domain: sde chim

text:
Fidji Sandre

Nina Huynh

Emilie Caupos

Lamyae El-Mrabet

Chandirane Partibane

Isabelle Lachaise

Christophe Pommier

Michael Rivard

Christophe Morin

Régis Moilleron

Julien Le Roux

Laure Garrigue-Antar
email: laure.garrigue-antar@u-pec.fr

Occurrence and fate of an emerging drug pollutant and its by-products during conventional and advanced wastewater treatment: Case study of furosemide

Keywords: Pharmaceutical, wastewater, residential care home, degradation products, oxidation

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

Introduction

In the context of a wide consumption of medicines in high income and emerging countries, increasing demographical urban pressure and population aging, hundreds of active substances used for therapeutic purposes and marketed worldwide end up in the wastewater system. Conventional wastewater treatment plant (WWTP) processes based on filtration, sand and grease removal, decantation and biological or physico-chemical treatments are not designed for the elimination of pharmaceutical compounds, so their removal can vary drastically depending on the molecule (e.g., its hydrophobicity) [START_REF] Chiffre | Occurrence of pharmaceuticals in WWTP effluents and their impact in a karstic rural catchment of Eastern France[END_REF]. The active substances of these drugs are discharged with WWTP effluents at concentrations ranging from ng.L -1 to µg.L -1 and end up in the aquatic environment, representing a risk for organisms [START_REF] Cizmas | Pharmaceuticals and personal care products in waters: occurrence, toxicity, and risk[END_REF], Obinwanne Okoye et al. 2022). To overcome this problem, the implementation of tertiary or quaternary treatments after conventional treatments is increasingly studied, and several countries (e.g., Switzerland, Germany) already regulate the removal of organic micropollutants from wastewater. The optimization of biological treatments, for example with the addition of fungus (Trametes versicolor) in bioreactors (Cruz-Morato et al. 2014;[START_REF] Badia-Fabregat | Identification of some factors affecting pharmaceutical active compounds (PhACs) removal in real wastewater. Case study of fungal treatment of reverse osmosis concentrate[END_REF], can be effective on potentially biodegradable compounds. However, the most frequent processes used to achieve a satisfactory removal of micropollutants include adsorption on activated carbon [START_REF] Guillossou | Organic micropollutants in a large wastewater treatment plant: What are the benefits of an advanced treatment by activated carbon adsorption in comparison to conventional treatment?[END_REF], membrane processes and oxidation processes such as ozonation [START_REF] Guillossou | Benefits of ozonation before activated carbon adsorption for the removal of organic micropollutants from wastewater effluents[END_REF].

Ozone has been used for a long time as both an oxidant and a disinfectant for drinking water production, and is increasingly applied for the treatment of wastewater effluents (Lim et al. 2022). It is a very powerful oxidant that can react directly with organic molecules, mainly by electrophilic reaction, and also indirectly by the production of radicals initiated by hydroxyl or hydroperoxide ions in the aqueous medium [START_REF] Guo | The Application and Reaction Mechanism of Catalytic Ozonation in Water Treatment[END_REF]. Ozonation has been shown to be effective in the degradation of hormones, antibiotics, antivirals and pesticides (Lim et al. 2022). Advanced oxidation processes (AOPs) are also very efficient for the degradation of organic molecules (Miklos et al. 2018). These processes are based on the production of highly reactive hydroxyl radicals from one or more primary oxidants (e.g., chlorine, ozone, hydrogen peroxide, etc.) [START_REF] Sarathy | An Overview of UV-based Advanced Oxidation Processes for Drinking Water Treatment[END_REF] or by photocatalysis [START_REF] Prieto-Rodriguez | Treatment of emerging contaminants in wastewater treatment plants (WWTP) effluents by solar photocatalysis using low TiO2 concentrations[END_REF]). Advanced oxidation processes in which Ultraviolet (UV) radiation is combined with ozone, chlorine, peroxodisulfate or hydrogen peroxide are efficient to reach extensive degradation. The most common combination is with H2O2 because it is much less energy-consuming and expensive than with O3, and it is also less sensitive to matrix changes and dissolved organic matter than sulfates. While advanced treatment processes for organic micropollutants are not widely used, many WWTP use a disinfection step before discharging effluents into the environment (e.g., in bathing areas). Another process commonly used for disinfection and inactivation of pathogenic microorganisms is based on UV irradiation [START_REF] Hijnen | Inactivation credit of UV radiation for viruses, bacteria and protozoan (oo)cysts in water: A review[END_REF]. Direct UV photolysis with UV-C (100-280 nm) shows good efficiency on pesticides [START_REF] Sanches | Drinking water treatment of priority pesticides using low pressure UV photolysis and advanced oxidation processes[END_REF]) but some pharmaceutical compounds like carbamazepine are more resistant to it [START_REF] Pereira | Evaluation of UV irradiation for photolytic and oxidative degradation of pharmaceutical compounds in water[END_REF]).

Although oxidation treatments can be quite efficient for the degradation of micropollutants, they often form oxidation by-products. Transformation products (e.g., metabolites) of pharmaceuticals can also be already present in the incoming water or be formed during conventional biological processes. The formation of oxidation by-products depends on the mode of oxidation, the medium (especially the presence of nitrogen, halogens, organic matter) and the type of radical produced in the case of advanced oxidation (Miklos et al. 2018). These degradation by-products present a hazard for health [START_REF] Stalter | Fingerprinting the reactive toxicity pathways of 50 drinking water disinfection by-products[END_REF]) and for the environment [START_REF] Wang | Potential risks from UV/H2O2 oxidation and UV photocatalysis: A review of toxic, assimilable, and sensory-unpleasant transformation products[END_REF]. Apart from the regulated disinfection by-products (e.g., trihalomethanes and haloacetic acids), hundreds of chlorination by-products have been identified [START_REF] Zhang | Study on Transformation of Natural Organic Matter in Source Water during Chlorination and Its Chlorinated Products using Ultrahigh Resolution Mass Spectrometry[END_REF] from the reaction of chlorine with organic matter and their potential toxicity can be a threat to human health [START_REF] Stefán | Formation of chlorination by-products in drinking water treatment plants using breakpoint chlorination[END_REF], Mazhar et al. 2020). Many products are also generated during UV-H2O2 oxidations depending on the dose of oxidant, the UV fluence and the pH of the medium. Oxidation by-products are often more toxic than the parent compounds on algae, daphnia, and medaka fish [START_REF] Wang | Potential risks from UV/H2O2 oxidation and UV photocatalysis: A review of toxic, assimilable, and sensory-unpleasant transformation products[END_REF], it is thus of critical importance to assess the toxicity of degradation products to fully establish the potential impacts of organic micropollutants on the environment.

To address the questions related to the presence of pharmaceuticals in the environment, we selected furosemide from a list of high-risk emerging pollutants [START_REF] Besse | Human pharmaceuticals in surface waters[END_REF] and subjected this drug to a predictive study aiming to anticipate the fate of organic contaminants (Laurence et al, 2011(Laurence et al, , 2014;;[START_REF] Olvera-Vargas | Microbial biotransformation of furosemide for environmental risk assessment: identification of metabolites and toxicological evaluation[END_REF]. Classified by the World Health Organization as an essential medicine, furosemide is a loop diuretic prescribed all over the world for hypertension and heart, liver, and kidney failure (Abbot and Kovacic 2008). Mostly eliminated unmodified by humans and moderately degraded in conventional treatment plants (between 8 and 54% in [START_REF] Castiglioni | Removal of Pharmaceuticals in Sewage Treatment Plants in Italy[END_REF], >65% in Matamoros et al. 2009, 74% in Kasprzyk-Hordern et al. 2009, or between 30-80% according to the WWTP considered in [START_REF] Ferreira Da Silva | Occurrence and distribution of pharmaceuticals in surface water, suspended solids and sediments of the Ebro river basin, Spain[END_REF], furosemide is also widely found in the environment from several tens to few thousand of ng.L -1 in rivers [START_REF] Banjac | Emission factor estimation of ca. 160 emerging organic microcontaminants by inverse modeling in a Mediterranean river basin (Llobregat, NE Spain)[END_REF][START_REF] Baken | Toxicological risk assessment and prioritization of drinking water relevant contaminants of emerging concern[END_REF][START_REF] Munro | Evaluation of combined sewer overflow impacts on short-term pharmaceutical and illicit drug occurrence in a heavily urbanised tidal river catchment (London, UK)[END_REF][START_REF] White | Tracking changes in the occurrence and source of pharmaceuticals within the River Thames, UK; from source to sea[END_REF][START_REF] Cantwell | Spatial patterns of pharmaceuticals and wastewater tracers in the Hudson River Estuary[END_REF]. Applied to furosemide, our predictive study confirmed that saluamine and furfural are two transformation products (TPs) of furosemide (Laurencé et al. 2014). More importantly, it also identified the pyridinium of furosemide as a TP and a human metabolite of this drug (Laurencé et al. 2019). Whereas furfural presents moderate acute toxicity in freshwater invertebrates (LC50 = 13 mg.L -1 for 72h exposure in [START_REF] Reed | Furfural[END_REF], saluamine shows acute toxicity in rats [START_REF] Al-Omar | Toxicity Studies on 4-Chloro-5-Sulfamoylanthranilic Acid the Degradation Product of a Loop Diuretic Furosemide[END_REF]). We also previously demonstrated that pyridinium of furosemide exhibits neurodegenerative properties in mice (Laurencé et al. 2019). Therefore, the fate of furosemide and its TPs in wastewater and in the environment needs to be assessed to fully characterize the potential impacts of this pharmaceutical on the aquatic ecosystems. Furosemide is also an interesting and highly relevant model to study the fate of pharmaceuticals along with their TPs. Especially, the production of saluamine and pyridinium of furosemide as well as their potential degradation during conventional and advanced wastewater treatment remains unknown to date. The purpose of this work was thus i) to assess the occurrence and removal of furosemide and its known TPs in a large WWTP and in wastewater from an elderly care facility, ii) to study their removal by several advanced oxidation processes (ozonation, UV/H2O2) and iii) to identify the subsequent formation of potential new TPs following these treatments.

Material & methods

Reagents

The furosemide-d5 standard was purchased from Cluzeau Info Labo. Internal standards (atenolol-d7 and sulfamethoxazole-d4) and saluamine (SAL) were purchased from Sigma-Aldrich. For the extraction and analysis, MS grade methanol was purchased from Fisher. Ultrapure water was produced by a Milli-Q® IQ 7000 ultrapure water system. Furosemide (FUR) was purchased from Sigma. Pyridinium of furosemide (PYR) was synthesized at ICMPE as previously described (Laurencé et al. 2011), and its structure has been confirmed by NMR with over 96% purity. Hydrogen peroxide 50% was purchased from CarlRoth.

Sampling sites

To assess the WWP efficiency on furosemide degradation, raw water (RW) and treated effluent water (TW) were collected on 2021-12-07 and 2021-12-08 from the Seine-Centre WWTP (Colombes, France), operated by the Paris public sanitation service (SIAAP) and treating wastewater of about 1,000,000 people equivalent with a flow of 240,000 m 3 /day. The Seine-Centre WWTP is mostly based on biofiltration stages ensuring the treatment of organic carbon as well as nitrification and denitrification processes. For the oxidation tests on raw sewerage water samples, wastewater from residential care homes (RCH) was chosen because of its presumably high concentration of furosemide, thus allowing a better identification of the possible degradation products. Three wastewater outlets of health care institutions were collected in Dordogne, France. The first sample (RCH1) was collected on 2021-05-26 in a RCH of 10,000 m 2 located in Bergerac, in an urban area. It has a capacity of 90 beds plus 15 beds for Alzheimer's patients, 10 places in daycare centers and 5 emergency beds. The sample was taken from a lift station which only collects water from the facility. The second sample (RCH2) was collected on 2021-06-09 from a reeducation center located in a national forest at Antonne-et-Trigonant with 120 places and 40 beds for senior residents. The third sample (RCH3) was collected on 2021-06-08 from a RCH at Lolme with 90 beds and 5 daycare places. Several physico-chemical parameters were recorded: pH, Dissolved Organic Carbon (DOC), Chemical Oxygen Demand (COD) and Biological Oxygen Demand at 5 days (DBO5), Suspended Matter (SM), Total Kjeldahl Nitrogen (TKN), ammoniacal nitrogen and organic nitrogen (table 1). The RCH outlet water samples were filtered on GF/D filters (2.7 µm pore size) and then on GF/F filters (0.7 µm pore size). pH and DOC were measured after filtration (table 1). Particulate phase samples (i.e., particles collected on filters) were stored at -20 °C and freeze-dried until treatment for analysis. For RCH samples, it is interesting to note the large gap between the Kjeldahl nitrogen and ammonia nitrogen values, which implies a high proportion of organic nitrogen, reflecting the short residence time of the effluent and confirming the relatively fresh effluent sampling

Oxidation assays

Furosemide degradation kinetics experiments were performed in ultrapure water spiked with 1 mg.L - 1 of furosemide. UV/H2O2 and ozonation were applied at high doses or exposure as described below, and chlorination was also conducted at high dose (35 mgCl2.L -1 ) to maximize the formation of degradation products (see Text S1). Oxidation processes were also applied on 200 mL filtered wastewater samples from RCH facilities.

Photodegradation under UV light with H2O2. The sample was placed in a beaker, under a UV lamp at 254 nm (UV-C ; 0.34 mW/cm²) with stirring. To establish degradation kinetics of furosemide in ultrapure water, 1 mg.L -1 of H2O2 was added and aliquots of 1 mL were taken at 2,5,10,15,20,30,45,60,90,120,210 and 600 minutes. For the oxidation of wastewater from RCH, the H2O2 dose was determined according to the DOC value by using 0.375 mg of H2O2 per mg of DOC, and the total reaction time under UV irradiation was 5 hours.

Ozonation. Ozone was produced by a generator with an external source (O2 gas cylinder). The bottle containing the sample to be oxidized was immersed in an ice bath throughout the experiment. For the ozonation of furosemide solutions in ultrapure water, a concentrated ozone solution was first produced (33 mg/L O3) and diluted in the furosemide samples at the required ozone concentrations, determined according to the DOC concentration of the sample (Equation 1).

[𝑂 3 ](𝑚𝑔𝑂 3 . 𝐿 -1 ) = 2 𝑥 𝐷𝑂𝐶 (𝑚𝑔𝐶. 𝐿 -1 ) (Eq. 1)

Aliquots of 1 mL were collected at 1, 2, 4, 5, 7, 10, 15, 20, 30, 45 and 60 minutes. For the oxidation of RCH wastewater, ozone was bubbled directly into the samples for 1.5 hours with an O2 flow to the ozonator of 2 L.h -1 . The residual O3 was measured by colorimetry with indigo carmine using a double beam spectrometer (UV6300 PC, VWR) at 600 nm.

Analytical procedures for quantification

After filtration, the residential care home samples are split into several aliquots of which 5 are spiked.

Furosemide and its TPs present in dissolved phases of wastewater samples were extracted through an automated extraction system (Thermo AutoTrace 280 SPE Instrument) on OASIS HLB cartridges (200 mg, 6 cc). After conditioning with methanol and ultrapure water, samples (200 mL) were loaded onto cartridges and eluted by 10 mL of methanol. The particulate phases of RCH1 and RCH2 samples were also analyzed. The filters (GF/D and GF/F) were frozen and freeze-dried to remove water and determine their dry mass. Furosemide and its TPs were extracted by assisted microwave extraction (Antoon Paar, Multiwave 3000). Two cycles of extractions of 30 minutes in a mixture of 60% methanol-40% dichloromethane (v/v) were performed (100 °C, 800 W). The extracts were filtered on folded filters washed with dichloromethane, evaporated to 1 mL under rotary evaporator and diluted in 100 mL ultrapure water to carry out a purification step on OASIS HLB cartridges. Cartridges were eluted with 10 mL of methanol, internal standards (Atenolol-d7 and Sulfamethoxazole-d4) were added to the purified extracts and the final volume was adjusted to 1 mL by evaporation. Extract concentrations were evaluated using internal calibration. Internal standards were chosen based on their similar retention time to the one of the targeted molecule (Table S2). An evaluation of matrix effects revealed their low variability for all molecules across 16 urine samples. Spiked samples were also extracted, leading to the evaluation of extraction yields of furosemide, saluamine and pyridinium. These extraction yields were used for the correction of concentrations in the samples. The detection and quantification limit of the instrument for each compound is presented in Table S3. Analyses were performed using Ultra Performance Liquid Chromatography coupled with a triple quadrupole detector (Acquity-TQD, Waters). Separation was carried out on an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1

x 100 mm) with a 15 min gradient elution from 90:10 ultra-pure water (A) and methanol (B) both acidified with 0.1% formic acid, to 0:100 (A:B) maintained for 5 min, before reequilibration of the column (0.4 mL.min -1 ). For the detection, furosemide was ionized in negative electrospray mode, and saluamine and pyridinium of furosemide were ionized in positive mode. MS/MS acquisition was used (Table S2).

The phase distribution (Kd value) was calculated according to equation (2) [START_REF] Park | Removal characteristics of pharmaceuticals and personal care products: Comparison between membrane bioreactor and various biological treatment processes[END_REF], where Kd is the solid-water distribution coefficient in L.kg -1 , Cs is the concentration of furosemide, saluamine or pyridinium of furosemide adsorbed onto MES ng.L -1 and Cw is the concentration on furosemide, saluamine or pyridinium of furosemide in the liquid phase in ng.L -1 , and SS is that of suspended matter in the mixture in mg.L -1 .

𝐾 𝑑 = 𝐶𝑠 𝑆𝑆 𝑥 𝐶𝑤 × 10 6 (Eq. 2)

Analytical procedures for the identification of TPs

Degradation kinetics experiments were analysed by ultra-performance liquid-chromatography coupled to ion-mobility time-of-flight mass spectrometry (UPLC-IMS-QTOF, Vion, Waters) to enable the tentative identification of degradation products. The separation was carried out on an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 x 100 mm) with a 25 min gradient from 98:2 ultra-pure water (A) and acetonitrile (B) both acidified with 0.1% formic acid, to 2:98 (A:B) maintained for 5 min, before reequilibration of the column. Ionization was performed by an electrospray source in both positive and negative mode, in low energy (6 V) and high energy ramp (20 to 56 V). Data was acquired and analyzed with the UNIFI software (Waters). Briefly, after 4D peak detection, the chromatograms at successive kinetics points were compared to spot potential TPs. Molecular formula attribution was performed with a restricted list of atoms (C, H, N, O, P, S and Cl) and several online libraries, as a part of Chemspider in UNIFI, were interrogated (Sigma-Aldrich, Drugbank, NIST, MassBank and LGC Standard)

to tentatively identify the detected products. In order to ensure mass precision lockspray infusion was used during all the injections.

Results & discussion

Occurrence and removal in wastewater treatment plants

First, the concentrations of furosemide were evaluated in the Seine-Centre WWTP, along with the possible presence of its known degradation products saluamine and pyridinium of furosemide. This WWTP was chosen because it is located directly downstream of the city of Paris, thus receiving most of Parisian wastewaters. The known degradation products of furosemide, saluamine, furfural and pyridinium of furosemide, have been searched for in wastewater. Furfural could not be analyzed with our method, the compound being possibly too small and/or too polar to be extracted and analyzed with the same method as the other three. Although described as a furosemide transformation product since long ago (Andreasen et al. 1942), reports on saluamine toxicity and occurrence in the environment are scarce. Nonetheless, we found saluamine and pyridinium of furosemide in WWTP raw water both at concentrations of a few thousands of ng.L -1 (table 2).

<Table 2>

Interestingly, the distribution between the three compounds in raw water was about one third for each, which means that the degradation products are formed upstream of the WWTP, probably during the journey through the water system as commonly observed. Indeed, other drug TPs have already been found in WWTP influents such as N4-Acetylsulfamethoxazole, 2-hydroxycarbamazepine, Odesmethylvenlafaxine, 4-hydroxydiclofenac which are TPs of sulfamethoxazole, carbamazepine, venlafaxine and diclofenac, respectively [START_REF] Aymerich | Attenuation of pharmaceuticals and their transformation products in a wastewater treatment plant and its receiving river ecosystem[END_REF]). Furosemide could have been hydrolyzed or biodegraded in the wastewater transport system. The hydrolysis of furosemide has been known since the 70's and leads to the formation of saluamine in an acidic environment [START_REF] Bundgaard | Photodegradation and hydrolysis of furosemide and furosemide esters in aqueous solutions[END_REF][START_REF] Andreasen | In vitro studies on the hydrolysis of frusemide in gastrointestinal juices[END_REF] and some studies showed that saluamine and pyridinium of furosemide can be produced by biotransformation of furosemide by several microorganisms (Hezari & Davis 1993;Laurencé et al. 2014;[START_REF] Olvera-Vargas | Microbial biotransformation of furosemide for environmental risk assessment: identification of metabolites and toxicological evaluation[END_REF]. These three compounds are still present in the two WWTP effluents samples at several hundred nanograms per liter (table 2). Saluamine can be formed in water sediments (Li et al. 2014) and by photolysis [START_REF] Bundgaard | Photodegradation and hydrolysis of furosemide and furosemide esters in aqueous solutions[END_REF]) but to our knowledge, this is the first evidence of saluamine and pyridinium of furosemide presence in wastewater. winter. [START_REF] Castiglioni | Removal of Pharmaceuticals in Sewage Treatment Plants in Italy[END_REF] showed that the elimination rate of furosemide was much lower in winter than in summer (8% versus 54%). These differences could be related to the lower temperatures in winter, which possibly attenuated the biological activity. However, in our case, the samples were collected in winter. Thus, the differences in efficiency of the treatment plants for furosemide removal could be explained by the different processes used in the plants. For example, the plant studied in Matamoros et al. (2009) included several types of wetland constructs that were shown to be relatively efficient for the elimination of furosemide in recent literature [START_REF] Ahmed | Progress in the biological and chemical treatment technologies for emerging contaminant removal from wastewater: A critical review[END_REF]Machado et al. 2017). In the case of Seine-Centre, the purification process, which includes biofiltration, nitrification and denitrification processes, also promoted an efficient elimination of furosemide. In spite of a good elimination rate of furosemide and its TPs saluamine and pyridinium of furosemide (70 to 90% removal), several hundreds of nanograms per liter still remained in the wastewater effluent.

Many authors reported significant concentrations of furosemide at the outlet of wastewater treatment plants (table 3). Concentrations exceeding 10 µg.L -1 were even found in a few studies (22300 ng. WWTP effluents concentrations not only depend on the influent concentrations, but also on the treatment processes used within each WWTP. In some cases, furosemide concentrations can be higher at the WWTP outlet than at the inlet (Kleywegt et al. 2016). Due to the fact that an important fraction of furosemide is excreted as a glucuro-conjugated form [START_REF] Yang | Drug Acyl Glucuronides: Reactivity and Analytical Implication[END_REF], and thus not taken into account during the analysis, this difference could be assigned to the deconjugation occurring during the WWTP treatment, leading to an increased concentration of the native, unchanged form of furosemide at the outlet. It cannot be excluded that in our raw samples, furosemide concentrations could be underestimated for similar reasons, and consequently, the removal percentage as well.

Overall, the WWTP appeared to be relatively effective in removing furosemide as well as its TPs, thus

showing that there was no additional production of the targeted TPs during the biological process.

Occurrence in residential care home wastewater

The concentration of furosemide and its TPs was then evaluated in RCH wastewater samples (Table 4).

We anticipated this type of sample to be highly-charged in furosemide, as this medication is frequently prescribed at high doses to elderly people affected by chronic and age-related diseases such as hypertension and heart failure. Effluents from three different RCH were analyzed. In all three samples, significant amounts of furosemide were found, and to a lesser extent, of pyridinium of furosemide and saluamine, but their concentrations and proportions relative to each other were variable. ).

<Table 4>

Furosemide concentration variation across the three sites could be directly related to their hosting capacity. The highest furosemide concentration was found in RCH2 which also had the highest hosting capacity and on the contrary, the lowest concentration was observed in RCH3 which also has the lowest number of beds. On the other hand, Gomez-Canela et al. (2019) showed that there were quite large furosemide concentration variations (of a few thousand nanograms per liter) within the same institution over several samples, thus making it more relevant in this case to discuss the orders of magnitude. The concentrations of furosemide in the RCH1 and RCH2 samples were quite close, but the concentration of saluamine was 2-fold higher in RCH2. The higher proportion of saluamine in RCH2 could be explained by a greater biological activity which could lead to a more important biodegradation of furosemide (Laurencé et al. 2014;[START_REF] Olvera-Vargas | Microbial biotransformation of furosemide for environmental risk assessment: identification of metabolites and toxicological evaluation[END_REF]. The proportion of pyridinium of furosemide was also much lower in RCH2, implying a preferential transformation of furosemide to saluamine in this case. This difference could be explained by the neutral pH (7.10 at 20°C) indeed the formation of saluamine is promoted in acidic conditions [START_REF] Bundgaard | Photodegradation and hydrolysis of furosemide and furosemide esters in aqueous solutions[END_REF][START_REF] Andreasen | In vitro studies on the hydrolysis of frusemide in gastrointestinal juices[END_REF].

Few studies have investigated the presence of furosemide in non-treated effluents of RCH. Gomez-Canela et al. ( 2019) reported concentrations up to 3200 ng.L -1 and 3400 ng.L -1 in two different senior residence effluents in Spain with a capacity of 103 and 96 beds, respectively, and Nagarnaik et al.

(2010) found 1031 ng.L -1 in nursing care facility effluents which were considerably lower than the concentrations in this study for an equivalent hosting capacity. Slightly higher concentrations (median 6120 ng.L -1 and maximum 25100 ng.L -1 ) were found by Kleywegt et al. ( 2016) in a long care center for senior residents and other clients in Canada but with a much larger capacity (289 beds).

Furosemide and its major degradation products were also analyzed in the solid fraction of RCH1 and RCH2 samples (Table 4). Although the three compounds were difficult to analyze because of an important matrix effect, significant concentrations (several hundreds of nanograms per gram) were found. ) was quite close to the one of furosemide, but was much lower in RCH2 (0.36 L.kg -1 ), which shows a much better affinity of furosemide for the solid phase in sample 1. Kd values for saluamine were more important (103.7 and 18.3 L.kg -1 ), meaning that a significant fraction of saluamine was adsorbed on the solid phase. Thus, total saluamine load in wastewater could be largely underestimated when only performing the conventional analysis of the dissolved phase.

Saluamine has only been

Advanced oxidation in pure water and in residential care houses wastewater

Oxidation of furosemide in ultrapure water

The partial resistance of hundreds of compounds to conventional wastewater treatments highlights the need to go further in the degradation processes. We chose two AOPs which have the potential to upgrade WWTPs worldwide and are already used in some countries like Japan or Switzerland [START_REF] Prasse | Spoilt for choice: A critical review on the chemical and biological assessment of current wastewater treatment technologies[END_REF]. Chlorination oxidation experiments were also performed (Figures S3, S4 and Table S1).

Both oxidation processes have been used for a long time for disinfection and the oxidants produced are likely to react with furosemide. The photodegradation of furosemide has been known for several decades. UV irradiation leads to the substitution of chlorine by a hydroxyl group, to the hydrolysis of the furfuryl group, that results in the production of furfural and saluamine, or the oxidation of the sulfamoyl group [START_REF] Bundgaard | Photodegradation and hydrolysis of furosemide and furosemide esters in aqueous solutions[END_REF]Moore & Burt 1981). Furosemide is also degraded in sunlight or artificial laboratory light which makes it a good candidate for UV degradation [START_REF] Starling | Intensification of UV-C treatment to remove emerging contaminants by UV-C/H2O2 and UV-C/S2O82-: Susceptibility to photolysis and investigation of acute toxicity[END_REF].

The use of ozonation has increased in recent years for the treatment of wastewaters due to its effectiveness in removing organic compounds. As an electrophilic agent, the presence of electron-rich moieties such as aromatic compounds determines the reactivity with ozone (Lim et al. 2022).

According to [START_REF] Zoumpouli | Aqueous ozonation of furans: Kinetics and transformation mechanisms leading to the formation of α,β-unsaturated dicarbonyl compounds[END_REF], furosemide is expected to present an important reactivity with ozone due to the presence of the furan ring and the aniline group. However, the potential formation of furosemide TPs, especially pyridinium of furosemide, remains unknown for AOPs. AOPs are known to generate a variety of TPs that are potentially hazardous to ecosystems, and whose identification is a challenge due to the lack of analytical standards. Furosemide degradation was thus first investigated in ultrapure water spiked with furosemide. Pyridinium of furosemide was quantified after the oxidation, and aliquots were also taken to monitor the formation of unknown TPs by high-resolution mass spectrometry.

As anticipated, the two AOPs were effective in degrading furosemide under our experimental conditions. Ozonation was the most efficient (see supplementary information Figures S1,S2).

<Table 5>

The pyridinium of furosemide was produced during both oxidation experiments (table 5). During the UV/H2O2 oxidation, the aliquot taken at 210 minutes already presented a few micrograms per liter of pyridinium with 1.5% conversion rate of furosemide. This rate reached 5.8% after 300 minutes.

However, during this 5h-time lapse, less than 10% of furosemide was degraded, which means that pyridinium formation is slow, probably because it involves reaction intermediates [START_REF] Olvera-Vargas | Microbial biotransformation of furosemide for environmental risk assessment: identification of metabolites and toxicological evaluation[END_REF][START_REF] Zoumpouli | Aqueous ozonation of furans: Kinetics and transformation mechanisms leading to the formation of α,β-unsaturated dicarbonyl compounds[END_REF]. Pyridinium of furosemide was also produced by ozonation within a few minutes. Its production started immediately after ozone addition, and its concentration increased gradually until 4 minutes, with a furosemide conversion rate of 3.2% which remained unchanged after 10 minutes, in conjunction with a total consumption of ozone and furosemide concentration reaching a plateau. As in the UV/H2O2 experiment, pyridinium of furosemide did not seem to be degraded by the ozone treatment. Pyridinium of furosemide was also detected after chlorination (35 mgCl2.L -1 ) of furosemide (3 mg.L -1 ), with a maximum conversion rate of 1.4% after only 2 min of reaction time, but contrary to the other oxidation processes, its concentration quickly decreased after 5 min, probably because of the formation of chlorinated derivatives (Table S1). This result might be of importance for the quality of drinking water produced from resources impacted by furosemide, even though the fast degradation of pyridinium of furosemide seems to indicate a low risk of exposure.

Oxidation of residential care houses wastewater

In order to get insights on the AOPs efficiencies with furosemide heavily-loaded real samples and on the formation of TPs in the presence of wastewater matrix components, we next subjected the effluents from the three RCH samples to the two AOPs (figure 2). The overall abatement was quite variable from one sample to another and for the different compounds, depending on the water parameters and the oxidation process used. Ozonation was very effective at removing the three pollutants, with an important reduction (below quantification limit) of furosemide and pyridinium of furosemide for the three tested samples (figure 2). As for chlorination (supplementary data, figure S4, SI), abatement rates of almost 100% were observed for furosemide and pyridinium. These results were thus consistent with the experiments conducted with ultrapure water. Although the lifetime of ozone in WWTP effluent is shorter because of background absorptions (Lim et al. 2022), ozone was injected continuously in the samples and therefore furosemide reached higher removals than in the ultra-pure water experiment (figure S2, SI). This could also explain why pyridinium of furosemide was not observed in the ozonated samples, a higher ozone dose probably leading to its decomposition. Error bars represent standard error. For Ozonation, the samples were exposed for 1.5h with 2L.h -1 O3.

For UV/H2O2, the samples were exposed for 5H to (0.375xCOD value)mg.L -1 H2O2. (BLACK AND WHITE Experiments combining ozonation with other treatments such as biological activated carbon [START_REF] Reungoat | Ozonation and biological activated carbon filtration of wastewater treatment plant effluents[END_REF] or ultrasonication (Ibanez et al. 2013) did not significantly improve the removal efficiency and showed that the ozone treatment is responsible for most of the elimination of furosemide.

Contrary to furosemide and pyridinium, saluamine was less removed and its removal rate was variable depending on the sample. Saluamine was well degraded in the RCH3 sample, but higher concentrations of saluamine were observed from RCH1 and RCH2 samples after ozonation. This production of saluamine could be explained by the conversion of both furosemide and pyridinium to saluamine in the conditions of these samples (e.g., different initial concentrations, competition reactions of ozone with wastewater constituents such as nitrites or organic matter leading to a decrease in saluamine decomposition). Contrary to experiments in ultrapure water, no additional formation of pyridinium was observed, and all pyridinium already present in the RCH samples was degraded by ozone.

Furosemide removal by UV/H2O2 was between 70 and 80% for all the samples, which is very close to the 82% removal found by Jie (2012) in hospital wastewater. However, the effectiveness of UV/H2O2 on furosemide elimination is still uncertain. [START_REF] Park | Removal characteristics of pharmaceuticals and personal care products: Comparison between membrane bioreactor and various biological treatment processes[END_REF] but with a lamp 8 times more powerful (Joule/cm 2 ). The efficiency of furosemide degradation by UV/H2O2 thus seems to be strongly dependent on the UV irradiation intensity. Similarly to ozonation and chlorination, large differences of removal were observed between the three samples for the two degradation products.

Generation of furosemide new transformation by-products

As these advanced oxidation processes have been reported to generate numerous by-products by reaction with organic contaminants, many of them being often potentially more toxic than their parent molecules (reviewed in [START_REF] Prasse | Spoilt for choice: A critical review on the chemical and biological assessment of current wastewater treatment technologies[END_REF], the formation of other transformation products of furosemide was investigated after the oxidation experiments by high-resolution mass spectrometry To our knowledge, the product 3.e. was not clearly mentioned in the literature. However, Aalizadeh et al. (2018) found a by-product of furosemide of similar mass (m/z 276.99) after ozonation but they did not identify the structure. On the other hand, the compound 3.f. from UV/H2O2 oxidation has already been observed as a photodegradation product of furosemide after exposure to fluorescent lamps, and its structure was identified with 1 H-NMR (Katsura et al. 2015). Its structure resembles the one of furosemide with m/z 311.03, except that the chlorine has been replaced by a hydroxyl group. In Jakimska et al. ( 2014), this product has been found after photodegradation experiments carried out on river water samples and was considered as one of the most persistent transformation products.

These authors then found it in WWTP influents and effluents. In our study, the 3.f product formation kinetics was studied (see supplementary data, figure S4), showing that its degradation occurred after one hour. (2004) dimer is larger than the other molecules and very polar: it is possible that our extraction method did not retain it. Moreover, these degradation products were observed after UV irradiation alone, thus it is conceivable that by combining UV with H2O2 treatment, they were further degraded as well, as described by [START_REF] Starling | Degradation of Losartan Potassium and Furosemide by UV and UV/H2O2 processes[END_REF]. Indeed, these authors found similar degradation products of furosemide after UV and UV/H2O2 treatment, but they did not remain stable after H2O2 addition. If these compounds were present in our experiments, they may be below the limit of detection.

The m/z 276 and m/z 288 transformation products obtained after ozonation have also been mentioned

in the literature but their structure could not be elucidated [START_REF] Aalizadeh | Development and application of retention time prediction models in the suspect and non-target screening of emerging contaminants[END_REF]. In Zoumpouli et al.

(2021), a molecule with a pyridinium structure (m/z 328) and a saluamine-like molecule (m/z 265) were also obtained by ozonation of the furan ring.

Taken together, our data show that ozonation, UV/H2O2 and chlorination degrade furosemide, generating several different by-products. High oxidant doses were chosen to maximize the formation of transformation products in order to enhance their analytical detection and identification, so their presence should still be investigated at realistic doses (e.g., at a specific ozone dose < 1 mgO3/mgDOC) and at the industrial scale [START_REF] Guillossou | Benefits of ozonation before activated carbon adsorption for the removal of organic micropollutants from wastewater effluents[END_REF]. Besides saluamine production, the pyridinium of furosemide was formed under all three conditions but was only degraded by chlorination.

Furthermore, chlorination also generated a product (m/z 276) never described before, but which was degraded after a few hours. Another product (m/z 311), resulting from photodegradation and previously reported (Katsura et al. 2015), was also identified.

Conclusion

This study provides new findings on the occurrence and fate of furosemide from its origin to wastewater treatment plants. The known degradation products of furosemide, saluamine and pyridinium of furosemide were searched and quantified for the first time in real samples (WWTP inlets and outlets and RCH wastewaters). The Seine-Centre WWTP showed a good removal of furosemide (>70%), saluamine (>80%) and pyridinium (>90%). However, concentrations of several hundred ng.L -1 were found after treatment and are released into the aquatic environment. These concentrations sometimes exceed 10 µg.L -1 in the literature for furosemide and may pose a risk to ecosystems.

To limit the release of these pharmaceutical compounds in the environment, we investigated AOPs and the possible production of TPs. UV/H2O2, chlorination and ozonation were first shown to be effective in degrading furosemide in ultrapure water. In RCH wastewater, ozonation and chlorination showed complete degradation of furosemide and pyridinium, but saluamine was still present in the samples after treatment due to a possible production. UV/H2O2 showed variable removal rates depending on the compounds. Although these treatments appeared quite effective, new TPs were identified following the chlorination (m/z 276) and ozonation (m/z 311) processes. The elimination of pollutants is used as a criterion for the evaluation of AOPs, but the presence of TPs should also be considered. Before concluding on the effectiveness of these advanced treatments, the toxicity and persistence of the end-products should be investigated. Thus, our study underlines the necessity and the relevance to use approaches such as non-targeted analysis for the detection of emerging pollutants, to unveil new TPs, combined with toxicity assessments in order to improve the characterization and design of wastewater treatment processes. Keeping in mind that these data alone do not provide information on their toxic risk and cocktail effects, our study highlights the need to develop an integrative strategy coupling state-of-the art chemical analysis techniques with ecotoxicological tests with defined end-points.

CRediT author statement

Fidji Sandre:

Figure 1 .

 1 Figure 1. Removal percentage of furosemide, saluamine and pyridinium of furosemide in Seine-Centre

  L -1 in De vieno et al. (2017), 26000 ng.L -1 in Vymazal et al. (2017), and 11000 ng.L -1 in Rozman et al. (2017)).

  detected once in natural sediments doped with furosemide(Li et al. 2014) but our present work uncovers for the first time the presence of pyridinium of furosemide in the solid fraction. In sediments, furosemide concentrations of 7, 98, and 350 ng.g -1 were reported[START_REF] Ferreira Da Silva | Occurrence and distribution of pharmaceuticals in surface water, suspended solids and sediments of the Ebro river basin, Spain[END_REF][START_REF] Ferrari | Exposure of the Main Italian River Basin to Pharmaceuticals[END_REF][START_REF] Björklund | Pharmaceutical Residues Affecting the UNESCO Biosphere Reserve Kristianstads Vattenrike Wetlands: Sources and Sinks[END_REF] respectively). The concentrations obtained in our study are closer to those obtained in WWTP sludge, where the concentrations reached up to 686 ng.g -1[START_REF] Huber | A first screening and risk assessment of pharmaceuticals and additives in personal care products in waste water, sludge, recipient water and sediment from Faroe Islands, Iceland and Greenland[END_REF]) and 3602 ng.g -1[START_REF] Salgado | Assessing the diurnal variability of pharmaceutical and personal care products in a full-scale activated sludge plant[END_REF]). These values indicate that a significant fraction of furosemide is adsorbed onto the particulate phase. The partition coefficients between the liquid and solid phase (Kd) are good indicators of the compounds affinity with the solid phase. The Kd values of the suspended matter of the samples RCH1 and RCH2 were 7.7 and 5.4 L.kg -1 respectively. Jelic et al. (2012) found a Kd of 43 L.kg -1 for furosemide in the solid phase of WWTP effluent which is 5 to 8 fold higher than the values found in this study. The Kd value is very dependent on the matrix considered. For example, in sediments,[START_REF] Björklund | Pharmaceutical Residues Affecting the UNESCO Biosphere Reserve Kristianstads Vattenrike Wetlands: Sources and Sinks[END_REF] found a Kd of 2517 L.kg -1 which shows a very good affinity of furosemide for sediments whereas Jelic et al. (2012) found a lower Kd in thickened sludge (127 L.kg -1 ) and digested sludge (110 L.kg -1 ). In a Membrane BioReactor (MBR) or Anaerobic-Anoxic-Aerobic system (A2O) -treated sludge , Park et al. (2017) obtained a Kd of 2.1, which is quite low in comparison. Narumya et al. (2013) showed a decrease of the Kd for furosemide before and after sludge digestion. In sample RCH1, the Kd value of pyridinium of furosemide (12.5 L.kg - 1

Figure 2 .

 2 Figure 2. Effectiveness of a) UV/H2O2 and b) ozonation on residential care home (RCH) discharge water.

  FIGURE)

  described the use of UV irradiation alone as not very effective on pharmaceuticals and personal care products, and only achieved 23% of removal of furosemide in WWTP post-treatment. However, as the treatment was performed during a purification process in a WWTP, the suspended particles can interfere and reduce the effectiveness of UV as compared to laboratory experiments performed with filtered samples. With UV/H2O2, Ikonen et al. (2021) found no removal of furosemide while Singh et al. (2015) observed a complete degradation,

(Figure 3 .

 3 Figure 3. Molecular Structure of a. furosemide (m/z 329.74) and its transformation products. b.

  photodegradation of furosemide. These by-products were not found in our oxidation experiments. The

Table 1 .

 1 Physico

	-chemical parameters of samples from WWTP raw wastewater (RW1 = 2021-12-07,
	RW2 = 2021-12-08), WWTP treated wastewater (TW1 = 2021-12-07, TW2 = 2021-12-08) and
	wastewater outlets of residential care homes (RCH). NA = Not available (not measured). (BLACK AND
	WHITE TABLE)
	<Table 1>

  .L -1 and 3410 ng.L -1 in Spain[START_REF] Collado | Pharmaceuticals occurrence in a WWTP with significant industrial contribution and its input into the river system[END_REF] Celic et al. 2019), 4577 ng.L -1 in Portugal[START_REF] Santos | Contribution of hospital effluents to the load of pharmaceuticals in urban wastewaters: Identification of ecologically relevant pharmaceuticals[END_REF], 1652 ng.L -1 in Italy[START_REF] Feo | Pharmaceuticals and other contaminants in waters and sediments from Augusta Bay (southern Italy)[END_REF], 2916 ng.L -1 and in Switzerland(Lee et al. 2014).

	Our samples concentrations at 24 hour interval were of the same order of magnitude, indicating little
	variation over this period. This result is in contrast to Gomez-Canela et al. (2019), who observed
	significant variations in furosemide concentrations over 5 consecutive days, but on nursing home
	effluents. However, for furosemide, other studies usually reported seasonal rather than weekly
	variations with lower concentrations in summer (Kot-Wasik et al. 2016; Delli Compagni et al. 2020).
	The two collected raw water samples (2021-12-07 and 2021-12-08 samples) exhibited substantial
	concentrations of furosemide of 3351 and 2819 ng.L -1 respectively (table 2). These concentrations
	were consistent with those found in recent literature for WWTPs raw water in other European
	countries. Concentrations between 1 to 5 µg.L -1 were found: 1491 ng.L -1 in Sweden (Baresel et al.

2019), 2625 ng.L -1 in Poland (Kot-Wasik et al. 2016), 2601 ng.L -1 in Greece (Papageorgiou et al. 2019), 1901 ng

Table 2 .

 2 Concentrations

(ng.L -1 ) of Furosemide, Pyridinium of Furosemide and Saluamine in WWTP water samples (RW = raw wastewater, TW = treated wastewater) (BLACK AND WHITE TABLE)

Table 3 .

 3 Concentrations (in ng.L -1 ) of furosemide in WWTP effluents. (BLACK AND WHITE TABLE)

	<Table 3>

Tracol & Duchemin 2007 1 ; Celic et al. 2019 2 ; Santos et al. 2013 3 ; Castiglioni et al. 2018 4 ; Giebułtowicz et al. 2016 5 ; Huber et al. 2016 6 ; Frieberg 2018 7 ; Diaz-Sosa et al. 2020 8 ; Papageorgiou et al. 2016 9 ; Kleywegt et al. 2016 10 ; Estrada-Arriaga et al. 2016 11 ; Afsa et al. 2020 12 ; Hanamoto et al. 2018 13 ; Al-Odaini et al. 2013 14 .

Table 4 .

 4 Concentration

of furosemide, saluamine and pyridinium of furosemide in residential care homes wastewater in dissolved and particulate phases. (BLACK AND WHITE TABLE

Table 5 .

 5 Pyridinium of furosemide concentration and furosemide percentage of conversion during oxidation experiments. (BLACK AND WHITE TABLE)

  Investigation, Visualization, Writing-original draft & Editing; Nina Huynh: Methodology, ://doi.org/10.1016/j.watres.2008.10.047 Katsura, S., Yamada, N., Nakashima, A., Shiraishi, S., Furuishi, T., Ueda, H., 2015. Identification of Furosemide Photodegradation Products in Water-Acetonitrile Mixture. Chemical and Pharmaceutical Bulletin 63, 617-627. https://doi.org/10.1248/cpb.c15-00122 Kleywegt, S., Pileggi, V., Lam, Y.M.,Elises, A., Puddicomb, A., Purba, G., Di Caro, J., Fletcher, T., 2016. The contribution of pharmaceutically active compounds from healthcare facilities to a receiving sewage treatment plant in Canada: Contribution of pharmaceutically active compounds to STP effluent. Environmental Toxicology and Chemistry 35, 850-862. https://doi.org/10.1002/etc.3124 Kot-Wasik, A., Jakimska, A., Śliwka-Kaszyńska, M., 2016. Occurrence and seasonal variations of 25 pharmaceutical residues in wastewater and drinking water treatment plants. Environmental Monitoring and Assessment 188, 661. https://doi.org/10.1007/s10661-016-5637-0 Laurencé, C., Rivard, M., Lachaise, I., Bensemhoun, J., Martens, T., 2011. Preparative access to transformation products (TPs) of furosemide: a versatile application of anodic oxidation. Tetrahedron 67, 9518-9521. https://doi.org/10.1016/j.tet.2011.10.006 Laurencé, C., Rivard, M., Martens, T., Morin, C., Buisson, D., Bourcier, S., Sablier, M., Oturan, M.A., 2014. ://doi.org/10.1016/j.chemosphere.2014.05.036 Laurencé, C., Zeghbib, N., Rivard, M., Lehri-Boufala, S., Lachaise, I., Barau, C., Le Corvoisier, P., Martens, T., Garrigue-Antar, L., Morin, C., 2019. A new human pyridinium metabolite of furosemide, inhibitor of mitochondrial complex I, is a candidate inducer of neurodegeneration. Biochemical Pharmacology 160, 14-23. https://doi.org/10.1016/j.bcp.2018.12.007 Lee, C.O., Howe, K.J., Thomson, B.M., 2012. Ozone and biofiltration as an alternative to reverse osmosis for removing PPCPs and micropollutants from treated wastewater. Water Research 46, 1005-1014. https://doi.org/10.1016/j.watres.2011.11.069

	quality	of	receiving	waters.	Water	Research	43,	363-380.
	Anticipating the fate and impact of organic environmental contaminants: A new approach applied
	to	the	pharmaceutical	furosemide.	Chemosphere	113,	193-199.
	Investigation, Review & Editing; Emilie Caupos: Methodology, Investigation, Review & Editing; Lamyae
	El-Mrabet: Investigation, Review & Editing; Chandirane Partibane: Investigation, Review & Editing;

Isabelle Lachaise: Investigation, Review & Editing; Christophe Pommier: Investigation, Review & Editing; Michael Rivard: Supervision, Review & Editing; Christophe Morin: Review & Editing; Régis httpshttps

Acknowledgments

Thanks to SIAAP for providing the WWTP inlet and outlet samples and to PRAMMICS platform (OSU-EFLUVE UMS 3563) for the analytical equipment (UPLC-IMS-QTOF and AutoTrace SPE). The funding was provided by the OPUR program and WaterOmics project (ANR-17-CE34-0009-01). Thanks to the LDAR sampling service and specifically Benoît CHIRON and Jérôme PRADES for the residential care homes samples. Thanks to Université Paris-Est Créteil and to the French ministry of higher education research and innovation for F. Sandre phD fellowship.

Moilleron: Review & Editing; Julien Le Roux: Supervision, Conceptualization, Methodology, Review & Editing; Laure Garrigue-Antar: Supervision, Conceptualization, Review & Editing.

Huerta-Fontela, M., Galceran, M.T., Ventura, F., 2011. Occurrence and removal of pharmaceuticals and hormones through drinking water treatment. Water Research 45, 1432Research 45, -1442. . https://doi.org/10.1016/j.watres.2010.10.036 Ibáñez, M., Gracia-Lor, E., Bijlsma, L., Morales, E., Pastor, L., Hernández, F., 2013


Daniela Bănaru
email: daniela.banaru@univ-amu.fr]

Laurence Le Diréach
email: laurence.ledireach@univ-amu.fr

Guillaume Crest
email: guillaume.crest@live.fr]

Melissa Tenaille
email: melissa.tenaille@etu.univ-amu.fr]

Mireille Harmelin-Vivien

M Harmelin

(NW Mediterranean)?

Keywords: Mullus surmuletus, total length and weight, relative body condition, gonadosomatic index, gonadal development stages, Port-Cros marine protected area, North-Western Mediterranean Mullus surmuletus, longueur et poids totaux, indice corporel relatif, indice gonado-somatique, stades de développement des gonades, Aire marine

come   L'archive ouverte pluridisciplinaire

INTRODUCTION

The surmullet Mullus surmuletus Linnaeus, 1758 (Osteichthyes, Perciformes, Mullidae) is a teleost fish species commonly found in the Mediterranean, the Black Sea and the East Atlantic [START_REF] Hureau | Mullidae. In : Fishes of the North-eastern Atlantic and the Mediterranean[END_REF]. This bentho-demersal species occurs from shallow waters down to 400 m, being more abundant between 50 and 200 m depth [START_REF] Hureau | Mullidae. In : Fishes of the North-eastern Atlantic and the Mediterranean[END_REF][START_REF] Tserpes | -Distribution of Mullus barbatus and M. surmuletus (Osteichthyes: Perciformes) in the Mediterranean continental shelf: implications for management[END_REF]. M. surmuletus juveniles settle in shallow coastal habitats and disperse in deeper waters when growing, while adults undergo seasonal migrations toward deep habitats for reproduction [START_REF] Macpherson | Substrate use and temporal pattern of recruitment in juvenile fishes of the Mediterranean littoral[END_REF][START_REF] Machias | Bathymetric distribution and movements of red mullet (Mullus surmuletus)[END_REF]. M. surmuletus is a major target species in the Mediterranean Sea, exploited by various artisanal small fisheries using trawls and nets. However, the General Fisheries Commission for the Mediterranean-GFCM stock assessment in the French Mediterranean area is performed only for the red mullet M. barbatus, a related species considered as over-exploited [START_REF] Biseau | Diagnostic halieutiques française 2019 sur les ressources halieutiques débarquées par la pêche française (métropolitaine)[END_REF]. No biomass data are available for the populations of the south-eastern coast of France, the scientific MEDITS trawling campaigns covering only the Gulf of Lion and the east coast of Corsica (Ifremer, 2021; https://campagnes.flotteoceanographique.fr). These two sympatric Mullidae species are among the 15 most important species in the French Mediterranean Sea in terms of landings and value [START_REF] Demaneche | Rapport R3 Méditerranée continentale, échantillonnage des marées au débarquement. Méthode d'élévation et évaluation des captures et de l'effort de pêche des flottilles de la façade Méditerranée continentale sur la période 2007-2008. Programme P6 AESYPECHE "Approche écosystémique de l'halieutique[END_REF]. FAO fishery databases reported 292 tonnes of catches for M. barbatus and 90 tonnes for M. surmuletus along the entire French Mediterranean coast in 2018 (www.fao.org). They represent together the third most important species groups in terms of annual average landings in 2008-2018 of the fleet of gillnetters [6-12m[ of the Var region (south-eastern coast of France). Their landings represent 6% of the average annual value in Euros and 3% (25 tonnes) of the average annual volume landed by the Var fleet (https://sih.ifremer.fr/; [START_REF] Sock | Exploitation et Gestion des ressources halieutiques par la pêche aux petits métiers dans le Var (en Méditerranée française) sur la période 2008-2018[END_REF]. In addition, fisheries landings in the south-east of France are probably largely underestimated due to the absence of auctions and direct sales at ports and to restaurants [START_REF] Paca | Etat des lieux et caractérisation de la pêche maritime professionnelle et des élevages marins en PACA. Amélioration de la connaissance sur la petite pêche artisanale et la pisciculture marine locale[END_REF].

By selecting individuals of a certain size and exploiting specific areas, fisheries may affect mean body length, population demography, sex-ratio and reproduction [START_REF] Froese R | Minimizing the impact of fishing[END_REF].

The gonado-somatic index and gonadal development are generally related to body length, with higher values in larger individuals [START_REF] Michaletz | Effect of body size on fecundity, the gonadosomatic index, egg size, and timing of spawning of gizzard shad[END_REF]. Fisheries regulation of fishing effort, nets and mesh size, etc) is shown to have positive effects on mean body length, population structure and reproduction indices [START_REF] Roberts | Are marine reserves effective in management of reef fisheries?[END_REF][START_REF] Lloret | Condition, feeding and reproductive potential of white seabream Diplodus sargus as indicators of habitat quality and the effect of reserve protection in the northwestern Mediterranean[END_REF]Boudouresque et al., 2021). It is thusessential to study species life-history traits in order to understand how fish populations are impacted by fisheries, and whether the existing fishery regulations would allow their sustainable exploitation [START_REF] King | Marine fish life history strategies: applications to fishery management[END_REF].

The morphometrics and growth of M. surmuletus have been studied in different Mediterranean areas, such as the Adriatic Sea (Jukic and [START_REF] Piccinetti | Quantitative and qualitative characteristics of demersal resources in the Adriatic Sea with some population dynamics estimates[END_REF], the Balearic Islands [START_REF] Reñones | Life history of the red mullet Mullus surmuletus from the bottom trawl fishery off the Island of Majorca (North-West Méditerranean)[END_REF] and the Aegean Sea [START_REF] Karakulak | Length-weight relationships for 47 coastal fish species from the northern Aegean Sea, Turkey[END_REF]Gökhan et al., 2007, Arslan andIsmen, 2013;[START_REF] Kousteni | Life-history traits of the striped red mullet Mullus surmuletus (Linnaeus, 1758) in the south Aegean Sea (eastern Mediterranean)[END_REF]. However, to our knowledge, the only available data concerning these parameters for M. surmuletus on the French Mediterranean coasts were provided by [START_REF] Campillo | Les pêcheries françaises de Méditerranée : synthèse des connaissances[END_REF] for the populations of the Gulf of Lion. [START_REF] Kousteni | Life-history traits of the striped red mullet Mullus surmuletus (Linnaeus, 1758) in the south Aegean Sea (eastern Mediterranean)[END_REF] indicated in the Aegean Sea a reproduction period between March and July, while in the Gulf of Lion it occurs between June and August [START_REF] Campillo | Les pêcheries françaises de Méditerranée : synthèse des connaissances[END_REF].The aim of this study was to characterize the main biological parameters (length-weight relationship, relative body condition, sex ratio, gonado-somatic index and gonadal development stage) of M. surmuletus, as well as their spatial and seasonal variations, for the south-eastern coasts of France, where it represents one of the major exploited fish species of high commercial value. As M. surmuletus is traditionally exploited by small scale fisheries mainly in spring and autumn in this area, when this species is present in the coastal area, these two seasons were chosen for sampling. By comparing data from fisheries regulated and non-regulated zones, it would be possible to provide evidence of the effects of the existing fisheries management on the life history traits of these populations, necessary for proper stock assessment.

MATERIAL AND METHODS

Sampling and study zones

Sampling was done in the Var region, on the south-eastern coast of France (North-Western Mediterranean Sea), at four sites located in a fisheries regulated zone, the adjacent marine area (AMA) of Port-Cros National Park (PCNP), and at four sites located in non-regulated zones to the west and east of this area (Fig. 1). The AMA in the central part of Var encompasses Hyères Bay and Lavandou Bay, with Port-Cros Island fishing regulated since 1963, Porquerolles island fishing regulated since 2007 and Le Levant Island which is a military zone with limited access for any activity. In this oldest marine protected area in Europe, regulations governing sites, fishing gear, catches, fish size, etc. are applied to both professional and recreational fishing (Boudouresque et al., 2005;[START_REF]Évolution de la gouvernance : l'exemple de la pêche dans les eaux du Parc national de Port-Cros[END_REF]Astruc et al., 2018;Barcelo et al., 2018). In addition to the regulations implemented by the PCNP, there are also regulations imposed by the local Prud'homies for access to some areas on certain dates with a limit on the number of authorized vessels or regulations on the duration of setting the nets and their mesh and length. These coasts are influenced by the Northern Current flowing in an east-west direction along the continental slope [START_REF] Millot | Circulation in the western Mediterranean sea[END_REF]. Sampling was done with professional fishermen partners using bottom-set trammel nets between 15 and 30 m depth.

Sampling was performed at the West and AMA sites in autumn 2019 (October to December) and at all sites in spring 2020 (April to June). A total number of 263 individuals were analyzed (Table 1).

Sample treatment

For each specimen, total length (LT) was recorded to the nearest millimetre (mm), while total weight (WT), eviscerated weight (WE) and gonad weight (WG) were recorded to the nearest gram (g). Sex was determined by macroscopic observation of gonads and gonadal development stages were assessed according to Nikolsky's scale (1963): I: immature, II: resting, III: developing, IV: maturing, V: mature, VI: spent. Sex ratio, expressed as the number of females per male, was calculated by size, by zone and by season.

Data analysis

Data analyses were performed using STATISTICA 12.7. Normality and homogeneity of variances were tested using Shapiro-Wilk and Levene tests prior to analyses [START_REF] Underwood | Experiments in ecology: their logical design and interpretation using analysis of variance[END_REF]. Power regression analysis was used to describe the total length-total weight relation according to the equation: TW = a × TL b [START_REF] Ricker | Computation and interpretation of biological statistics of fish populations[END_REF]. Differences in length between sexes, zones and seasons were tested using Kruskal-Wallis non-parametric tests and multiple comparisons of average rank. To examine the variation in fish condition with zone and season, while avoiding the effect of size, the relative condition factor Kn was used [START_REF] Cren | The length-weight relationship and seasonal cycle in gonad weight and condition in the perch (Perca fluviatilis)[END_REF]. Kn was computed with the formula: Kn =WE/WE', here WE is the observed individual eviscerated weight and WE' is the estimated eviscerated weight from the Log 10 WE-Log 10 TL relationship (WE' = 10 -intercept × TL slope ). The gonado-somatic index (GSI) was calculated according to the equation: GSI = (WG / WE) × 100, where WG is the gonad weight and WE the eviscerated weight of specimen. Both GSI and Kn were calculated per sex, per zone and per season. Differences in mean gonado-somatic index for both sexes were analyzed by ANCOVA with TL as covariate to remove the effect of individual size, and zone and season as factors.

RESULTS

Morphometry

Of a total number of 263 individuals measured, 179 were females, 66 males, and 18 were undetermined, including mostly immature specimens. Their size (TL) ranged between a minimum of 12.2 cm and a maximum of 32.0 cm, with most individuals measuring between 17 and 23 cm (Fig. 2).

Mean (± SD) total lengths of females (21.5 ± 3.8 cm) and males (20.5 ± 2.9 cm) did not differ statistically (p > 0.05) and were significantly greater than immature individuals (18.1 ± 3.3 cm) (H = 13.65; p = 0.001). The relationship between TL and WT indicated an isometric growth (a = 0.011; b = 3.080), similar between females (a = 0.011; b = 3.074) and males (a = 0.077; b = 3.180).

The surmullet sampled in the AMA zone were significantly larger than those sampled in the West and East zones (H = 46.41; p < 0.0001) (Fig. 3). Between-zones differences were significantly higher for females (H = 31.06 p < 0.0001) than for males (H = 6.85 p = 0.032).

Females measured 23.0 ± 0.4 cm TL in the AMA compared to 20.4 ± 0.5 and 19.5 ± 0.6 cm in the West and East zones respectively. Males were larger (21.3 ± 0.5 cm) in the AMA than in the East zone (19.2 ± 0.8 cm), but did not significantly differ from those of the West zone (20.2 ± 0.6 cm) (Fig. 3). On average larger individuals were sampled in autumn than in spring (H = 10.59; p < 0.001), for both females (22.1 ± 0.5 and 21.1 ± 0.4 cm, respectively) and males (22.5 ± 0.8 and 19.6 ± 0.3 cm, and respectively) (H = 4.63 p = 0.031 and H = 14.26 p < 0.0001, respectively).

Relative body condition

No difference in relative body condition (Kn) was found between males and females (p > 0.05) or between study zones (p > 0.05). Kn was higher in autumn (1.04 ± 0.09) than in spring (0.99 ± 0.10) for the whole population (H = 7.88; p = 0.005). However, contrasted seasonal tendencies occurred between zones with higher mean Kn values in the AMA in spring and in the West zone in autumn (Table 2).

Reproduction

Sex-ratio

Juveniles dominated the 12 cm TL size class and mature individuals started to dominate from the 14 cm size class (> 75%). Females strongly dominated in all size classes with an increasing female-male sex-ratio with size (Fig. 2). The sex-ratio was 1:1 in the 15-16 cm size class and reached 10:1 in the 26 cm size class. Some size classes larger than 27 cm were even exclusively composed of females.

Higher sex-ratio values were measured in the West and East zones (3:1) than in the AMA (2:1) (Fig. 4A), and in autumn than in spring (4:1 and 2:1, respectively) (Fig. 4B).

Gonado-somatic index

The gonado-somatic index increased with size for both females and males (Fig. 5). Females showed a higher increase in GSI with size than males. The linear relationships between individual size class and GSI by sex presented a higher slope for females (0.248) than for males (0.030). The highest GSI values were found in the 31 cm size class for females and in the 24 cm size class for males (Fig. 5).

Mean GSI differed among zones only for males (F = 5.39; p = 0.007), with higher values in the AMA compared to the West and East zones (Fig. 78A). The apparently higher GSI for females in the AMA was not significant and was related to the greater individual size of females in this zone (F = 24.35; p < 0.0001). Season had a significant effect on GSI independently of size, which was a significant covariate for females and males (F = 54.30; p < 0.0001 and F = 10.14; p = 0.002, respectively). Both females and males showed a higher GSI in spring than in autumn (F = 31.35; p < 0.0001 and F = 9.19; p = 0.004, respectively) (Fig. 6B). Gonad weight and gonado-somatic index were positively correlated with Kn (p < 0.0001; r = 0.28 and respectively p = 0.004; r = 0.18).

Gonadal development stages

The gonadal development stages increased with size for both females (Fig. 7A) and males (Fig. 7B), but there were no significant differences between females and males (p > 0.05). For females, stage I dominated in the 14 cm size class, stage II in 20 cm, stage III between 22 and 28 cm, while stage V showed its highest percentage in the 30 cm size class (Fig. 7A). For males stage II dominated in size class between 14 and 18 cm, III between 20 and 22 cm, IV in 24 cm, stage V in 26 cm, while stages III and IV were equally represented in 30 cm size class (Fig. 7B). For females, gonadal development stage III dominated in all zones, while stage VI was observed only in the AMA and West zones (Fig. 8A). For males, stage II dominated in all zones (Fig. 8B). For both females and males individuals in stage V were more numerous in the AMA. However, differences in gonadal development stage between zones were not significant (p > 0.05) and were related only to differences in the individual size for both females and males (F = 44.20; p < 0.0001 and respectively F = 9.16; p = 0.004).

Lower gonadal development stages dominated in autumn for both females (II and III) (F = 50.12; p < 0.0001) (Fig. 8C) and males (II and III) (F = 7.00; p = 0.010) (Fig. 8D). Significant seasonal differences, independent of size, were also observed for both females (F = 70.63; p < 0.0001) and males (F = 18.21; p < 0.0001). However, development stages in males seemed to be in advance compared to females in autumn, as stage IV and V were observed for males, while stage I were still present in females. More advanced gonadal development stages occurred in spring, with stages IV, V and VI observed in similar percentages in both sexes.

DISCUSSION

The results of this article provide new original data on life-history traits of Mullus surmuletus in the Var region, South-Eastern France (NW Mediterranean), and enabled the comparison of life-history traits of Mullus surmuletus in fisheries regulated and non-regulated zones in the Port Cros National Park and neighbouring areas.

The total length and total weight of Mullus surmuletus were within the range of those recorded in other Mediterranean regions [START_REF] Campillo | Les pêcheries françaises de Méditerranée : synthèse des connaissances[END_REF]; Arslan and Ismen, 2013 and references therein; [START_REF] Kousteni | Life-history traits of the striped red mullet Mullus surmuletus (Linnaeus, 1758) in the south Aegean Sea (eastern Mediterranean)[END_REF]. In 2019 and 2020, their mean total length was in the upper range or higher that those sampled in the Gulf of Lion during summer (https://campagnes.flotteoceanographique.fr; www.ifremer.fr). In spring, new cohorts of young individuals were observed in the study area, which lowered the mean total length of the analyzed individuals.

In this study no significant differences between the growth curves of male and female M. surmuletus were found, in accordance with the results of [START_REF] Machias | Bathymetric distribution and movements of red mullet (Mullus surmuletus)[END_REF], but in contrast to those of other authors [START_REF] Reñones | Life history of the red mullet Mullus surmuletus from the bottom trawl fishery off the Island of Majorca (North-West Méditerranean)[END_REF][START_REF] Karakulak | Length-weight relationships for 47 coastal fish species from the northern Aegean Sea, Turkey[END_REF]Arslan and Ismen, 2013). The functional regression b value showed an isometric growth for both females and males, as also reported in previous studies [START_REF] Campillo | Les pêcheries françaises de Méditerranée : synthèse des connaissances[END_REF]Bensahla-Talet et al., 2014). This result differed from studies carried out in Majorca [START_REF] Reñones | Life history of the red mullet Mullus surmuletus from the bottom trawl fishery off the Island of Majorca (North-West Méditerranean)[END_REF], Morocco [START_REF] Lamrini | Croissance et reproduction du rouget barbet de roche (Mullus surmuletus L. 1758) dans la baie de M'diq (Maroc)[END_REF], the Aegean Sea (Arslan and Ismen, 2013 and references therein) and in the Bay of Biscay (N' Da et al., 2006) which indicated a positive allometric growth.

Differences in the relationship between length and weight may be related to environmental and trophic factors as well as reproduction and stage of maturity [START_REF]Some Simple Methods for the Assessment of Tropical Fish Stocks[END_REF][START_REF] Cherif | Le rouget de roche (Mullus surmuletus) des côtes nord tunisiennes : reproduction, sexualité et croissance[END_REF]. The study area is impacted by the oligotrophic Northern Mediterranean current [START_REF] Millot | Circulation in the western Mediterranean sea[END_REF] and is probably less productive than other marine coastal areas [START_REF] Diaz | Early spring phosphorus limitation of primary productivity in a NW Mediterranean coastal zone (Gulf of Lions)[END_REF].

Moreover, trophic conditions and diet may influence the relative body condition. Condition factors compare the well-being of fish based on the assumption that heavier fish of a given length are in better condition [START_REF] Froese | Cube law, condition factor and weight-length relationships: history, meta-analysis and recommendations[END_REF]. Fish condition is relevant because it influences growth, reproduction and survival (Lambert and [START_REF] Dutil | Can simple condition indices be used to monitor and quantify seasonal changes in the energy reserves of Atlantic cod (Gadus morhua)? Can[END_REF][START_REF] Shulman | The biochemical ecology of marine fishes[END_REF]. In this study relative body condition did not differ significantly between sexes, in accordance with the results published by [START_REF] Lloret | Condition, feeding and reproductive potential of white seabream Diplodus sargus as indicators of habitat quality and the effect of reserve protection in the northwestern Mediterranean[END_REF]. However, body condition was positively correlated with gonad mass and gonado-somatic index, confirming the influence of body condition on reproductive success.

Previous studies showed that the adults of Mullus surmuletus are carnivores rather opportunistic feeders and feed on diverse benthic prey according to their size, but also to zones and seasons (Bautista-Vega et al., 2008). Their main prey are amphipods, decapods and annelids, but they also feed on mysids, brachyurans, euphausiids, mollusks, and ophiurids (Bautista-Vega et al., 2008 and references therein). Spatial differences in relative body condition potentially related to diet were also discussed by Bautista-Vega (2007) for different areas of the Gulf of Lion. In contrast, no significant differences in Le Cren relative body condition were observed between zones in our study. However, Bautista-Vega (2007) used Fulton index, which may be influenced by stomach content and gonad weight. In our study, a lower relative body condition was observed in spring, that may be related to a higher reproduction investment for gonad development during this season, as previously discussed by [START_REF] Shulman | The biochemical ecology of marine fishes[END_REF].

In this study the size at first maturity was 14 cm for females and 15 cm for males. This size should correspond to the first year of life, as maturation during the first year was apparently a common trait in the two Mullus species (Reňones et al. 1995). These results were similar to those of previous studies, which reported that the mean length at first maturity was 14 cm in the Gulf of Lion [START_REF] Campillo | Les pêcheries françaises de Méditerranée : synthèse des connaissances[END_REF] and between 13 and 15 cm in Mallorca and on the Egyptian Mediterranean coast [START_REF] Morales-Nin B | Parametros biologicos del salmonete de roca Mullus surmuletus (L. 1758)[END_REF][START_REF] Reñones | Life history of the red mullet Mullus surmuletus from the bottom trawl fishery off the Island of Majorca (North-West Méditerranean)[END_REF][START_REF] Mehanna | Growth, mortality and spawning stock biomass of the striped red mullet Mullus surmuletus, in the Egyptian Mediterranean waters[END_REF].

In the south-east of France, the percentage of females increased with size and size classes > 27 cm were sometimes exclusively composed of females, as recently observed by [START_REF] Kousteni | Life-history traits of the striped red mullet Mullus surmuletus (Linnaeus, 1758) in the south Aegean Sea (eastern Mediterranean)[END_REF] in the Aegean Sea. This was also observed by [START_REF] Reñones | Life history of the red mullet Mullus surmuletus from the bottom trawl fishery off the Island of Majorca (North-West Méditerranean)[END_REF] in Mallorca, where individuals > 23 cm presented very low proportions of males. Females largely dominated in all zones and in both seasons. A sex-ratio in favour of females in M. surmuletus was recorded in previous studies in the Eastern Channel and the North Sea [START_REF] Mahé | L. 1758) en Manche orientale et mer du Nord[END_REF], but also in the Mediterranean Sea (Kousteni et al., 2019 and references therein), Morocco [START_REF] Lamrini | Croissance et reproduction du rouget barbet de roche (Mullus surmuletus L. 1758) dans la baie de M'diq (Maroc)[END_REF] and Spain [START_REF] Reñones | Life history of the red mullet Mullus surmuletus from the bottom trawl fishery off the Island of Majorca (North-West Méditerranean)[END_REF]. This might be attributed to differences in spatial distribution between sexes. Coastal areas, probably more productive, could be more favourable for feeding for females preparing for reproduction. The high proportions of females in our samples might thus be due to the females coming close to the coast for feeding, before and after reproduction, which takes place in deeper waters [START_REF] Machias | Bathymetric distribution and movements of red mullet (Mullus surmuletus)[END_REF]. The observed imbalance in the relative proportions of sexes in our samples suggested that males and females displayed different spatial behaviour, with males living offshore most of the time [START_REF] Machias | Bathymetric distribution and movements of red mullet (Mullus surmuletus)[END_REF]. In our study, the sex-ratio was less unbalanced in the AMA (2:1) than in the West and East areas (3:1). Marine protected areas are known to have positive effects on population demography and sex-ratio compared to fisheries non-regulated areas [START_REF] Grüss | Conservation and fisheries effects of spawning aggregation marine protected areas: What we know, where we should go, and what we need to get there[END_REF]Boudouresque et al., 2021). The sex-ratio of the Var M. surmuletus population differed from those of the Gulf of Lion, where males generally dominated and the sex-ratio females-males reached 1:8 in 2011. However, annual variations were observed in the Gulf of Lion, with the sex-ratio females-males varying from 1:2 in 2019 to 1:1 in 2020. [START_REF] Reñones | Life history of the red mullet Mullus surmuletus from the bottom trawl fishery off the Island of Majorca (North-West Méditerranean)[END_REF] showed that gonad maturity occurs earlier in the year for males (December to June) compared to females (March to June). They also indicated that for females the period of reproductive activity was shorter than for males and reached a maximum in April and May. [START_REF] Kousteni | Life-history traits of the striped red mullet Mullus surmuletus (Linnaeus, 1758) in the south Aegean Sea (eastern Mediterranean)[END_REF] indicated in the Aegean Sea a maximum in the spawning period for both sexes between March and July. This is in agreement with the results of our study, since the highest gonado-somatic index and the highest percentage of mature gonads for both females and males were observed during spring (May to June). Some authors reported a maximum spawning period in May and June [START_REF] Papaconstantinou | Age, growth and reproduction of red mullet (Mullus surmuletus Linnaeus, 1758) in the Gulf of Seronikos and Thermaikos[END_REF]. [START_REF] Campillo | Les pêcheries françaises de Méditerranée : synthèse des connaissances[END_REF] indicated that reproduction occurs from June to August in the Gulf of Lion (NW Mediterranean), while in the Aegean Sea Arslan and Ismen (2013) observed the highest GSI between January and May. However, increasing seawater temperature related to global change may impact the annual reproduction cycle of marine species [START_REF] Wootton R | Ecology of Teleost Fishes[END_REF][START_REF] Kousteni | Life-history traits of the striped red mullet Mullus surmuletus (Linnaeus, 1758) in the south Aegean Sea (eastern Mediterranean)[END_REF]. This might explain the gonad development stages (developing and maturing) observed during autumn (October to December) in this study. In the future, sampling should be completed with a larger number of individuals and cover all seasons at a larger spatial scale, from coastal to deeper waters, in order to confirm the annual reproduction cycle of M. surmuletus in the Var region.

In our study higher percentages of M. surmuletus reproducers were found in the AMA for both females and males compared to adjacent areas. Several studies reported the effects of marine reserve protection on the spawning biomass of different marine species (e.g. review by [START_REF] Roberts | Are marine reserves effective in management of reef fisheries?[END_REF]. By selecting size and targeting specific zones, fishing may influence population mean size, size distribution, sex ratio, age and size at maturity, and reproduction indices [START_REF] Rochet | Short-term effects of fishing on life history traits of fishes[END_REF]. For species such as M. surmuletus, highly targeted by commercial fisheries, information on morphometrics, population demography, growth and reproduction are essential for population regulation [START_REF] King | Marine fish life history strategies: applications to fishery management[END_REF][START_REF] Winemiller | Life history strategies, population regulation, and implications for fisheries management[END_REF]. Generally, improving knowledge of growth and the reproduction cycle as well as migration related to reproduction is necessary for sustainable fisheries management [START_REF] Mullen | Effects of movement on stock assessment in a restricted-range fishery[END_REF][START_REF] Machias | Bathymetric distribution and movements of red mullet (Mullus surmuletus)[END_REF].

The combination of the quality of the coastal and island habitats of the PCNP and the management measures and fishing practices in the PCNP AMA thus appear to have a positive effect on the mean total length of the surmullet population, its sex-ratio, and the gonadosomatic indices, potentially inducing a positive effect on reproduction and stock enhancement.

  This study was funded by the EMFF (European Fund for Maritime Fisheries Affairs), within the framework of the PACHA program. This study was performed in collaboration with professional fishermen and Port-Cros National Park. Thanks are due to Port-Cros National Park, Toulon fishing Prud'homies, Giens, Salins and Lavandou sections, and Stéphanie Joubert from the Var Departmental Maritime Fisheries Committee. Thanks are addressed to the association Planète Mer and the CDPMEM Var, within the framework of the PELA-Méd program (Fishermen Engaged for the Future of the Mediterranean), who contributed to this study by providing samples from the West and East zones of the PCNP AMA. D. Bănaru benefited from leave within the ANR CONTAMPUMP project (no ANR-19-CE34-0001). Thanks are also addressed to Michael Paul for English proofreading and to the two reviewers who helped us to improve the manuscript. REFERENCES ARSLAN M. & İŞMEN A., 2013. -Age, growth and reproduction of Mullus surmuletus (Linnaeus, 1758) in Saros Bay (Northern Aegean Sea). J. Black Sea / Medit. Environ., 19(2): 217-233. ASTRUCH P., BOUDOURESQUE C.-F., ROUANET É., LE DIREACH L., BONHOMME P., et al. 2018. -A quantitative and functional assessment of fish assemblages of the Port-Cros Archipelago (Port-Cros National Park, north-western Mediterranean Sea). Sci. Rep. Port-Cros Natl. Park, 32: 17-82. BARCELO A., BERNARDI P., BUZAUD C., CREUSEFOND M., DESPINOY P., GABRIEL J., HILY G., MAZZELLA C., MILLIER L., CRESP G., RIDOLFI J., COUPEZ L., CROSSETTI G., RENOUX S. & PRODROMIDES M., 2018. -Mode de gouvernance pour la gestion concertée du coeur marin de l'île de Porquerolles, au sein du Parc national de Port-Cros (Provence, France) : retour des acteurs sur 10 années de pratique. Sci. Rep. Port-Cros Natl. Park, 32: 83-111. BAUTISTA-VEGA A., 2007. -Etude des réseaux trophiques aboutissant aux rougets méditerranéens (Mullus barbatus et M. surmuletus) par l'utilisation des isotopes stables. Thèse Doctorat Sciences de l'Environnement marin, Université de la Méditerranée Aix-Marseille 2, 156 p. BAUTISTA-VEGA A.A., LETOURNEUR Y., HARMELIN-VIVIEN M. & SALEN-PICARD C., 2008. -Difference in diet and size-related trophic level in two sympatric fish species, the red mullets Mullus barbatus and Mullus surmuletus, in the Gulf of Lions (north-west Mediterranean Sea). J. Fish Biol., 73: 2402-2420. https://doi.org/10.1111/j.1095-8649.2008.02093. BENSAHLA-TALET L., BENSAHLA-TALET A. & ZITOUNI B., 2014. -Croissance relative pour quatre poissons (téléostéens : Sparidae, Mullidae) pêchés dans la baie d'Oran, Algérie (Méditerranée ouest). In : Troisième colloque international sur la biodiversité et écosystèmes littoraux (Chouikhi A., Boutiba Z., Mouffok S., Bouderbala M. éds.), Université d'Oran publ., Algérie, p 399-407. BOUDOURESQUE C.F., CADIOU G. & LE DIRÉACH L., 2005. -Marine protected areas: a tool for coastal areas management. In: Strategic management of marine ecosystems (Levner E., Linkov I. & Proth J.M. eds.), Springer publ., Dordrecht: 29-52. BOUDOURESQUE C.-F., DOMINICI J.-M., DURIEZ O., ASTRUCH P., LE DIRÉACH L, MÉDAIL F., SALA E., SCHOHN T. & VICENTE N., 2021. -A terrestrial and marine nature reserve in the NW Mediterranean, Scàndula (Corsica): Biodiversity and lessons from 46 years of management. Sci. Rep. Port-Cros Natl. Park, 35: 43-181.

Figures and tables legends

  Figures and tables legends

Figure 1 .

 1 Figure 1. -Sampling sites in West, AMA (Adjacent Marine Protected Area of Hyères Bay) and East zones on the south-eastern coast of France, NW Mediterranean sea.

Figure 2 .

 2 Figure 2. -Percentage of individuals of M. surmuletus analyzed by two-cm size class (total length in cm) and by sex. N = number of individuals, F = females, M = males, Unidentif. = unidentified group includes immature individuals and those whose sex could not be identified.

Figure 3 .

 3 Figure 3. -Mean (± SE) total length (TL, cm) of M. surmuletus in West, AMA and East zones. N = number of analyzed individuals per sex in zones.

Figure 4 .

 4 Figure 4. -Percentages of both sexes and females:males sex-ratio of Mullus surmuletus by zone (A) and season (B).

Figure 5 .

 5 Figure 5. -Mean gonado-somatic index (GSI, %) of Mullus surmuletus with size (LT, cm) by sex.

Figure 6 .

 6 Figure 6. -Mean gonado-somatic index (GSI, %) of males and females of Mullus surmuletus by A) zone and B) season.

Figure 7 .

 7 Figure 7. -Mean percentage of gonadal development stages of Mullus surmuletus by 2-cm size class (LT, cm) and sex for A) females and B) males. N = number of individuals.

Figure 8 .

 8 Figure 8. -Mean percentages of gonadal development stages (GDS) of Mullus surmuletus A) by zone for females B) by zone for males C) by season for females and D) by season for males. N = number of individuals.

  Figure 1

Table 1 .

 1 -Number of individuals analyzed by zone and season. For each zone and season, the number of individuals by month is detailed in italic characters.Table 2. -Mean (± SE) relative body condition (Kn) by sex, zone and season.

		Percentage
		100%
		90%
		80%
		70%
		50% 60%				Undet.
		40%				Females
		20% 30%				Males
		10%
		0%
			12 14 16 18 20 22 24 26 28 30 32
					Size classes (Total Length, cm)
		Sex-ratio
		F:M		4:1 2:1 2:1 2:1 3:1 5:1 15:1	2:1
		N =	2	6	26 65 58 47 24 16 12 6	1
	535	Figure 2

WEST AMA EAST