Guy Gorochov
email: guy.gorochov@sorbonne-universite.fr

Stephan Von Gunten
email: stephan.vongunten@pki.unibe.ch

Diversification of IgA Antibody Specificities by Mild Chemical Modification?

Intravenous immunoglobulin (IVIG) or subcutaneous immunoglobulin (SCIG) consisting mainly of IgG antibodies are used for substitution therapy in humoral immunodeficiency or as a high-dose treatment for inflammatory and autoimmune conditions. The antibodies in these preparations are derived from thousands of blood donors and collectively represent the donors' IgG repertoires, which in humans is both public and private [START_REF] Schneider | The human IgG anti-carbohydrate repertoire exhibits a universal architecture and contains specificity for microbial attachment sites[END_REF][START_REF] Luetscher | Unique repertoire of anti-carbohydrate antibodies in individual human serum[END_REF]. Mild chemical modification of antibodies has been proposed as one of several strategies to improve the efficacy of polyclonal immunoglobulin preparations [START_REF] Späth | Clinical Use and Therapeutic Potential of IVIG/SCIG, Plasma-Derived IgA or IgM, and Other Alternative Immunoglobulin Preparations[END_REF], as this approach can lead to further diversification of antibody specificities beyond somatic recombination, junctional diversity and somatic hypermutation [START_REF] Kanyavuz | Breaking the law: unconventional strategies for antibody diversification[END_REF]. Indeed, mild chemical modification of IVIG has shown to improve survival in experimental models of sepsis and aseptic systemic inflammatory response syndromes [START_REF] Djoumerska-Alexieva | Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes[END_REF], and to enhance pro-apoptotic effects on leukocytes under inflammatory conditions [START_REF] Graeter | Enhanced Pro-apoptotic Effects of Fe(II)-Modified IVIG on Human Neutrophils[END_REF].

While IVIG and SCIG preparations mainly consist of IgG antibodies, evidence suggests that IgA formulations might also be beneficial for clinical applications and exhibit idiosyncratic advantages [START_REF] Späth | Clinical Use and Therapeutic Potential of IVIG/SCIG, Plasma-Derived IgA or IgM, and Other Alternative Immunoglobulin Preparations[END_REF][START_REF] Sterlin | When Therapeutic IgA Antibodies Might Come of Age[END_REF]. The heavily N-glycosylated secretory component (SC) that stabilizes IgA also prevents rapid proteolysis of IgA dimers and multimers in the hostile environment of the digestive tract. Therefore, the oral route might one day be used for passive digestive transfer of specific or polyclonal IgA that could be very beneficial to IgA-deficient patients that presently do not benefit from any specific treatment [START_REF] Sterlin | The antibody/microbiota interface in health and disease[END_REF]. Any technical clue, as the one tested by Gorshkova et al. in this issue of Pharmacology [START_REF] Gorshkova | Induced polyspecificity of human secretory immunoglobulin A antibodies: Is it possible to improve their ability to bind pathogens?[END_REF], to further diversify anti-microbiota reactivity of future therapeutic IgA preparations, would therefore be more than welcome. Indeed, IgA polyreactivity appears to represent a key biological feature of this antibody, eventually accounting for its ability to regulate immune/microbiota homeostasis [START_REF] Sterlin | The antibody/microbiota interface in health and disease[END_REF][START_REF] Pabst | IgA and the intestinal microbiota: the importance of being specific[END_REF].

Gorshkova et al. report that protein-modifying agents can alter the repertoire of secretory IgA and that exposure to acidic pH broadens the range of reactivities to common and distinct pathogens [START_REF] Gorshkova | Induced polyspecificity of human secretory immunoglobulin A antibodies: Is it possible to improve their ability to bind pathogens?[END_REF]. Specifically, low pH treatment of colostrum and breast milk IgA broadened the recognition of bacterial antigens from E. coli and S.

aureus, but also Colo205 cell antigens, and increased IgA binding to viral antigens from hepatitis C or D virus, human immunodeficiency virus type 2 and norovirus.

However, the recognition of select bacterial antigens was also lost by low pH treatment. It remains unclear whether low pH treatment might partially denature some IgA, potentially leading to non-specific interactions. Conversely, it is also possible that bound materials that might co-purify with antibodies are removed by acid treatment, thereby offering more options for specific interactions. As opposed to low pH, treatment with ferrous ions had little or no effect on IgA binding to these antigens, while heme exposure increased reactivities of colostrum, but not breast milk, secretory IgA. Components of milk might affect the integrity of antibodies or block antibody reactivities, eventually requiring material processing prior to analysis [START_REF] Schneider | Isolation of antibodies from human plasma, saliva, breast milk, and gastrointestinal fluid[END_REF], such as casein depletion by sodium phosphates employed in the study by Gorshkova. However, the divergent effect of modifying treatment on colostrum and milk IgA by heme, highlights the potential importance of co-factors that might directly influence IgA reactivities or effects of mild protein-modifying agents on antibodies in a biological context. Microarray technology allows for the high-throughput analysis of antibody immunosignatures [START_REF] Schneider | The human IgG anti-carbohydrate repertoire exhibits a universal architecture and contains specificity for microbial attachment sites[END_REF][START_REF] Luetscher | Unique repertoire of anti-carbohydrate antibodies in individual human serum[END_REF][START_REF] Pashova | Antibody repertoire profiling with mimotope arrays[END_REF], and has been used to analyse reactivity profiles of IgA [START_REF] Sterlin | Human IgA binds a diverse array of commensal bacteria[END_REF][START_REF] Rollenske | Parallelism of intestinal secretory IgA shapes functional microbial fitness[END_REF]. Gorshkova et al. tested native or modified secretory IgA against an array of 4'345 linear B cell epitopes, and while decreased or increased binding patterns were observed upon modification by different agents, treatment with acidic pH enhanced the polyspecificity of IgA in particular for common pathogens [START_REF] Gorshkova | Induced polyspecificity of human secretory immunoglobulin A antibodies: Is it possible to improve their ability to bind pathogens?[END_REF]. It is therefore tempting to speculate from the results of Gorshkova al. that mere gastric exposure of IgA preparations might improve their efficacy. Further in vivo studies will now be needed to document how such modified IgA preparations might impact functional microbial fitness and host-microbial mutualism [START_REF] Rollenske | Parallelism of intestinal secretory IgA shapes functional microbial fitness[END_REF], in any sort of way. However, it is clear that that the report of Gorshkova and colleagues in this issue of Pharmacology is an important first step.

FIGURE LEGEND

Fig. 1 .

 1 Fig. 1. Proposed concept of enhanced reactivities of IgA under mild protein-

ACKNOWLEDGMENT

The authors thank Aldona von Gunten for the illustration.

DISCLOSURES

The authors have no conflicts of interest to declare.


Melina Abdou
email: melina.abdou@unige.ch

Jörg Schäfer

Teba Gil-Díaz

Mary-Lou Tercier-Waeber

Charlotte Catrouillet

Francesco Massa

Michela Castellano

Emanuele Magi

Paolo Povero

Gérard Blanc

Spatial variability and sources of platinum in a contaminated harbor -tracing coastal urban inputs

Keywords: Platinum, Technology Critical Element, emerging contaminant, seawater, suspended particulate matter, distribution coefficient, urban tracer, coastal environment

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

Introduction

Platinum (Pt) is a Technology Critical Element (TCE) and an emerging contaminant of growing concern according to evidences of anthropogenic disturbance on its global biogeochemical cycle [START_REF] Rudnick | Composition of the Continental Crust[END_REF][START_REF] Cobelo-García | COST action TD1407: network on technology-critical elements (NOTICE)--from environmental processes to human health threats[END_REF]. Among its specific properties, Pt is an excellent catalyst used in industrial processes, such as petroleum chemistry, but more importantly in vehicle catalytic converters to reduce gaseous pollutants in exhaust fumes.

Strongly increasing Pt demand for this technology results in growing anthropogenic Pt inputs into the environment, leading to a global increase of Pt concentrations in all environmental compartments including soils, atmosphere and aquatic systems (e.g. [START_REF] Rauch | Platinum Group Elements in Airborne Particles in Mexico City[END_REF][START_REF] Rauch | Platinum uptake by the freshwater isopod Asellus aquaticus in urban rivers[END_REF][START_REF] Schäfer | Time-Dependent Increase of Traffic-Emitted Platinum-Group Elements (PGE) in Different Environmental Compartments[END_REF]. However, other anthropogenic Pt sources (potentially implying the release of different Pt species to the environment) may occur in aquatic environments including Pt-derived anti-cancer drugs (e.g. [START_REF] Vyas | Platinum-based anticancer drugs in waste waters of a major UK hospital and predicted concentrations in recipient surface waters[END_REF].

Coastal areas are facing high anthropogenic pressure due to the increasing density of human population in such attractive zones [START_REF] Small | A global analysis of human settlement in coastal zones[END_REF]. Monitoring of trace metal concentrations in these sites is therefore essential to detect potential contamination and prevent associated adverse consequences on the ecosystem, especially for emerging contaminants.

Platinum concentration in seawater is difficult to quantify precisely, in particular in less contaminated sites, because of extremely low environmental levels (sub ng.L -1 ; [START_REF] Mashio | Dissolved Platinum Concentrations in Coastal Seawater: Boso to Sanriku Areas, Japan[END_REF]. Existing studies on marine coastal areas, including saline estuaries, report dissolved Pt concentrations ranging from 0.05 to 0.30 ng.L -1 [START_REF] Cobelo-García | Behavior of platinum during estuarine mixing (Pontevedra Ria, NW Iberian Peninsula)[END_REF], 2014a;[START_REF] Goldberg | Some comparative marine chemistries of platinum and iridium[END_REF][START_REF] Mashio | Dissolved platinum in rainwater, river water and seawater around Tokyo Bay and Otsuchi Bay in Japan[END_REF][START_REF] Mashio | Dissolved Platinum Concentrations in Coastal Seawater: Boso to Sanriku Areas, Japan[END_REF][START_REF] Obata | Determination of picomolar levels of platinum in estuarine waters: A comparison of cathodic stripping voltammetry and isotope dilutioninductively coupled plasma mass spectrometry[END_REF][START_REF] Turetta | Platinum group elements determination in seawater by ICP-SFMS: Initial results[END_REF][START_REF] Van Den Berg | The determination of platinum in sea water by adsorptive cathodic stripping voltammetry[END_REF]. A small number of studies report Pt concentrations in Suspended Particulate Matter (SPM) ranging from ~ 2 to 10 ng.g -1 in the Lérez Estuary and from ~ 0.2 to 1 ng.g -1 in the Gironde Estuary [START_REF] Cobelo-García | Behavior of platinum during estuarine mixing (Pontevedra Ria, NW Iberian Peninsula)[END_REF], 2014a). Variations in Pt concentrations and possible Pt enrichment in those coastal sites have been attributed to Pt inputs from human activities, especially in systems receiving anthropogenically-impa cted freshwater inputs, as observed in the Tokyo Bay (e.g. [START_REF] Obata | Determination of picomolar levels of platinum in estuarine waters: A comparison of cathodic stripping voltammetry and isotope dilutioninductively coupled plasma mass spectrometry[END_REF]. Several studies have addressed bioaccumulation of Pt in wild marine bivalves, such as oysters and mussels [START_REF] Abdou | Past and present platinum contamination of a major European fluvial-estuarine system: Insights from river sediments and estuarine oysters[END_REF][START_REF] Neira | Evidence of increased anthropogenic emissions of platinum: Time-series analysis of mussels (1991-2011) of an urban beach[END_REF]. However, there is a lack of data on the comprehensive characterization of the factors controlling the behavior of Pt in coastal seawater, including its partitioning between suspended particles and seawater, and huge uncertainties remain concerning its transport, bioavailability and fate in the coastal ocean [START_REF] Cobelo-García | Behavior of platinum during estuarine mixing (Pontevedra Ria, NW Iberian Peninsula)[END_REF].

The Mediterranean Sea is a semi-enclosed system under ever-growing anthropogenic pressure along its northwestern coast, comprising several highly populated cities with extensively urbanized and industrialized areas [START_REF] Martín | Recent accumulation of trace metals in sediments at the DYFAMED site (Northwestern Mediterranean Sea)[END_REF]. Considering the possible changes in seawater chemistry due to additional anthropogenic inputs, previous studies have addressed environmental quality in historical and industrial harbors such as the Toulon Bay or the Genoa Harbor, reflecting intense pollution by metals, (e.g. [START_REF] Oursel | Dynamics and fates of trace metals chronically input in a Mediterranean coastal zone impacted by a large urban area[END_REF][START_REF] Tessier | Study of the spatial and historical distribution of sediment inorganic contamination in the Toulon bay (France)[END_REF], metal-organic components [START_REF] Pougnet | Sources and historical record of tin and butyl-tin species in a Mediterranean bay (Toulon Bay, France)[END_REF] and nutrients [START_REF] Ruggieri | Seasonal and spatial variability of water quality parameters in the Port of Genoa, Italy, from 2000 to 2007[END_REF]. The Genoa Harbor on the northwest coast of Italy (Ligurian Sea) is one of the most important ports of the Mediterranean Sea, known for heavy contamination due to a variety of human activities.

For many years, industrial effluents were the major cause of water pollution in this site, before the closure of the main heavy industries adjacent to the harbor during the 1990s. Nowadays, discharges from sewage treatment plants and from creeks account for generally high levels of nutrients, organic matter and faecal pollution in the harbor waters [START_REF] Ruggieri | Seasonal and spatial variability of water quality parameters in the Port of Genoa, Italy, from 2000 to 2007[END_REF].

The present work provides first information on Pt levels and spatial distribution in this major Mediterranean harbor combined with physical-chemical parameters. Such findings may help to improve our understanding on Pt sources, other than the commonly assumed influence from catalytic converter emissions, and Pt cycling in an anthropogenically impacted coastal site. The objectives are therefore: (i) to quantify Pt levels and spatial distribution in a marine system under strong anthropogenic influence, involving different Pt sources, (ii) to characterize the factors controlling Pt distribution and partitioning between seawater and suspended particles considered a prerequisite for a better understanding of its biogeochemical cycle in coastal environments, and (iii) to investigate the potential use of Pt as an innovative tracer of human inputs (traffic, wastewaters, industrial activities) in coastal environments.

Experimental

Study area and sampling strategy

The coastline of Genoa

The Genoa Harbor is an artificial harbor on the northwest coast of Italy (44°24'39.8"N 8°55'44.8"E). The port infrastructures extend over approximately 7 million m² along about 20 km of coastline, protected by sea defenses and artificial breakwaters. The innermost, natural part of the Genoa Harbor is the so-called Old Port hosting a ferry terminal, several marinas and receiving urban treated sewage effluents [START_REF] Ruggieri | Seasonal and spatial variability of water quality parameters in the Port of Genoa, Italy, from 2000 to 2007[END_REF]Fig. 1). Outside the Old Port a wide, less confined area protected by artificial breakwaters extends to the east and to the west, hosting diverse industrial activities and receiving discharges from a coal-fired power plant, treated urban wastewater and untreated surface run-off [START_REF] Ruggieri | Seasonal and spatial variability of water quality parameters in the Port of Genoa, Italy, from 2000 to 2007[END_REF]. In addition, important dredging has affected the area in the last decade [START_REF] Cutroneo | The use of a combined monitoring system for following a turbid plume generated by dredging activities in a port[END_REF]. Two seasonally active rivers discharge freshwater and treated wastewater into the harbor area: the Polcevera River to the west and the Bisagno River to the east (Fig. 1; catchment surface area of 93 km 2 and 140 km 2 , respectively; [START_REF] Cutroneo | Environmental complexity of a port: Evidence from circulation of the water masses, and composition and contamination of bottom sediments[END_REF]). Both rivers, characterized by irregular and harsh streamflow fluctuations, run through the city of Genoa and are surrounded by dense urban structures hosting a variety of commercial and industrial activities.

Sampling campaigns

Two longitudinal profiles were performed to collect surface seawater and SPM samples inside and outside the Genoa Harbor on-board the Research Vessel MASO (3 rd -6 th April 2017; Fig. 1). The first profile was carried out from the inner part of the port towards the eastern entrance (sampling sites E1 to E6; Fig. 1). This represents a supposed decreasing contamination gradient covering the strongly confined Old Port (sites E1 and E2), a less confined zone inside the breakwaters (site E3), the eastern Inlet (site E4) close to the mouth of the Bisagno River and two sites outside the Harbor, directly in contact with open Mediterranean water, i.e. the Ligurian Current (sites E5 and E6). This profile is performed along the less industrialized section of the harbor. The second profile addressed the western part of the Genoa Harbor (sampling sites W1 to W6, Fig. 1) hosting coal deposits, a terminal for oil products (no storage tanks in the harbor), and receiving power plant discharges and inputs from the Polcevera River transporting sewage treatment plant effluents [START_REF] Ruggieri | Seasonal and spatial variability of water quality parameters in the Port of Genoa, Italy, from 2000 to 2007[END_REF].

Eastward profile was performed on April 3 rd under north-northeast wind with an average speed of 16 km.h -1 (Table 1; timeanddate.com). Westward profile was done the following day with weaker wind (~ 9 km.h -1 ) originating from south-southeast. Due to complex harbor shape, station bottom depth differs according to the location. Within the Old Port, site E1 has a bottom depth of 6 m (Table 1). Other sites within the breakwaters, show average depth of 11 m while it is of 16 m for E4 at the eastern inlet. We observe average depth of 14 m for external sites E5 and E6, and 26 m for W5 and W6. 1) on April 6 th 2017 at three different times of the day (10 am, 12 pm, 2 pm). The bottom depth of this sampling site is of 6 m (Table 1). This sampling site is located at the limit of the confined area of the harbor, supporting diverse harbor activities such as navigation, ship construction and wrecking industries. Main hospitals with oncology ward of Genoa are located rather close to this area, including an Oncology Clinic (~ 3 km).

Sample collection

Seawater and SPM samples were collected at all sites (i.e. on-board the R/V MASO or from the CNR Platform) by pumping surface water (~ 5 m depth) with a purpose-built low voltage (12 V) peristaltic pump connected to Teflon tubing. The latter was rinsed with seawater, accounting for more than three times the tubing volume (seawater pumping time of 10 min) prior to sample collection. Seawater was collected into acid-washed 500 mL Teflon bottles (Nalgene®), previously rinsed with pumped water at each site. Water samples for analyses of dissolved Pt (PtD) were filtered immediately through 0.2 μm polycarbonate filters (Nucleopore®) with a filter-syringe (Sartorius®) into acid-washed 60 mL Teflon bottles (Nalgene®), previously rinsed with an aliquot of the filtrate, then acidified to pH = 1 (36.5-38 % HCl Baker Instra) and stored in the dark at 4 °C pending analysis. Water aliquots from the 500 mL Teflon bottle were collected to quantify ammonium (NH4 + ) and dissolved organic carbon (DOC) concentrations. The samples for NH4 + analysis were filtered as aforementioned into pre-cleaned 15 mL polypropylene (PP) tubes (Kartell®), and stored frozen pending analysis. Aliquots for DOC were filtered through pre-combusted (500 °C, 4 h) glass-fiber filters (0.7 µm, Whatman® GF/F) using a pre-cleaned (RBS detergent, thoroughly rinsed with ultrapure deionised water, Milli-Q® water) and pre-combusted glass syringe. Filtrates were collected in pre-cleaned and pre-combusted glass bottles, acidified (HCl 0.3 % v/v; Baker, Analyzed) and then stored in the dark at 4 °C until analysis.

Particulate samples were collected for analyses of particulate platinum (PtP) and particulate organic carbon (POC). In both cases, the sampling bottle was carefully shaken and seawater was placed in a pre-rinsed filtration unit covered by a lid. For PtP, SPM were collected on preweighed Teflon filters (47 mm, 0.45 µm FHLC filter, Merck Millipore®), whereas for POC, SPM were collected on pre-combusted and pre-weighed glass-fiber filters (0.7 µm, Whatman® GF/F). Once in the laboratory, both filters were then oven-dried (48 h at 50 °C) to constant weight, re-weighed (to determine SPM content) and kept in the dark at room temperature pending analysis. A field blank was performed for all sampled parameters and processed in the same way as seawater samples but using Milli-Q water.

Analytical methods

Platinum in seawater

Seawater samples were analyzed by Adsorptive Cathodic Stripping Voltammetry (AdCSV) as described in Cobelo-García et al. (2014b). Measurements were carried out using a μAutolab Type III potentiostat (Metrohm®) connected to a polarographic stand (Metrohm® 663 VA Stand) equipped with three electrodes: (i) a hanging mercury drop electrode (HMDE, the working electrode), (ii) a silver/silver-chloride (Ag/AgCl) reference electrode, and (iii) a glassy carbon auxiliary electrode. A polytetrafluoroethylene (PTFE) voltammetric cell served in all experiments and the potentiostat was controlled using the NOVA 2.1® software. Elimination of organic matter by UV oxidation was performed by placing sample aliquots in capped Teflon bottles with overnight irradiation using two 64 W UV lamps (NIQ 60/35 XL, Heraeus) under a fume hood. Aliquots (10 mL) of UV-digested sample were pipetted into the voltammetric cell together with 30 µL of 3.3 mM formaldehyde (37-41 % Analytical Reagent Grade, Fisher Chemical®), 30 µL of 0.45 mM hydrazine sulfate (Analytical Reagent Grade, Fisher Chemical®), and 300 µL of sulfuric acid (H2SO4, 93 -98 % Trace metal grade, Fisher Chemical®). Platinum concentrations were quantified by standard internal addition method, adding a controlled amount of a Pt stock solution freshly prepared daily from a monoelementary Pt standard solution (1000 μg.mL -1 PLASMACAL, SCP Science®). We applied a deposition time of 300 s and experimental parameters as described elsewhere (Cobelo-García et al., 2014b). In the absence of Certified Reference Material (CRM) for dissolved Pt in seawater, analytical quality of the voltammetric procedure was evaluated by means of Pt spiked CRM seawater quantification (CASS-6, NRCC) giving recoveries > 80% and precision expressed as Relative Standard Deviation (RSD) lower than 10 % (n = 5) at the 0.5 ng.L -1 range.

The detection limit (D.L.) for dissolved Pt measured by AdCSV (calculated as 3 x blank standard deviation, n = 20) was estimated to 0.03 ng.L -1 . Satisfactory results were obtained for field blank with PtD of 0.05 ng.L -1 .

Platinum in SPM

Particulate platinum concentrations (PtP) in Suspended Particulate Matter samples were analyzed by Triple Quadrupole-Inductively Coupled Plasma-Mass Spectrometry (TQ-ICP-MS, Thermo® iCAP TQ). Teflon filters with SPM (typical mass of ~ 3 -5 mg) were acid digested as described in [START_REF] Abdou | Tracing platinum accumulation kinetics in oyster Crassostrea gigas, a sentinel species in coastal marine environments[END_REF]. Briefly, 2 mL HCl and 1 mL HNO3 (30 % HCl and 65 % HNO3 Suprapur, Merck®) were added to the samples in polypropylene (PP) tubes (DigiTUBEs, SCP SCIENCE®) and closed with caps. Samples were placed in a Teflon-coated heating block, and digested at 110 °C for 3 h. Cooled contents were then diluted in 10 mL Milli-Q water, centrifuged at 4000 rpm for 10 min (20 °C) and filters were discarded. Analyses were performed applying the standard internal addition method (as previously explained). After obtaining the raw signal, Hafnium-oxide (HfO + ) interferences were monitored using the 193 Ir signal that is highly interfered by HfO + [START_REF] Djingova | On the determination of platinum group elements in environmental materials by inductively coupled plasma mass spectrometry and microwave digestion[END_REF]. Raw signal was thereafter mathematically corrected using 194 Pt / 195 Pt natural ratio and assuming that HfO + is the only/dominant spectral interference on those selected isotopes. Analytical quality control was performed using the BCR®-723 road dust (Institute for Reference Materials and Measurements) and Jsd-2 sedimentary rocks (indicative value from Geological Survey of Japan) as available CRMs for Pt concentrations. Analyses of these CRMs by TQ-ICP-MS gave satisfactory recovery values of 87% and 101% respectively (n = 3) and precision expressed as RSD of ~ 5 %. The detection limit (D.L.) for PtP (n = 10) was estimated to 0.34 ng.g -1 for typical sediment sample mass of 4 mg. Satisfactory results were obtained for field blank with PtP below detection limit.

Complementary measurements

Parallel to sampling, other master variables (i.e. temperature, dissolved oxygen, salinity, and turbidity) as well as chlorophyll-a (Chl-a) concentrations were measured in situ using a submersible probe (OS316Plus, Idronaut®), previously calibrated on-board the R/V MASO or in an on-site laboratory on the CNR Platform. Salinity was measured using the Practical Salinity Scale and Turbidity with Nephelometric Turbidity Units. Seawater pH and redox potential (Eh) were determined with a Sentix® 41 probe (PROFILINE, WTW) and a PH-25 probe (CRISON®), both previously calibrated using the manufacturer's specifications and supplied reagents (NIST pH standards).

Dissolved organic carbon (DOC) was analyzed with an automated analyzer (TOC-L, Shimadzu®) as described in [START_REF] Sharp | Reevaluation of high temperature combustion and chemical oxidation measurements of dissolved organic carbon in seawater[END_REF]. Accuracy of DOC measurements was tested using reference material (Deep Sea Reference, DSR, University of Miami) providing recovery results > 90 %. Analytical precision expressed as %RSD was 5 % at the 0.5 mg.L -1 range. The D.L. (n = 10) was estimated to 0.06 mg.L -1 . Particulate organic carbon (POC) was analyzed with a LECO© CS-125 after carbonate elimination as described elsewhere (e.g. [START_REF] Schäfer | Ten-year observation of the Gironde tributary fluvial system: Fluxes of suspended matter, particulate organic carbon and cadmium[END_REF]. Analytical quality was checked by measuring CRMs (e.g. LECO 501-503). Accuracy was within 5 % of the certified values and the analytical error generally < 5 % (% RSD). Ammonium concentrations were determined by colorimetric procedure (visible-UV spectrophotometer, Thermo®, Genesys 20, [START_REF] Aminot | Determination of ammonia in seawater by the indophenol-blue method: Evaluation of the ICES NUTS I/C 5 questionnaire[END_REF]. Analytical error of the method is < 5 % (% RSD), accuracy within 5 % of the reference values, and the D.L. of 18 µg.L -1 . Satisfactory results were obtained for field blank values of DOC, POC and NH4 + (~ 0.2 mg.L -1 , 0.04 %, and ~ 30 µg.L -1 respectively).

Data treatment

The correlation matrix for the different parameters was produced using the software PAST® applying the linear r (Pearson) parametric correlation coefficient. The statistical significance (p-value) was set to 0.05. Surfer® software (Golden Software Inc., ver. 13) was used to produce the distribution maps of the analytical results and the data interpolation was performed using Minimum Curvature algorithm. Due to the complexity of the port area, a fault traces file of the port shape (Fig. 1) and a spatial resolution of 5 m were used to perform the calculation gridding.

The interpolated variable limits were fixed to minimum/maximum of the measured data for the PtD map; and from 0 until the maximum of the measured data for the PtP map. For each interpolation map, the software returns the coefficient of multiple determination (R 2 ), which represents the goodness of fit of the model. When the regression equation fits well to the data, R 2 is large and close to 1.

Particle-water distribution coefficients, KD, defining the concentration ratios of PtP over PtD were calculated according to the equation ( 1)

KD = [PtP] / [PtD] x 10 3
(1)

Results

The results obtained for major physical-chemical parameters and complementary measurements are represented for two distinct longitudinal profiles, both starting from inside the Genoa harbor and going towards the open Mediterranean Sea along the east and west part of the coastline (Table 1) 8.14 and 8.24, with highest values for external sites in both profiles (Table 1). Oxygen saturation levels, DOC, and POC ranged between ~ 70 % and 98 %; 0.7 and 2.0 mg.L -1 ; and 1 % to 5 % respectively. An increasing gradient occurred for O2 saturation eastward from inner to outer sites while levels of DOC, POC%, Chl-a and NH4 + decreased (except for one higher POC% value at site E5). Chlorophyll-a and NH4 + values at sites E5 and E6 were of ~ 0.3 µg.L -1 and below the D.L. (18 µg.L -1 ), respectively. In contrast, the westward profile showed relatively constant values for O2 saturation (~ 90 %). Elevated Chl-a, DOC, and POC % levels tended to occur for stations inside the breakwaters, while most NH4 + concentrations were below the D.L.

Dissolved Pt concentrations ranged from 0.08 ng.L -1 to 0.18 ng.L -1 (Table 1) and showed clear concentration differences between sites inside the harbor and sites outside the breakwaters (Fig. 2A). Despite the distance from the stations, a coefficient of multiple determination of 0.51 was obtained for PtD map that clearly confirms the observed gradient (Fig. 2A). Westward profile showed somewhat similar PtD concentrations at the sites W1 to W3, all inside the breakwaters, and a decrease towards the most distant site of this profile (~ 0.10 ng.L -1 at W6).

The eastward profile displayed an increasing PtD gradient from ~ 0.10 ng.L -1 at E1 to 0.18 ng.L -1 at E4 and a sharp drop towards lower values at sites E5 and E6 (0.08 ng.L -1 ;

Table 1).

A B

The observed PtP concentrations ranged from below D.L. (< 0.34 ng.g -1 ) up to ~ 3 ng.g -1 (Table 1). A coefficient of multiple determination of 0.61 was obtained for the PtP map. Along the westward profile, PtP concentrations were relatively constant and low (< 1 ng.g -1 ; Fig. 2C) with values below D.L. in sites W4 and W6. In the eastward profile, PtP were highest in the Old Port (site E1), and showed an overall decreasing trend towards the outer sites reaching values below D.L. at sites E5 and E6 (PtP level at site E3 was also below the D.L.).

Calculated particle-water distribution coefficients varied from 2 x 10 3 to 23 x 10 3 and showed eastward and westward gradients similar to those observed for PtP concentration variations (Table 1).

The relationships between spatial variations of PtD and PtP concentrations and the other parameters measured are displayed separately for each profile only for the sites within the breakwaters (Table 2). Despite the low number of samples, results of the correlation matrix suggest that linear correlations exist between selected master variables and Pt levels in both the dissolved and particulate phases along the profiles (Table 2). Eastward, PtD concentrations seemed to be slightly positively correlated (r ~ 0.6) with wind speed and pH, and significantly positively correlated (r > 0.9) with Eh and O2. These concentrations were significantly negatively correlated with turbidity, DOC, POC Chl-a, and with NH4 + levels (r < -0.9).

Opposite behavior occurred for PtP concentrations. In the westward profile, PtD concentrations which were weakly positively correlated with turbidity, Eh, (r > 0.8) and pH (r ~ 0.6) and negatively correlated with O2% (r ~ -0.6). Again PtP concentrations showed opposite behavior with significant correlations (Table 2). . Platinum concentrations at the CNR Platform (fixed site PF; Fig. 1) measured at three different times of the day showed variations for both PtD (0.09 to 0.15 ng.L -1 ) and PtP (3.1 to 14 ng.g -1 ;

Table 3), with highest concentrations at 12 pm. Dissolved Pt concentrations were in the same range as those measured along the profiles, whereas relatively higher PtP levels occurred, leading to higher KD values. During the measurements at the CNR Platform, wind direction changed from north to south with relatively weak wind speed, similar to those during the sampling of the westward profile. The master variables at the CNR Platform were in the same range as levels observed at other internal sites (Tables 1 and3). Only dissolved O2 saturation values were somewhat lower than those measured along the longitudinal profiles and were close to those in the Old Port site (E1; Table 1). Dissolved Organic Carbon was also in the lower range compared to longitudinal profiles, with values similar to those observed at site E4.

Ammonium values were close to those quantified during the westward profile.

Discussion

Water masses in the harbor and Pt distribution

Inside the harbor, water mass characteristics are supposedly strongly influenced by the wind/wave conditions, the freshwater supply by the two main water courses (the Polcevera and Bisagno Rivers), and run-off in the port area [START_REF] Capello | Physical and sedimentological characterisation of dredged sediments[END_REF]. Such physical parameters may influence Pt distribution in the Genoa Harbor. [START_REF] Cutroneo | The use of a combined monitoring system for following a turbid plume generated by dredging activities in a port[END_REF] report the formation of eddies in the current formed by wind in the Genoa area. The relative shallowness of the port area (Table 1) further accentuates the influence of the wind on the entire water column [START_REF] Capello | Physical and sedimentological characterisation of dredged sediments[END_REF]. Higher wind speed as recorded during the first day (Table 1) was correlated with at all internal sites and behind the breakwaters (except site E3), whereas for the external sites from outside the breakwaters PtP were below D.L. (< 0.34 ng.g -1 ; Fig. 2B). The PtP levels inside the Genoa Harbor were similar to those reported for the Lérez Estuary (~ 1 to 15 ng.g -1 ), interpreted as anthropogenic Pt enrichment [START_REF] Cobelo-García | Behavior of platinum during estuarine mixing (Pontevedra Ria, NW Iberian Peninsula)[END_REF]. Such enrichment may partly originate from small watercourses flowing into the harbor (Fig. 1), even though salinity measurements show little influence of freshwater inputs. Since sampling took place at ~ 5 m depth i.e. below the main influence of freshwater torrents [START_REF] Capello | Changes in the physical characteristics of the water column at the mouth of a torrent during an extreme rainfall event[END_REF] and no strong rainfall occurred the previous days (only 3.8 mm on April 2 nd ), our salinity records mainly reflect seawater influence. In the inner part of the Old Port, the semi closed shape and shallow bottom (< 10 m depth; Table 1) of the harbor make the water exchange times rather long, in particular close to the bottom depth (corresponding to E1 sampling point). [START_REF] Povero | Ports of Genoa: Old Port, Multedo Oil Terminal and Voltri Container Terminal[END_REF] calculated a mean of 20 days for a complete water exchange in this inner part and 7 days in the outer system (central basin and eastern entrance), which is wider and ~ 10 m deeper. Although both calculated water residence times are not representative of the whole area (because they do not consider its irregular shape) and refer mainly to the eastward transect, they can help explaining the Pt enrichment in the inner part of the harbor. They may also explain the decreasing gradient towards the outer basin and the open sea, together with dilution affecting the generally stagnant harbor waters when flowing outside. Moreover, inside the harbor, important dredging activities were undertaken from 2009 to 2014 and are, to a lesser extent, still carried out in the recent years. Since then, turbid plumes, influenced by wind/wave conditions, have been recorded [START_REF] Cutroneo | The use of a combined monitoring system for following a turbid plume generated by dredging activities in a port[END_REF]. Intensification of big ships and ferry transit and berthing increase the presence of turbid plumes [START_REF] Capello | Physical and sedimentological characterisation of dredged sediments[END_REF], suggesting that sediment resuspension may represent a source of trace metals, including Pt, in the system.

Anthropogenic Pt sources

Industrial activities

Along the eastern profile, from the inner to the outer part of the harbor, PtD concentrations tended to increase with a maximum at site E4, close to the mouth of the Bisagno River (Fig. 2A), while the highest PtD in the westward profile occurred inside the breakwaters, including site W3 (0.17 ng.L -1 ), i.e. near the Polcevera River mouth. The western part of the harbor hosts coal deposits and a terminal for oil products (Fig. 1). Indeed, westward the Polcevera River mouth, the Multedo oil terminal is one of the most important in Italy and Europe. It handles crude oil, final products (petrol, gasoline, fuel oil) semi-manufactured, and basic petrochemical products, for a maximum total capacity of 30 million tons per year [START_REF] Recchia | Risk Communication and Public Perception of Technological Hazards (Part Two)[END_REF]. Platinum is commonly used as a catalytic agent in oil industry (e.g. catalytic cracking of crude petroleum; [START_REF] Peavy | The importance of platinum in petroleum refining[END_REF] and such activities could therefore be a source of Pt inputs into the coastal system.

Built in 1952, the coal power plant of Genoa is also located on the west side of the harbor. Coal industry may contribute to local Pt concentrations, since high Pt concentrations have been reported for coal samples (~ 5 ng.g -1 , Finkelman and Aruscavage, 1981; up to 70 ng.g -1 , [START_REF] Dai | Concentrations and origins of platinum group elements in Late Paleozoic coals of China[END_REF]. The bulk terminal of the harbor containing coal (dry bulk) and oil (liquid bulk) is also located in the western area [START_REF] Cutroneo | Environmental complexity of a port: Evidence from circulation of the water masses, and composition and contamination of bottom sediments[END_REF]. Historical intense oil and coal-based industrial activities in the Genoa Harbor and erosion/leaching of such deposits stored uncovered against wind and/or rain might lead to the presence of industrial coal particles in the suspended particulate matter around the Polcevera River mouth and in the Sampierdarena channel that connects the outer basin of the Old Port and Polcevera River mouth. Although the Pt concentrations in this oil or coal are unknown, one cannot exclude that oil inputs, industrial coal and/or fly ashes could contribute to the Pt enrichment at this sampling site given the range of values reported in the literature. Since 1953, a steel mill has also been present in the western area and related activities cause the release of particulate matter of different sizes [START_REF] Mazzei | Elemental composition and source apportionment of particulate matter near a steel plant in Genoa (Italy)[END_REF]. Metallurgic activities can be a source of Pt release into aquatic environments as recorded in other systems (e.g. Gironde fluvial estuarine continuum; [START_REF] Abdou | Past and present platinum contamination of a major European fluvial-estuarine system: Insights from river sediments and estuarine oysters[END_REF].

Furthermore, in urban systems, other possible Pt sources may arise from direct inputs of dental laboratories, electronic industries, glass manufacturing, Pt-containing pharmaceuticals or industrial catalyst productions [START_REF] Kümmerer | European hospitals as a source for platinum in the environment in comparison with other sources[END_REF].

Hospital and sewage effluents

Platinum-based compounds have been successfully applied in anti-cancer chemotherapy for decades (e.g. [START_REF] Lenz | Fate of cancerostatic platinum compounds in biological wastewater treatment of hospital effluents[END_REF]. Surface waters draining effluents from hospital with oncology ward contain patient excretion of Pt-based anticancer drugs and display Pt concentrations that could exceed natural inputs [START_REF] Vyas | Platinum-based anticancer drugs in waste waters of a major UK hospital and predicted concentrations in recipient surface waters[END_REF]. [START_REF] Monteiro | Improved voltammetric method for simultaneous determination of Pt and Rh using second derivative signal transformationapplication to environmental samples[END_REF] evaluated PtD concentrations in the inflow and outflow of a wastewater treatment plant (WWTP) in Lisbon to ~ 5 and 15 ng.L -1 respectively after a dry weather period and ~ 10 and 25 ng.L -1 after a raining event. In the western part of the Genoa Harbor, Pt inputs from sewage treatment plants reach the Polcevera river mouth (Fig. 1). Despite probable dilution in natural waters compared to levels previously reported, such inputs may add to Pt sources affecting the sampling site W3

(PtD ~ 0.18 ng.L -1 ). Small sewage effluents also occur directly in the Old Port (Fig. 1).

Considering only the sites E1 to E4 located inside the relatively stagnant harbor waterbody (due to confinement and low tidal amplitudes; [START_REF] Cutroneo | Environmental complexity of a port: Evidence from circulation of the water masses, and composition and contamination of bottom sediments[END_REF][START_REF] Cutroneo | The use of a combined monitoring system for following a turbid plume generated by dredging activities in a port[END_REF], the eastward gradients of Pt concentrations have been correlated with parameters reflecting sewage effluent signature.

Inside the harbor, PtD concentrations were positively correlated with dissolved oxygen (r > 0.9) and pH (r > 0.6), and negatively correlated with turbidity, Chl-a, % POC, DOC, and NH4 + (r < -0.9). The opposite trends occurred for PtP concentrations, which were positively correlated with Chl-a, % POC, DOC, and NH4 + . Assuming that these major variables serve as proxies for wastewater inputs and degradation [START_REF] Ruggieri | Seasonal and spatial variability of water quality parameters in the Port of Genoa, Italy, from 2000 to 2007[END_REF], this could explain Pt distribution inside the Old Port. Accordingly, supply of discharged sewage rich in organic matter and poor in oxygen content typically occur into the harbor basins [START_REF] Ruggieri | Seasonal and spatial variability of water quality parameters in the Port of Genoa, Italy, from 2000 to 2007[END_REF]. The CNR platform (site PF) and surrounding waters are close to the urban area hosting a relevant center for Medical Oncology (red cross/blue circle). Highest levels of PtP (14 ng.g -1 ) are recorded at this sampling site and relatively higher levels of PtD (0.15 ng.L -1 ) both at 12 pm.

Traffic-related emissions

The Genoa Harbor is in direct vicinity (< 5 km distance) from the urban highway and very close to urban heavy traffic roads, a likely source for Pt bearing particles. Automobile catalysts, which are thought to be the principal Pt anthropogenic source, have atmospheric residence times ranging from hours to days depending on particle size and density and can be transported from regional to longer distances [START_REF] Rauch | Platinum Group Elements in Airborne Particles in Mexico City[END_REF][START_REF] Rauch | Source characterisation of atmospheric platinum group element deposition into an ombrotrophic peat bog[END_REF]. Dust and suspended matter with Pt particles are thereafter removed from the atmosphere by wet (rainfall) or dry (gravitation) deposition [START_REF] Pawlak | Fate of platinum metals in the environment[END_REF]. For other trace metals such as copper (Cu), enhanced atmospheric input of Cu-rich urban particles showing great water solubility may result in the predominance of the atmospheric input of Cu over the river input [START_REF] El Sayed | Nutrients and trace metals in the northwestern Mediterranean under coastal upwelling conditions[END_REF].

Therefore, in addition to causing water mixing, wind can be a vector of Pt into aquatic systems.

The two wind situations observed during our study are typical of the Genoa area. Winds from the N-NE (0-30° N) and SE (135-150° N) are two of the most frequent wind situations in winter and summer, respectively [START_REF] Cutroneo | The use of a combined monitoring system for following a turbid plume generated by dredging activities in a port[END_REF]. Our field campaign took place in spring, a period of the year generally featuring the two situations. The eastward profile was sampled under relatively strong N-NE winds causing the currents in the entire harbor to flow outwards [START_REF] Cutroneo | The use of a combined monitoring system for following a turbid plume generated by dredging activities in a port[END_REF]. Relatively clear gradients of all monitored parameters can be explained by such conditions. Recent data confirm the importance of atmospheric deposition in Pt enrichment of oceanic surface waters [START_REF] López-Sánchez | New insights on the dissolved platinum behavior in the Atlantic Ocean[END_REF]. Automobile catalyst exhaust Pt inputs come from the land and Pt-bearing particles can be transported by the wind causing a gradient of PtP concentrations eastward. In the Genoa Harbor system, such particles could originate from catalyst exhaust fumes and/or industrial fly ashes. On the reverse, the westward profile was carried out under weaker, SE winds and showed less clear parameter variations. The E and SE winds generate a westward current forcing the surface harbor waters inside, while only in the bottom layer a weak outflow can occur [START_REF] Cutroneo | Environmental complexity of a port: Evidence from circulation of the water masses, and composition and contamination of bottom sediments[END_REF][START_REF] Cutroneo | The use of a combined monitoring system for following a turbid plume generated by dredging activities in a port[END_REF].

However, due to the morphological complexity and the port structures jutting out in all directions, there is an extreme variability in the current direction in the port [START_REF] Capello | Physical and sedimentological characterisation of dredged sediments[END_REF] and more data would be needed covering longer time-scales to confirm the present assumptions. If such source types influence Pt partitioning and its spatial distribution, they also may affect Pt levels both in historical records (e.g. [START_REF] Abdou | Platinum in sediments and mussels from the northwestern Mediterranean coast: Temporal and spatial aspects[END_REF][START_REF] Abdou | Past and present platinum contamination of a major European fluvial-estuarine system: Insights from river sediments and estuarine oysters[END_REF] and at short temporal scales as observed in site PF.

Platinum reactivity and partitioning

Platinum seawater speciation

Platinum concentrations in both dissolved and particulate phases are controlled by several factors implying various biogeochemical processes. Assuming that Pt originates from multiple potential point sources prevailing at different sites it is likely that Pt speciation differs from site to site. Organic forms might be represented by the three Pt-based drugs mainly used in the hospital: cisplatin, carboplatin and oxaliplatin along with their metabolites, while Pt emitted from vehicles or industrial activities may be under inorganic forms. Hospital effluents transport dissolved Pt ranging from < 10 ng.L -1 to 250 µg.L -1 [START_REF] Rauch | Sources of Platinum Group Elements in the Environment[END_REF][START_REF] Vyas | Platinum-based anticancer drugs in waste waters of a major UK hospital and predicted concentrations in recipient surface waters[END_REF], mainly excreted from these anticancer drugs and/or derived metabolites.

Such excretion also continues outside the hospital, as more and more patients go home directly after the treatment to the extent that the majority of Pt is emitted from outpatients at home [START_REF] Lenz | Fate of cancerostatic platinum compounds in biological wastewater treatment of hospital effluents[END_REF][START_REF] Vyas | Platinum-based anticancer drugs in waste waters of a major UK hospital and predicted concentrations in recipient surface waters[END_REF]. The phase in which Pt species are released as well as the dissolved/particulate partitioning in sewage and the related removal efficiency in WWTPs remain widely unknown. Previous studies suppose that municipal and hospital effluents contain carboplatin (the most stable drug) as well as degradation products being the monoaquacisplatin and the diaquacisplatin, from anti-cancer drug administration [START_REF] Lenz | Fate of cancerostatic platinum compounds in biological wastewater treatment of hospital effluents[END_REF][START_REF] Vyas | Platinum-based anticancer drugs in waste waters of a major UK hospital and predicted concentrations in recipient surface waters[END_REF]. Recent work reports that the behavior of organic Pt species in recipient waters is governed by chloride (Cl -) concentrations with, for cisplatin, decreasing proportion of reactive species with increasing salinity [START_REF] Curtis | Speciation and Reactivity of Cisplatin in River Water and Seawater[END_REF][START_REF] Turner | Particle-water interactions of platinum-based anticancer drugs in river water and estuarine water[END_REF]. [START_REF] Curtis | Speciation and Reactivity of Cisplatin in River Water and Seawater[END_REF] proved that environmental conditions favoring cisplatin retention and its adsorption on particles are turbid waters of low chlorinity and long residence time. Dispersal is likely to occur in rapidly flushed waters under low turbidity conditions and high chlorinity. Our study site is characterized by high salinities that may limit cisplatin adsorption, but turbid plumes arriving from sewage effluents and long water and particle residence times inside the breakwaters may in turn favor such process. More investigation is needed under similar environmental conditions.

Both run-off and sewage also contain PGE-bearing catalytic converter fragments with intact PGE-load [START_REF] Prichard | Identification of Platinum and Palladium Particles Emitted from Vehicles and Dispersed into the Surface Environment[END_REF], which may undergo partial dissolution in contact with seawater. The few data on Pt speciation in seawater suggest that Pt(IV) is the most important oxidation state [START_REF] Cobelo-García | Behavior of platinum during estuarine mixing (Pontevedra Ria, NW Iberian Peninsula)[END_REF]. Laboratory works showed that its inorganic equilibrium speciation is dominated by the stable PtCl5(OH) 2-complex [START_REF] Gammons | Experimental investigations of the hydrothermal geochemistry of platinum and palladium: V. Equilibria between platinum metal, Pt(II), and Pt(IV) chloride complexes at 25 to 300°C[END_REF]Cobelo-García et al., 2014a). Laboratory and field observations report decreasing particulateaqueous distribution coefficient for inorganic forms with increasing salinity (Cobelo-García et al., 2014, 2008;[START_REF] Turner | Particle-water interactions of platinum group elements under estuarine conditions[END_REF], suggesting increasing Pt levels in the dissolved phase as Cl - concentrations increase. The observed eastward particulate/dissolved gradients suggest Pt dissolution from solid carrier phases, either road dust [START_REF] Sebek | Dissolution kinetics of Pd and Pt from automobile catalysts by naturally occurring complexing agents[END_REF] or small sewage particles, and subsequent dilution of PtD by seawater that would be favored by Pt-Cl complex formation.

Potential carrier phases of Pt in sites with anthropogenic influence

Given the above, one would expect inputs of different co-existing Pt forms from different overlapping sources into the marine environment with wastewater, run-off of the surrounding area and atmospheric deposition. Numerous discharges occur in the Genoa Harbor area, comprising release of unknown and potentially dangerous substances (Fig. 1) that may contribute to Pt contamination. A rough mass balance (PtD = PtP x SPM x 10 -3 ) for the eastern profile inside the harbor suggests that Pt release from suspended particles would account for a PtD addition of ~ 0.02 ng.L -1 , whereas the observed PtD values increased by ~ 0.06 ng.L -1 . The amount of Pt presumably transferred from the particulate to the dissolved phase is not contradictory to the field data but does not explain all the PtD addition observed, supporting the idea of additional PtD inputs through the different discharges (Fig. 1). Outside the breakwaters, biogeochemical equilibration processes and the resulting higher PtD would be masked by

massive dilution with open Mediterranean water. Biogeochemical processes, inherent to anthropogenic discharges and the mixing of environmental waters can widely modify the original Pt phase. Previous work has shown that relatively high dissolved metal loads in wastewater inputs equilibrate in the recipient waters. Efficient adsorption on high complexation site particles induces profound changes in element partitioning between the dissolved and particulate phases (e.g. [START_REF] Deycard | Contributions and potential impacts of seven priority substances (As, Cd, Cu, Cr, Ni, Pb, and Zn) to a major European Estuary (Gironde Estuary, France) from urban wastewater[END_REF]. Indeed, the residual POC in effluent wastewater mainly consists of reactive (degradable) organic matter, once exposed to natural microorganism communities in the receiving waterbody [START_REF] Deycard | Inputs, dynamics and potential impacts of silver (Ag) from urban wastewater to a highly turbid estuary (SW France)[END_REF]. Such organic matter and especially that containing thiols and reduced sulfur groups, may strongly bind soft metals and potentially compete with inorganic ligands such as Cl -from the recipient waters [START_REF] Levard | Effect of Chloride on the Dissolution Rate of Silver Nanoparticles and Toxicity to E. coli[END_REF][START_REF] Wood | The interaction of dissolved platinum with fulvic acid and simple organic acid analogues in aqueous solutions[END_REF]. Literature suggests that, considering cisplatin flocculation in natural water, complexation with specific organic ligands containing nitrogen or sulphur is likely (e.g. [START_REF] Curtis | Speciation and Reactivity of Cisplatin in River Water and Seawater[END_REF][START_REF] Turner | Particle-water interactions of platinum-based anticancer drugs in river water and estuarine water[END_REF]. This means that Pt partitioning between the two phases might be partly influenced by interactions with POC from wastewater discharges when mixing with coastal waters, although the degree of surface complexation with organic matter remains unknown [START_REF] Couceiro | Adsorption and desorption kinetics of rhodium (III) and platinum (IV) in turbid suspensions: Potential tracers for sediment transport in estuarine flumes[END_REF]. Natural sediments may also contain POC and laboratory experiments have proven that Pt adsorption kinetics onto estuarine sediments is greatest for cohesive sediments with relatively high total carbon contents [START_REF] Couceiro | Adsorption and desorption kinetics of rhodium (III) and platinum (IV) in turbid suspensions: Potential tracers for sediment transport in estuarine flumes[END_REF]. Another experimental study showed that complexation with organic ligands can be critical to particle-water fractionation of Pt discharged into aquatic systems, removal from the dissolved phase being most favorable when entering a turbid watercourse [START_REF] Turner | Adsorption Kinetics of Platinum Group Elements in River Water[END_REF].

All of these observations seem in line with the reported highest PtP levels co-occurring with high turbidity sewage inputs in the Genoa Harbor (Tables 1 and3; Fig. 2B). Depth profiles of PtD concentrations in the Atlantic Ocean also showed a higher particle-reactivity or lower solubility of PtD with decreasing oxygen levels [START_REF] López-Sánchez | New insights on the dissolved platinum behavior in the Atlantic Ocean[END_REF].

The related daily variations in KD values at site PF (Table 3) suggest that the observed KD might not describe equilibrium conditions. However, the average KD values obtained for the Genoa Harbor (~ 8 x 10 3 ) are consistent with values obtained for the seawater end-member of the Gironde Estuary (~ 4 x 10 3 , Cobelo-García et al., 2014a), and the maximum KD value recorded at the CNR Platform (~ 1 x 10 5 ) is similar to those found in the seawater end-member of the Lérez Estuary [START_REF] Cobelo-García | Behavior of platinum during estuarine mixing (Pontevedra Ria, NW Iberian Peninsula)[END_REF]. Outside the Old Port, but still inside the harbor, the growing influence of open seawater with a decrease in turbidity, as well as change in the nature of SPM surfaces (decrease in %POC) could explain low PtP concentrations (below detection limit), going along with increasing PtD. Considering possible wind effect and water column mixing, remobilization of Pt from particles through the degradation of organic matter and redox reactions (correlation of Pt levels with pH and Eh parameters; Table 2) seems to contribute to PtD enrichment at those sampling sites, as observed elsewhere for Pt and other trace metals (Almécija et al., 2016;[START_REF] El Sayed | Nutrients and trace metals in the northwestern Mediterranean under coastal upwelling conditions[END_REF]. However, multiple solid phases other than solely POC can also decrease PtD concentrations. Inorganic mineral phases such as iron and manganese oxides may also be important Pt carrier phases [START_REF] Colodner | Post-depositional mobility of platinum, iridium and rhenium in marine sediments[END_REF]. In fact, those phases could be more important than organic material for the removal of cisplatin degradation products [START_REF] Turner | Particle-water interactions of platinum-based anticancer drugs in river water and estuarine water[END_REF].

Whatever the dissolved or particulate phase transporting Pt, this element has proven to be bioavailable to marine organisms (e.g. [START_REF] Rauch | Platinum uptake by the freshwater isopod Asellus aquaticus in urban rivers[END_REF]. It is crucial to better understand Pt speciation and distribution, since the physical and chemical forms of this emerging contaminant are expected to considerably influence its biological availability. In fact, previous work on Pt toxicity in marine bivalves has shown strong accumulation and the onset of defense mechanisms, for exposure levels that might be reached in case of acute pollution, especially in semi-closed coastal systems [START_REF] Abdou | Tracing platinum accumulation kinetics in oyster Crassostrea gigas, a sentinel species in coastal marine environments[END_REF].

Coastal anthropogenic contamination -Pt as a novel urban tracer

Although the geochemical cycles of Pt in coastal environments under high urban/industrial pressure are far from being fully understood (due to the limited number of data), our study, combined to already existing works, allows to draw perspectives for future research in this field.

Assuming the dominance of Pt organic forms released by hospital effluents, monitoring of Pt concentrations may serve as a tracer for these effluents release and spreading in the environment together with other proxies commonly used such as POC% or nutrient levels. Main substances expected in effluents are the most stable anti-cancer compounds (including carboplatin) and degradation products from the different drugs [START_REF] Lenz | Fate of cancerostatic platinum compounds in biological wastewater treatment of hospital effluents[END_REF][START_REF] Vyas | Platinum-based anticancer drugs in waste waters of a major UK hospital and predicted concentrations in recipient surface waters[END_REF]. However, important behavior differences (reactivity, phase affinity, removal…) are observed among Ptbased drugs and their various degradation products [START_REF] Turner | Particle-water interactions of platinum-based anticancer drugs in river water and estuarine water[END_REF]. Such organic Pt sources need monitoring and characterization, especially considering the increasing cancer incidence and the related increasing demand for chemotherapy treatment in developed countries [START_REF] Johnson | Do cytotoxic chemotherapy drugs discharged into rivers pose a risk to the environment and human health? An overview and UK case study[END_REF]. Inorganic forms of Pt are assumed to dominate in catalytic converters emissions. High resolution monitoring of concentrations of those forms could therefore reflect atmospheric particle deposition and traffic-related urban contamination in coastal areas.

Inorganic Pt forms may also reflect atmospheric contamination related to industrial activities including oil and coal-based industries. The lack of knowledge regarding Pt speciation is directly related to analytical challenges inherent to the ultra-trace environmental levels of this emerging metallic contaminant. Future work should focus on developing analytical techniques for routine Pt quantification in seawater for both dissolved and particulate phases including speciation analyses.

Spatial distribution of Pt concentrations in the Genoa Harbor showed disparities partly depending on urban source proximity, yet, temporal aspects also may be crucial. Dredging activities, daily cycles in traffic intensity, industrial emissions, and/or wastewater composition [START_REF] Deycard | Contributions and potential impacts of seven priority substances (As, Cd, Cu, Cr, Ni, Pb, and Zn) to a major European Estuary (Gironde Estuary, France) from urban wastewater[END_REF][START_REF] Mazzei | Elemental composition and source apportionment of particulate matter near a steel plant in Genoa (Italy)[END_REF][START_REF] Kümmerer | Hospital effluents as a source for platinum in the environment[END_REF], as well as wind conditions could lead to variable fluxes of Pt into the harbor waters explaining why PtP varied by a factor of ~ 5 (i.e. from 3 to 14 ng.g -1 at site PF) within few hours. Spatial and temporal variations in Pt levels inside the Genoa Harbor may therefore reflect both distance from the source and release dynamics of a wide range of different potential sources.

Platinum distribution therefore appears to vary according to urban pollution type, source and intensity, suggesting that this element can serve as an innovative and sensitive tracer of anthropogenic impact from multiple urban sources (traffic, sewage, hospital effluents). The present dataset suggests that such anthropogenic inputs supplied to rivers or directly in the harbor by urban and/or industrial wastewater discharges and by atmospheric deposition may represent important local Pt point sources to coastal waters. Platinum levels observed in the Genoa Harbor waters are far lower than concentrations measured in highly contaminated waters including polluted rivers (up to ~ 7 ng.L -1 in Tokyo Bay urban rivers; [START_REF] Obata | Determination of picomolar levels of platinum in estuarine waters: A comparison of cathodic stripping voltammetry and isotope dilutioninductively coupled plasma mass spectrometry[END_REF] or even hospital effluents (up to 250 µg.L -1 ; [START_REF] Lenz | Fate of cancerostatic platinum compounds in biological wastewater treatment of hospital effluents[END_REF][START_REF] Vyas | Platinum-based anticancer drugs in waste waters of a major UK hospital and predicted concentrations in recipient surface waters[END_REF]

Fig. 1 :

 1 Fig. 1: Sampling sites in the Genoa Harbor. Two sampling profiles were performed with an eastward profile (sites E1 to E6) and a westward profile (sites W1 to W6). Sampling site PF (PlatForm) corresponds to the CNR Platform. Numbers correspond to main urban/industrial activities with 1: Steel mill, 2: Bulk containers, 3: Coal power plant, 4: Oil storage, 5: Ferry terminal, 6: Shipyards/marinas. Red crosses indicate hospitals with oncology ward with the Oncology Clinics highlighted in blue. Colored circles show discharges from various sources. Geospatial data provided by the Ligurian Administrative Region via own Geoportal -WFS service (https://geoportal.regiosne.liguria.it/). Satellite image provided by ESRI© (ArgGIS/Imagery)

  . A clear decreasing gradient occurred for turbidity values from inside towards the open sea eastward while westward profile featured no clear trend. Temperature values varied between 14.5 °C and 15.0 °C with a trend for higher temperature for sampling sites located inside the breakwaters. For both profiles, lowest temperatures occurred for external sites being E6 and W6. For the eastern profile, salinity (S) of ~ 37.7 in the inner harbor, compared to S = 38.0 in the outmost sites, suggested a ~ 1 % mixing of freshwater. Westward, only the site W1 showed lower salinity (37.8), whereas other sites showed average salinity of ~ 38. Redox potential (Eh) showed an increasing trend for eastward inner sites with highest value of 211 mV for site E4, while at external sites E5 and E6, lower values (~ 190 mV) occurred. Westward, Eh values did not show any particular trend (~ 180 mV), yet slightly higher values at external sites W5 and W6 (~ 204 mV). Measurements of pH varied between

Fig. 2 :

 2 Fig. 2: Dissolved and particulate Pt concentrations along the two longitudinal profiles in the Genoa Harbor. A: spatial distribution of dissolved Pt concentrations (PtD; ng.L -1 ); and B: spatial distribution of particulate Pt concentrations (PtP; ng.g -1 ).

  O2 saturation and turbidity (r ~ 0.8 and -0.8 respectively; data not shown). This result suggests an influence of wind speed on water mixing along the eastward profile. Along the westward profile, weaker winds (coming from the south and probably stopped by breakwaters) showed no correlation with O2 saturation levels. Considering only the eastward profile, increasing wind and subsequent water mixing is correlated with increasing PtD and decreasing PtP levels (r ~ 0.6 and -0.9 respectively). In the inner harbor, strong wind could lead to vertical and horizontal seawater mixing, sediment resuspension, and redistribution of dissolved and especially particulate components throughout the water column as observed in other studies(e.g. El Sayed et al., 1994; see Section 4.3.). The two profiles sampled westward and eastward from the Old Port show elevated PtD concentrations within the whole harbor area, compared to the external sites (E5, E6 and W5, W6). The lowest PtD values occurred at the sites E5 and E6 (Fig. 2A) mainly influenced by open Mediterranean water transported with the east-west oriented northern Ligurian Current.Therefore, the PtD concentrations in seawater from these sites (~ 0.08 ng.L -1 ) may be considered as representative of surface water in the northwestern Mediterranean Sea. This assumption is supported by identical PtD levels of 0.08 ng.L -1 in surface waters from an open-sea site, south from the Balearic Islands[START_REF] Abdou | Tracing platinum accumulation kinetics in oyster Crassostrea gigas, a sentinel species in coastal marine environments[END_REF]. Open Mediterranean surface water PtD concentrations appear somewhat higher than average PtD in the North Atlantic seawater reported in the early nineties (~ 0.05 ng.L -1 ;[START_REF] Colodner | The Marine Geochemistry of Rhenium, Iridium and Platinum[END_REF] and recently confirmed by[START_REF] López-Sánchez | New insights on the dissolved platinum behavior in the Atlantic Ocean[END_REF] for North, Central and South West Atlantic Ocean. Values are also higher than those reported in the northwest Pacific Ocean (~ 0.04 ng.L -1 ;[START_REF] Suzuki | Precise determination of dissolved platinum in seawater of the Japan Sea, Sea of Okhotsk and western North Pacific Ocean[END_REF]. However, the supposed Mediterranean surface water PtD is similar to values in the coastal North Atlantic Ocean off the slightly Pt-contaminated Gironde Estuary (0.08 ng.L -1 ;Cobelo-García et al., 2014a) and lies within the range of values reported for the Lérez Estuary seawater end-member (from 0.08 to 0.10 g.L -1 ;[START_REF] Cobelo-García | Behavior of platinum during estuarine mixing (Pontevedra Ria, NW Iberian Peninsula)[END_REF]. These observations are consistent with expected relatively higher Pt inputs into the semi-enclosed Mediterranean Sea due to the strong urbanization of its coastline. Similarly, the western profile shows the lowest PtD values (i.e. ~ 0.10 ng.L -1 ; Fig.2A) at the sites located outside the breakwaters (W5 and W6). These concentrations are slightly higher than open Mediterranean PtD, suggesting minor local contamination off the harbor and 'downstream' from the sites E5 and E6 (with respect to the dominant westward Ligurian Current). Conversely, inside the breakwaters, contamination is consistently higher with PtD concentrations up to more than 2-fold the open Mediterranean value (Fig.2A).Particulate Pt concentrations in suspended particles partly reflect the general concentration gradient from inside the harbor towards the open sea. They were measurable (~ 0.4 to 3 ng.g -1 )

  For the same sample, sewage-related signals are recorded, with clearly lower O2 saturation and highest POC levels compared to the other sites. Sites E1 and PF are also characterized by a more confined area with shallower bottom depth (~ 6 m depth). High levels of PtP and relatively low levels of PtD, where sewage inputs are at maximum (site E1) might be related to: (i) the presence of originally high PtP concentrations in wastewaters or run-off, and/or (ii) the adsorption of PtD on organic-rich ligands present in wastewaters (high concentrations of dissolved and particulate organic carbon in the turbid sewage plume). The effect of sediment resuspension in these shallower sampling sites is not discarded. Kinetics of suspected adsorption/desorption processes may explain the observed differences in PtD and PtP distributions between sites (see Section 4.3.).

Figure captions Fig. 1 :

 captions1 Figure captions

Fig. 2 :

 2 Fig. 2: Dissolved and particulate Pt concentrations along the two longitudinal profiles in the Genoa Harbor. A: spatial distribution of dissolved Pt concentrations (PtD; ng.L -1 ); and B: spatial distribution of particulate Pt concentrations (PtP; ng.g -1 ).

Table 1 :

 1 . It remains unclear to what extent dilution of Pt concentrations by recipient seawater or the absence of rainy events during our sample collection could have resulted in relatively low Pt levels. Future research and field sampling should therefore consider sample collection both near pollution sources (i.e.rivers, urban discharges, sewage effluents) and in recipient coastal waters in order to perform relevant mass balance calculations. This should be done at high spatial as well as high temporal resolutions in order to fully understand Pt cycles in urban coastal environments.With TCE increasing environmental release being a global concern, this work provides knowledge on the extent of Pt contamination and biogeochemical processes occurring in a representative urban coastal site. The first information on Pt distribution and reactivity of both dissolved and particulate phases in an industrialized and urbanized Mediterranean harbor is given. Measured concentrations for PtD (~ 0.08 to 0.18 ng.L -1 ) and PtP (< 0.34 to 14 ng.g -1 ) allowed proposing PtD ~ 0.08 ng.L -1 (as measured in the Ligurian Current and south from the Balearic Islands) as a first reference value for western Mediterranean surface seawater. There is a clear evidence of anthropogenic Pt contamination at all sites studied within the Genoa Harbor showing distinct concentration gradients. They are attributed to different overlapping local sources such as industry, run-off, hospital effluents, domestic wastewater as well as physical parameters such as wind and dust / catalyst particles atmospheric deposition. Gradients in both PtD and PtP as well as major variables attributed to input and degradation of urban sewage inside the relatively stagnant harbor waters reflect biogeochemical processes modifying the nature, surface properties and abundance of suspended particles, leading to profound changes in Pt partitioning and carrier phases. While quantification of Pt emitted from vehicle exhaust, supposedly under inorganic form, rise attention, organic forms may also be of major concern in urbanized coastal systems. Such mobilization of anthropogenic Pt can enhance its bioavailability, bio-uptake and induce adverse effects on marine organisms. The role of biogeochemical processes and responses from marine wildlife to contamination, including potential Pt complexation to biologically-derived organic ligands such as algae exudates, are still unknown and need further investigation. On the other hand, this study opens the perspective of using Pt as an innovative tracer of urban inputs that should be applied in other urbanized coastal areas of the Mediterranean Sea and worldwide. Dissolved and particulate Pt concentrations, Pt distribution coefficients, and master variables monitored along two longitudinal profiles in the Genoa Harbor. The eastward profile corresponds to sites E1 to E6, with E1 being located in the Old Port, while the westward profile corresponds to sites W1 to W6. With: Dissolved Pt concentration (PtD; ng.L -1 ), Particulate Pt concentration (PtP; ng.g -1 ), Particle-water distribution coefficient (KD), Wind direction (Wind dir.), Wind speed (Wind sp.; km.h -1 ), Bottom depth (m), Turbidity (Turb.; NTU), Temperature (Temp; °C), Salinity, Redox potential (Eh; mV), pH, dissolved oxygen saturation (O2%), Dissolved Organic Carbon (DOC; mg/L), Particulate Organic Carbon (POC%), Chlorophyll-a (Chl-a; µg/L), ammonium (NH4 + ; µg/L), d.l.: detection limits, n.d.: not determined. Turb. 2.23 7.38 2.89 5.17 2.51 4.17 6.47 4.85 3.88 3.52 2.10 2.13 Temp. 14.61 14.82 14.88 14.80 14.84 15.02 14.94 14.84 14.85 14.96 14.50 14.88 Salinity 38.0 37.9 38.0 38.0 38.0 37.8 37.8 37.7 37.8 37.8 38.1 38.0

	5. Conclusions

Table 2 :

 2 Correlation table of PtD and PtP concentrations with master variables. Values correspond to Pearson's r coefficient. n.d.: not determined. Stars correspond to a significant level of 0.05 (p-value<0.05). Note that only the sites inside the breakwaters are considered.

		Eastward profile (E1 to E4) Westward profile (W1 to W4)
		PtD	PtP	PtD	PtP
	Wind sp.	0.59	-0.90	0.43	0.52
	Turb.	-0.95*	1.00*	0.80	-0.12
	Temp.	0.15	-0.14	-0.07	-0.99*
	Salinity	0.43	-0.34	-0.39	0.99*
	Eh	0.96*	-0.93	0.83	1.00*
	pH	0.63	-0.86	0.63	1.00*
	O2%	0.92*	-0.98*	-0.62	0.10
	DOC	-0.99*	1.00*	-0.35	-0.87
	POC%	-0.90*	0.94	0.36	0.79
	Chl-a	-0.91*	1.00*	0.33	0.12
	NH4 +	-0.97*	0.99*	n.d.	n.d.

Table 3 :

 3 Dissolved and particulate Pt concentrations, particle-water distribution coefficients, and master variables at the CNR Platform (site PF) at 10 am, 12 pm, and 2 pm. Units of the various parameters are as reported in Table1.

	Param./Time	10 am	12 pm	2 pm
	PtD	0.09	0.15	0.11
	PtP	7.7	14	3.1
	KD (x10 3 )	82	94	27
	Wind dir.	N	SSE	S
	Wind sp.	4	9	7
	Bottom depth	6	6	6
	Turb.	4.00	2.60	4.60
	Temp.	14.76	14.66	14.73
	Salinity	38.0	38.0	38.0
	Eh	190	182	191
	pH	8.20	8.20	8.21
	O2%	81.9	82.8	87.1
	DOC	1.01	0.68	0.63
	POC%	2.50	5.15	2.02
	Chl-a	1.00	1.40	1.60
	NH4 +	47.0	48.1	44.8

Table 2 :

 2 Correlation table of PtD and PtP concentrations with master variables. Values correspond to Pearson's r coefficient. n.d.: not determined. Stars correspond to a significant level of 0.05 (p-value<0.05). Note that only the sites inside the breakwaterss are considered.

Table 3 :

 3 Dissolved and particulate Pt concentrations, particle-water distribution coefficients, and master variables at the CNR Platform (site PF) at 10 am, 12 pm, and 2 pm. Turb.: turbidity. Units of the various parameters are as reported in Table1.

Acknowledgements

This work has greatly benefited from the financial support by the FEDER Aquitaine-1999-Z0061, the COST Action TD1407, and the EU FP7 Ocean 2013.2 Project SCHeMA (Project-Grant Agreement 614002), which is gratefully acknowledged. Authors are also thankful for the help of the UNIGE-It Team, the DISTAV, and the RV/MASO crew for sampling and assistance. Analytical support from Dominique Poirier, Céline Charbonnier and Aude Charrier is greatly acknowledged.

Conflicts of interest

The authors declare no conflicts of interest.