Daniel F Araujo

Emmanuel Ponzevera

Nicolas Briant

Joel Knoery

Teddy Sireau

Meryem Mojtahid

Édouard Metzger

Christophe Brach-Papa

Joël Knoery

Meryem Mojtahid²

; Edouard Metzger

Assessment of the metal contamination evolution in the Loire estuary using Cu and Zn stable isotopes and geochemical data in sediments

Keywords: coastal pollution, coastal biogeochemistry, geochemical tracers, metal isotopes, trace metal, anthropogenic contamination

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

Introduction

Estuarine ecosystems are of great importance to human development and human wellbeing.

However the increasing population and intense exchange of material and energy in the coastal zones make them sensitive and vulnerable to the chemical contamination and environmental degradation [START_REF] Bianchi | Biogeochemistry of Estuaries[END_REF]Agardy and Alder, 2008;[START_REF] De Souza Machado | Metal fate and effects in estuaries: A review and conceptual model for better understanding of toxicity[END_REF][START_REF] Lu | Major threats of pollution and climate change to global coastal ecosystems and enhanced management for sustainability[END_REF].

Particularly, the anthropogenic contamination of trace metals such as copper (Cu), zinc (Zn), lead (Pb), cadmium (Cd), silver (Ag) and mercury (Hg) is of great concern in conjunction with their concentrations and speciation, which could confer them toxicity and impose major risks for human and biota health [START_REF] Adriano | Trace Elements in Terrestrial Environments[END_REF][START_REF] Hardy | Technical aspects of trace element supplementation[END_REF][START_REF] Long | Incidence of adverse biological effects within ranges of chemical concentrations in marine and estuarine sediments[END_REF]. Trace metals are released by anthropogenic activities (e.g. agriculture, industry, mining, metallurgy, energy production and transport) and dispersed among the different natural compartments such as the soil [START_REF] Babcsányi | Copper in soil fractions and runoff in a vineyard catchment: Insights from copper stable isotopes[END_REF][START_REF] Bigalke | Stable Cu and Zn isotope ratios as tracers of sources and transport of Cu and Zn in contaminated soil[END_REF]Fekiacova et al., 2015a;[START_REF] Juillot | Contrasting isotopic signatures between anthropogenic and geogenic Zn and evidence for post-depositional fractionation processes in smelter-impacted soils from Northern France[END_REF], the water (Araújo et al., 2017a;[START_REF] Borrok | Isotopic variations of dissolved copper and zinc in stream waters affected by historical mining[END_REF][START_REF] Chen | Zinc isotopes in the Seine River waters, France: A probe of anthropogenic contamination[END_REF][START_REF] Skierszkan | Molybdenum and zinc stable isotope variation in mining waste rock drainage and waste rock at the Antamina mine[END_REF]Thapalia et al., 2015) and the atmosphere [START_REF] Dong | Isotopic signatures suggest important contributions from recycled gasoline, road dust and non-exhaust traffic sources for copper, zinc and lead in PM10in London, United Kingdom[END_REF][START_REF] Gonzalez | New Insights from Zinc and Copper Isotopic Compositions into the Sources of Atmospheric Particulate Matter from Two Major European Cities[END_REF][START_REF] Novak | Cu-Zn isotope constraints on the provenance of air pollution in Central Europe: Using soluble and insoluble particles in snow and rime[END_REF][START_REF] Souto-Oliveira | Multi-isotopic fingerprints (Pb, Zn, Cu) applied for urban aerosol source apportionment and discrimination[END_REF]. To develop correct environmental management strategies, either for remediation actions appropriately targeted as well as to maintain emissions at sustainable levels, it is imperative to identify their sources, to understand their transport and fates in the environment and to examine at what extent the anthropogenic activities affect their biogeochemical cycling.

To this end, the studies of Cu and Zn isotope biogeochemistry have been promising [START_REF] Weiss | Atmospheric deposition and isotope biogeochemistry of zinc in ombrotrophic peat[END_REF]. In surface environments, the relative abundance of these isotopes can change during several biogeochemical processes including weathering and pedogenesis [START_REF] Bigalke | Stable Cu isotope fractionation in soils during oxic weathering and podzolization[END_REF][START_REF] Fernandez | Fractionation of Cu, Fe, and Zn isotopes during the oxidative weathering of sulfide-rich rocks[END_REF][START_REF] Suhr | Elemental and isotopic behaviour of Zn in Deccan basalt weathering profiles: Chemical weathering from bedrock to laterite and links to Zn deficiency in tropical soils[END_REF][START_REF] Vance | The behaviour of Cu and Zn isotopes during soil development: Controls on the dissolved load of rivers[END_REF], mineral precipitation/dissolution [START_REF] Veeramani | Zinc isotope fractionation as an indicator of geochemical attenuation processes[END_REF][START_REF] Wall | Cu isotope fractionation during bornite dissolution: An in situ X-ray diffraction analysis[END_REF], adsorption on solid surfaces [START_REF] Bryan | Zinc isotope fractionation during adsorption onto Mn oxyhydroxide at low and high ionic strength[END_REF][START_REF] Dong | Zinc isotope fractionation during adsorption to calcite at high and low ionic strength[END_REF][START_REF] Guinoiseau | Zn Isotope Fractionation during Sorption onto Kaolinite[END_REF], ionexchange [START_REF] Maréchal | Ion-exchange fractionation of copper and zinc isotopes[END_REF], cellular homeostasis [START_REF] Caldelas | Zinc isotopic fractionation in Phragmites australis in response to toxic levels of zinc[END_REF][START_REF] Wanty | In vivo isotopic fractionation of zinc and biodynamic modeling yield insights into detoxification mechanisms in the mayfly Neocloeon triangulifer[END_REF], redox reactions and ore refining (smelting and electroplating, [START_REF] Kavner | Redox-driven stable isotope fractionation in transition metals: Application to Zn electroplating[END_REF]Sivry et al., 2008a;[START_REF] Sonke | A chemical and mineralogical reconstruction of Zn-smelter emissions in the Kempen region (Belgium), based on organic pool sediment cores[END_REF][START_REF] Yin | Zn isotopes fractionation during slags' weathering: One source of contamination, multiple isotopic signatures[END_REF][START_REF] Yin | Application of Zn isotopes in environmental impact assessment of Zn-Pb metallurgical industries: A mini review[END_REF]. The different mechanisms of Cu and Zn isotope fractionation, e.g, via kinetic or thermodynamic equilibrium, and the significant isotopic signature variability observed among different natural compartments and materials, enable these isotopes to be potential environmental indicators useful to detect trends of pollution over time periods, identifying sources of contamination and elucidate contaminant transport mechanisms [START_REF] Babcsányi | Copper in soil fractions and runoff in a vineyard catchment: Insights from copper stable isotopes[END_REF][START_REF] El Azzi | Origin and fate of copper in a small Mediterranean vineyard catchment: New insights from combined chemical extraction and δ65Cu isotopic composition[END_REF][START_REF] Petit | The estuarine geochemical reactivity of Zn isotopes and its relevance for the biomonitoring of anthropogenic Zn and Cd contaminations from metallurgical activities: Example of the Gironde fluvial-estuarine system, France[END_REF]Sivry et al., 2008;Thapalia et al., 2015).

Sediments are the main reservoir of trace metal contaminants in aquatic environments, and thus, frequently used to diagnostic the degree of anthropogenic inputs. In coastal areas, Cu and Zn isotope compositions of sediments are mainly controlled by mixing source processes of detrital-continental, marine-autochthonous and anthropogenic materials (Araújo et al., 2017b;[START_REF] Zhang | Historical records, distributions and sources of mercury and zinc in sediments of East China sea: Implication from stable isotopic compositions[END_REF]. In Sepetiba bay (Brazil), a site historically contaminated by electroplating wastes, Zn isotope signatures of sediment samples fit well in a ternary mixing model involving detrital terrigenous, anthropogenic and marine authigenic sources (Araújo et al., 2017a,b). In the Gironde fluvial-estuarine system (France), Cu isotope compositions of suspended particulate matter were consistent with a binary mixing processes involving geogenic and anthropogenic inputs, this last one possibly related to the use of Cu sulfate fungicides in the vineyards surrounding the fluvial-estuarine system [START_REF] Petit | Anthropogenic sources and biogeochemical reactivity of particulate and dissolved Cu isotopes in the turbidity gradient of the Garonne River (France)[END_REF].

In this work, we investigate spatial and temporal variations on Cu and Zn isotope compositions and trace metal concentrations of bulk sediments in the Loire estuary (France).

This estuarine system has been a receptacle of chronic metal and nutrients inputs of multiple diffuse sources hosted in its fluvial-estuarine watershed [START_REF] Moatar | La Loire fluviale et estuarinne: un milieu en évolution[END_REF].

Consequently, increases in metal concentration and eutrophication processes have been well documented [START_REF] Briant | Spatial and temporal distribution of mercury and methylmercury in bivalves from the French coastline[END_REF][START_REF] Dhivert | Dynamics of metallic contaminants at a basin scale -Spatial and temporal reconstruction from four sediment cores (Loire fluvial system, France)[END_REF][START_REF] Minaudo | Eutrophication mitigation in rivers: 30 Years of trends in spatial and seasonal patterns of biogeochemistry of the Loire River[END_REF]. Despite a tendency of attenuation of anthropogenic inputs in the Loire river watershed in the last years [START_REF] Dhivert | Dynamics of metallic contaminants at a basin scale -Spatial and temporal reconstruction from four sediment cores (Loire fluvial system, France)[END_REF], metal source identification and apportionment remain unknown or incipient for most part of metal contaminants. This is in part due to the complexity of discriminating multiple sources that emerged in the past, as it is the case for Cu and Zn [START_REF] Grosbois | Severe and contrasted polymetallic contamination patterns (1900-2009) in the Loire River sediments (France)[END_REF], and also due to the difficulty to differentiate between natural and anthropogenically-influenced elements at concentrations close to geochemical baselines.

To overcome such limitations and drawbacks, we propose a combination of Cu and Zn isotope information and a multi-elemental "fingerprinting" approach of trace elements (Pb, Cd, Hg, Ag, Cu and Zn) to assess the trace metal contamination levels and evolution in the Loire estuary. We expect that spatial and temporal patterns of trace metal concentrations coupled to Cu and Zn isotope ratios on sediments can be useful to deconvolve past and current anthropogenic inputs to the estuarine system. Copper is redox sensitive and widely used as a component of agricultural fungicides [START_REF] Babcsányi | Copper in soil fractions and runoff in a vineyard catchment: Insights from copper stable isotopes[END_REF][START_REF] Babcsányi | Copper stable isotopes to trace copper behavior in wetland systems[END_REF][START_REF] El Azzi | Origin and fate of copper in a small Mediterranean vineyard catchment: New insights from combined chemical extraction and δ65Cu isotopic composition[END_REF], while Zn is redox-insensitive and a contaminant frequently associated to urban and industrial contamination [START_REF] Chen | Zn isotopes in the suspended load of the Seine River, France: Isotopic variations and source determination[END_REF][START_REF] Souto-Oliveira | Multi-isotopic fingerprints (Pb, Zn, Cu) applied for urban aerosol source apportionment and discrimination[END_REF].

Methods

Study area

Located in the North Bay of Biscay continental shelf, the Loire estuary (~106 km long) is the outlet the Loire River (1012 Km, surface area of 117,054 km 2 , mean annual water discharge of 846 m 3 /s), one of the largest river systems in Western Europe flowing into the Atlantic ocean (Grobois et al., 2012;[START_REF] Coynel | Spatial distribution of trace elements in the surface sediments of a major European estuary (Loire Estuary, France): Source identification and evaluation of anthropogenic contribution[END_REF]. It drains crystalline and volcanic bedrocks of the southern central and western Armorican massifs, and the sedimentary bedrocks of the central Paris Basin Unit. The Loire basin has integrated along its secular history several economical activities. Firstly, based on textile, leather and fur tanning in the 16 th century, and then based on exploitation of geological resources (mining and metallurgy), raw materials and energy production from the industrial revolution in the 19 th century until present.

The Loire Estuary is a macrotidal system with a maximum tidal amplitude of ~6 m at the river mouth (Saint Nazaire; Fig. 1) and a water residence time estimated between 3 and 30 days during flood and low water discharge periods, respectively [START_REF] Coynel | Spatial distribution of trace elements in the surface sediments of a major European estuary (Loire Estuary, France): Source identification and evaluation of anthropogenic contribution[END_REF][START_REF] Gallenne | Study of fine material in suspension in the estuary of the Loire and its dynamic grading[END_REF]. The Loire Estuary basin is highly industrialized and urbanized comprising (i) the metropole region of Nantes and Saint-Nazaire cities with its more than 800,000 inhabitants, (ii) the 4 th French seaport (Nantes-Saint-Nazaire harbour), (iii) one of the biggest French refinery of crude oil (Donges with a capacity of 11.5 million tons/year), (iii) the largest coal-fired thermal electricity production in France (Cordemais), and (iv) various industrial activities such as gas storage, aircraft industry, shipyards, etc. [START_REF] Coynel | Spatial distribution of trace elements in the surface sediments of a major European estuary (Loire Estuary, France): Source identification and evaluation of anthropogenic contribution[END_REF][START_REF] Dhivert | Dynamics of metallic contaminants at a basin scale -Spatial and temporal reconstruction from four sediment cores (Loire fluvial system, France)[END_REF]. In addition, along the Loire river and its tributaries (Sèvre river), extend one of the main wine producers regions of France ("Loire Valley"). This overall growing economic activity has been driving a continuous industrial, agricultural and urban expansion and, consequently, imposing major risks to the coastal ecosystem [START_REF] Moatar | La Loire fluviale et estuarinne: un milieu en évolution[END_REF].

Sampling and sample preparation

Seventeen surface sediments sites along the Loire estuary were considered for this study (Fig. 1). The corresponding surface sediments were acquired from the sample bank of the french monitoring framework ROCCH "Réseau d'Observation de la Contamination Chimique" (see ROCCH-RNO website) handled by the Institut Français de Recherche pour l'Exploitation de la Mer (Ifremer). The surface (1-2cm) sediment samples were collected using an Ekman grab in 2014. After collection, surface sediment samples were stored in plastic bags, deep-frozen, lyophilized, homogenized and sieved at 2 mm.

A sediment core labeled "PV1" (300 cm length) was sampled on the intertidal mudflat "Les Brillantes" located in the inner Loire estuary, on the bank opposite to Donges refinery plant (Fig. 1), during the Paleovase mission in Mars 2015. The vibracore was collected using aluminum tubes (6 m long, 74 mm inside diameter and 1 mm thick) driven into the sediment from a barge resting on the sediment at low tide. Core PV1 was subsampled into 2 cm-thick sediment slices every 5 cm in the upper meter and every 10 cm for the rest of the sediment core. A sedimentation rate of approximately 0.7 cm/year was estimated using the 210 Pb technique in the upper 35 cm of the sediment core following the method of [START_REF] Schmidt | Recent sediment transport and deposition in the Cap-Ferret Canyon, South-East margin of Bay of Biscay[END_REF]. 14 C dating was discarded because it has been proven to give unreliable ages (inversions and inaccuracies), due to multiple factors linked mostly to estuarine hydrodynamics (e.g. erosion, remobilization of sediments, contamination by younger carbon, transport of vegetal material) [START_REF] Durand | Midto late-Holocene environmental evolution of the Loire estuary as observed from sedimentary characteristics and benthic foraminiferal assemblages[END_REF]. Despite assuming a constant sedimentation rate through the entire core is very approximate, considering the inherent complications in dating estuarine systems, we are at least sure that we record the pre-industrial era. After collection, subsamples of core PV1 followed the same sample preparation procedure as for surface sediments.

All reagents, labware acid cleaning and solution dilutions used in sample preparation and elemental and isotope analysis were performed using high purity water (18.2 MΩ.cm H 2 O, Nanop System®) and ultra-pure acids (PlasmaPure Plus grade, SCP science®). For sediment digestion, about 200 mg aliquots of dry sediments were weighted in Teflon® bombs and heated on a coated graphite block using multiple-step acid concentrated procedure with HF, HCl and HNO 3 . Firstly, 250 µl of HNO 3, 750 µl of HCl and 6 ml of HF were added and heated at 130 °C for 4 hours. Then, samples were evaporated to dryness at the same temperature. A second acid-step with 4 ml of HNO 3 and 20 ml of high purity water was added and heated at 130 °C for 6 hours. Then, acid extracts were transferred to Falcon® tubes and diluted to a final volume of 50 ml with high purity water.

Aliquots of the final extract solution were split for subsequent elemental and isotope analysis. Three procedural blanks and three international certified reference materials (MESS-3 and PACS-2 from NRC -CNRC) were processed with each batch of sediment samples (n= 15 samples) and followed the complete procedure (digestion, elemental analysis, ion chromatography and isotope determination).

Analytical methods

Elemental determination of Cu, Zn, Pb, Cd, Hg, Ag and Al were conducted by inductively coupled plasma mass spectrometry for the core (Q ICP-MS, iCAP Qc, KED mode, using He gas; Thermo Fisher Scientific) and surface sediments samples (Q ICP-MS, X-Series; Thermo Fisher Scientific), externally calibrated, and using internal standards (Rh and In). The accuracy and average bias were always within 10% of the certified values of both reference materials for considered elements.

Prior to the isotope analysis, Zn and Cu were separated from matrix elements by ionexchange chromatography employing a home-made Teflon® columns filled with 2.0 ml of the anion exchange resin AG-MP1 (100-200 mesh) and a modified protocol of Maréchal et al.(1999), Chapman et al. (2006) and Borrok et al. (2007). The entire chromatography procedure was performed in an overpressured air-filtered cleanroom (Class 100) at the Laboratoire de Biogéochimie des Contaminants Métalliques (LBCM, Ifremer, Nantes).

Recovery yielding measured by ICP-MS of chromatographic protocol was 100 ± 5% and the total procedural blanks were below 1% of Zn and Cu mass content of the samples. For subsequent mass bias corrections, the Zn ETH standard (Archer et al., 2017) was added to the Cu purified samples after the ion exchange procedure and concentration matched with the ratio 1:1, at concentrations between 100 and 300 µg ml -1 . Similarly, Cu NIST-976 standard was added to the Zn purified samples.

Copper and Zinc isotopes were measured at the Pôle Spectrométrie Océan (PSO, Ifremer, Brest, France) by MC-ICP-MS (Neptune, Thermo Scientific). Samples were dissolved in 2% (v/v) HNO 3 and introduced into MC-ICP-MS with a stable introduction system (SIS: cyclonic spray chamber) coupled with a low flow PFA nebulizer (50 µL min -1 ).

The MC-ICPMS was equipped with X skimmer cone and jet sample cone and run in low resolution mode. Copper and Zinc isotope ratios were corrected for instrumental mass bias using the sample standard bracketing method combined to an exponential law. The final isotope compositions are expressed as δ 65/63 Cu and δ 66/64 Zn values reported to NIST SRM 976 and to the "JMC-Lyon" (Johnson Matthey Company, lot 3-0749-L), respectively, as follow below:

(‰) = -1 ! " # $$/$&
' 10000 + 0.28‰ (eq.1)

-. /012 34$ (‰) = (eq.2)

The Zn ETH standard data are converted to the JMC Lyon values by adding a factor of 0.28‰, as recommended by Archer et al. (2017). The use of data reported against JMC standard is advantageous because it allows to compare to most of Zn isotope published data (Araújo et al., 2017c). The error introduced by this conversion is smaller than analytical uncertainty.

For analytical quality control, replicate of digested and purified reference materials were measured two or three times along each analytical session. The δ 65 Cu NIST and δ 66 Zn JMC average values obtained for marine sediments (MESS-3 and PACS-2) are listed in the Table 1. External precision (2s) average obtained from two to three measurements of reference materials and unknown samples was ±0.05‰ and ±0.04‰ for δ 65 Cu NIST and δ 66 Zn JMC, respectively. Uncertainty bars reported in the figures correspond to the external precision.

Enrichment Factor

The Enrichment Factor (EF) of trace metals is an index used to estimate anthropogenic inputs discounting dilution effects related to grain size and mineralogical heterogeneities by the normalization of a metal concentration in the sample to the concentration of a reference element [START_REF] Chen | Zn isotopes in the suspended load of the Seine River, France: Isotopic variations and source determination[END_REF]. A reference element should be associated with finer particles (related to grain size) and its concentration without significant anthropogenic influence [START_REF] Barbieri | The Importance of Enrichment Factor (EF) and Geoaccumulation Index (Igeo) to Evaluate the Soil Contamination[END_REF]. Aluminum is widely used to normalize the metal concentrations in coastal sediments since it represents the alumina-silicates (clay minerals), important bearing phase of trace elements in these systems [START_REF] Chen | Distribution and accumulation of heavy metals in the sediments of Kaohsiung Harbor, Taiwan[END_REF]. In this work, EF values were calculated as follows:

@A = B (C/DE) FGHIJK (C/DE) LMM N (eq.3)
where "M" is the concentration of the metal of interest and "Al" is the reference element concentration, whereas (M/Al sample ) represents the concentration ratio in the sample and (M/Al) UCC refers to the concentration ratio reported for the Upper Continental Crust [START_REF] Rudnick | Composition of the Continental Crust[END_REF], Table 1). Enrichment factor values were interpreted as suggested by [START_REF] Chen | Distribution and accumulation of heavy metals in the sediments of Kaohsiung Harbor, Taiwan[END_REF] EF < 1 indicates no enrichment; EF < 3 is minor enrichment; EF = 3-5 is moderate enrichment; EF = 5-10 is moderately severe enrichment; EF = 10-25 is severe enrichment; EF = 25-50 is very severe enrichment; and EF > 50 is extremely severe enrichment.

Statistics

All descriptive and exploratory statistics were performed using the SPSS® software v.18.0 (IBM®, Armonk, NY, USA). The normality distribution of data was verified using Kolmogorov Smirnov and Shapiro-Wilk tests. A multivariate approach (Principal Component Analysis [PCA]) was used to examine and identify the main interrelations among the isotope and elemental variables. A correlation matrix with varimax rotation was used to calculate the factor loads (Araújo et al., 2017d;[START_REF] Mar Da Costa | Biogeochemical mechanisms controlling trophic state and micropollutant concentrations in a tropical artificial lake[END_REF]. The principal components (PCs) that had eigenvalues higher than one were selected for discussion [START_REF] Mulholland | Geological and anthropogenic influences on sediment metal composition in the upper Paracatu River Basin, Brazil[END_REF].

The commonality values of all variables equal or higher than 0.5 were used as criterions for PCA results acceptance.

Results

Isotope and geochemical data of sediments

All elemental and isotope data of sediment samples are presented in Table 1. Calculated enrichment factor are provided in the Table 2. All measured trace metal concentration depth profiles of core PV1 display a similar pattern with proximity to the sediment-water interface.

There is a trend of general increase from the ~96 cm deep horizon, followed by a peak at ~14 cm and then a sharp decline from 14 cm deep to the top of the core (Fig. 2a). In the case of Hg, it is also observed a marked increase from 296 to 163 cm and occurrence of maxima at 69 and 163 cm deep. Considering a mean sedimentation rate of 0.7 cm/year along the sediment core, the 96 cm deep horizon corresponds approximately, in years, to the end of the XIX th century (~1880), and hence marks the beginning of the pre-industrial period. In turn, 14 cm deep corresponds to the mid-1990's. Enrichment factor profiles of trace metals present similar patterns as bulk concentrations data (Fig. 2b). Among all studied trace metals in the sedimentary archive and surface sediments, the highest enrichment factors were observed for Ag (from 5 to 21), while lower EFs were obtained for Cd (from 1 to 10), Pb (from 3 to 9), Hg (from 0 to 9), Zn (from 1 to 4) and Cu (from 0 to 2). All trace metal concentrations are correlated with each other (Pearson correlation coefficient r = ~ 0.85, in average, n = 31) considering the whole sediment dataset.

PCA analysis were performed separately in the PV1 core and surface sediments samples to verify interrelations of the dataset in temporal and spatial scales (Fig. 3a Zinc and copper isotopes present different spatial and temporal behaviors in relation to their enrichment factors. For Zn, low and moderate correlations between Zn EF and δ 66 Zn values are observed in the PV1 core profile (R 2 = 0.43, n = 11, p < 0.05, Fig. 4). In surface sediments were weakly correlated but not statistically significant (R 2 = 0.28, n= 14, p > 0.05, Fig. 3). For Cu, surface sediments showed a weak correlation between Cu EF and δ 65 Cu (R 2 = 0.27, n= 17, p < 0.05, Fig. 4). In the PV1 core profile, these variables were uncorrelated (R 2 = 0.14, n= 12, p > 0.05). Despite this, it is noticed that before the pre-industrial period (from the base core to 95 cm), sub-samples of PV1 core present an average δ 65 Cu NIST of -0.05 ±0.02‰ (2s, n=3), whereas sub-samples corresponding to industrial period (96 cm deep upward),

show an average of -0.11 ±0.01‰ (2s, n= 9). These δ 65 Cu NIST averages are statistically different (test t, p < 0.05).

Discussion

Assessing temporal and spatial contamination of trace metals

The noticeable increase of trace metal concentrations above 96 cm deep horizon probably In the core PV1, Cu, Zn, Cd, Pb, Ag and Hg concentrations upward of 96 cm display similar temporal trends to the sedimentary geochemical records obtained upstream along the Loire River [START_REF] Dhivert | Dynamics of metallic contaminants at a basin scale -Spatial and temporal reconstruction from four sediment cores (Loire fluvial system, France)[END_REF][START_REF] Grosbois | Severe and contrasted polymetallic contamination patterns (1900-2009) in the Loire River sediments (France)[END_REF], although with relatively lower concentrations. Since a high proportion of sediment particles of the Loire estuary is of fluvial origin [START_REF] Negrel | Multi-element chemistry of Loire estuary sediments: Anthropogenic vs. natural sources[END_REF], it is likely that part of anthropogenic metals reach the estuary via fluvial transport associated to particulate material. It is also likely that they reach the estuary after a certain time from their emission, resulting in similar but time-shifted sedimentary geochemical profiles between estuarine and fluvial systems. Human induced erosion and remobilization of deeper horizons may have mixed between layers corresponding to before the industrial period with initial coal mining activities in the Loire watershed. This possible perturbation to the progressive layering of sediment along the length of the core is likely due to dredging linked to a small boat harbor nearby. In the case of Hg and Ag, the high single point maxima at 69 and 163 cm deep may be related to such sediment reworking.

In a spatial analysis, considering the sum of all trace metal enrichment factors for each sample (Table 2, Fig. 5a), it is identified an overall enrichment of trace metal in the upper estuary (downstream and upstream of Nantes (samples 1 and 2) and in one sample at the mouth of the estuary (close St Nazaire city, sample 4). This spatial pattern of enrichment is similar for Ag (Fig. 5b), the most enriched trace element of the Loire estuary. The metal trace enrichment in these zones have attributed to urban inputs, including effluents from the highly urbanized sub-watersheds and to domestic and industrial effluents from waste water treatment plants [START_REF] Coynel | Spatial distribution of trace elements in the surface sediments of a major European estuary (Loire Estuary, France): Source identification and evaluation of anthropogenic contribution[END_REF]. The mixing of trace metal contaminated particles coming from different watershed sources along the course of the Loire river towards the estuary suggest a common mechanism of transport and dispersion in the estuarine system, which explain the fact of all trace metals are correlated among them. The PCA analysis of surface sediments (Fig. 4b) also confirm a grouping of all trace elements, including Hg. In the inner estuary, fine-grained (< 4µm) particles and metal concentrations are highly correlated in the Loire estuary [START_REF] Coynel | Spatial distribution of trace elements in the surface sediments of a major European estuary (Loire Estuary, France): Source identification and evaluation of anthropogenic contribution[END_REF], indicating that grain-size distribution and hydrodynamic features constitute the main controlling factors in the spatial distribution of these elements.

The long-distance transport of fine-grain particles coming from the Loire estuary mouth to open ocean and posteriorly to Bourgneuf bay can explain the enrichment in metals in the samples 11, 14 and 18 (Fig. 5a). No major river drains this bay, but it is surrounded by large marshes, drained by dozens of creeks. A second source of metals could be associated to potential local anthropogenic inputs involving shipyard activity and the use of antifouling paints.

As discussed here, trace metal source identification in the Loire estuary is complex due to the presence of multi-anthropogenic activities of which the relative contributions seem to change in spatial and temporal scales [START_REF] Grosbois | Severe and contrasted polymetallic contamination patterns (1900-2009) in the Loire River sediments (France)[END_REF]. In addition, both intraestuarine and watershed-derived anthropogenic source inputs are mixed in the inner-estuary, limiting the discrimination of mixing components by elemental concentration data.

Alternatives approaches using geochemical ratios (eg., Ag/Pb; Cd/Zn) have been useful to highlight the main mixing end-members in the upper Loire river, and to evidence dominance of urban sources over mining sources for some elements, such as Ag and Zn, during the period 1950-1980 [START_REF] Dhivert | Dynamics of metallic contaminants at a basin scale -Spatial and temporal reconstruction from four sediment cores (Loire fluvial system, France)[END_REF][START_REF] Grosbois | Severe and contrasted polymetallic contamination patterns (1900-2009) in the Loire River sediments (France)[END_REF].

Identifying anthropogenic and natural sources using Cu and Zn isotopes

Here we discuss Cu and Zn isotope compositions to infer possible source mixing processes of these elements in the Loire estuary. In turn, the PV1 core profile indicated minor enrichment for Cu and moderate enrichment for Zn (Fig. 2ab, Table 2) and a well-marked anthropogenic fingerprint characterized by a multi-metallic enrichment (including Cu and Zn) above 96 cm with a peak maximum of enrichment at ~14 cm (Fig. 2a, 2b, Table 2). Taking into account the EF values of Cu and Zn, the anthropogenic inputs of these metals are more evident in a temporal scale.

A moderate correlation between Zn EF and δ 66 Zn values were observed in the core PV1 (R 2 = 0.43, n = 11, p < 0.05), with an overall trend toward lighter Zn isotope compositions with enrichment factor increasing (Fig. 4). This trend can be interpreted as a binary mixing process between Zn derived from terrigenous material and multiple anthropogenic Zn sources. Since sediment particle transport from rivers to oceans does not alter significantly Zn isotope compositions of the particulate phase [START_REF] Araújo | Zinc isotopes as tracers of anthropogenic sources and biogeochemical processes in contaminated mangroves[END_REF][START_REF] Guinoiseau | Fate of particulate copper and zinc isotopes at the Solimões-Negro river confluence, Amazon Basin, Brazil[END_REF], isotope ratios in the suspended sediments behave conservatively, reflecting the relative Zn contributions of natural and anthropogenic sources.

In the Loire estuary, sediments are dominantly composed of detrital minerals derived mainly from silicate rocks of the basin. Mineralogical characterization of SPM and bed sediments in the Loire estuary show an assembly of clay minerals dominated by smectites (30%), illites (33%), kaolinites (27%) and chlorites (10%) with feldspars, calcite, biotites, zircons and other accessory minerals originated from the weathering processes of intrusive rocks in the upper Loire watershed (Roger, 1988). Zinc isotope composition of the Upper Continental Crust ranges between +0.2 and +0.3‰ [START_REF] Moynier | The Isotope Geochemistry of Zinc and Copper[END_REF], with an estimated average value of +0.28 ±0.05‰ (2s, [START_REF] Chen | Zinc isotope fractionation during magmatic differentiation and the isotopic composition of the bulk Earth[END_REF] Thapalia et al., 2015), vehicle emissions (-0.87 to +0.07‰, Gioia et al., 2008), urban runoff (from +0.13 to +0.15‰, [START_REF] Chen | Zn isotopes in the suspended load of the Seine River, France: Isotopic variations and source determination[END_REF], wastewaters and sludge (~0.1‰; [START_REF] Chen | Zn isotopes in the suspended load of the Seine River, France: Isotopic variations and source determination[END_REF] and atmospheric industrial emissions (< -0.6‰, Souto-Oliveira et al., 2018; from -0.52 to +0.02‰, [START_REF] Mattielli | Zn isotope study of atmospheric emissions and dry depositions within a 5 km radius of a Pb-Zn refinery[END_REF]from -0.27 to +0.15‰, Ochoa et al., 2016). The large variability of isotope compositions of urban sources is due to different isotope fractionation mechanisms in the anthropogenic Zn cycling involving the ore refining, where hightemperature processes employed during roasting and smelting of ores tend to fractionate zinc isotopes in the gas phase, with an enrichment of the lightest isotopes in air emissions and heavier isotopes in slags and effluents [START_REF] Yin | Application of Zn isotopes in environmental impact assessment of Zn-Pb metallurgical industries: A mini review[END_REF]. The final metallic Zn product (Zn 0 ) remain with signatures similar to the average of original concentrated ores (Sivry et al., 2008b;[START_REF] Sonke | Historical variations in the isotopic composition of atmospheric zinc deposition from a zinc smelter[END_REF][START_REF] Yin | Application of Zn isotopes in environmental impact assessment of Zn-Pb metallurgical industries: A mini review[END_REF]. Other studies show that electroplating solid wastes are also enriched in the heaviest isotopes (Araújo et al., 2017a,b).

In the urban environment, runoff on streets, commercial areas and roofs, atmospheric leaching by rainwaters and inputs from domestic and industrial wastewaters are connected to urban sewage network [START_REF] Grosbois | Severe and contrasted polymetallic contamination patterns (1900-2009) in the Loire River sediments (France)[END_REF]. The mixing of multi-diffuse sources of Zn in the sewage effluents can lead to a homogenous "Zn urban fingerprint", which would explain the identification of a single anthropogenic end-member in the Loire estuary, in both temporal and spatial scales. Analogous Zn binary source mixing setting was observed in the Seine river (France, Chen et al., 2009) involving natural silicate particles transported by river (~0.3‰), and anthropogenic particles derived from wastewater treatment plants or combined sewer overflows (~0.1‰). Likewise for urban lakes of United States of America, Zn source mixing processes comprise a natural end-member (+0.35‰) and other anthropogenic (+0.10‰), related to vehicle sources (tire wear and emissions, Thapalia et al., 2015).

Considering the punctual anomalous enrichment factor of Zn close to waste water treatment plants [START_REF] Coynel | Spatial distribution of trace elements in the surface sediments of a major European estuary (Loire Estuary, France): Source identification and evaluation of anthropogenic contribution[END_REF] and the evidence for a homogenous "Zn urban fingerprint", we propose a representative signature of +0.1‰ to be the main urban anthropogenic source in the Loire estuary. The value of +0.1‰ as suggested for the anthropogenic end-member in the Loire estuary and Seine River are in line with the ranges from +0.1‰ to +0.2‰ attributed to "common" anthropogenic Zn proposed by John et al.

(2007), based on measurement of a wide variety of materials (electrochemically purified Zn, galvanized steel, electroplated materials, vitamins, laboratorial standards, health products, etc.).

Because natural and anthropogenic ranges of Zn signatures in the Loire estuary are relatively small, δ 66 Zn JMC values of some samples overlap partially with both sources, taking into account analytical uncertainties. This partial overlap can lead to a decreasing of resolution for source discrimination and thereby, compromising a precise quantification of source contributions. A theoretical mass balance with different anthropogenic sources reported in the literature illustrate how source discrimination resolution can vary depending of Zn contamination context (Fig. 6). This theoretical model estimates that natural terrigenous and urban anthropogenic end-members of Zn overlap for an anthropogenic contribution lesser than 70% of Zn (total mass) in the sample. Therefore, the identification, discrimination and quantification of anthropogenic Zn is difficult due the low degree of Zn enrichment in the investigated samples. This is the case of surface sediments, which were collected in 2014 year, in a period of attenuated Zn contamination. The weak and not significant correlation between δ 66 Zn JMC values and Zn EF values (R 2 = 0.28, n= 14, p > 0.05, Fig. 4) exemplify the difficulty of determining the anthropogenic or natural origin of Zn in these samples. Anyway, a light isotope composition in the sample 1, located in the upper estuary (Fig. 5d) is consistent with the urban Zn isotope signature of proposed binary source model.

Copper concentrations were low and variations of Cu isotope compositions on spatial and temporal scale were weakly or no-correlated with Cu EF values. Then, identification of source mixing processes using the Cu isotope dataset is not evident. This may be result of: (i) a possible overlap between anthropogenic vs. natural isotope signatures or, (ii) the predominance of natural Cu origin over anthropogenic one, as supported by Cu EF values.

The overall δ 65 Cu NIST values of the studied sediment samples (-0.04 ±0.18, 2s, n= 31) are close to the average of the Upper Continental Crust (+0.07± 0.10‰, [START_REF] Liu | Copper isotopic composition of the silicate Earth[END_REF][START_REF] Savage | Copper isotope evidence for large-scale sulphide fractionation during Earth's differentiation[END_REF] and fell in the range reported for sedimentary materials from estuaries and river worldwide [START_REF] Guinoiseau | Fate of particulate copper and zinc isotopes at the Solimões-Negro river confluence, Amazon Basin, Brazil[END_REF][START_REF] Vance | The behaviour of Cu and Zn isotopes during soil development: Controls on the dissolved load of rivers[END_REF][START_REF] Vance | The copper isotope geochemistry of rivers and the oceans[END_REF]). An overall tendency of enrichment in the light isotope of sediments in relation to the UCC have been attributed to an isotope partitioning between dissolved and particulate phase during weathering processes, involving the formation of aqueous organic complexes enriched in the heavy isotope and residual particulate phases (Fe-Mn oxyhydroxides, clay minerals) enriched in the light isotopes [START_REF] Guinoiseau | Fate of particulate copper and zinc isotopes at the Solimões-Negro river confluence, Amazon Basin, Brazil[END_REF][START_REF] Vance | The behaviour of Cu and Zn isotopes during soil development: Controls on the dissolved load of rivers[END_REF][START_REF] Vance | The copper isotope geochemistry of rivers and the oceans[END_REF]. Bordeaux city, France (δ 65 Cu NIST =-0.37‰ and -0.28‰, [START_REF] Petit | Anthropogenic sources and biogeochemical reactivity of particulate and dissolved Cu isotopes in the turbidity gradient of the Garonne River (France)[END_REF]. Considering this small dataset available in the literature, and in analogy with the previous Zn discussion, positive δ 65 Cu NIST values of urban sources and use of antifouling paints could explain the positive trend between Cu isotope compositions and Cu EF (Fig. 3). In turn, run-off of contaminated vineyard soils in the Loire catchment could explain the small shift toward lighter isotope compositions above 96 cm depth in the PV1 core. However, the range of δ 65 Cu NIST values of fungicides is wide enough to overlap with those reported for unpolluted soils and sediments, making source discrimination using Cu isotopes uneasy (Fekiacova et al., 2015). Similar drawbacks were verified in soils contaminated by Cu-fungicide, where anthropogenic, pedogenic and geogenic isotope signatures overlapped partially among them [START_REF] Babcsányi | Copper in soil fractions and runoff in a vineyard catchment: Insights from copper stable isotopes[END_REF][START_REF] El Azzi | Origin and fate of copper in a small Mediterranean vineyard catchment: New insights from combined chemical extraction and δ65Cu isotopic composition[END_REF].

Considering the exclusive natural Cu origin, the subtle spatial and temporal changes on Cu isotope ratios could be associated to relative variations on distribution of sediment components such as clay minerals, amorphous solids and organic matter. Other controlling factors could be associated with hydrological sorting of particle-size along the estuary. Analysis of silt and fine clays fractions of soils showed distinct Cu isotope compositions (+0.23 ± 0.07‰ and -0.37 ± 0.10‰, respectively, [START_REF] Babcsányi | Copper in soil fractions and runoff in a vineyard catchment: Insights from copper stable isotopes[END_REF]. In the Loire estuary, particle sorting and grain-size compositions of surface sediments are highly variable with the proportion of mud (silt+clay) and sand ranging from 0% to 89% and from 3% to 100%, respectively [START_REF] Coynel | Spatial distribution of trace elements in the surface sediments of a major European estuary (Loire Estuary, France): Source identification and evaluation of anthropogenic contribution[END_REF]. In the inner-estuary, there is a general trend to increasing grain sizes from upstream to downstream, despite coarse can occur in areas affected by buildings of channels navigation close to urban zones [START_REF] Coynel | Spatial distribution of trace elements in the surface sediments of a major European estuary (Loire Estuary, France): Source identification and evaluation of anthropogenic contribution[END_REF]. Thus, an open question remains whether anthropogenic actions (i.e. dredging of navigation channel and urban development, landscape use changes) modify particles-size and mineralogy distribution, and hence induce changes on Cu isotope composition of sediments. Further investigations probably could elucidate the apparently contrasted isotope signatures obtained for samples 1 and 18.

Considering the low Cu EF values, the hypothesis of a predominant natural origin of Cu over anthropogenic one seems more plausible. As proposed by [START_REF] Coynel | Spatial distribution of trace elements in the surface sediments of a major European estuary (Loire Estuary, France): Source identification and evaluation of anthropogenic contribution[END_REF], anthropogenic influence on the Cu and Zn concentrations in the Loire estuary is restricted to punctual sites. Further studies that increase sampling sites are required to better constrain anthropogenic sources inputs in the estuary and test these models. Here we suggest that the Cu isotope compositions of sediments in Loire estuary, at least at the studied stations reflect mostly the signature of weathered silicate particles. They are derived from soils and rocks of fluvial-estuarine watershed, with their isotope ratios close to those reported for Upper Continental Crust, but with a slight overall tendency of enrichment in the light isotope.

Conclusions

Metal trace concentrations and Cu and Zn isotopes were used to identify past and current anthropogenic inputs in the Loire estuary. The order of metal enrichment in the sedimentary archive and surface sediments follow Ag > Pb > Cd > Hg > Zn > Cu. In a temporal scale, it recorded an increase of metal concentrations from the beginning of the industrial period and an ongoing attenuation of metal contamination inputs in the Loire estuary in the 1990's. In the inner-estuary, grain-size distribution and hydrodynamic features constitute the main controlling factors in the spatial distribution of these elements.

Zinc isotopes can be used to identify a binary mixing process between Zn derived from terrigenous material, and Zn issued from multi-urban anthropogenic sources. However, because natural and anthropogenic ranges of Zn signatures in the Loire estuary are relatively narrow, a precise quantification of anthropogenic contribution in the samples is difficult, if samples present a very low degree of Zn enrichment. In turn, Cu isotope compositions of sediments in the Loire estuary are controlled by natural sources, reflecting the signature of weathered silicate particles derived from soils and rocks of the Loire river watershed.

In conclusion, this work demonstrates the applicability of Zn isotopes to detect anthropogenic sources in coastal systems, even under a relatively low degree of contamination. Increasing the details of sampling and additional characterization parameters (e.g., sequential leaching, mineralogy, granulometry, fine solid speciation, etc…) in the Loire estuary may help to detect possible diffuse anthropogenic sources of Cu, as well as to better understand the role of grain-size and adsorption processes on mineral surfaces in the isotope fractionation along the estuary.

Fig. 1 The Loire estuary location and sampling sites of surface sediments (star) and a sediment core (PV1 core, pentagon). * Copper isotope determination were performed in several replicates for MESS-3 (n =6) and PACS-2 (n = 2), while zinc isotope compositions were determined exclusively in replicates of MESS-3 (n = 4). For certified reference materials, each replicate was prepared separately with different digestions and chromatographic separation for isotope determinations. **Elemental concentrations reported by [START_REF] Rudnick | Composition of the Continental Crust[END_REF]. Copper and zinc isotope compositions reported by [START_REF] Savage | Copper isotope evidence for large-scale sulphide fractionation during Earth's differentiation[END_REF] and [START_REF] Chen | Zinc isotope fractionation during magmatic differentiation and the isotopic composition of the bulk Earth[END_REF], respectively.

Table 2. Enrichment factors of sediments in the Loire estuary.

  reflects the strong industrial development occurring during the 1880-1980 period in the Loire river watershed, while the decreasing trend from 14 cm deep upward shows the attenuation of metal contamination inputs in the 1990's (Fig.2a,b). Previous studies revealed different polymetallic contaminations along the time in the Loire river watershed[START_REF] Dhivert | Dynamics of metallic contaminants at a basin scale -Spatial and temporal reconstruction from four sediment cores (Loire fluvial system, France)[END_REF][START_REF] Grosbois | Severe and contrasted polymetallic contamination patterns (1900-2009) in the Loire River sediments (France)[END_REF]: (i) the 1900-1950 period, marked by a severe contamination of Hg, Au and Ag associated to mining activities; (ii) the 1950-1980 period, characterized by a pronounced increase in concentrations of Hg, Bi, Cd, Ag, Zn, Cu, Pb, Sb attributed to several sources including smelting, industrial and urban sources; and lastly, and (iii) the period 1980currently, with a noticeable trend of decreasing in metal contaminants. This decrease results of de-industrialization and industry closure in the Loire watershed[START_REF] Dhivert | Dynamics of metallic contaminants at a basin scale -Spatial and temporal reconstruction from four sediment cores (Loire fluvial system, France)[END_REF], increasing of the rate of recycling and/or treatment of metals within the anthroposphere[START_REF] Meybeck | Historical perspective of heavy metals contamination (Cd, Cr, Cu, Hg, Pb, Zn) in the Seine River basin (France) following a DPSIR approach (1950-2005[END_REF] and implementation of environmental regulation (e.g., the 2000 European water framework directive) on solid waste management and wastewater treatment in the local municipalities[START_REF] Grosbois | Severe and contrasted polymetallic contamination patterns (1900-2009) in the Loire River sediments (France)[END_REF]. Important coal mining and burning districts in the Furan sub-basin (St. Etienne coal mining basin) and in the Bourbince-Arroux subbasins (Blanzy-Montceau coal-basin) stopped their operations in the late 1990's[START_REF] Dhivert | Dynamics of metallic contaminants at a basin scale -Spatial and temporal reconstruction from four sediment cores (Loire fluvial system, France)[END_REF]. In the Loire estuary, decreasing of anthropogenic Pb can also be associated to the closing of the Pb refinery (Tréfimétaux) in 1988 near Nantes, and the closing of the lead tetraethyl manufacturing plant (Kuhlmann-Octel) in mid-1990's in Paimboeuf, immediately near the location of PV1.

  The main potential anthropogenic Cu sources in the Loire river watershed include urban sources, run-off from vineyard soils and anti-fouling paints employed into embarkations.Souto-Olveira et al. (2018) reported several values for urban sources with positive δ 65 Cu NIST values: cement (δ 65 Cu NIST = +0.30 to +0.61‰); road dust (δ 65 Cu NIST = +0.08 to +0.25‰), and vehicular traffic (δ 65 Cu NIST = +0.46 to +0.59‰); similar enrichment in the heavy isotope have been reported in antifouling paint (+0.54 ±0.03) and contaminated sediments in the Port Camargue (French mediterranean sea,Briant et al., 2013). Commercial Cu-based fungicides applied in vineyards and remaining in their soils range from -0.35 to +0.91‰[START_REF] Babcsányi | Copper in soil fractions and runoff in a vineyard catchment: Insights from copper stable isotopes[END_REF][START_REF] El Azzi | Origin and fate of copper in a small Mediterranean vineyard catchment: New insights from combined chemical extraction and δ65Cu isotopic composition[END_REF]. The widely applied fungicide Bordeaux mixture (CuSO 4 +Ca(OH) 2 ) has light isotope values (δ 65 Cu NIST = -0.35‰, El Azzi et al., 2013; δ 65 Cu NIST = -0.06‰,[START_REF] Babcsányi | Copper in soil fractions and runoff in a vineyard catchment: Insights from copper stable isotopes[END_REF] close to the vineyard soils around

  Determination of isotope compositions in specific phases of sediment using traditional sequential extractions protocols have demonstrated the isotope heterogeneity of different geochemical components in vineyard soils (El Azzi et al., 2013): copper in silicate residual fractions was close to local bedrock (δ 65 Cu NIST +0.07‰), while in water soluble fraction of SPM was similar to dissolved river Cu (δ 65 Cu NIST = +0.26‰). Copper from fungicide treatment (δ 65 Cu = -0.35‰) was bound to organic matter (δ 65 Cu NIST = -0.20‰) without or with slight isotopic fractioning, while Cu adsorbed onto iron oxides were enriched in the heavy isotope (δ 65 Cu = +0.5‰).

Fig 2 .Fig. 3 Fig. 4 Fig. 5 Fig. 6

 23456 Fig 2. Profiles of trace element concentrations and Cu and Zn isotope compositions in the PV1 core. The Cu and Zn isotope composition ranges of Upper Continental Crust for Cu and Zn are compiled in Table 1. Zinc urban anthropogenic source refers to values reported by John et al.(2008) and Chen et al.(2009). The value of the pesticide namely "Bordeaux mixture" is reported by (El Azzi et al., 2013, δ 65 Cu NIST = -0.34‰)

  and 3b, respectively). In the PV1 core, the two extracted main components explained 88% of the variance dataset: the first component (66% of variance) encompasses the Zn, Cu, Ag, Pb, Al variables, while the second component (22% of variance) grouped Hg, Cd and δ 65 Cu values.In the surface sediments dataset, PCA analysis provided two main components accounting for almost 83% of the variance: the first component (69% of variance) associated Zn, Cu, Ag, Pb, Hg, Cd and Al. The second component (14% of variance) included Cd and δ 65 Cu variables. In similar way, the PCA analysis of PV1 core and surface sediment no appointed any correlation between the variable δ 66 Zn value and the other elements.The average of Cu and Zn isotope compositions (δ 65 Cu and δ 66 Zn) of sediments are -0.04 ±0.18 (2s, n= 31) and +0.24 ±0.11 (2s, n= 26), respectively. In core PV1 profile, the δ 65 Cu NIST values vary between -0.02 and +0.08‰ (average of +0.04 ±0.07‰, 2s, n =12, Fig.

2c), while the δ 66 Zn JMC values are ranged between +0.20 to +0.32‰ (average of +0.26 ±0.07‰, 2s, n=11, Fig.

2d

). Despite these narrow ranges of isotope values, they correspond to more than two and threefold the external precision (2s), indicating detectable but subtle isotope changes in the profile core. In the surface sediments, δ 65 Cu NIST values are centered on -0.08 ±0.17‰ (2s, n= 17), while δ 66 Zn JMC values are around +0.24 ±0.13‰ (2s, n= 15).

  Surface sediments presented minor enrichment for Cu and Zn (EF< 3). It was observed a major Zn enrichment in the estuary mouth (samples 4, 5, 7, 8), Bourgneuf bay (samples 12, 17 and 18) and in the sample 11, while for Cu the most enriched samples were restricted to the samples 4 (estuary mouth) and samples 12 and 14 (Bourgneuf bay). It was not identified a clear relationship between δ 66 Zn values and δ 65 Cu values,

	enrichment factor and sample localizations for surface sediments. A study of spatial
	distribution of metals performed by Coynel et al. (2016) in surface sediments showed only
	high enrichment factor values (5 < EF < 10) for Zn, Pb, Cu in several sites near Nantes city,
	especially close to waste water treatment plants. Instead, Negrel et al. (1997) verified a
	gradual enrichment of Zn (~33%) and Pb (~40%) in surface sediments from river outlet
	toward to marine environment, possibly due to contaminant emissions of inner-anthropogenic
	sources.

  . The heaviest δ 66 Zn JMC values closer to +0.3‰ of low Zn enriched-sediments reflect the natural end-member represented by silicate rocks of the Loire watershed. This value is also very similar to unpolluted fluvial and estuarine sedimentary material (local natural background) reported in many environmental studies: (+0.28 ±0.05‰, 2s, Chen et al. 2013; δ 66 Zn =+0.31 ±0.04‰, 2s,Sivry et al., 2008; δ 66 Zn =+0.31 ±0.09, 2s, Sonke et al., 2008).Common urban anthropogenic Zn present lighter δ 66 Zn JMC values than Upper Continental Crust, such as tire wear (from +0.00 to +0.20‰,[START_REF] Souto-Oliveira | Multi-isotopic fingerprints (Pb, Zn, Cu) applied for urban aerosol source apportionment and discrimination[END_REF]

Table 1 .

 1 Geochemical data and Cu and Zn isotope compositions of sediments in the Loire estuary.

	[Al]		(%)		6.2	6.2	4.9	6.8	7.4	4.6
	[Ag]	) (mg.kg -1		0.45	0.45	0.34	0.54	0.48	0.15
	[Hg]	) (mg.kg -1		0.09	0.09	0.06	0.14	0.09	0.02
	[Pb]	) (mg.kg -1		64.3	64.3	45.1	65.7	63.4	38.0
	[Cd]	) (mg.kg -1		0.24	0.24	0.14	0.21	0.16	0.03	0.17
	[Zn]	) (mg.kg -1		126	126	63	179	168	40	127
	[Cu]	) (mg.kg -1		26.2	26.2	11.7	35.8	30.9	6.2	23.1
	2s		0.05	0.02	0.01	-	0.05	0.00	0.19
	δ 66 Zn JMC	(‰)		0.16	0.27	0.31	-	0.15	0.33	0.29
	2s		0.04	0.04	0.06	0.05	0.02	0.03	0.01
	δ 65 Cu NIST	(‰)		0.08	-0.06	-0.24	-0.10	-0.15	-0.16	-0.15
	Code		1	2	3	4	5	6	7
	Sample/sampling site	surface sediment	Sainte Luce	Indre	Pont de Saint-Nazaire	Face pointe de Mindin	St Brévin Mindin	Villes -Martin	Face St Brévin

Acknowledgments

This work was conducted within the POLLUSOLS project framework and was financed by the regional government of the Pays de la Loire (France). The authors acknowledge the Ifremer teams for supporting and providing sediment samples of the French monitoring framework ROCCH "Réseau d'Observation de la Contamination Chimique"). We thank all laboratorial support of LBCM technician team (Ifremer, Nantes): Sandrine Bruzac, Emmanuelle Rozuel, Bastien Thomas and Sylvette Crochet. We thank Sabine Schmidt for 210 Pb analysis. We would like to thank also the Grand Port Maritime of Nantes Saint-Nazaire (GPMNSN) for financial support (project HISTLOIRE; 2012-2015; PI: Mojtahid M.) and particularly Didier Lehay for his logistic help (mooring barge "Willendeavour"). The OSUNA has also financially supported the coring through the project PALEOVASE (2015; PI: Mojtahid M.). Finally, the authors would like to warmly thank Sylvain Haquin and Franck Lelong, technicians in the M2C laboratory of Caen, UMR CNRS 6143, who brought all their technical facilities to perform long coring on an intertidal mudflat.