Nicolas Layglon
email: nicolas.layglon@univ-tln.fr

Benjamin Misson

Gaël Durieu

Clément Coclet

Sébastien D'onofrio

Huy Duc

David Dang

Jean-Ulrich François

Stéphane Mullot

Véronique Mounier

Lenoble

Sébastien D'onofrio

Huy Duc Dang

David François

Jean-Ulrich Mullot

Stéphane Mounier

Véronique Lenoble

Dario Omanović

Cédric Garnier

Sébastien D ' Onofrio

Long-term monitoring emphasizes impacts of the dredging on

Keywords: sediment resuspension, dissolved metals, Chelex-DGT samplers, Toulon Bay Investigation, Writingreview&editing, WritingOriginal Draft, Validation Writingreview&editing, formal analysis, WritingOriginal

come

Introduction

Coastal areas are of great interest for Humanity. Harbors, located at the continentocean interface, represent hotspots of copper (Cu) contamination on the coast, both in water column and sediment [START_REF] Cindrić | Evidencing the natural and anthropogenic processes controlling trace metals dynamic in a highly stratified estuary: The Krka River estuary (Adriatic, Croatia)[END_REF][START_REF] Coclet | Changes in Bacterioplankton Communities Resulting From Direct and Indirect Interactions With Trace Metal Gradients in an Urbanized Marine Coastal Area[END_REF][START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF][START_REF] Tessier | Study of the spatial and historical distribution of sediment inorganic contamination in the Toulon bay (France)[END_REF].

Copper contamination from harbors has been proved to be highly toxic for planktonic microorganisms [START_REF] Coclet | Impacts of copper and lead exposure on prokaryotic communities from contaminated contrasted coastal seawaters: the influence of previous metal exposure[END_REF][START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF]. Lead (Pb) contamination is mostly archived in harbors sediment, often originating from older sources such as coal combustion, battery factories, metal smelting, leaded gasoline. However, Pb can be remobilized into the water column and be transferred to aquatic resources consumed by humans [START_REF] Dang | Evidencing the Impact of Coastal Contaminated Sediments on Mussels Through Pb Stable Isotopes Composition[END_REF].

Due to sedimentation, harbors become less accessible and therefore need to be dredged to preserve or increase their level of activity. During dredging activities, sediment can become a major source of chemical contamination for the water column [START_REF] Torres | Effects of dredging operations on sediment quality: contaminant mobilization in dredged sediments from the Port of Santos, SP, Brazil[END_REF]. For example, sediment resuspension could lead to an important release of metals in the water column, both in the particulate and dissolved phases [START_REF] Dang | Evidencing the Impact of Coastal Contaminated Sediments on Mussels Through Pb Stable Isotopes Composition[END_REF].

The ecologically-meaningful planktonic microbial realm responds quickly to dredging and more generally to sediment resuspension. Thus, it represents a useful tool to evaluate the extent of the impact of such human-induced perturbations on the ecosystem. While physiological responses have been identified for long [START_REF] Borgmann | A new standardized sediment bioassay protocol using the amphipod Hyalella azteca (Saussure)[END_REF][START_REF] Munawar | Toxicity of Detroit River Sediment-Bound Contaminants to Ultraplankton[END_REF][START_REF] Nayar | Environmental impact of heavy metals from dredged and resuspended sediments on phytoplankton and bacteria assessed in in situ mesocosms[END_REF] and suggest the stimulation of heterotrophic growth by increased resource availability, community structure shifts have scarcely been studied with respect to this specific kind of perturbation. Recent experimental work suggested that sediment resuspension could be responsible of large changes in ultraphytoplanktonic groups'

relative abundances [START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF]. However, the in situ response of ultraphytoplankton to dredging operations monitored remains to be evaluated.

The influence of an 18-month long dredging operation on Cu and Pb contamination and ultraplankton structure and abundance was studied in Toulon Bay (NW Mediterranean Sea, France). Due to historical (World War II) as well as current activities, Toulon Bay is subjected to strong anthropogenic pressure resulting in a strong multi-contamination in metals [START_REF] Tessier | Study of the spatial and historical distribution of sediment inorganic contamination in the Toulon bay (France)[END_REF], metalloids [START_REF] Dang | Key parameters controlling arsenic dynamics in coastal sediments: An analytical and modeling approach[END_REF], organo-metals [START_REF] Pougnet | Sources and historical record of tin and butyl-tin species in a Mediterranean bay (Toulon Bay, France)[END_REF] and organic contaminants [START_REF] Misson | Chemical multi-contamination drives benthic prokaryotic diversity in the anthropized Toulon Bay[END_REF]. On the other hand, natural variations commonly observed in other coastal areas, such as salinity, appear to be of relatively low amplitude [START_REF] Coclet | Changes in Bacterioplankton Communities Resulting From Direct and Indirect Interactions With Trace Metal Gradients in an Urbanized Marine Coastal Area[END_REF][START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF]. A gradient of distribution and structure of ultraplankton have been recently observed [START_REF] Coclet | Changes in Bacterioplankton Communities Resulting From Direct and Indirect Interactions With Trace Metal Gradients in an Urbanized Marine Coastal Area[END_REF][START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF]) correlated with the already-known chemical contamination.

Through sediment resuspension, we hypothesized that this dredging operation would lead to a general increase in Cu and Pb contamination in the particulate fraction, to an increase in dissolved Pb, to a development of heterotrophic prokaryotes and to a strong dominance of eukaryotic ultraphytoplankton in the bay. The spatial extent of this phenomenon could be limited by efforts made to contain suspended particles, and its significance could be challenged by the frequency of other human-induced sediment resuspension events in this harbor. In order to test this hypothesis, a long-term monitoring was performed. The dredging site, the dumping site and a control reference site located close to an aquaculture area were monthly monitored. Discrete water sampling was used to evaluate ultraplankton abundance as well as Cu and Pb concentrations partitioning between the dissolved phase and the particles. A continuous weekly monitoring of labile Pb was also weekly conducted using DGT samplers.

In addition to focusing on the dredging period, common spatial and temporal variations of the different parameters were recorded up to 1 year before dredging and for almost 2 years afterwards, in order to be able to describe a reference period.

Experimental

Dredging operation and sampling sites

A long-term monitoring covering dredging and non-dredging periods was performed in Toulon Bay, known to be highly contaminated in both sediment and water compartments [START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF][START_REF] Tessier | Study of the spatial and historical distribution of sediment inorganic contamination in the Toulon bay (France)[END_REF].

The dredging operation consisted in sediment suction (manually driven) at the dredging site (DS) located in the northern part of Small Bay, in an area well exposed to water currents (Figure 1). Dredged material was stored underwater in a geotextile bag of ~ 60 x 20 m at a dumping site (IDuS) located ~ 800 m away from DS, in an enclosed docking area. This bag was supposed to retain particles, but it was water permeable. Dredging operation occurred from September 2014 until January 2016. Once dredging operations were complete, the geotextile bag and the sediment it contained were removed from the dock and transferred to land for treatment of dredge spoil.

In order to evaluate the impact at short distance and taking into account a potential difference between DS and IDuS, a third site (ODuS), located few meters away for IDuS in the same enclosed dock was also sampled. Furthermore, a control reference site (RS) located 3 km away from DS, in the southern part of Small Bay (Figure 1), close to the aquaculture area was studied. DS was monitored to evaluate the potential impact of the dredging at the dredging site. IDuS was studied to assess the supposed impact of sediment resuspension.

ODuS was examined to measure the impact of sediment resuspension at short distance, as well as the effectiveness off the geotextile bag in relation to both particulate and dissolved contamination export. Concerning RS, the authors are aware that it is not a pristine site since it is near the aquaculture. Nevertheless, it was considered as a reference site since is closed to the main water renewal chanel, with a huge influence of the open sea water. These four sites were monthly monitored.

Seawater sampling and samples pretreatment

Seawater was monthly sampled from September 2013 to November 2017. The monitoring of ODuS only started in July 2014. Samples were collected at 5m depth using a 4.2 L horizontal van Dorn type water sampler (Wildco, model Beta) previously acid-cleaned (10 % v/v HNO 3 , pro analysis, Fisher Scientific) and fully rinsed with Milli-Q water (18.2 MΩ, Millipore) in the laboratory. On field, the water sampler was washed with acidified Milli-Q water (0.1 % v/v HCl, TraceSelect, Fluka) and then rinsed three times with seawater from the site to be sampled. One liter of seawater was stored in fluorinated ethylene propylene (FEP) bottles (Nalgene), which had previously been carefully cleaned (10 % HNO 3 (Fisher Scientific Pro), rinsed three times with Milli-Q water, filled with 0.1 % v/v HCl (TraceSelect, Fluka) and rinsed three times with seawater from the site. Back to the laboratory, these 1 L samples were immediately split under a metal-clean laminar hood: (i) one part to analyze the acid leachable metals concentrations, and (2) another part filtered over 0.2 µm (cellulose acetate syringe filter (Sartorius) pre-rinsed and conditioned following the same protocol as described for 1 L FEP bottles). To avoid adsorption onto vessels, each fraction was stored into metals clean FEP bottles. All samples (acid leachable samples and filtered samples) were then acidified with 0.2 % v/v HNO 3 s.p (Merck) before being UV-irradiated (150-W mercury lamp, Hanau, Germany) for at least 24h in order to completely decompose organic matter [START_REF] Omanović | Trace metal detection in Šibenik Bay, Croatia: Cadmium, lead and copper with anodic stripping voltammetry and manganese via sonoelectrochemistry. A case study[END_REF] and assess the concentration of metals by stripping voltammetry. The total acid leachable metals concentration will further be referred as total concentrations (Cu T and Pb T ) in this manuscript.

In addition, from January 2015 to November 2017, 10 mL seawater samples were filtered on field through a 25 mm Swinnex filter holder (Millipore, USA) equipped with a 90 µm nylon mesh. The filtrate was fixed with 0.25 % glutaraldehyde (final concentration) and frozen at -80°C until flow cytometry analyses.

Because of the heterogeneity of the monitoring period for the biological and chemical analyses, and the absence of biological data before the beginning of the dredging operation, the data will not be presented in 3 periods representing the situation before, during and after the dredging but only in 2 periods representing on the one hand the dredging period and on the other hand the reference period, which includes all the other sampling dates.

The number of samples analyzed as well as the beginning and the end of each type of sampling are summarized in Supplementary Information 1.

DGT passive sampling

In addition to discrete water sampling, DGT passive samplers were deployed at three sites (DS, ODuS, RS) in order to constantly monitor time-weighted average The dissolved and total concentrations of Cu and Pb were determined by Differential Pulse Anodic Stripping Voltammetry (DPASV). The measurements were carried out using an AutolabIII potentiostat (EcoChemie) equipped with an autosampler. A three-electrode system (663 VA Stand, Metrohm) consisting of Hg working electrode (size 3), Ag/AgCl/3M KCl reference electrode and Pt counter electrode was used for measurements. Deposition potential was -0.8 V during 600 s. The initial and final scanning potential were set to -0.75 to 0.02 V. The peak positions were -0.15 ± 0.03 V and -0.37 ± 0.03 V for Cu and Pb respectively. Automated measurements were controlled by the GPES 4.9 software (EcoChemie). Quantification of the metals concentrations was performed using the standard additions method, automated using syringe burettes (Pump XE 1000, TECAN).

Voltammograms were processed (peak height of 2 nd derivative as analytical signal characteristic) using ECDSOFT software [START_REF] Omanović | Automation of Voltammetric Measurements by Polarographic Analyser PAR 384B[END_REF]. Additional information can be find elsewhere [START_REF] Cindrić | Evidencing the natural and anthropogenic processes controlling trace metals dynamic in a highly stratified estuary: The Krka River estuary (Adriatic, Croatia)[END_REF][START_REF] Louis | Characterisation and modelling of marine dissolved organic matter interactions with major and trace cations[END_REF][START_REF] Oursel | Dynamics and fates of trace metals chronically input in a Mediterranean coastal zone impacted by a large urban area[END_REF]. All Pb and Cu results were above the limit of detection (LOD) value (0.004 and 0.04 nM, respectively).

Ultraplankton characterization and enumeration

Autotrophic prokaryotes (Synechococcus-like SYN) and photosynthetic eukaryotes were characterized and enumerated using a BD AccuryTM C6 (BD Biosciences) flow cytometer, as previously described [START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF][START_REF] Marie | An improved protocol for flow cytometry analysis of phytoplankton cultures and natural samples: An Improved Protocol for Flow Cytometry Analysis[END_REF]. The differentiation between pico-and nano-planktonic cells was performed with the help of non-fluorescent size calibration beads (ThermoFisher). One photosynthetic picoeukaryotic groups (PPE) and two photosynthetic nanoeukaryotic groups (PNE) were differentiated. PNE were indexed according to their autofluorescence into highly red fluorescent photosynthetic nanoeukaryotes (HR PNE) and highly orange fluorescent photosynthetic nanoeukaryotes (HO PNE, corresponding to putative cryptophytes). Free-living heterotrophic prokaryotes (HP) were enumerated after staining with SYBR Green as previously described [START_REF] Cabrol | Inhibitory effects of sodium azide on microbial growth in experimental resuspension of marine sediment[END_REF].

2.6.

Statistical analyses

Differences in metals concentrations or in microbial groups' abundances were statistically tested through Kruskal-Wallis tests and Tukey post-hoc comparisons. These analyses were performed by using RStudio (RStudio team, packages stats and PMCMR).

In order to evaluate the links between dissolved and total concentrations of metals, Spearman's correlation analyses were performed using a homemade software named Corrdata (developed by Dr. Omanović).

Meteorological data

In order to evaluate the potential influence of wind onto Cu and Pb concentration in the Bay, wind characteristics were collected from a nearby weather station (Toulon -La Mitre station, 43.104065 N, 5.931631E). Daily average wind intensity and direction were recovered from Meteo France. More information on the treatment of these data and on the comparison with Cu and Pb data is available in Supplementary Information SI 2.

Results and discussion

3.1. Metal concentrations in the Bay during the reference period A north-south decreasing trend (i.e., IDuS, ODuS, DS, RS) of both total and dissolved metal concentrations was observed in the Bay for the non-dredging period (Figure 2), as previously observed either punctually or during a one-month monitoring [START_REF] Coclet | Changes in Bacterioplankton Communities Resulting From Direct and Indirect Interactions With Trace Metal Gradients in an Urbanized Marine Coastal Area[END_REF][START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF]. Cu T and Pb T as well as Cu D (dissolved Cu) and Pb D (dissolved Pb) concentrations were higher in the northern part of the Bay with a 4-or 5-fold increase in ODuS when compared to RS (Table 1). It should be underlined that Pb T increased in a higher extend than Cu T . The present study clearly demonstrates that this spatial trend of water column contamination remains stable at a pluri-annual time scale (Figure 3). For all sampling dates, PbD was higher at ODuS than DS, and concentration at RS were always the lowest. Concerning Pb T , Cu D and Cu T this observation was true 20 times out of 21. Moreover, they were always higher in RS

when compared to open Mediterranean seawater (Table 1). DS presented intermediate concentrations between RS and ODuS (Table 1). This spatial trend clearly reflects the anthropization of the northern part of the Bay in which various industrial and harbor sources contributing to the contamination are located [START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF][START_REF] Dang | Evidencing the Impact of Coastal Contaminated Sediments on Mussels Through Pb Stable Isotopes Composition[END_REF][START_REF] Tessier | Study of the spatial and historical distribution of sediment inorganic contamination in the Toulon bay (France)[END_REF]. The difference between DS and ODuS could be linked to their respective hydrodynamics, DS being located in a rather open part whereas ODuS was located at the bottom of a very enclosed dead-end, thus submitted to limited water exchanges. Furthermore, the enclosed dock of ODuS is subjected to high maritime traffic of numerous navy boats, thus potentially exposed to higher contamination by sediment resuspension (Supplementary Information 3). Similar concentration gradients were observed in other studies of semi-enclosed Bays and were explained by combinations of point contaminant sources, the hydrodynamics of the zone, diffusive fluxes from sediment, as well as sediment resuspension events [START_REF] Cindrić | Evidencing the natural and anthropogenic processes controlling trace metals dynamic in a highly stratified estuary: The Krka River estuary (Adriatic, Croatia)[END_REF][START_REF] Lares | Spatial Variability of Trace Metals and Inorganic Nutrients in Surface Waters of Todos Santos Bay, México in the Summer of 2005 During a Red Tide Algal Bloom[END_REF][START_REF] Sañudo-Wilhelmy | Trace Metal Concentrations in the Surf Zone and in Coastal Waters off Baja California, Mexico[END_REF].

In the studied area, the proportion of Pb D in the water column was steady (56 ± 3 % on average), demonstrating the absence of any spatial trend in Pb partitioning between the dissolved and particulate phases. On the contrary, Cu D proportion exhibited variations among sites and was lower in the northern part (68 % at DS and 70 % at ODuS) compared to RS (81 %). Dissolved concentrations for both metals were within the range found for anthropized costal zones [START_REF] Cindrić | Evidencing the natural and anthropogenic processes controlling trace metals dynamic in a highly stratified estuary: The Krka River estuary (Adriatic, Croatia)[END_REF][START_REF] Oursel | Behaviour and fate of urban particles in coastal waters: Settling rate, size distribution and metals contamination characterization[END_REF][START_REF] Oursel | Dynamics and fates of trace metals chronically input in a Mediterranean coastal zone impacted by a large urban area[END_REF]. Furthermore, Cu D /Pb D ratio was higher at RS (20) compared to DS and ODuS (respectively 16 and 15). Cu T /Pb T ratio was also higher at RS ( 14) than in DS and ODuS (12). Due to the higher Pb T increase (as quoted before), this ratio decrease tends to point out sediment as the main source of Pb contamination at every sites, resulting in a different contamination level of the water due to different sediment contamination levels [START_REF] Tessier | Study of the spatial and historical distribution of sediment inorganic contamination in the Toulon bay (France)[END_REF] and/or sediment grain size distribution [START_REF] Wafo | A chronicle of the changes undergone by a maritime territory, the Bay of Toulon (Var Coast, France), and their consequences on PCB contamination[END_REF] which impacts resuspension. The contribution of a higher maritime traffic in the northern part of Small Bay to the frequency of sediment resuspension remains to be determined. Furthermore, a probable higher dilution of resuspended sediment and diffusive flux can be hypothesized in RS compared to DS and in DS compared to ODuS according to hydrodynamics, ODuS being very enclosed and RS being close to the main connection between Small Bay and Large Bay.

As observed for Pb D and Pb T concentrations, DGT-labile Pb concentrations showed a decreasing trend from ODuS towards RS (Figure 2). DGT-labile Pb concentration is considered as the potentially bioavailable fraction. This relation is supported by a few studies which found out that the accumulated metal (including Pb) in biota are better correlated with DGT-labile fraction than with dissolved fraction [START_REF] Kim | Monitoring trace metals in seawater using a diffusive gradient in thin film probe in Ulsan Bay, East Sea, Korea: Comparison with transplanted mussels[END_REF].

The main benefit of using DGT passive samplers is to provide continuous and time integrated level of metals concentration over the deployment period. No correlation was found between DGT-labile Pb and Pb D (Spearman's correlation test, p > 0.05) in each single site, whereas DGT-labile Pb and Pb D were correlated when all sites were considered together (Spearman's correlation test, ρ = 0.565, p < 0.05). Moreover, the concentration of DGT-labile Pb for some periods was higher than the corresponding Pb D concentration. Taken together, these observations demonstrate that monthly discrete sampling was too occasional compared to one-week-long DGT deployments and not adequate to represent the DGT-deployment period. Such discrepancy was already observed for other dynamic systems [START_REF] Cindrić | Evaluation of Discrete and Passive Sampling (Diffusive Gradients in Thin-films -DGT) Approach for the Assessment of Trace Metal Dynamics in Marine Watersa Case Study in a Small Harbor[END_REF]. It suggests a strong short-term variability of Pb concentration within the Bay, which is also obvious on temporal profiles of DGT-labile Pb concentrations (Figure 3). Considering that one of the main Pb sources for the water column in the Bay appears to be sediment resuspension [START_REF] Dang | Evidencing the Impact of Coastal Contaminated Sediments on Mussels Through Pb Stable Isotopes Composition[END_REF], this high short-term variability could reflect the very punctual character of sediment resuspension (in both space and time). No link between Pb concentration increases and meteorological features could be highlighted during this monitoring (Figure SI2. 2) and the influence of river inputs can be neglected according to previous studies and very low discharge [START_REF] Nicolau | Base flow and stormwater net fluxes of carbon and trace metals to the Mediterranean sea by an urbanized small river[END_REF]. As a result, it could be hypothesized that Pb contamination events of the water column by sediment resuspension occurs along with human activities rather than natural climatic events in Toulon Bay, even in the absence of any dredging operation. Further work is needed to identify which activity could be responsible. The link with the gathering of most of the maritime traffic in the northern part of the Bay should be investigated in priority.

Distribution and structure of ultraplankton during the reference period

The abundance of heterotrophic prokaryotes (HP) ranged between 0.9 x 10 5 and 2.1 x 10 6 cells.mL -1 . These abundances are in the classical range generally observed for coastal Mediterranean sites [START_REF] Gregori | Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining[END_REF][START_REF] Šantić | HNA and LNA bacteria in relation to the activity of heterotrophic bacteria 16[END_REF][START_REF] Suffridge | B Vitamins and Their Congeners as Potential Drivers of Microbial Community Composition in an Oligotrophic Marine Ecosystem[END_REF] and of previous observations in Toulon Bay [START_REF] Coclet | Changes in Bacterioplankton Communities Resulting From Direct and Indirect Interactions With Trace Metal Gradients in an Urbanized Marine Coastal Area[END_REF]. Neither a clear seasonal trend nor a temporally stable difference between sites as demonstrated for metals could be observed.

Indeed, although HP abundance was sometimes enriched in the northern enclosed area when compared to the southern site as previously observed during a short-term monitoring [START_REF] Coclet | Changes in Bacterioplankton Communities Resulting From Direct and Indirect Interactions With Trace Metal Gradients in an Urbanized Marine Coastal Area[END_REF] where similar HP abundances were also regularly observed at all sites during our longer term monitoring. As a consequence, the average HP abundance was not statistically different between sites (Kruskal-Wallis tests, p > 0.05, Figure 4). Since this situation is quite different from the metals' contamination spatial trend, which was observed at almost all the sampling dates, the metals' contamination trend can clearly not be the main driver of HP abundance dynamics in Toulon Bay. Longer-term temporal processes such as seasonal variations in water-temperature [START_REF] Fuhrman | A latitudinal diversity gradient in planktonic marine bacteria[END_REF][START_REF] Gilbert | Defining seasonal marine microbial community dynamics[END_REF], resource availability [START_REF] Chow | Temporal variability and coherence of euphotic zone bacterial communities over a decade in the Southern California Bight[END_REF][START_REF] Gilbert | Defining seasonal marine microbial community dynamics[END_REF] or biotic interactions related to the structure of both phytoplanktonic and zooplanktonic communities [START_REF] Gilbert | Defining seasonal marine microbial community dynamics[END_REF][START_REF] Goni-Urriza | Consequences of contamination on the interactions between phytoplankton and bacterioplankton[END_REF] could represent more important drivers of HP abundance in Toulon Bay.

Similarly, range of ultraphytoplanktonic community's abundance (4.5 x 10 3 to 6.8 x 10 4 cell.mL -1 ) was similar to other observations reported for Mediterranean coastal sites [START_REF] Gregori | Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining[END_REF][START_REF] Thyssen | Onset of the spring bloom in the northwestern Mediterranean Sea: influence of environmental pulse events on the in situ hourly-scale dynamics of the phytoplankton community structure[END_REF] or previously in Toulon Bay [START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF] and did not show any clear temporal or spatial trend. When considering the different phytoplanktonic subgroups, SYN (Synechococcus-like) and PPE (picoeukaryotes-like) were the most abundant, representing at least 70 % of the community in 95 % samples. SYN abundance demonstrated a decreasing trend towards the northern enclosed ODuS whereas PPE abundance appeared stable across sites (Figure 4). As a consequence, a notable community structure shift was observed from a SYN-dominated (49.1 % on average) ultraphytoplanktonic community in RS to a PPE-dominated (56.3 % on average) community in ODuS. The proportion of these two most abundant ultraphytoplanktonic groups was highly variable through time. However, the proportion of SYN was higher in RS than in ODuS for 13 of the 16 sampling dates. Similarly, the proportion of PPE was lower in RS than in ODuS for 75 % sampling dates. Concerning nanophytoplanktonic groups, HR PNE (highly red fluorescent photosynthetic nanoeukaryotes) represented on average 10 % of the community's abundance and appeared to be slightly more abundant in ODuS than in RS. Finally, HO PNE (highly orange fluorescent photosynthetic nanoeukaryotes) was the least abundant group (6 % on average) and displayed no spatial trend. The spatial differences mainly driven by picophytoplanktonic groups are in agreement with previous observations in Toulon Bay either at high-spatial but low-temporal frequency [START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF] or low-spatial and similar temporal frequency [START_REF] Delpy | Pico-and Nanophytoplankton Dynamics in Two Coupled but Contrasting Coastal Bays in the NW Mediterranean Sea (France)[END_REF].

Dredging significantly affected Cu and Pb contamination in the water column at large spatial scale

The spatial trend of Cu and Pb concentrations during the dredging period was similar to the one observed during the reference period (Figure 2). A significant increase in Pb T , Pb D and DGT-labile Pb concentrations between the dredging period and the reference period was observed in all sites (Kruskal-Wallis tests, p < 0.05). RS site was subjected to the highest relative increases in Pb concentrations (Table 2). [START_REF] Van Den Berg | Dredging-related mobilisation of trace metals: a case study in The Netherlands[END_REF] and [START_REF] Slotton | Heavy metals in intact and resuspended sediments of a California reservoir, with emphasis on potential bioavailability of copper and zinc[END_REF] showed that there was no increased Pb D during their monitoring of a dredging activity, hypothesizing a relatively slow oxidation of metal sulfides and a fast re-oxydation of Mn and Fe which precipitate Cu and Pb under oxy-hydroxides particulate forms. Based on their hypothesis, during this present monitoring it could be assumed that metal sulfides undergone a fast oxidation which was not counterbalanced by Mn and Fe re-oxydation or that the amount of oxy-hydroxides formed were not sufficient to precipitate all the remobilized Pb.

Contribution of the organic matter on this remobilization remains to be determined.

When comparing the dredging period to the reference period, the increase in Pb D and Pb T concentrations ranged between 0.2 and 2.28 nM for RS, DS and ODuS (Table 2). During the dredging period, the increases in Pb concentrations between ODuS and IDuS were far higher, with around 26 nM for Pb D and around 356 nM for Pb T . Since an increase of Pb concentrations between the dredging and the reference period was observed, along with an increase in Pb concentrations between IDuS and ODuS, it can be concluded that sediment resuspension by the dredging operation increased water column Pb contamination at the scale of the whole studied area. The dispersion of remobilized Pb appeared nevertheless strongly limited by the use of the geotextile bag. This is in agreement with the hypothesis of sediment resuspension as the major source of Pb contamination in the water column of Toulon Bay previously proposed by [START_REF] Dang | Evidencing the Impact of Coastal Contaminated Sediments on Mussels Through Pb Stable Isotopes Composition[END_REF].

Pb D fraction was globally homogeneous during the dredging period in RS, DS and ODuS (58 ± 3 % on average). It was the same to that determined during the reference period (56 ± 3 % on average). However, at IDuS, Pb D percentage dropped down to 8.1 %. This could be explained by a far higher particles concentration inside the dumping site, for which Pb is known to have a high affinity. Moreover, the increase in Pb D concentrations in ODuS and at the other sites suggests a Pb D export outside of the geotextile bag. This export at rather long distance is not surprising, considering the main hydrological features of the Bay as previously detailed, RS being localized on the main water exchange area with the open coastal part [START_REF] Dufresne | Hydro-sedimentary model as a postaccidental management tool: Application to radionuclide marine dispersion in the Bay of Toulon (France)[END_REF].

No significant difference in Cu T and Cu D concentrations was observed between the dredging and the reference period in all sites (Kruskal-Wallis tests, p > 0.05) ( 2 and Table 2). This might correspond to very important quantities of suspended matter at IDuS site but, contrarily to what was observed for Pb, no export of Cu D outside of the geotextile bag could be detected. This could be explained by Cu higher affinity (compared to Pb) to organic ligands, which could have trapped Cu in IDuS because of more numerous organic active sites for Cu surface complexation/adsorption.

Cu D /Pb D was higher at RS (15) than in DS (13) and ODuS (11) (Table 2). Contrarily, Cu T /Pb T was homogeneous at RS, DS and ODuS (10 ± 0.5) and lower compared to the reference period. These ratios were lower during the dredging period compared to the reference period due to higher Pb concentrations during the dredging period. This observation confirms the different impacts of sediment resuspension on Cu and Pb contamination of the water column, in both the dissolved and particulate phases.

In spite of homogeneous Cu T /Pb T, Cu T and Pb T were spatially highly variable during the investigated period. It could increase up to 90-fold for Cu and to 1,600-fold for Pb between RS and IDuS (Figure 3). Although suspended matter was not evaluated, such increase was likely to be due to the increase of suspended sediment particles since the difference between IDuS and ODuS in average reached a 7.5 factor for Cu and a 55 factor for Pb, respectively, although the two sites were only separated by a few meters and a geotextile bag. In plus of being spatially variable during this period, Cu T, Pb T and Pb D concentrations were highly variable in time (Figure 3). As an example, for the most extreme variations at IDuS, Cu T , Pb D or Pb T could increase by a 35-fold, 16-fold or even 645-fold between two consecutive samplings, respectively. These increases could sometimes be synchronized at all sites, suggesting possible short-term water exchange between sites through hydrodynamics.

However, those synchronized periods were quite rare, suggesting the predominance of local impact in the short-term. The significant increase of all Pb concentrations during the dredging period nonetheless suggests a cumulative effect of the dredging operation in the long-term. It should be noticed that there were more strong wind events during the dredging period (around 7 by month in average) than during the reference period (around 3 by months in average) (Supplementary Information 2). Nevertheless, no co-variation was found between wind direction or intensity and Cu or Pb concentration, suggesting that the wind was not responsible for the high variability of those concentrations in the Bay during the dredging period and emphasize the influence of the dredging onto the water column contamination.

Thus, although precautions were taken to limit contaminated particles dispersion at a large scale, the underwater storage of dredged sediment in a permeable compartment still lead to a significant environmental impact, especially since DGT-labile Pb, and thus potentially bioavailable Pb, was released in the water column.

Dredging accentuated the spatial trends of ultraplankton structure

The highest HP abundances observed during the whole monitoring were recorded within the geotextile bag, at IDuS. Moreover, at all sites, a slight but significant HP enrichment (Kruskal-Wallis tests, p < 0.05) was recorded during the dredging period when compared to the reference period (Figure 4). HP enrichment was previously observed along with sediment resuspension in experimental conditions [START_REF] Goni-Urriza | Consequences of contamination on the interactions between phytoplankton and bacterioplankton[END_REF]). Yet, the present work transposes this observation in situ and demonstrates that although precautions were taken, the dredging methodology used was not enough to avoid impact on the biota.

Regarding the ultraphytoplanktonic community, no significant difference in total abundance was observed and none of the four considered subgroups demonstrated noticeable variation during the dredging (Kruskal-Wallis tests, p > 0.05). At IDuS, while the mean total abundance was not different from the other sites (Kruskal-Wallis tests, p > 0.05), PPE abundance was on average the highest among all sites whereas SYN abundance was on average the lowest (Figure 4). As a consequence, the shift in biological structure observed from RS to ODuS during the reference period was still observed during the dredging and even accentuated within the geotextile bag where the highest proportion of PPE was observed 65% of the sampling dates. This suggests that sediment resuspension and particles concentration in IDuS accentuated the structure shift from the reference situation in RS. [START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF] demonstrated by experimental approaches that such structure shift could be linked to metals' contamination as well as to sediment resuspension. While metals' addition led to a strong decay of the ultraphytoplanktonic community, exposition to sediment elutriate temporally promoted phytoplanktonic growth [START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF]. [START_REF] Lafabrie | Impact of contaminated-sediment resuspension on phytoplankton in the Biguglia lagoon (Corsica, Mediterranean Sea)[END_REF] also reported similar temporal stimulation of pico and nanoeukaryotic phytoplanktonic groups by sediment resuspension. Hence, the spatial community structure shift observed during this long-term study in the shallow and enclosed Toulon Bay could well be linked to sediment resuspension by different human activities.

Although not determined in this study, the ultraphytoplanktonic biovolume must have been higher in the northern enclosed area and especially within the geotextile bag since the dominance of smaller SYN cells (≤ 1µm) was replaced by the dominance of larger PPE cells (≥ 2,5 µm during this monitoring) without any variation in total cells' number. As a consequence, higher primary production could explain the larger abundances of HP, which are known to strongly rely on phytoplanktonic resources in the marine environment [START_REF] Buchan | Master recyclers: features and functions of bacteria associated with phytoplankton blooms[END_REF][START_REF] Luria | Seasonal Shifts in Bacterial Community Responses to Phytoplankton-Derived Dissolved Organic Matter in the Western Antarctic Peninsula[END_REF][START_REF] Williams | The role of planktonic Flavobacteria in processing algal organic matter in coastal East Antarctica revealed using metagenomics and metaproteomics: Metaproteomics of marine Antarctic Flavobacteria[END_REF]. The role of sediment resuspension in providing organic resources for the HP community cannot be excluded since sediment resuspension can release significant organic carbon amounts [START_REF] Guigue | Remobilization of polycyclic aromatic hydrocarbons and organic matter in seawater during sediment resuspension experiments from a polluted coastal environment: Insights from Toulon Bay (France)[END_REF], fueling HP growth at short time scales [START_REF] Goni-Urriza | Consequences of contamination on the interactions between phytoplankton and bacterioplankton[END_REF]. Moreover, the mixotrophic potential of PPE [START_REF] Hartmann | In situ interactions between photosynthetic picoeukaryotes and bacterioplankton in the Atlantic Ocean: evidence for mixotrophy: Bacterivory by marine phototrophic picoeukaryotes[END_REF] could also contribute to their success in case of sedimentresuspension-related HP growth, like the one observed in the present study. The respective influences of phytoplanktonic structure shifts and organic carbon release by sediment resuspension requires further investigation to assess a potential trophic alteration of the planktonic food web by dredging activities, as observed in other coastal contexts [START_REF] Jonsson | Terrestrial discharges mediate trophic shifts and enhance methylmercury accumulation in estuarine biota[END_REF].

Conclusion

The long-term monitoring during the reference period emphasized the consistency of the previously-observed spatial gradient of metals' contamination in Toulon Bay. Moreover, although investigated here on a smaller area, the dominance of PPE over SYN in ultraphytoplanktonic community of the most contaminated sites also appeared recurrent in the long term. Temporal ultraphytoplankton and HP abundance dynamics however appeared to be driven by other parameters than metals' contamination.

During a rather long dredging operation, the use of a combination of sediment aspiration and immersion of dredged material underwater in a geotextile bag appeared efficient in preventing dumped sediment dispersion and water column contamination by contaminated particles at a large spatial scale. However, in spite of the use of this protocol, statistically significant increases in Pb concentrations and lability were observed at large spatial scale (several km away from the dredging and dumping sites) during the dredging operation under the probable influence of hydrodynamics in the area. Impacts on the biota of the dredging operation consisted in stimulating HP abundance through ecological processes that remain to determine.

In the dumping site created for this dredging operation, the highest metals concentrations, HP abundances and PPE proportion were observed thus appearing to extend the limit of both chemical and biological spatial trends observed during the reference period.

Taken together, these observations point toward a strong potential influence of sediment resuspension in the ecological functioning of the highly anthropized Toulon Bay.

The simultaneous characterization of energetic (light, O 2 ) and nutritive (inorganic and organic nutrients) resources as well as a better characterization of the speciation of Pb controlling its lability represent further direction to better evaluate the ecological processes underlying these observations. Moreover, evaluating the quantity, mineralogy, grain size distribution and contamination of particles along with physico-chemical characteristics of the water (redox potential, pH and oxygen availability) at a higher temporal resolution are needed for a better understanding of the processes leading to metals' remobilization.

This present study clearly points out the necessity to take precautions during dredging activities in order to minimize the contamination in the dissolved fraction which is often disregarded. Indeed, the impact in the particulate phase seemed to have been under control, nevertheless, the dissolved phase was impacted by export of contaminant through the geotextile bag. Therefore, techniques to reduce the impact of harbors activities onto the environment need to be improved. at the three sites monitored apart from the dredging period.

Figures and tables captions

Table 2 The ranges and average values of dissolved and total Cu and Pb concentrations (in nM) at the four sites monitored during the dredging period. Values in parentheses correspond to percentage and absolute (in nM) concentrations' increase compared to the reference period.

Note that for IDuS no DGT were deployed. The stars (*) mean that there is a significant difference between the reference and the dredging period. ND stands for non-determined. Table 2 The ranges and average values of dissolved and total Cu and Pb concentrations (in nM) at the four sites monitored during the dredging period. Values in parentheses correspond to percentage and absolute (in nM) concentrations' increase compared to the reference period.

Supplementary material

Note that for IDuS no DGT were deployed. The stars (*) mean that there is a significant difference between the reference and the dredging period. ND stands for non-determined.

Figure. 1

 1 Figure. 1 Location of the monitoring sites and the main anthropogenic activities in Toulon

Figure. 2

 2 Figure. 2 Spatial variations of Cu and Pb concentrations (nM) during the two define periods

Figure. 3

 3 Figure. 3 Spatial and temporal variation of Cu T (A), Pb T (B), Cu D (C), Pb D (D) and Pb L (E).

Figure. 4

 4 Figure. 4 Spatial variations of microbial groups' abundance (cell.mL -1 ) during the two define

Figure. 1

 1 Figure. 1 Location of the monitoring sites and the main anthropogenic activities in Toulon Bay. Sampling sites are represented with the following marking, cross: dredging site (DS), closed star: inside the dumping site (IDuS) (inside the geotextile bag), closed circle: outside the dumping site (ODuS) (outside the geotextile bag), closed triangle: reference site (RS) (near the fish farming area). Note that ODuS is deliberately moved to the right for more visibility on the map, however only a few meters separate it from IDuS.

FigureFigure. 2

 2 Figure(s)

FigureFigure. 3

 3 Figure(s)

FigureFigure. 4

 4 Figure(s)

Figure

  Figure(s)

  Table2). Cu T concentrations appeared slightly higher and highly variable in IDuS compared to ODuS during the dredging period. Taken altogether, these observations indicate that sediment resuspension can be a source of Cu contamination in Toulon Bay water column, but this contamination is lower than that of Pb and the used geotextile bag seems efficient in limiting the dispersion of Cu remobilized during the dredging operation. As observed for Pb, Cu D fraction was globally homogeneous in RS, DS and ODuS (77 ± 6 %) during the dredging period and close to that determined during the reference period (around 70 %). However, at IDuS, the percentage of Cu D dropped down to 7.2 % but no significant correlation could be observed between Cu D and Cu T . On the contrary, Cu D was quite well correlated to Cu T at all other sites (0.961, 0.939 and 0.718 at RS, DS and ODuS respectively) during both reference and dredging periods. At last, Cu D decreased in IDuS compared to ODuS during the dredging, whereas Cu T increased (Figure

Table 1

 1 Ranges and average values of dissolved and total Cu and Pb concentrations (in nM)

Table 1

 1 Ranges and average values of dissolved and total Cu and Pb concentrations (in nM) at the three sites monitored apart from the dredging period.

	Element	Site	Min	Average	Max
		RS	3.40	9.35 (a)	19.97
	CuD	DS	5.50	15.33 (b)	28.88
		ODuS	12.93	36.20 (c)	73.16
		RS	4.52	11.57 (a)	22.16
	CuT	DS	10.43	22.58 (b)	37.83
		ODuS	18.11	51.83 (c)	95.14
		RS	0.13	0.48 (a)	1.05
	PbD	DS	0.36	0.98 (b)	1.66
		ODuS	1.65	2.37 (c)	3.90
		RS	0.30	0.82 (a)	1.42
	PbT	DS	0.75	1.83 (b)	3.70
		ODuS	2.15	4.27 (c)	9.74
		RS	0.12	0.77 (a)	1.71
	PbL	DrS	0.65	1.44 (b)	3.47
		ODuS	2.22	2.75 (c)	3.98

Table ( s)

 (

	Fraction	Site	Min	Average	Max
		RS	4.78	12.74 (36%; 3.39)	31.98
	CuD	DS ODuS	5.75 18.63	20.72 (35%; 5.39) 41.17 (14%; 4.97)	37.52 60.70
		IDuS	17.63	31.84	65.60
		RS	6.24	15.25 (32%; 3.68)	36.24
	CuT	DS ODuS	7.19 25.54	25.23 (12%; 2.65) 59.34 (14%; 7.51)	41.54 109.85
		IDuS	34.01	440.00	3362.34
		RS	0.35	0.86 (80%; 0.38) *	1.81
		DS	0.52	1.58 (61%; 0.6) *	2.60
	PbD	ODuS	1.49	3.66 (54%; 1.29) *	7.79
		IDuS	2.22	29.32	81.31
		RS	0.47	1.49 (82%; 0.67) *	3.12
	PbT	DS ODuS	0.73 1.91	2.59 (42%; 0.76) * 6.55 (53%; 2.28) *	5.38 14.86
		IDuS	3.99	362.39	2978.76
		RS	0.45	0.97 (26%; 0.20) *	3.09
	PbL	DS ODuS	0.79 2.00	1.65 (15%; 0.21) * 4.63 (68%; 1.88) *	3.09 10.45
		IDuS	ND	ND	ND

Table(s)

Acknowledgments

This study was supported by SE.D.RI.PORT. (SEdimenti, Dragagi e RIschi PORTuali, Interreg Marittimo IT/FR 2017-2020, co-funded by European Regional Development Fund) and MISTRALS-MerMex-WP3-C3A research programs, by LASEM Toulon and CARTT of the Institut Universitaire Technologique from Toulon University. N. Layglon PhD was partly funded by Sud region and supported by LASEM Toulon. C. Coclet PhD was funded by Sud region and supported by Toulon Provence Méditerranée. The authors wish to thank French Navy for sampling assistance as well as Christian Martino for all instrument repairs and care.

In addition, the authors would like to thank Camille Mazoyer for discussions on Toulon Bay hydrodynamics.

This paper is dedicated to our friend and mentor, Cédric Garnier, an outstanding scientist who tragically passed away and without whom this work would not have been possible.

Supplementary Data

Click here to download Supplementary Data: Supplementary material.pdf

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: