) and nutrient data [START_REF] Mackas | Least squares multiple tracer analysis of water mass composition[END_REF], in this study, we used SP, the titration alkalinity (TA), and δ 18 O(H2O) as conservative tracers as well as temperature and dissolved O2 (DO) concentrations as non-conservative tracers, to constrain the water mass analysis. Non-conservative tracers should be applied with caution in OMP, because of temporal and spatial variability, particularly in the surface waters. For example, the temperature of the Saguenay River water ranges from 0°C in winter to +16°C in summer whereas the temperature of the St. Lawrence River ranges from 0°C in winter to >20°C in summer. Dissolved O2 concentration was also used as a non-conservative tracer, because it is a function of temperature (e.g. O2 solubility increases with decreasing temperature and salinity) and, like ΣSi and SRP, also a function of primary production and biological respiration. Consequently, end-member matrix and sample observations were multiplied by a diagonal weight matrix to account for differences in tracer reliability, environmental variability, and precision of the data (Table 2). The error associated with the water mass fraction analysis was estimated to be about ±10% of the fractional values [START_REF] Macdonald | Composition and modification of water masses in the Mackenzie shelf estuary[END_REF][START_REF] Lansard | Seasonal variability of water mass distribution in the southeastern Beaufort Sea determined by total alkalinity and δ 18 O[END_REF]. 5a-d.

Results and discussion

Variations of the water properties over two tidal cycles

The tides are very strong in the fixed station region. The impact of tides on the local circulation is described in detail in [START_REF] Saucier | Tidal circulation and buoyancy effects in the St. Lawrence Estuary[END_REF]. Near the SF, the tidal elevation in the channel can be larger than 5 m (unpublished data) at the spring tide. [START_REF] Mertz | Topographic waves and topographically induced motion in the St. Lawrence Estuary[END_REF] reported internal oscillations on the order of 40-60 m over a tidal semi-diurnal cycle. Similar oscillations can be observed on Figures 4a,b,c. The cold, upwelled water can reach the surface and be observed flowing downstream as on Figure 3. As observed on this figure, our SF is located at the edge of these cold surface waters.

Surface salinities decrease and temperatures increase during ebb tide (Figs. 4a,b), as freshwaters from the Saguenay and the St. Lawrence Rivers, but mostly of the latter (Figs. 5a,b), intrude into the Lower St. Lawrence Estuary (LSLE) and at our fixed station. The intrusion is detectable to depths of about 30 m and accompanied by a slight (less than 0.1 pH unit) decrease in pH (Fig. 4f). Higher salinity, lower temperature and higher pH waters impinge closer to the surface during the flood tide whereas patches of low pH waters from the Saguenay Fjord persist at the surface well into flood tide (Figs. 4a,b,f). The former correspond to the upwelling/invasion of the cold intermediate layer (CIL) nearly up to the surface where it accounts for more than 80% of the mixture (Fig. 5c). Temporal variations of the surface-water titration alkalinity (TA) and dissolved inorganic carbon (DIC) concentrations are almost perfectly correlated with those of the salinity (Figs. 4e,g). Temporal variations of the surface-water major nutrient concentrations are more diffuse, but are nonetheless either correlated (SRP (r 2 = 0.63); Fig. S1) or anti-correlated (DIN (r 2 = 0.46) and ΣSi (r 2 = 0.62); Fig. S1) to salinity in the top 3 m, reflecting the differential input from the freshwaters (Saguenay and St. Lawrence Rivers) and the CIL.

Irrespective of the tide, dissolved oxygen concentrations remain high (> 260 µM, Fig. 4d) throughout the top 80-100 m of the water column. Likewise, the computed pCO2 in the top 80 m of the water column is slightly supersaturated with respect to the atmosphere (~ 400 µatm) and shows few discernible features, except for a greater pCO2 supersaturation at low tide when the contribution of freshwaters from the St. Lawrence River and Saguenay Fjord, both of which are rich in metabolic CO2 and dissolved silicate (as well as ammonium in the latter case) relative to the CIL, are maximum (Fig. 5a,b).

As expected, below 150 m depth, hypoxic bottom waters are dominated by Atlantic waters that enter the Gulf and Estuary through Cabot Strait. These waters intrude to depths as shallow as 80 m at high tide (Fig. 5d).

pH in the surface waters

The pHT of the surface waters (top 3 m) varied from 7.855 to 7.934 during the 50-hour survey, nearly as much as the world's oceans have experienced as a result anthropogenic CO2 uptake over the last century [START_REF] Caldeira | Ocean model predictions of chemistry changes from carbon dioxide emissions to the atmosphere and ocean[END_REF]. The pHT of surface waters is determined by the relative contribution of the three source-water types (SLW, SRW, and CIL), itself determined in great part by tidal mixing, as well as by gas exchange with the atmosphere, the addition of metabolic CO2 through bacterial respiration and biological uptake of CO2 through photosynthesis. [START_REF] Mucci | Acidification of Lower St. Lawrence Estuary bottom waters[END_REF] reported on the variation of surface-water (0-16 m depth interval) pH over the period 1933-2009 (also limited to the ice-free season) in the Lower St.

Lawrence Estuary (LSLE). Unlike the deeper waters that have become hypoxic and acidified, the pH of surface waters shows no systematic variations over this period. Although the intra-annual variability reaches ~0.1 pH unit, the inter-annual variability is nearly null (slope = 0, p > 0.05; i.e., no statistically significant temporal variation). Along the Estuary and Laurentian Channel, pH variations can exceed the intra-annual variability in the LSLE as the salinity and balance between respiration and photosynthetic rates vary [START_REF] Dinauer | Spatial variability of surface-water pCO2 and gas exchange in the world's largest semi-enclosed estuarine system: St. Lawrence Estuary (Canada)[END_REF].

Throughout the three-day survey, waters in the surface layer (0-3 m) at the sampling sites were always supersaturated in CO2 (443-550 μatm) with respect to the atmosphere at the time of sampling. The saturation state of the surface mixed layer water with respect to aragonite, given by:

ΩA = [Ca 2+ ][CO3 2-]/K * A (1)
where K * A is the stoichiometric solubility of aragonite at the in-situ temperature and salinity and 1 atmosphere total pressure [START_REF] Mucci | The solubility of calcite and aragonite in seawater at various salinities, temperatures and one atmosphere total pressure[END_REF], varied little during the sampling period and was close to saturation (ΩA = 0.88 to 1.02, 0.95 ± 0.03, n=27, Fig. 6), but fell slightly below saturation (as low as ΩA = 0.88) at low tide. Given the ratio of the stoichiometric solubility constants (K * A/K * C = 1.5; [START_REF] Mucci | The solubility of calcite and aragonite in seawater at various salinities, temperatures and one atmosphere total pressure[END_REF], the saturation state of the surface waters with respect to calcite was close to 1.5. Deeper waters became undersaturated with respect to aragonite below 60-80 m reaching saturations as low as 0.70, but remained supersaturated with respect to calcite to the bottom at the fixed station.

Validation of the OMP analysis

Using the results of the OMP analysis and the source-water type properties in Table 1, the total alkalinity (TA), DIC and nutrient concentrations of each discrete water sample at the fixed station was calculated, over the whole time series, assuming conservative mixing (i.e., closed system). These computed values were then used as input parameters into CO2SYS to calculate the pHT of the mixture and results compared to measured values. Differences between the calculated and observed surface waters properties are compiled in Table 3; these reflect changes to the water properties beyond those that can be accounted for by mixing of the source waters. The ΔpHcalcmeas values (thereafter referred to as ΔpHT) of the surface waters (1-3 m) are all negative (-0.004 to -0.066), indicating that either CO2 was lost by ventilation to the atmosphere or taken up by photosynthesis during our observation period. As indicated above, surface waters remained supersaturated with respect to the atmosphere throughout the sampling period and thus served as a net source to the atmosphere. CO2 exchange across the air-sea interface is, however, a slow process, much slower than for oxygen and most other gases [START_REF] Zeebe | CO2 in Seawater: Equilibrium, Kinetics, Isotopes[END_REF], particularly under the low wind conditions (~5-28 knots) experienced during the cruise. Given these wind speeds, the parameterizations of [START_REF] Raymond | Gas exchange in rivers and estuaries: Choosing a gas transfer velocity[END_REF] and [START_REF] Wanninkhof | Relationship between wind speed and gas exchange over the ocean revisited[END_REF] for the gas transfer velocity, an average surface water pCO2 of 499 (± 26 μatm) during the sampling period, and a local atmospheric pCO2 of 391 μatm, we estimated the air-sea CO2 efflux (loss of CO2 to the atmosphere) to range between 2.6 and 4.8 mmol m -2 d -1 . Over our sampling period, the loss of CO2 to the atmosphere did not affect the DIC budget significantly (less than 1 μmol/kg) and would have increased the surface-water pH by at most 0.003 unit, slightly below the uncertainty of our field measurements. The positive ΔpCO2, ΔDIN and ΔSRP values (net DIC and nutrient uptake) as well as negative ΔDO values (net oxygen production) in the surface waters are clear evidence that photosynthesis (CO2 uptake, pH increase) dominated over respiration (CO2 production and pH decrease) and ventilation over the whole sampling period (Table 3).

Unfortunately, in the absence of independent estimates of the absolute photosynthetic and/or respiration rates, we can only determine which of these two processes dominates. It is also interesting to note that, despite the large uncertainties in the model and the intrinsic patchiness of our measurements, the average ΔDIN:ΔSRP ratio (11 ± 4) nearly corresponds to the Redfield stoichiometry of 15-16 (Redfield et al. 1963) and the DIN: SRP ratio (12, r 2 = 0.79) below 30 m depth in the LSLE. When the surface-water ΔpHT, ΔDIN, ΔSRP, ΔΣSi and ΔDO values are plotted as function of the time of the day, no systematic variation with daylight is apparent (Fig. 7). The study region is known to be an area of high primary productivity, notably during the spring tide periods when upwelling and internal waves amplitude are maximum (Therriault and[START_REF] Therriault | Control of phytoplankton production in the lower St. Lawrence estuary: Light and freshwater runoff[END_REF][START_REF] Levasseur | Phytoplankton biomass and nutrient dynamics in a tidally induced upwelling: the role of the NO3:SiO4 ratio[END_REF]), but can be strongly suppressed spring fresh-water runoff [START_REF] Zakardjian | Late spring phytoplankton bloom in the Lower St. Lawrence Estuary: the flushing hypothesis revisited[END_REF]. Hence, it is not surprising that we observe net autotrophy in the surface waters but our conservative mixing model is not able to resolve the diurnal cycle of photosynthesis and respiration, likely because it is not sensitive enough. Note that we have not modeled temporal variations of pH below the surface mixed layer (SML) because, as shown in Figure 5, below ~30m, the water column is entirely dominated by two water masses, the CIL between 30 and 50 m and Atlantic (deep LSLE) waters below 150 m. In other words, with the exception of the SML, significant variations in water column composition in response to the tide are only found between 50 and 150 m. Given that these waters are isolated from the atmosphere (no gas exchange), below the euphotic zone (no photosynthesis), and respiration rates are negligible on the time scale of the experiments, temporal variations are simply due to tidal oscillations.

Susceptibility of estuarine waters to acidification resulting from direct anthropogenic CO2 uptake

As noted above, most inner (river-dominated) estuaries are net sources of CO2 to the atmosphere [START_REF] Cai | The chemistry, fluxes, and sources of carbon dioxide in the estuarine waters of the Satiala and Altamaha Rivers, Georgia[END_REF][START_REF] Chen | Reconciling opposing views on carbon cycling in the coastal ocean: Continental shelves as sinks and near-shore ecosystems as sources of atmospheric CO2[END_REF][START_REF] Regnier | Anthropogenic perturbation of the carbon fluxes from land to ocean[END_REF]. Their elevated DIC enrichments and pCO2 supersaturations can mainly be attributed to the in situ microbial degradation of internally and externally supplied organic carbon and the lateral transport of inorganic carbon from rivers, coastal wetlands and ground waters [START_REF] Barwell-Clarke | Institute of Ocean Sciences Nutrient Methods and Analysis[END_REF]. Thus, these waters do not directly take up anthropogenic CO2 from the atmosphere. Nonetheless, there have been several reports of weak CO2 uptake by strongly stratified and/or marine-dominated (outer) estuaries [START_REF] Koné | Seasonal variability of carbon dioxide in the rivers and lagoons of Ivory Coast (West Africa)[END_REF][START_REF] Maher | Carbon budgets for three autotrophic Australian estuaries: Implications for global estimates of the coastal air-water CO2 flux[END_REF][START_REF] Cotovicz | A strong CO2 sink enhanced by eutrophication in a tropical coastal embayment (Guanabara Bay, Rio de Janeiro, Brazil)[END_REF], including the Lower St. Lawrence Estuary [START_REF] Dinauer | Spatial variability of surface-water pCO2 and gas exchange in the world's largest semi-enclosed estuarine system: St. Lawrence Estuary (Canada)[END_REF]. The resistance of these estuarine waters to a decrease in pH in response to CO2 uptake is equated to a multiple of the carbonate ion concentration ([CO3 2-]) [START_REF] Stumm | Aquatic Chemistry: Chemical Equilibria and Rates in Natural Waters[END_REF], and the latter, within the pH range of most of these (7.5 < pHT <8.1), is approximated by TA -DIC [START_REF] Broecker | Tracers in the Sea[END_REF].

Variations of the saturation state of waters with respect to aragonite (or calcite) depend on the calcium and carbonate ion concentration product and the mineral solubility under in-situ conditions (Eqn. 1). The [Ca 2+ ] in estuarine waters is determined by the composition and mixing ratio of the freshwater and seawater end-members, but given its high and conservative concentration in seawater, the freshwater signature typically becomes indistinguishable (within a 1% deviation of the diluted seawater value) from that of seawater above a salinity of ~14. The minimum salinity measured during our survey was 24.82. Hence, the [Ca 2+ ] can accurately be estimated from the measured salinities, i.e., [Ca 2+ ] = SP * 0.01028/35 [START_REF] Millero | Chemical Oceanography, 4 th Edition[END_REF]. Both [Ca 2+ ] and K * A increase with salinity and to a similar extent (31% and 32.5%, respectively, over the range of salinities encountered during our study), so that ΩA varies almost exactly with the [CO3 2-].

Hence, again, the impact of CO2 uptake on the saturation state of most marine-dominated estuaries is almost exclusively related to changes in [CO3 2-] which, as noted above, can be approximated by (TA -DIC).

Biological response of some local CaCO3-secreting invertebrates to ocean acidification

Atmospheric CO2 uptake lowers the pH, the carbonate ion concentration and saturation state of natural waters with respect to calcite and aragonite (ΩC and ΩA, respectively).

Consequently, the ability of many organisms to calcify is reduced by a decrease in the saturation state of the waters and sediments they inhabit [START_REF] Gazeau | Impacts of ocean acidification on marine shelled molluscs[END_REF][START_REF] Clements | Effects of CO2-driven sediment acidification on infaunal marine bivalves: A synthesis[END_REF].

Aragonite being 50% more soluble than calcite (Mucci, 1981), organisms whose shells or skeletons are partially or wholly aragonitic will be more susceptible to acidification.

Aragonite-undersaturated waters (ΩA < 1) dominate the top 20 metres of the water column at the head of the Laurentian Channel except when the CIL intrudes almost up to the surface at high tide. Below the CIL, the aragonite saturation depth (ΩA = 1) oscillates between 40 and 90 m reaching the shallowest depths at high tide when these waters are brought closer to the surface. Hence, the recruitment, growth, metabolism and survival of calcifying invertebrates and other benthic organisms exposed to these waters could be deleteriously affected [START_REF] Parker | Adult exposure influences offspring response to ocean acidification in oysters[END_REF][START_REF] Galbraith | Effect of ocean acidification on the early life stages of the blue mussel Mytilus edulis[END_REF]Talmage et al. 2010;Kuhihara 2008).

Commercial harvesting of bivalve shellfish (scallops, soft-shell clams, mussels, Atlantic surf-clams, Atlantic razor clams), echinoids (green sea urchin), and gastropods (common whelk)

in Quebec sustains several coastal communities with annual landings in the range of 2.5-4 million dollars (DFO; http://www.qc.dfo-mpo.gc.ca/peches-fisheries/recreative-recreational/mollusquemollusc-eng.asp?p=/peches-fisheries/recreative-recreational/mollusque-mollusc-eng.html).

Scallops (e.g., Chlamys islandica), soft-shell clams (e.g., Mya arenaria), the common whelk (e.g.,

Buccinum undatum) and the green sea urchin (e.g., Stronylocentrotus droebachiensis) are found on the seafloor near the head of the Laurentian Channel and, with the exception of scallops [START_REF] Hutin | Acoustic detection of a scallop bed from a singlebeam echosounder in the St. Lawrence[END_REF], are mostly harvested from no more than 20 m depth (Bernard Saint-Marie, DFO, pers. comm.). Except for the green sea urchin, harvesting of the other species has declined steadily since the beginning of the new millennium and there are currently no active commercial shellfish fisheries in this region while recreational harvesting of scallops is now forbidden. The shells of most bivalves and whelks found in the study area are composed of a combination of calcite and aragonite (micro-) structural components (groups and layers) whereas the body and spines of sea urchins are typically composed of high-magnesium calcite (more soluble than aragonite) and, thus, their constant exposure to the aragonite-undersaturated (corrosive) waters in the top 20 m of the water column and along the shorelines in the vicinity of the study area could possibly have contributed, along with overfishing [START_REF] Archambault | Effect of the commercial fishery on the Ile Rouge Iceland scallop (Chlamys islandica) bed in the St. Lawrence estuary: assessment of the impacts on scallops and the benthic community[END_REF], to their decline. Since the habitat of scallops extends to depths of 80 meters (the largest scallop bed in the vicinity of Tadoussac is situated 5-10 km downstream of Ile Rouge in 40-80 m of water; [START_REF] Hutin | Acoustic detection of a scallop bed from a singlebeam echosounder in the St. Lawrence[END_REF], this would put them well into the aragonite-undersaturated (corrosive) waters found below the CIL.

To our knowledge, no specific study of the impacts of elevated pCO2s (lower pH and ΩA) has been carried out on Chlamys islandica and Buccinum undatum, but studies conducted on other species of scallops and whelk have reported deleterious effects on calcification and growth of the former and the nutritional properties of the latter [START_REF] Tate | Ocean acidification and warming impacts the nutritional properties of the predatory whelk, Dicathais orbita[END_REF][START_REF] Andersen | Effect of increased pCO2 levels on early shell development in great scallop (Pectin maximus Lamarck) larvae[END_REF].

Conversely, in addition to lowered calcification rates, the impacts of elevated pCO2 on Mya arenaria were found to extend to their burrowing behavior, post-settlement dispersal (Clements et al. 2016) as well as predator-prey interactions (Glaspie et al. 2017) whereas, in the case of Stronylocentrotus droebachiensis, the effects extend to the fecundity, gonad growth, feeding rates and susceptibility to metal toxicity (Lewis et al. 2016;[START_REF] Dupont | Long-term and translife-cycle effects of exposure to ocean acidification in the green sea urchin Strongylocentrotus droebachiensis[END_REF]Siikavuopio et al. 2007).

Notwithstanding the potential impact of ocean acidification (OA) on the organisms listed above, one should consider that these organisms may have, over time, developed a tolerance or acclimatized to the high-amplitude and high-frequency (diurnal, seasonal) variations of environmental variables (T, S, pH) encountered in dynamic estuaries such as the USLE, as has been demonstrated in long-term exposures to elevated pCO2 [START_REF] Dupont | Long-term and translife-cycle effects of exposure to ocean acidification in the green sea urchin Strongylocentrotus droebachiensis[END_REF]Moulin et al. 2015;[START_REF] Uthicke | Echinometra sea urchins acclimatized to elevated pCO2 at volcanic vents outperform those under present-day pCO2 conditions[END_REF]). Whereas the above discussion is limited to commercially-harvested CaCO3-secreting benthic organisms, OA may impact on the metabolic activity (e.g., survival, growth, reproduction) of many other organisms, but an expanded discussion of these would be well beyond the scope of this paper given the range of responses documented in the literature (e.g., [START_REF] Doney | Ocean acidification: A critical emerging problem for ocean sciences[END_REF].

In summary, the pH of surface waters at the head of the Laurentian Channel is modulated by tides and mixing of three source-water types, the relatively high alkalinity (TA) and dissolved 2 for details).

Errors originate mostly from the variance on the fractional contributions obtained from the OMP analysis and, in this specific situation on the fractional contribution of the Saguenay River water (most divergent TA and pH). Although typically on the order of 10% [START_REF] Lansard | Seasonal variability of water mass distribution in the southeastern Beaufort Sea determined by total alkalinity and δ 18 O[END_REF], we applied a +50% error to this value and the difference in computed pHT was less than 0.003 unit, below the uncertainty of our field measurements and smaller than the size of the symbols.

-0. TA -titration alkalinity, DIC -dissolved inorganic carbon, DO -dissolved oxygen, ΣSi -dissolved silicate, SRP -soluble reactive phosphate, DIN -dissolved inorganic nitrogen 1 DIC values were not measured directly, but calculated from pH and TA using the Excel version of CO2SYS [START_REF] Pierrot | MS Excel Program Developed for CO2 System Calculations[END_REF] for the marine waters and the web-based version of PHREEQC (www.ndsu.edu/webphreeq/) for the freshwaters. 2 Values were not measured at the time of sampling, they are averages of measurements carried out in the previous 5 years at similar times of the year. 3 pH of freshwaters were not measured at the time of sampling, values that appear in the table are averages of measurements carried out in the previous 5 years at similar times of the year on the NBS scale (infinite dilution convention). 3. Reconstructed surface water properties at the fixed station (SF) (conservative mixing of source-water types according to the OMP results) over two full tidal cycles (June 13-15, 2013) and differences between the calculated and observed values. ________________________________________________________________________ Time Depth pHT DIN SRP ΣSi DO ΔpHT ΔDIN ΔSRP ΔSi ΔDO (m) ---------µmol/L -------------------µmol/L ----------16:15 3.2 7.833 13.5 1.06 18.5 297 -0.021 -5.1 0.21 -3.9 -10 18:20 3. 2 for details). Errors originate mostly from the variance on the fractional contributions obtained from the OMP analysis and, in this specific situation on the fractional contribution of the Saguenay River water (most divergent TA and pH). Although typically on the order of 10% [START_REF] Lansard | Seasonal variability of water mass distribution in the southeastern Beaufort Sea determined by total alkalinity and δ 18 O[END_REF], we applied a +50% error to this value and the difference in computed pHT was less than 0.003 unit, below the uncertainty of our field measurements and smaller than the size of the symbols.

  St. Lawrence Estuary (LSLE) is the Laurentian Channel, a 1240 km long, 250-500 m deep submarine valley that extends from the edge of the eastern Canadian continental shelf, through Cabot Strait, to the confluence of the St. Lawrence Estuary and the Saguenay Fjord at Tadoussac. The water column in the LSLE is strongly stratified and, throughout most of the year, is characterized by three distinct layers (Dickie and Trites 1983): (1) a thin surface layer (25-50 m deep) of low salinity (27-32) flowing seaward, (2) an intermediate cold (-1 to 2°C) and saline (31.5-33) layer (CIL) that originates in the Gulf in the winter, extends to about 150 m depth, and flows landward, and (3) a warmer (4-6°C) and saltier (34-34.6) deep layer (150+ m) that flows landward (El-Sabh and Silverberg 1990). Two typical vertical profiles taken at one of our sampling stations, station 1B (Figure 1b: 48°19.8'N, 069°17.4'W; June 11, 2013; 16:48GMT) and at station 18 (Figure 1a: 49° 16.0, 064° 16.5; June 7, 2013; 16:01 GMT) are reproduced in Figure

Figures

  Figures 4a-l show time-series of water property profiles from the CTD (1-m resolution) at

  inorganic carbon (DIC)-rich water of the St. Lawrence River (TA-DIC <0), the TA and DIC-poor water of the Saguenay River (TA-DIC) <0) and saline waters of the TA and DIC-rich cold intermediate layer (CIL) of the St. Lawrence Estuary (TA-DIC) >0) that upwell in this region.Consequently, in-situ pHT values lower than 8.0 are found below the CIL or 80 m depth and in the top 30 m of the water column. Nevertheless, upwelling of the TA-rich CIL and mixing with lower salinity waters near the surface limit pH excursions within a narrow range (7.86 to 7.93) as the (TA-DIC) of the mixture at our study site (head of the Laurentian Channel) was always positive.The presence of the CIL also appears to impede the upwelling of the hypoxic, CO2-rich and aragonite-undersaturated bottom waters of the Lower St. Lawrence Estuary (LSLE) to depths shallower than about 80 m (Figure5), although the large density gradient, as well as the tidal (~5 m) and internal-wave amplitudes (maximum 50-60 m), would not allow bottom waters to intrude at the surface. The CIL is not unique to the SLE as similar layers are common in other subpolar basins[START_REF] Osafune | Bidecadal variability in the intermediate waters of the northwestern subarctic Pacific and the Okhotsk Sea in relation to 18.6-year period nodal tidal cycle[END_REF][START_REF] Cyr | Behavior and mixing of a cold intermediate layer near a sloping boundary[END_REF]. Organisms living within the first 20 meters of the water column at the head of the Laurentian Channel are bathed, at low tide, by aragoniteundersaturated waters (ΩA < 1) intruding from the St. Lawrence and Saguenay Rivers. Likewise, those that live below 80 meters depth, below the CIL, such as the largest scallop bed in the area, are continuously exposed to the aragonite-undersaturated bottom waters (ΩA < 1) of the LSLE that upwell up to nearly 40 metres from the surface at high tide. Whereas the growth, recruitment, and other metabolic functions of CaCO3-secreting invertebrates exposed to these aragoniteundersaturated waters may be deleteriously affected, some of these organisms may have, over time, developed a tolerance or acclimatized to the high-amplitude and high-frequency (diurnal, seasonal) variations of environmental variables (T, S, pH) encountered in this dynamic environment.

Figure

  Figure 1-a)

Figure 3 -Figure 3 .

 33 Figure 3 -

Figure 5 .

 5 Figure 5 -

Figure 6 .

 6 Figure 6-

Figure 7 -Figure 7 -

 77 Figure 7-

Figure 1 .

 1 Figure captions -

Figure 2 .

 2 Figure 2. a) Vertical profiles of the salinity (red line) and temperature (blue line) throughout the

Figure 3 .

 3 Figure 3. Three-day (June 13-15, 2013) average sea surface temperature during the fixed station

Figure 4 .

 4 Figure 4. Water properties measured at the fixed station (48°11.2'N, 69°34.4'W) over two tidal

Figure 5 .

 5 Figure 5. Distribution (%) of water-source types at the fixed station (48°11.2'N, 69°34.4'W) over

Figure 6 .

 6 Figure 6. Saturation state of the water column with respect to a) calcite and b) aragonite at the

Figure 7 -

 7 Figure 7 -Difference between calculated (based on conservative mixing of the four source water

Time of day pH calc-meas vs time of day

		080
		-0.070
		-0.060
	calc-meas	-0.050 -0.040				Day 1 Day 2
	pH	-0.030				Day 3
		-0.020
		-0.010
		0.000
		0:00	6:00	12:00	18:00	0:00

Table 1 .

 1 Source water types and their characteristic properties used in the OMP analysis

	______________________________________________________________________________
	Water types	T	SP δ 18 O	TA	DIC 1 DO ΣSi SRP DIN pCO2 pHt
		(°C)		(‰)	---(μmol kg -1 )----------(μmol L -1 ) -------(μatm)
	______________________________________________________________________________
	St. Lawrence	15	0	-9.61 1000 1022 300 45 2	0.7 2	575 8.11 3
	River
	Saguenay	12	0 -13.14	80	97 320 78 2	1.2 2	340 7.12 3
	River
	CIL	-0.3 32.13 -1.64	2183 2108 295 10.5 1.1	9.8 450 7.97
	LSLE	5.2 34.51 -0.28	2296 2165 63.5 52.2 2.4 22.8 1400 7.62
	Bottom water
	______________________________________________________________________________

Table 2 .

 2 Weight applied to each parameter used in the OMP analysis

	_____________________________________________________________________
			Weights used in OMP analysis
			Above the CIL	Below the CIL
	Parameters	Precision
	_____________________________________________________________________
	Temperature (°C)	0.01	1	25
	Practical salinity	0.01	25	25
	Dissolved O2 (µmol L -1 )	2.0	1	5
	Total alkalinity (µmol kg -1 )	2.0	25	25
	δ 18 O(H2O) (‰)	0.05	25	25
	Mass conservation		25	25
	____________________________________________________________________

Table

Can. J. Fish. Aquat. Sci. Downloaded from www.nrcresearchpress.com by GOTEBORGS UNIV on 09/20/17For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

Acknowledgements-

We wish to thank the Captain and crew of the Coriolis II for their help during a trying research cruise. We also wish to acknowledge the help of Gilles Desmeules and Sylvain Blondeau for their dedicated electronic and field sampling support as well as Dr. Michel Starr, Qiang Chen, Liliane St-Amand, Sonia Michaud, Hourssem Gaalloul, Jory Cabrol, Robin Bénard, Honghai Zhang for their help in field data acquisition. Most of the plots in this study were created with the ODV Software [Schlitzer, 2009]. This research was funded by a Team grant from the Fonds Québécois de recherche nature et technologies (FQRNT-Équipe-165335) and a Ship-Time Allocation grant from the Natural Sciences and Engineering Research Council of Canada (NSERC-STAC-436804-2013). Finally, we would like to thank the journal reviewers for their constructive comments.

Supplemental material -


Nicolas Peilleron

Arnaud Seigneurin

Caroline Herault

Camille Verry

Michel Bolla

Jean-Jacques Rambeaud

Jean-Luc Descotes

Jean-Alexandre Long

Gaelle Fiard
email: gfiard@chu-grenoble.fr

External evaluation of the Briganti nomogram to predict lymph node metastases in intermediate-risk prostate cancer patients

Keywords: Prostate cancer, lymph node dissection, nomogram, radical prostatectomy prostate cancer, nomogram, staging, lymph node involvement, radical prostatectomy

Purpose:

The Briganti nomogram can be used with a threshold of 5% to decide when to offer lymph node dissection during radical prostatectomy. The objective of the study was to assess the accuracy of the Briganti nomogram on intermediate-risk prostate cancer patients managed in a single academic department.

Methods:

We retrospectively reviewed the files of all patients managed by radical prostatectomy (RP) and bilateral pelvic lymph node dissection (BPLND) in our center between 2005 and 2017. The overall accuracy of the model in predicting metastatic lymph node disease was quantified by the construction of a receiver-operator characteristic (ROC) curve. A calibration plot was drawn to represent the relationship between the predicted and observed frequencies.

Results:

We included 285 patients, among whom 175 (61.4%) were classified as intermediate risk as defined by D'Amico. The median follow-up was 60 (34-93) months. Twenty-seven patients (9.5%) were diagnosed with lymph node metastases. The median number of lymph nodes removed was 10 (7-14). The mean Briganti score was 19.3% in patients with lymph node involvement (LNI) and 6.3% in patients without LNI. Focusing on intermediate-risk patients, 91(52%) and 84 (48%) had a Briganti score <5% and ³5%, respectively, among whom 6 (6.6%) and 7(8.3%) had lymph node metastases. The accuracy of the score was low for intermediate risk patients with an area under the curve (AUC) of 53.1% (95% CI 0.45-0.61).

Conclusion:

The Briganti nomogram in our retrospective cohort showed low accuracy for the prediction of lymph node involvement in an intermediate-risk prostate cancer population.

Introduction

Thanks to the widespread use of PSA screening, over 90% prostate cancer (PCa) cases are nowadays diagnosed at a localized stage. Among these patients, using the D'Amico classification, around 25% of the patients are in the intermediate-risk group [START_REF] Schröder | Screening and prostate cancer mortality: results of the European Randomised Study of Screening for Prostate Cancer (ERSPC) at 13 years of follow-up[END_REF].

Radical prostatectomy (RP) and external-beam radiation therapy (EBRT) combined with short-term androgen-deprivation therapy (ADT) are the two standard radical treatment options for this group of patients [START_REF] Bill-Axelson | Radical Prostatectomy or Watchful Waiting in Early Prostate Cancer[END_REF][START_REF] Jones | Radiotherapy and Short-Term Androgen Deprivation for Localized Prostate Cancer[END_REF]. Nowadays, when RP is chosen, the EAU guidelines recommend the performance of extended bilateral pelvic lymph node dissection (eBPLND) if the risk of lymph node metastases is greater than 5% [START_REF] Rozet | Recommandations en onco-urologie 2016-2018 du CCAFU : Cancer de la prostate[END_REF][START_REF] Mottet | EAU-ESTRO-SIOG Guidelines on Prostate Cancer. Part 1: Screening, Diagnosis, and Local Treatment with Curative Intent[END_REF].

The probability of lymph node involvement (LNI) can be estimated via the use of nomograms including that of Briganti and al [START_REF] Briganti | Validation of a Nomogram Predicting the Probability of Lymph Node Invasion among Patients Undergoing Radical Prostatectomy and an Extended Pelvic Lymphadenectomy[END_REF][START_REF] Briganti | Updated nomogram predicting lymph node invasion in patients with prostate cancer undergoing extended pelvic lymph node dissection: the essential importance of percentage of positive cores[END_REF]. It was initially developed and recently validated in high-risk patients to identify those who would benefit the most from RP or EBRT with short-term ADT based on their lymph node status [START_REF] Roumiguié | External validation of the Briganti nomogram to estimate the probability of specimen-confined disease in patients with high-risk prostate cancer[END_REF][START_REF] Briganti | Identifying the Best Candidate for Radical Prostatectomy Among Patients with High-Risk Prostate Cancer[END_REF].

Intermediate-risk patients are probably the most heterogeneous group. The lymph node metastases rate within this group may range from 3.7% to 20.1% [START_REF] Briganti | Updated nomogram predicting lymph node invasion in patients with prostate cancer undergoing extended pelvic lymph node dissection: the essential importance of percentage of positive cores[END_REF]. Although the eBPLND is the gold standard for nodal staging, it is associated with a significant morbidity rate up to 19.8% [START_REF] Heidenreich | Anatomical Extent of Pelvic Lymphadenectomy in Patients Undergoing Radical Prostatectomy[END_REF]. The use of nomograms therefore aims at selecting patients at a higher risk of LNI to reduce morbidity. However, nomograms are usually developed from a very selected cohort of patients and can prove less appropriate in different settings.

Our primary objective was to evaluate the accuracy of the Briganti score (SB) in predicting the risk of LNI in a cohort of intermediate-risk patients treated with RP and BPLND.

Our secondary objectives were to explore potential predictive factors of nomogram inaccuracy and lymph node metastases, to report the biochemical recurrence-free survival of patients with or without LNI, and to assess the consequences of the systematic application of the nomogram using various thresholds to our cohort. All patients had a preoperative pelvic MRI to assess lymph node metastatic status.

Material and Methods

Patients and methods

We

The Briganti score was calculated for all patients using the 2012 version. PSA dosage was obtained 4-6 weeks after surgery. Persistent detectable PSA was defined by a dosage > 0.2ng/ml. Open, laparoscopic and robot assisted (using the DaVinci Si system) laparoscopic RP were performed by senior and junior surgeons. A standard extended BPLND template was used including external, internal iliac up to the ureteral crossing, and obturator lymph nodes.

The learning curve of the supervising surgeon performing the robotic procedures started in 2014 and ended in 2016, based on procedure duration. Complications related to BPLND were noted and rated using the Clavien classification.

The study received institutional review board approval and was registered with the CNIL reference number: 2216943v0 (French National Agency regulating Data Protection).

Follow-up

All patients were reviewed in outpatient clinic 6 weeks after surgery with a first PSA dosage. In case of undetectable PSA, new appointments were scheduled every 6 months during the first 2 years of follow-up, then on a yearly basis up to 5 years, with PSA dosage still performed on a 6-monthly basis. Patients were then usually referred back to their GP with a recommendation of annual PSA testing provided that the PSA had remained undetectable. Biochemical recurrence (BCR) was defined by a PSA level > 0.2 ng/ml confirmed on 2 successive postoperative dosages. The threshold was set at 0.4 ng/ml when adjuvant radiotherapy was performed.

Patients with LNI and undetectable post-operative PSA were given the option of surveillance and early salvage treatment in case of rising PSA values with salvage radiotherapy and concomitant androgen deprivation therapy. In case of detectable post-operative PSA, adjuvant radiotherapy with a dose of 66 Gy and optional concomitant androgen deprivation therapy was offered. All cases were discussed in a dedicated multidisciplinary meeting.

Statistical analysis

Continuous variables were reported using median values with interquartile ranges (IQR) and compared with the Kruskal-Wallis test. Categorical variables were reported as counts and proportions (%) and compared using the chi-2 test or Fisher's exact test, as appropriate.

The accuracy of the Briganti score was evaluated using Receiver Operating Characteristics (ROC) curves, analyzed by calculating the Area Under Curve (AUC). The AUC tells us about the discrimination of the model, i.e. its ability to distinguish patients with LNI and those without LNI. We also used a calibration curve, representing the relation between the predicted frequencies on the x-axis and the observed frequencies on the y-axis. Calibration is the concordance between the predictions and the observations of the model. If the model predicts perfectly, all points must be on a 45° line. Each point corresponds to a group of patients for which the observed proportion of LNI and the probability predicted by the model were calculated.

We performed two multivariate analyses, using logistic regression, for estimating adjusted Odds Ratio on potential predictive factors of lymph node metastases and Briganti score inaccuracy.

BCR-free survival rates were calculated from the date of surgery, which was the date of origin, to the date of diagnosis of BCR or death or last follow-up. Survival curves were performed according to the Kaplan-Meier method, and comparison between groups using the log-rank test.

All reported p-values were two-sided. Significance level was defined at 0,05 for all tests. The Bonferroni correction was applied for the multiple tests performed to limit the risk of alpha risk inflation. In our study 19 tests were performed, which explains a significance level set at 0.002632. Statistical analyses were performed with STATA® 16.0 and R® 3.6.1 softwares.

Results

Population and surgery outcomes

Between 2005 and 2017, 285 patients were treated by RP and BPLND for localized prostate cancer in our center. Among them, 175 patients were in the intermediate-risk group.

Patient characteristics are detailed in Table 1.

Lymph node involvement was present among 7.4% (N=13) of intermediate-risk patients and 9.5% (N=27) of all patients.

The rate of positive surgical margins was 36.6% (N=64) in the intermediate group and 38.6% (N=110) for the entire cohort.

After a median follow-up of 60 (34-93) months, the rate of BCR was 30.9% (N=88) and was higher in the group with LNI (66.7%(N=18) vs 27.1% (N=58); p<0.001). The rate of complications related to BPLND was 10.9% (N=19) in the intermediate group and 9.1% (N=26) in the entire cohort. Among these 4.6% (N=13) were rated Clavien 3

or greater (lymphocele requiring drainage or surgery n= 9; ureteral injury n= 2; iliac injury with hemorrhagic shock n= 1; iliac thrombosis with massive pulmonary embolism and death n=1).

Accuracy of the Briganti score

ROC curve analysis (Figure 1 The proportion of high-risk patients was greater in the group with LNI (51.8%(N=14) vs 20.2%(N=52); p<0.001). The proportion of cT3 was higher in the group with LNI (11.1%(N=3) vs 0.8%(N=2); p<0.001). The rate of positive Briganti Score and the average Briganti Score were greater in the LNI group (77.8%(N=21) and 19.3% vs 47.7%(N=123) and 6.3%; p<0.001).

The distribution of pathological stages was significantly different between patients with and without LNI (pT2: 11.1%(N=3) vs 62.4%(N=161); pT3: 85.2%(N=23) vs 36.8%(N=95); pT4: 3.7%(N=1) vs 0.4%(N=1); p<0.001). The number of nodes removed was not statistically different between the two groups.

The proportion of adjuvant treatment was greater in the group of LNI patients (37%(N=10) vs 10.1%(N=26); p<0.001).

Using multivariate logistic analysis, the PSA level (OR 4.9, 95% CI 2.1-11.8), percentage of positives biopsies (OR 4.7, 95% CI 1.4-16) and T3 stage on MRI (OR 2.4, 95% CI 1-5.8) were independent predictive factors of LNI (Table 2). However, none of the factors studied proved predictive of Briganti score inaccuracy (Table 3).

Biochemical recurrence-free survival

5-year BCR-free survival rates were 74% (IC95%: 66%-81%) for intermediate-risk patients, and 70% (IC95%: 64%-76%) for the entire cohort. Figure 3 represents Kaplan-Meier curves for the BCR-free survival depending on LNI status, respectively for intermediate-risk patients (Figure 3a) and all patients (Figure 3b). BCR-free survival rate at 5 years was significantly lower for patients with LNI than those without LNI, for the intermediate-risk group as well as for the entire cohort (Intermediate-risk patients: 37%(IC95%: 13%-62%) vs 77%(IC95%: 69%-84%), all patients: 33%(IC95%: 15%-22%) vs 75%(IC95%: 68%-80%) ; Log rank test, p<0.05). Briganti score < 5%. 6.6%(N=6) of those patients had a LNI (Figure 4a). 141 (49.5%) patients had a negative Briganti Score and among them 4.3%(N=6) had a LNI (Figure 4b).

Among these patients with avoidable BPLND, the rate of complications ³ Clavien 3

(including 1 death) was respectively 2.1%(N=6) and 2.3%(N=4).

In order to precise the clinical relevance of various thresholds, we analyzed the consequences of the application of a different threshold on the number of BPLNDs to perform in order to detect 1 LNI.

Discussion

In this study, we report the results of its application on a separate cohort of 285 patients with localized prostate cancer including 175 intermediate-risk patients. The Briganti nomogram has previously been tested among patients of high risk group with an AUC between 65% and 72% [START_REF] Roumiguié | External validation of the Briganti nomogram to estimate the probability of specimen-confined disease in patients with high-risk prostate cancer[END_REF][START_REF] Briganti | Identifying the Best Candidate for Radical Prostatectomy Among Patients with High-Risk Prostate Cancer[END_REF][START_REF] Mearini | External validation of nomogram predicting the probability of specimen-confined disease (pT2-3a, R0N0) in patients undergoing radical prostatectomy and pelvic lymph node dissection[END_REF][START_REF] Hansen | Assessing the clinical benefit of a nomogram to predict specimen-confined disease at radical prostatectomy in patients with high-risk prostate cancer: An external validation[END_REF]. It has also been validated on cohorts of D'Amico all risk patients with an AUC between 79% and 83% [START_REF] Dell'oglio | External validation of the European association of urology recommendations for pelvic lymph node dissection in patients treated with robot-assisted radical prostatectomy[END_REF][START_REF] Gacci | External validation of the updated nomogram predicting lymph node invasion in patients with prostate cancer undergoing extended pelvic lymph node dissection[END_REF][START_REF] Hansen | External validation of the updated Briganti nomogram to predict lymph node invasion in prostate cancer patients undergoing extended lymph node dissection[END_REF]. Nevertheless, Roumiguie and al [START_REF] Roumiguié | External validation of the Briganti nomogram to estimate the probability of specimen-confined disease in patients with high-risk prostate cancer[END_REF] have more recently expressed reservations about the disappointing results they obtained with their group of intermediate-risk patients.

The accuracy of the Briganti score proved relevant for our entire cohort but insufficiently accurate for the population of interest, i.e. intermediate-risk patients, with respective AUC equal to 75.2% and 53.1%. For comparison, Briganti et al. reported an AUC equal to 76.2% [START_REF] Briganti | Validation of a Nomogram Predicting the Probability of Lymph Node Invasion among Patients Undergoing Radical Prostatectomy and an Extended Pelvic Lymphadenectomy[END_REF] and 87.6% [START_REF] Briganti | Updated nomogram predicting lymph node invasion in patients with prostate cancer undergoing extended pelvic lymph node dissection: the essential importance of percentage of positive cores[END_REF] in their initial validation cohort. In our cohort composed mainly of intermediate-risk patients, median PSA level, proportion of clinical T3 and percentage of positive biopsies were close to those of the internal validation cohort of Briganti (respectively 7.8 ng/ml vs 6.3 ng/ml, 1.8% vs 5.3%, and 36.1% vs 35.5%) [START_REF] Briganti | Updated nomogram predicting lymph node invasion in patients with prostate cancer undergoing extended pelvic lymph node dissection: the essential importance of percentage of positive cores[END_REF]. Of note, primary and secondary Gleason pattern >=4 was more frequently in our study (respectively 37.5% and 47.3% vs 17% and 31%). The Briganti nomogram was validated using a cohort of patients diagnosed with extended biopsy protocols and BPLND. Indeed the number of total biopsy cores taken and lymph nodes retrieved in our study were lower (respectively 10 vs 19 and 12 vs 17) [START_REF] Briganti | Updated nomogram predicting lymph node invasion in patients with prostate cancer undergoing extended pelvic lymph node dissection: the essential importance of percentage of positive cores[END_REF].

We acknowledge that the robot-assisted laparoscopic approach appeared, in this study, to retrieve a median number of lymph nodes inferior to the open approach, 9 [START_REF] Briganti | Updated nomogram predicting lymph node invasion in patients with prostate cancer undergoing extended pelvic lymph node dissection: the essential importance of percentage of positive cores[END_REF][START_REF] Roumiguié | External validation of the Briganti nomogram to estimate the probability of specimen-confined disease in patients with high-risk prostate cancer[END_REF][START_REF] Briganti | Identifying the Best Candidate for Radical Prostatectomy Among Patients with High-Risk Prostate Cancer[END_REF][START_REF] Heidenreich | Anatomical Extent of Pelvic Lymphadenectomy in Patients Undergoing Radical Prostatectomy[END_REF][START_REF] Mearini | External validation of nomogram predicting the probability of specimen-confined disease (pT2-3a, R0N0) in patients undergoing radical prostatectomy and pelvic lymph node dissection[END_REF][START_REF] Hansen | Assessing the clinical benefit of a nomogram to predict specimen-confined disease at radical prostatectomy in patients with high-risk prostate cancer: An external validation[END_REF] versus 11 [START_REF] Roumiguié | External validation of the Briganti nomogram to estimate the probability of specimen-confined disease in patients with high-risk prostate cancer[END_REF][START_REF] Briganti | Identifying the Best Candidate for Radical Prostatectomy Among Patients with High-Risk Prostate Cancer[END_REF][START_REF] Heidenreich | Anatomical Extent of Pelvic Lymphadenectomy in Patients Undergoing Radical Prostatectomy[END_REF][START_REF] Mearini | External validation of nomogram predicting the probability of specimen-confined disease (pT2-3a, R0N0) in patients undergoing radical prostatectomy and pelvic lymph node dissection[END_REF][START_REF] Hansen | Assessing the clinical benefit of a nomogram to predict specimen-confined disease at radical prostatectomy in patients with high-risk prostate cancer: An external validation[END_REF][START_REF] Dell'oglio | External validation of the European association of urology recommendations for pelvic lymph node dissection in patients treated with robot-assisted radical prostatectomy[END_REF][START_REF] Gacci | External validation of the updated nomogram predicting lymph node invasion in patients with prostate cancer undergoing extended pelvic lymph node dissection[END_REF][START_REF] Hansen | External validation of the updated Briganti nomogram to predict lymph node invasion in prostate cancer patients undergoing extended lymph node dissection[END_REF][START_REF] Gandaglia | Comparative Effectiveness of Robot-Assisted and Open Radical Prostatectomy in the Postdissemination Era[END_REF]. These results are similar to other series of robotic BPLND [START_REF] Dell'oglio | External validation of the European association of urology recommendations for pelvic lymph node dissection in patients treated with robot-assisted radical prostatectomy[END_REF][START_REF] Gandaglia | Comparative Effectiveness of Robot-Assisted and Open Radical Prostatectomy in the Postdissemination Era[END_REF]. This could be explained by learning curve of the supervising surgeon, and the involvement of junior surgeons, as outlined by a relatively high positive surgical margin rate. The lymph node retrieval technique and conditioning (separated into several flasks or monobloc) could also have influenced the number of nodes. Finally, the lymph node enumeration by the pathologist is subject to great inter-individual variability [START_REF] Sherbeck | High Variability in Lymph Node Counts Among an International Cohort of Pathologists: Questioning the Scientific Validity of Node Counts[END_REF].

The morbidity rate of BPLND in this study was 9.1%, lower than expected from previous publications. Indeed, series of eBPLND report complications rates varying from 10.5% to 19.8% [START_REF] Heidenreich | Anatomical Extent of Pelvic Lymphadenectomy in Patients Undergoing Radical Prostatectomy[END_REF]. A smaller number of nodes removed, consistent with a less-extensive dissection could be an explanation, although under-reporting of complications is also likely in this retrospective study. An attempt was made to evaluate the morbidity avoided if the score had been applied pre-operatively. For the entire cohort, 49.5%(N=141) of BPLND would have been omitted, preventing 2.1%(N=6) of complications ³ Clavien 3, including one death. For the intermediaterisk group, 52%(N=91) of avoidable BPLND and 2.3%(N=4) of complications ³ Clavien 3, including 1 death, would have been prevented.

The PSA level and the percentage of positive biopsies were identified as predictors of LNI in our study. These results confirm those of the literature [START_REF] Briganti | Updated nomogram predicting lymph node invasion in patients with prostate cancer undergoing extended pelvic lymph node dissection: the essential importance of percentage of positive cores[END_REF][START_REF] Roumiguié | External validation of the Briganti nomogram to estimate the probability of specimen-confined disease in patients with high-risk prostate cancer[END_REF][START_REF] Heidenreich | Percentage of positive biopsies predicts lymph node involvement in men with low-risk prostate cancer undergoing radical prostatectomy and extended pelvic lymphadenectomy[END_REF]. The percentage of positive biopsies also seems to be one of the key elements. Absent from the first nomogram in 2006, it was added in the calculation of the score in the updated version of 2012 based on the results of Heidenreich and al. [START_REF] Briganti | Updated nomogram predicting lymph node invasion in patients with prostate cancer undergoing extended pelvic lymph node dissection: the essential importance of percentage of positive cores[END_REF][START_REF] Heidenreich | Percentage of positive biopsies predicts lymph node involvement in men with low-risk prostate cancer undergoing radical prostatectomy and extended pelvic lymphadenectomy[END_REF]. Although not significant in our study probably due to lack of power explaining a wider confidence interval, the presence of T3 disease on MRI seems an interesting prognostic factor.

We report a BCR rate of 30.9% in this series mostly composed of intermediate and high-risk patients, probably linked with a rate of positive surgical margins on the high side of reported rates. Despite this, the 5-year BCR-free survival rates of our entire cohort and intermediate-risk, respectively equal to 70% and 74% were comparable to those of literature, probably thanks to prompt adjuvant or salvage treatment [START_REF] Beauval | External validation of a nomogram for identification of pathologically favorable disease in intermediate risk prostate cancer patients[END_REF][START_REF] D'amico | Biochemical outcome after radical prostatectomy or external beam radiation therapy for patients with clinically localized prostate carcinoma in the prostate specific antigen era[END_REF].

The inaccuracy of the nomogram in our cohort was in part due to failure to detect patients with LNI, but also because many patients with a Briganti score above 5% did not happen to have LNI. With a cut-off set at 5%, half of intermediate-risk patients (N=91) would have been spared BPLND and 6 LNI (6.6%) would have been missed. Increasing the threshold to 6% or 7% would have resulted in a higher number of patients avoiding BPLND (N=110 and 123, respectively), with a good negative predictive value over 91% for this rare event, but looking at the number of BPLNDs needed to detect 1 LNI, the threshold of 5% proved more clinically relevant.

We must acknowledge many limitations of the present study. Its unicentric and retrospective character obviously limits the generalizability of its results. The long inclusion time over 12 years and a small number of patients included per year contributed to a lack of power, especially for subgroup analyses. We chose to report only BPLND-related complications as we believe these could have been prevented in patients with a low Briganti score, although it leads to an underestimation of the overall complication rate of the procedure. The relatively low median number of nodes retrieved, in part explained by the learning curves of the laparoscopic, and subsequently robotic techniques, could contribute to the lack of accuracy of the nomogram in this cohort, even though the proportion of patients with LNI seems in keeping with previously published studies [START_REF] Dell'oglio | External validation of the European association of urology recommendations for pelvic lymph node dissection in patients treated with robot-assisted radical prostatectomy[END_REF][START_REF] Gacci | External validation of the updated nomogram predicting lymph node invasion in patients with prostate cancer undergoing extended pelvic lymph node dissection[END_REF][START_REF] Hansen | External validation of the updated Briganti nomogram to predict lymph node invasion in prostate cancer patients undergoing extended lymph node dissection[END_REF]. This limitation had been identified during the setting-up phase of the study and we tried to mitigate it by providing the BCR-free survival curves for both cohorts with and without LNI identified at BPLND. The different survival profiles proved in favor of a limited contamination of the N0 group. The use of a nomogram developed from a cohort including all risk categories on a subset of patients at intermediate risk could also explain the low accuracy obtained in this study. However, since BPLND is not recommended for low-risk patients, and should be performed for high-risk patients, it is toward this population that efforts to develop and validate prediction tools should be directed.

Conclusion

The

  retrospectively collected perioperative and follow-up data of consecutive patients treated by RP and BPLND for localized prostate cancer in our center between 2005 and 2017. Preoperative data included PSA level, clinical stage, Gleason score and percentage of positive biopsies. The clinical stage was determined according to the cTNM 2002 classification updated in 2010. Prostate biopsies were performed according to the currently recommended regimen of 12 randomized, ultrasound-guided biopsies, with the use since 2011 of an MRIultrasound fusion system (Koelis, France) allowing the realization of targeted biopsies in addition. The Gleason score was determined on biopsies and the prostatectomy specimen by pathologists specialized in urology, according to the grading system defined by Gleason in 1966 and reviewed by the ISUP (International Society of Urological Pathology) in 2005 and 2014. Pathologists reported the pathological stage with reference to the 2002 pTNM classification updated in 2009.

)

  respectively showed low and fairly relevant accuracy of the score for intermediate risk patients (AUC 53.1%, 95% CI 0.45-0.61) (Fig 1a) and all patients (AUC 75.2%, 95% CI 0.7-0.8) (Fig 1b), respectively. The calibration plot revealed a good correlation between the predicted probabilities of the score on the x axis and the observed proportion of LNI on the y axis across the 45° slope (Figure 2).

Figure 1 .Figure 2 .

 12 Figure 1. ROC and area under curve

Figure 3 .Figure 4

 34 Figure 3. Comparative survival curves between patients with and without lymph node invasion for intermediate-risk patients (3a. N=175) and the entire cohort (3b. N=285) at 5 years

Figure 4 .

 4 Figure 4. Results of Briganti Score application

Table 1 . Patient characteristics

 1

	Intermediate-risk	All patients
	patients (N=175)	(N=285)

Table 2 . Predictive factors of lymph node metastases

 2

		OR (IC95%)
		Univariate	Multivariate	p value
		analysis	analysis
	PSA
	<10	1	1
	≥10	4.1 (1.8-9.5)	4.9 (2.1-11.8)	<0.0001*
	Clinical stage
	T1	1	-
	T2	1.4 (0.6-3.2)	-
	T3	19.2 (2.8-129.9)	-
	Gleason score
	5-6	1	-
	7	2.4 (0.8-7.2)	-
	8-9	2.3 (0.6-10.0)	-
	Percentage of positives
	biopsies
	<33%	1	1
	≥33% -<66%	3.1 (1.1-8.3)	3.6 (1.3-10.4)	0.016*
	≥66%	5.2 (1.6-16.7)	4.7 (1.4-16.0)	0.013*
	T3 stage on MRI
	No	1	1
	Yes	2.9 (1.3-6.6)	2.4 (1.0-5.8)	0.046
	N nodes removed
	1-4	1	-
	5-9	0.8 (0.2-3.2)	-
	10-19	1.2 (0.3-4.3)	-
	≥20	0.8 (0.1-5.0)	-

Table 3 . Potential predictive factors of Briganti score inaccuracy Adjusted OR IC 95% p value T3 stage on MRI

 3

	No

Table 4 . Performance of the Briganti score according to the threshold chosen to discriminate patients with and without LNI -intermediate-risk patients

 4 The results summarizing the interest of the Briganti score according to the cut-off value used are shown in Table4. With a threshold set at 4%/5%, we would need to perform a total of 108/84 BPLNDs and detect 9/7 patients with LNI, resulting in 12 BPLNDs needed to detect 1 LNI while missing 4 patients with LNI and a Briganti score under the threshold. Although reducing the overall number of BPLNDs performed, by increasing the threshold up to 7%, the number of BPLNDs to perform to detect 1 patient with LNI would be 26, while missing 11 patients with LNI and a score under the threshold.

	Threshold	BPLND not recommended	BPLND recommended	NPV % (95%CI)
	(%)	(below threshold) N(%)	(above threshold) N(%)
		All	LNI -	LNI+	All	LNI -	LNI +
	4	67(38.3%)	63(94.0%)	4(6.0%)	108(61.7%)	99(91.7%) 9(8.3%)	94.0% (85.4 -98.3)
	5	91(52.0%)	85(93.4%)	6(6.6%)	84(48.0%)	77(91.7%) 7(8.3%)	93.4% (86.2 -97.5)
	6	110(62.9%)	101(91.8%) 9(8.2%)	65(37.1%)	61(93.8%) 4(6.2%)	91.8% (85.0 -96.2)
	7	123(70.3%)	112(91.1%) 11(8.9%) 52(29.7%)	50(96.2%) 2(3.8%)	91.1% (84.6 -95.5)

  Briganti nomogram applied to this cohort was not sufficiently accurate in predicting the risk of lymph node involvement in an intermediate-risk population. Still, its application before deciding to perform BPLND among intermediate-risk patients would have avoided 91 procedures (52%) and failed to detect only 6 cases (6.6%) of lymph node metastases. More precise tools are needed to precisely assess the LNI risk before surgery. The new version of a nomogram taking into account multiparametric MRI results could improve the accuracy and will also have to be validated.

Acknowledgments

Gaelle Fiard receives funding from and wishes to thank the Fondation de France and the European Urology Scholarship Program.

Availability of data and material

De-identified data will be made readily available to reviewers upon request

Code availability

Statistical software code will be made readily available to reviewers upon request

Declarations