Maria Kazour
email: maria.kazour@univ-littoral.fr

Sharif Jemaa

Christelle Issa

Gaby Khalaf

Rachid Amara

Microplastics pollution along the Lebanese coast (Eastern Mediterranean Basin): Occurrence in surface water, sediments and biota samples

Keywords: Microplastics, Levantine Basin, coastal landfill, filter-feeders, micro-Raman

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

Introduction

The world plastic production is continuously increasing, reaching 350 million tons in 2017 [START_REF] Plasticseurope | Plastics -the Facts 2017[END_REF]. Among the produced plastics, only 6-26% are recycled. Landfills receive 21-42% of wastes, whereas the rest are wildly released into the environment due to mismanagement through a variety of pathways [START_REF] Macarthur | The New Plastics Economy-Rethinking the Future of Plastics[END_REF][START_REF] O'connor | Review of the partitioning of chemicals into different plastics: Consequences for the risk assessment of marine plastic debris[END_REF]. Most of the manufactured plastics will eventually end up in the ocean. By the year 2025, about 250 million tons of wastes are expected to enter the ocean water [START_REF] Jambeck | Plastic waste inputs from land into the ocean[END_REF] leading to their accumulation in the marine environment [START_REF] Barnes | Accumulation and fragmentation of plastic debris in global environments[END_REF]UNEP, 2014). Plastic pollution in the marine environment is becoming a problem of global concern.

These plastics are capable of transforming into dangerous small particles known as microplastics (MPs) existing as particles of a size below 5 mm ubiquitously available in the aquatic environments [START_REF] Thompson | Lost at Sea: Where Is All the Plastic?[END_REF][START_REF] Andrady | Microplastics in the marine environment[END_REF]. They are either intentionally produced in small sizes such as polystyrene beads for example, or they are the resultant of macroplastics degradation (whether by Photochemical reaction, or mechanical actions.).

The Mediterranean Sea is prone to several anthropic pressures and has recently been described as one of the most affected marine environments with regards to marine litter and microplastics [START_REF] Lebreton | Numerical modelling of floating debris in the world's oceans[END_REF][START_REF] Cincinelli | A potpourri of microplastics in the sea surface and water column of the Mediterranean Sea[END_REF]. Mainly owing to the semi-enclosed situation and limited outflow of surface waters, a densely populated coastline and intensive fishing, shipping, touristic and industrial activities, substantial amounts of marine litter are accumulating in the Mediterranean Basin. Also, most studies on microplastics have been conducted in the northwestern Mediterranean Sea (e.g. [START_REF] Collignon | Annual variation in neustonic micro-and meso-plastic particles and zooplankton in the Bay of Calvi (Mediterranean-Corsica)[END_REF][START_REF] Fossi | Fin whales and microplastics: The Mediterranean Sea and the Sea of Cortez scenarios[END_REF][START_REF] Ruiz-Orejón | Floating plastic debris in the Central and Western Mediterranean Sea[END_REF]Schmidt el al., 2018;[START_REF] Lefebvre | Microplastics FTIR characterisation and distribution in the water column and digestive tracts of small pelagic fish in the Gulf of Lions[END_REF]. Results showed that their distribution and composition are not homogeneous and geographical differences exist between Mediterranean sub-basins [START_REF] Suaria | The Mediterranean Plastic Soup: Synthetic polymers in Mediterranean surface waters[END_REF][START_REF] Cincinelli | A potpourri of microplastics in the sea surface and water column of the Mediterranean Sea[END_REF]. Such heterogenicity in MPs distribution and of its concentration levels are likely reflecting a complex interplay between pollution sources, hydrodynamic features such as currents, up and down-welling, gyres, fronts and the shape of the coastline [START_REF] Suaria | The Mediterranean Plastic Soup: Synthetic polymers in Mediterranean surface waters[END_REF][START_REF] Schmidt | Occurrence of microplastics in surface waters of the Gulf of Lion (NW Mediterranean Sea)[END_REF][START_REF] Cincinelli | A potpourri of microplastics in the sea surface and water column of the Mediterranean Sea[END_REF]. In addition, the absence of a standardized sampling protocol for the identification and quantification levels of MPs could also be responsible for this heterogeneity in results.

Plastics in general, and microplastics specifically, are prone to be ingested by a wide range of organisms [START_REF] Lusher | Microplastic interactions with North Atlantic mesopelagic fish[END_REF] and about one hundred species in the Mediterranean have been affected either via ingestion or entanglement [START_REF] Deudero | Mediterranean marine biodiversity under threat: Reviewing influence of marine litter on species[END_REF]. Microplastics were already found in several Mediterranean fish species such as Boops boops, Sardina pilchardus, Mullus barbatus and Merluccius merluccius [START_REF] Giani | Microplastics occurrence in edible fish species (Mullus barbatus and Merluccius merluccius) collected in three different geographical sub-areas of the Mediterranean Sea[END_REF][START_REF] Bellas | Ingestion of microplastics by demersal fish from the Spanish Atlantic and Mediterranean coasts[END_REF][START_REF] Rios-Fuster | Anthropogenic particles ingestion in fish species from two areas of the western Mediterranean Sea[END_REF] and in wild mussels (Mytilus galloprovincialis) caught off the Adriatic Sea and central Tyrrhenian Sea [START_REF] Gomiero | First occurrence and composition assessment of microplastics in native mussels collected from coastal and offshore areas of the northern and central Adriatic Sea[END_REF]Renzi et al., 2018a).

Although it is suggested that it is the most polluted region, few studies have been conducted in the Eastern part of the Mediterranean Basin ( [START_REF] Van Der Hal | Exceptionally high abundances of microplastics in the oligotrophic Israeli Mediterranean coastal waters[END_REF][START_REF] Güven | Microplastic litter composition of the Turkish territorial waters of the Mediterranean Sea, and its occurrence in the gastrointestinal tract of fish[END_REF][START_REF] Gündoğdu | Micro-and mesoplastics in Northeast Levantine coast of Turkey: The preliminary results from surface samples[END_REF]. In the Levantine basin, the Lebanese coast is highly affected by numerous anthropogenic pressures such as harbor activities, fishing, domestic and industrial wastewater discharges (generally without prior treatment). Waste production in Lebanon has increased significantly during the last decade and the country has experienced waste crises. The regulated dumping sites could no longer manage the tremendous quantity of municipal garbage generated and, therefore, numerous unregulated and open dumping sites were created throughout the country mainly along the coast [START_REF] Khalil | Municipal leachates health risks: Chemical and cytotoxicity assessment from regulated and unregulated municipal dumpsites in Lebanon[END_REF]. The average composition of municipal solid waste generated shows an important percentage of plastics (15%) which is two to three times higher than that found in Sweden, France and the United States [START_REF] El-Hoz | A Qualitative-Quantitative Methodological Approach for Sustainable Reclamation of Open Dumps: The Case of the Controlled Dump of Tripoli[END_REF].

Though the processes may be slow, landfills are affected by UV radiation and mechanical abrasions leading to the fragmentation of plastics to microplastics, and small plastic debris would find their way into the aquatic environment via leachates discharges [START_REF] Alimi | Microplastics and nanoplastics in aquatic environments: aggregation, deposition, and enhanced contaminant transport[END_REF]. Despite these concerns, there have been no studies specifically examining the occurrence and abundance of microplastics along the Lebanese coastal environment. The aim of this study was to investigate the quantity and types of microplastics found in three matrices (sea water, sediments and marine biota) along the Lebanese coast. All suspected MPs were characterized by size, color and identified according to their polymer nature using micro-Raman spectroscopy.

Fish and bivalves are known to ingest microplastics [START_REF] Collard | Detection of Anthropogenic Particles in Fish Stomachs: An Isolation Method Adapted to Identification by Raman Spectroscopy[END_REF][START_REF] Phuong | Quantification and characterization of microplastics in blue mussels (Mytilus edulis): protocol setup and preliminary data on the contamination of the French Atlantic coast[END_REF][START_REF] Kazour | Sources of microplastics pollution in the marine environment: Importance of wastewater treatment plant and coastal landfill[END_REF], making them a potential vector of toxic chemicals through food chains and into human diets. In the present study we analyzed two marine species that are wholly consumed by the Lebanese community: one pelagic fish species: the European anchovy, engraulis encrasicolus (Linneaus, 1758) and a bivalve species, the spiny oyster, Spondylus spinosus (Schreibers, 1793).

Materials and Methods

Study and sampling area

Lebanon is located in the Eastern Mediterranean (Levantine Basin) with a coastal length of 220 km (Figure 1). Three sampling sites were chosen along the coast according to their degree of anthropogenic pressures making them pollution hotspots (Shaban, 2008). The first site, Tripoli, located in the north, is facing the town's public beaches, characterized by the presence of a fishing harbor, and the existence of a coastal landfill. This latter was constructed in the year 1999 and reached today 36 m height receiving about 400 tons of wastes/day. The second site Beirut, located in the middle, is the country's capital. This site is near the most important harbor in Lebanon and surrounded by three coastal landfills: One closed and two active landfills that reached their maximum capacities (55 meters of height) in July 2019. The third site Sidon, located in the south, is facing a river's mouth and is near a closed landfill operated between 1982 and 2013. With a height of 58 m, this landfill was the biggest in the country receiving 300 tons of wastes/day. Actually, its transformation to a public park led to the burying of all wastes underground; traces of these wastes can always be observed underwater. These landfills lack an appropriate leachates treatment or it is not operational, and leachates are directly dumped to the sea. The sampling for all three matrices was done in May 2018 (spring).

Contamination control

Several precautions were followed to minimize air-born contamination during sampling, samples preparation and analysis. Cotton lab coats were worn and different procedures (filtration, measurements and digestion) were done under a laminar flow hood. All surfaces and equipment were rinsed before usage with filtered ethanol 70% and distilled water. Laboratory supplies were made of glass (bottles, Petri dishes, Erlenmeyers, filtration system) and they were covered immediately after manipulation. All solutions (distilled water, Ethanol 70%, potassium hydroxide KOH 10% and zinc chloride ZnCl2) were filtered 3 times on glass fibers filter (GF/A filters, 1.6 µm porosity, Whatman, France) to eliminate unwanted particles or fibers. Controls were made to follow up the contamination occurring with every step of preparation and observation.

Water and sediments sampling and analyses

Sea surface water and sediments samples were collected during spring 2018. Sea surface water was collected using a manta trawl net (52 µm mesh size) with an opening of 0.63 m and a length of 0.255 m equipped with a flowmeter. The net was trawled for 10 minutes parallel to the coast at about 500 m from the coast with a small artisanal vessel (3 m of length) at a speed of 2 knots.

On average, 100 m 3 of sea water was sampled at each site (Supplementary Table 1). The net was washed with filtered distilled water and all collected residues were rinsed inside clean glass bottles. The collected water was filtered on a stainless-steel sieve of a mesh size of 850 µm and the recovered water was vacuum filtered on GF/A filters. The suspected MPs present on the sieve were sorted visually under the stereomicroscope, fixed on a microscopic slide and conserved inside a clean glass Petri dish.

The first two centimeters of sublittoral sediments were sampled using a steel ring with two stainless-steel plates, rinsed with filtered distilled water before usage. The samples were put inside clean glass jars and stored at -4°C. Samples were thawed at room temperature 12 hours prior to analysis. Then particles with a size range of 1 -5 mm were visually sorted and the remaining sediments underwent a density separation using filtered ZnCl2 (d= 1.8 g/cm 3 ) [START_REF] Imhof | Contamination of beach sediments of a subalpine lake with microplastic particles[END_REF]Nuelle et al., 2014). Briefly, 50 mL of filtered ZnCl2 was added to the sample; the solution was then shaken for 5 minutes twice (once every 4 hours) and let rest for 16 hours. The supernatant was then recovered and filtered on GF/A filters. Microplastics found were reported per gram of sediments sample and per m 3 in sea water samples.

Biota sampling and analyses

Anchovies and spiny oysters were also collected during spring 2018. Anchovies were caught using purse seine nets. Ten individuals of approximately the same size (9 cm ± 0.9) were chosen from each sampling site and were immediately individually conserved in aluminum foil and conserved at -4°C. In the laboratory, their mormophetric characteristics (total length and weight, digestive tube weight) were measured and their digestive tracts were dissected, conserved in aluminum foil and frozen at -4°C. For the spiny oysters, ten individuals were collected by fishermen from each sampling site. In the laboratory, the whole individual was removed from its shell and was conserved in aluminum foil. The net weight of the animal ranged between 4.8 g to 38.2 g -YY with an average of 20.2 g ± 9.4.

All samples were defrosted 12 hours prior to digestion. Fish digestive tracts and oyster soft tissues were digested using potassium hydroxide 10% KOH (Dehaut et al., 2016). In short, the organic material of each individual was put inside separate Erlenmeyer containing solution of KOH 10% and then they were incubated at 60°C for 24 hours. Then, the digestat was recovered and vacuum filtered on GF/A filters and put inside clean glass Petri dishes.

Stereomicroscope and micro-Raman spectroscopy

All samples were first observed under a Leica M165 C Stereomicroscope with a 120x magnification. Photos of items were captured using a Leica M170 HD camera connected to LAS (Leica Application Suite) software. They were listed, measured, and their morphology, color and shape were noted. Several criteria were taken into consideration while counting the suspected items: (1) absence of cellular or organic structures; (2) a homogenous thickness across the particles; and, (3) homogenous colors (Hidalgo-Ruz et al. 2012). Suspected microplastics were divided into 6 size classes: <200 µm, 200-400 µm, 400-600 µm, 600-800 µm, 800-1000µm, and > 1000 µm.

Suspected microplastics were analyzed using Micro-Raman Xplora Plus (HORIBA Scientific® France). This machine is equipped with a closing chamber that isolates the filter from its surrounding, hence minimizing any airborne contamination.

Due to the high amount of time it takes when analyzing filters under micro-Raman, five filters of anchovies and five filters of spiny oysters from each site containing the highest number of potential MPs were identified. Subsamples of sediments and water were analyzed in a way that 25% of particles were analyzed. For MPs polymers identification, two lasers were used with a wavelength of 532 nm and 785 nm with a range of 200-3400 cm -1 . Two objectives (Olympus, Rungis, France) were used: x10 and x100. Filters were either analyzed manually or using ParticleFinder module for LabSpec 6. Each particle spectrum is compared to a database polymer identification software (KnowItAll, BioRad®) and a personal library made with specific polymers obtained from Goodfellow (France).

Data and Statistical analysis

Anchovy Fulton's K condition index was calculated as an indicator of the fish general well-being: K= (Body weight/total length 3 )*100. The feeding intensity for the fish was calculated using the Gastro Somatic Index (GaSI) (Desai 1970): GasI = stomach weight/ (body weight -stomach weight). The mean (±SD) abundances of microplastics were calculated for anchovies (MPs/ind) and spiny oysters (MPs/g). After a Shapiro-Wilk test, data distribution of ingested MPs were non normal and a non-parametric tests (Kruskal-Wallis and Mann-Whitney U tests) were used to determine differences in microplastic numbers among species and sites. GaSI values followed a normal distribution and a one way analysis of variance (ANOVA) test with a post-hoc Tukey test was used. Spearman's rank correlation was used to test any correlation between the number of ingested MPs and the fish condition index or GasI. Non-metric Multidimensional Scaling (n-MDS) was performed on a Bray-Curtis similarity matrix created from transformed data (square root). This test was used as an ordination method alongside ANOSIM and SIMPER tests to compare the contribution of each observed polymers in sea water, sediments and biota to the similarity and dissimilarity between sites. Significance level was set at p < 0.05. All statistical tests were done using SPSS software (IBM SPSS STATISTICS 20) and PRIMER v7.0 (Primer-E Ltd., Plymouth Marine Laboratory, UK).

Results and Discussion

Microplastics concentrations in sea surface waters and sediments

Microplastics found along the Lebanese coastal waters consisted mainly of fragments (77.5%), fibers (15.25%) and microbeads (7.25%) (Figure 2-A). For all sites, blue and red colors were the most abundant representing 70% of all MPs. Concentration in the surface waters (4.3 MPs/m 3 ) were high compared to other Mediterranean regions and particularly the western part where MPs concentrations vary between 0.17 and 0.62 MPs/m 3 (Table 1). In the Eastern Mediterranean, the MPs concentration fluctuates importantly across regions, ranging from 0.7 items/m 3 in Northeast Levantine coast of Turkey [START_REF] Güven | Microplastic litter composition of the Turkish territorial waters of the Mediterranean Sea, and its occurrence in the gastrointestinal tract of fish[END_REF] to 7.68 ± 2.38 MP/m 3 in the Israeli surface water [START_REF] Van Der Hal | Exceptionally high abundances of microplastics in the oligotrophic Israeli Mediterranean coastal waters[END_REF].

The heterogenicity of the MPs distribution and of its concentration levels could be related to several factors. The mesh size of the net used could play an important role on the number of microplastics caught during sampling. The high concentration of MPs found in the present study maybe due to the low mesh size manta trawl net used (52 µm) compared to other studies (generally between 200 and 500 µm). Also, the different methodological analyses and the lack of a standard protocol could also be the cause of that difference. In fact, the water samples in this study were filtered on GF/A filters and all suspected items were observed; whereas other studies used organic material digestion [START_REF] Gündoğdu | Micro-and mesoplastics in Northeast Levantine coast of Turkey: The preliminary results from surface samples[END_REF][START_REF] Güven | Microplastic litter composition of the Turkish territorial waters of the Mediterranean Sea, and its occurrence in the gastrointestinal tract of fish[END_REF] or just visual observation [START_REF] Fossi | Fin whales and microplastics: The Mediterranean Sea and the Sea of Cortez scenarios[END_REF][START_REF] Lefebvre | Microplastics FTIR characterisation and distribution in the water column and digestive tracts of small pelagic fish in the Gulf of Lions[END_REF] which can lead to an underestimation and a bias in microplastics counting. In addition, the distance of the sampling location from the coast may also influence the concentration of MPs found. Our samples were collected in the coastal waters (about 500 m from the shore). MPs concentration might have differed if the collected samples came from the open sea ( [START_REF] Van Der Hal | Exceptionally high abundances of microplastics in the oligotrophic Israeli Mediterranean coastal waters[END_REF][START_REF] Pedrotti | Changes in the floating plastic pollution of the mediterranean sea in relation to the distance to land[END_REF]. On the other hand, the influence of hydrodynamic features such as currents, up and down-welling, gyres and fronts could also be responsible for this heterogeneity in MPs concentrations. The Eastern Mediterranean has been reported for its high contamination of floating plastic debris with a dominance of small fragments < 2.5 cm in comparison to the Western part [START_REF] Cózar | Plastic accumulation in the mediterranean sea[END_REF]. High fluxes of plastic debris (10-30 kg/km.day) were observed along the Levantine Basin coast from Egypt, Lebanon, Syria and Turkey [START_REF] Constantino | Abundance and composition of floating marine macro litter on the eastern sector of the Mediterranean Sea[END_REF]. Differences between the two parts of the Mediterranean Sea can be explained by the different circulation patterns: water currents in the Western part promote circulation; whereas currents in the Eastern part are somewhat attractors leading to the accumulation of floating debris [START_REF] Mansui | Modelling the transport and accumulation of floating marine debris in the Mediterranean basin[END_REF]. In addition, the complexity of circulation with the existence of mesoscale and sub-basin scale eddies in the Levantine basin (most specifically Shikmona anticyclonic eddy) may also explain the high number of MPs found on the Lebanese coast [START_REF] Menna | On the surface circulation of the Levantine sub-basin derived from Lagrangian drifters and satellite altimetry data[END_REF].

High concentrations of MPs in sediments were also found along the Lebanese coast (2433 ± 2000 MPs/Kg d.w.). These concentrations were higher than those found in others Mediterranean regions (Supplementary Table 2). This important difference can be either caused by the different density separation solution used (ZnCl2 used in our study is of higher density than NaCl used in other studies and more microplastics can be identified) or by the mismanaged waste along the Lebanese coast where high plastic items were visually observed during sampling. Both micro and macroplastics (MaPs) were present in all collected sediments samples.

Lebanon is a potential landfall of plastics along with other Eastern Mediterranean regions [START_REF] Duncan | The true depth of the Mediterranean plastic problem: Extreme microplastic pollution on marine turtle nesting beaches in Cyprus[END_REF] explaining the high abundance of macroplastics found in our samples (consisting of half of the observed anthropogenic materials found). All kind of shapes were found: square, rectangular, spherical and cylindrical (Figure 3). White was the dominant color in Sidon and Tripoli (46.3% and 36.8%, respectively); blue color was the most abundant in Beirut (23.7%). Pellets were highly abundant in two of our sampling sites (Tripoli and Sidon) which explain the high abundance of white color in these two locations. Several studies investigating MPs abundance in the Mediterranean sediments also reported the high presence of plastic pellets among their samples [START_REF] Karkanorachaki | Plastic pellets, meso-and microplastics on the coastline of Northern Crete: Distribution and organic pollution[END_REF][START_REF] Fanini | Dynamics of plastic resin pellets deposition on a microtidal sandy beach: Informative variables and potential integration into sandy beach studies[END_REF][START_REF] Kaberi | Microplastics along the shoreline of a Greek island (Kea isl., Aegean Sea): types and densities in relation to beach orientation, characteristics and proximity to sources[END_REF]Turner and Holmes, 2011). The first signaled pellets on the Lebanese coast were due to direct waste discharge of plastic factories on the beaches or in the rivers [START_REF] Shiber | Plastic pellets on the coast of Lebanon[END_REF]. To this day, and with the lack of an appropriate treatment, these industries are still releasing their wastes into the aquatic system and are susceptible to unintentional losses leading to this high accumulation of plastic pellets. All of which indicate their persistence whether in the Eastern or Western Mediterranean and they (pellets and fragments) consist potential harm to the benthic organisms inhabiting sandy beaches [START_REF] Carson | Small plastic debris changes water movement and heat transfer through beach sediments[END_REF][START_REF] Nelms | Plastic and marine turtles: a review and call for research[END_REF].

Along the Lebanese coast, inter-site differences in MPs concentrations were observed. Surface water samples showed that Sidon was the most contaminated site with 6.7 MPs/m 3 (Figure 2-A). This site is characterized by a high presence of microbeads with 1.16 microbeads/m 3 . Beirut had the lowest concentration of MPs with 2.35 MPs/m 3 consisting mainly (90%) of fragments.

The high number of MPs and pellets observed in the south of Lebanon can be explained by site's localization near the biggest landfill. Although this landfill is closed since 2013, results of our study provide preliminary evidence that landfill isn't the final sink of plastics, but a potential source of microplastics [START_REF] He | Municipal solid waste (MSW) landfill: A source of microplastics? -Evidence of microplastics in landfill leachate[END_REF]. Fragmentation and release of microplastics is a longterm process. Microplastics inside leachates from active and closed landfills have been identified [START_REF] He | Municipal solid waste (MSW) landfill: A source of microplastics? -Evidence of microplastics in landfill leachate[END_REF]. After the stop of post-closure care, microplastics in leachate would be discharged directly into coastal waters. This can pose serious environmental problems since microplastic is not listed as pollutant in the any country's regulations of landfill. This area is also characterized by the presence of the mouth of a major river, Al-Awali River. During sampling, we observed fragments and other wastes being brought into the coastal waters via the river runoff. This river extends for 48 km of length and collects all the urban water from nearby villages due to the non-existence of sewage treatment system. These microplastics are prone to be washed from surface waters onto beaches and, therefore, integrate within the sediments [START_REF] Barnes | Accumulation and fragmentation of plastic debris in global environments[END_REF][START_REF] Poeta | Marine litter in Mediterranean sandy littorals: Spatial distribution patterns along central Italy coastal dunes[END_REF]. The general circulation along the coast of Lebanon is dominant northwards and the water movements are strongly related to surface currents and seasonal metrological factors [START_REF] Goedicke | Submarine canyons on the central continental shelf of Lebanon[END_REF]. This northwards movement might explain why MPs and MaPs were the most abundant in sediments samples of Tripoli (4.68 MPs/g and 4.08 MaPs/g, respectively) (Figure 2-B). The high levels of MPs found in the surface water in Sidon and Beirut might have been carried by surface circulation ending up washed on Tripoli beaches.

Microplastics in anchovies and spiny oysters

Microplastics can be ingested by a large variety of marine species and more specifically by filterfeeders [START_REF] Van Cauwenberghe | Microplastics in bivalves cultured for human consumption[END_REF][START_REF] Catarino | Low levels of microplastics (MP) in wild mussels indicate that MP ingestion by humans is minimal compared to exposure via household fibres fallout during a meal[END_REF][START_REF] Collard | Detection of Anthropogenic Particles in Fish Stomachs: An Isolation Method Adapted to Identification by Raman Spectroscopy[END_REF] raising the alarm for commercial species that are highly consumed by humans. In our samples, 83.3% of European anchovies analyzed had ingested MPs. Such high level of ingestion is in agreement with what was described for anchovy from the Adriatic Sea (90%) (Renzi et al., 2019a). However, in the Western Mediterranean Sea, lower occurrences of MPs were described in anchovy digestive tracts (11 to 40 %) [START_REF] Collard | Detection of Anthropogenic Particles in Fish Stomachs: An Isolation Method Adapted to Identification by Raman Spectroscopy[END_REF][START_REF] Compa | Ingestion of microplastics and natural fibres in Sardina pilchardus (Walbaum, 1792) and Engraulis encrasicolus (Linnaeus, 1758) along the Spanish Mediterranean coast[END_REF][START_REF] Lefebvre | Microplastics FTIR characterisation and distribution in the water column and digestive tracts of small pelagic fish in the Gulf of Lions[END_REF] (Table 2). In the present study, anchovies ingested an average of 2.5 ± 0.3 MPs/individual which was higher than that found in anchovies collected on the Northwestern Mediterranean coasts such as Spanish coasts (0.18 ± 0.20 items/individual) [START_REF] Compa | Ingestion of microplastics and natural fibres in Sardina pilchardus (Walbaum, 1792) and Engraulis encrasicolus (Linnaeus, 1758) along the Spanish Mediterranean coast[END_REF] and Gulf of Lions (0.11 to 0.85 MPs/individual) [START_REF] Collard | Detection of Anthropogenic Particles in Fish Stomachs: An Isolation Method Adapted to Identification by Raman Spectroscopy[END_REF][START_REF] Lefebvre | Microplastics FTIR characterisation and distribution in the water column and digestive tracts of small pelagic fish in the Gulf of Lions[END_REF] but lower than those collected in the Adriatic Sea (6.62 ± 3.67 items/individual) (Renzi et al., 2019a). Anchovies caught in the Western Mediterranean Sea had lower ingested MPs than those caught in the Eastern and Central Mediterranean confirming the higher pollution of these regions (Table 2).

For instance, Japanese anchovies, Engraulis japonicus from the heavy polluted area of Tokyo bay displayed the same MPs ingestion (2.30 ± 2.50 MP/ind Tanaka and Takada, 2016) as in our study.

Three shapes of plastics were found: fragments, films and fibers (Figure 2 -C; Figure 3). There was a slight dominance of fragments (1.3 ± 0.36 fragments/fish) over of fibers (1.1 ± 0.17 fragment/fish), whereas films had the lowest occurrence (0.1 ± 0.1 films/fish). The items had different colors: black, blue, red, white, transparent, and green. For the three sites, blue fragments were the most abundant representing more than 50%; whereas for the fibers, black was the dominant color. This predominance of blue plastic items has been previously reported in fish and bivalves [START_REF] Romeo | First evidence of presence of plastic debris in stomach of large pelagic fish in the Mediterranean Sea[END_REF][START_REF] Güven | Microplastic litter composition of the Turkish territorial waters of the Mediterranean Sea, and its occurrence in the gastrointestinal tract of fish[END_REF][START_REF] Compa | Ingestion of microplastics and natural fibres in Sardina pilchardus (Walbaum, 1792) and Engraulis encrasicolus (Linnaeus, 1758) along the Spanish Mediterranean coast[END_REF][START_REF] Digka | Microplastics in mussels and fish from the Northern Ionian Sea[END_REF].

Along the Lebanese coast, anchovies from Beirut had ingested the highest number of MPs items (2.9 ± 1.9 MPs/individual) and those from Tripoli the lowest number (2.3 ± 1.6 items/individual) (Supplementary Table 3). However, there were no significant differences between the numbers of ingested items at the three analyzed sites (P > 0.05). No relationship was highlighted between the concentration of MPs in anchovy digestive tracts and in the surface water as it has been described in the western Mediterranean [START_REF] Compa | Ingestion of microplastics and natural fibres in Sardina pilchardus (Walbaum, 1792) and Engraulis encrasicolus (Linnaeus, 1758) along the Spanish Mediterranean coast[END_REF][START_REF] Lefebvre | Microplastics FTIR characterisation and distribution in the water column and digestive tracts of small pelagic fish in the Gulf of Lions[END_REF]. As a pelagic species, anchovy are highly moving fish so they potentially did not ingest MPs at the site they were collected, making correlations harder to underline. Gastro-Somatic Index (GaSI) and Fulton condition index were higher in Beirut yet not significantly different (Supplementary Table 3).There was no correlation between GaSI and the number of ingested items (r = 0.067; P> 0.05). Fulton condition index and the number of ingested items showed a weak but not significant correlation (r = 0.316; P = 0.089 > 0.05). In the western Mediterranean Sea, recent studies also revealed that anchovies' MPs ingestion was not related to the body condition [START_REF] Compa | Ingestion of microplastics and natural fibres in Sardina pilchardus (Walbaum, 1792) and Engraulis encrasicolus (Linnaeus, 1758) along the Spanish Mediterranean coast[END_REF][START_REF] Lefebvre | Microplastics FTIR characterisation and distribution in the water column and digestive tracts of small pelagic fish in the Gulf of Lions[END_REF].

Spiny oyster is a Lessepsian species of high commercial value in Lebanon. The collected individuals had an average of 7.2 ± 1.4 items/individual and 0.45 ± 0.3 items/g ww. To date, this is the first study done on MPs ingestion in S. spinosus in the Mediterranean and worldwide. Therefore, our results can only be compared to other oysters or bivalves species. Oysters from the present study had the same average of items found in Crassostrea gigas collected from the Atlantic Ocean with an average of 0.47 ± 0.16 particles/g ww (Van Cauwenberghe and Janssen, 2014), yet lower than that found in wild mussels (Mytilus galloprovincialis) collected from the central Tyrrhenian Sea (Mediterranean) with a an average of 7.2 items/g ww (Renzi et al., 2018a) and from the Adriatic Sea with an average of 1.06-1.3 items/g ww [START_REF] Gomiero | First occurrence and composition assessment of microplastics in native mussels collected from coastal and offshore areas of the northern and central Adriatic Sea[END_REF]. Our samples were less contaminated that those collected from the Mediterranean which is probably due to the difference in the analyzed species and not the geographical MPs concentration in the environment. Oysters tend to have more flesh than mussels explaining the difference in MPs ingestion per gram of ww.

Oysters from all sites had the presence of fragments, films and fibers with 86.3% of analyzed individuals having suspected MPs in their soft tissue (Figure 2-C). Due to a difference in individual total body soft tissue weight, the number of items was also reported to the number of items per gram. Oysters from Beirut had the highest items concentration with 8.3 ± 4.4 items/individual (or 0.78 ± 0.62 items/g) out of which fibers constituted more than 50%. Sidon had the highest number of fragments/individual reaching 4.3 ± 4.4 fragments/individual. Tripoli had the lowest number of items/g with 0.22 ± 0.13 items/g. Beirut was significantly different when comparing to the other sites (P < 0.05). The observed items had six different colors similar to those found in the anchovies; with blue color being dominant in the oysters' samples.

Size class distribution and polymers characterization

In the water samples, the size class < 200 µm dominated in Tripoli (48.78%) and Sidon (51.8%) whereas for Beirut, the size class > 1000 µm dominated (31.7%) (Figure 3 -A). Suspected items < 1000 µm represented a percentage of 88%, 69.3% and 87% at Tripoli, Beirut and Sidon, respectively. All sediment samples had a high abundance of the size class > 1000 µm representing more than 70% of the samples (Supplementary Figure 1). For the biota, the smallest size class < 200 µm was the most abundant in all sites (Figure3 -B). For anchovies, the ingested size class 200-400 µm was the second most dominant in Tripoli and Beirut with a percentage of 34%. The size class 600-800 µm was only present in Sidon's anchovies, whereas the size class > 1000 µm was absent in Tripoli. These results agree with the feeding ecology of the European anchovy which is a filter-feeder that feeds on planktonic organisms generally <2 mm [START_REF] Bacha | Spatial, temporal and ontogenetic variation in diet of anchovy (Engraulis encrasicolus) on the Algerian coast (SW Mediterranean)[END_REF]. No relationship between the size of the anchovies and the size of the ingested items was observed. Although Beirut's anchovies were significantly (P <0.05) bigger than those from the other two sites, they ingested items of a similar size than those ingested in Tripoli and Sidon. This indicates that the size of ingested items may be related to the available particles during feeding rather than the morphometric characteristics of organisms (i.e. mouth opening size). Plastics of all size classes were present inside the oyster's body soft tissue. The smallest size class (0-200 µm) was the most abundant for all sites. The two size classes: 200-400 µm and > 1000 µm were the second most abundant between the ingested MPs. Even though there was a significant difference in spiny oyster's net weight (P<0.05) between sites, all MPs items size classes were present in all collected individuals. This confirms that the size of ingested items is not related to the individual weight, but to the available particles existing in the surrounding water.

Among the suspected MPs analyzed, 94% were identified as microplastics. Nine types of polymers were identified in our samples (Figure 4). Six types were common to all three matrices (water, sediments and biota): polypropylene (PP), polyethylene (PE), polystyrene (PS), polyamide (PA), polyethylene terephthalate (PET) and polyurethane (PUR). In addition to these polymers, polyvinyl chloride (PVC) was present in water and sediments; polylactic acid (PLA) was only present in the water whereas acrylonitrile butadiene styrene (ABS) was only present in biota. Each of the three matrices had a specific polymer that dominated over the others: PE was the most abundant in water samples (70.83%, 50% and 47.37% at Tripoli, Beirut and Sidon, respectively). Polyethylene is the most common plastic on a global scale and consequently the most dominant plastic debris in the Mediterranean Sea and worldwide, deriving mainly from plastic bags and bottles [START_REF] Suaria | The Mediterranean Plastic Soup: Synthetic polymers in Mediterranean surface waters[END_REF]. It is well documented that plastics as PE float in the water surface, as its density is lower than this of the water. Polymers with a density higher than that of water (PVC, for example) were observed in the surface water. The existence of polymers denser than seawater have been already reported [START_REF] Suaria | The Mediterranean Plastic Soup: Synthetic polymers in Mediterranean surface waters[END_REF]Enders et al., 2015) with no clear mechanism leading to their existence. During sampling, there was strong waves and the water was agitated which might explain their presence due to the high velocity and turbulent mixing which increases the chances of denser polymers to persist on the sea surface [START_REF] Moritomi | A comprehensive interpretation of solid layer inversion in liquid fluidised beds[END_REF][START_REF] Suaria | The Mediterranean Plastic Soup: Synthetic polymers in Mediterranean surface waters[END_REF]. Polypropylene was the most abundant in sediments (48.11%,55.88% and 43.33% at Tripoli,Beirut and Sidon,respectively), and polystyrene dominated in the biota specimens (having a percentage equal to or above 25% for anchovies and spiny oysters).

Polypropylene was observed in all anchovies and spiny oysters of all sites except in the anchovies of Beirut where PE was observed instead. Polymers distribution didn't have a pattern with shapes or colors. For example, pellets found were mostly white (several were colored) yet their polymer type was of PE, PA, and PP. The same for fragments, different colors were related to different polymers with no similar pattern.

Comparison of the polymers composition identified in water, sediments and oysters indicated a dissimilarity between groups (ANOSIM, R=0.796, significance level = 0.2%). Cluster dendrogram led to the existence of two distinct groups (a and b) regrouping on the one hand the water and sediment samples and on the other hand the biota (anchovies and oysters) (Supplementary Figure 2). Non-metric multidimensional scaling (n-MDS) showed the dimensional distance separating these groups (a and b) based on their polymer composition Water and sediments formed 'group a' and were characterized by the high presence of PP and PE. Whereas 'group b' composed by biota samples, and were characterized by the high abundance of PS. Similarity of Percentages (SIMPER) indicated that PE, PP, PA and PUR contributed to 72.12% of dissimilarity between these two groups.

Microplastics potential entry into the Mediterranean Basin via coastal landfills, and their fate and distribution in the coastal environment

This study showed a high microplastics pollution in areas located in the vicinity of coastal landfills. Even though some of them are no longer active, but their waste continues to find its way into the sea due to mismanagement and lack of proper treatments. Some of the polymers found in our samples such as polyethylene, polypropylene and polystyrene were observed in transparent, white and different colors. They are widely used in packaging, consumer products, pipes and fitting, and are the common polymers found in landfills and their leachates [START_REF] Kazour | Sources of microplastics pollution in the marine environment: Importance of wastewater treatment plant and coastal landfill[END_REF][START_REF] He | Municipal solid waste (MSW) landfill: A source of microplastics? -Evidence of microplastics in landfill leachate[END_REF][START_REF] Praagh | Microplastics in Landfill Leachates in the Nordic Countries[END_REF]. The high abundance of these types of polymers in sea surface water and sediment samples can be traced back to mismanaged landfills along with accidental garbage disposal. Even though Lebanon is considered as a potential sink of plastics in the Eastern Mediterranean [START_REF] Duncan | The true depth of the Mediterranean plastic problem: Extreme microplastic pollution on marine turtle nesting beaches in Cyprus[END_REF], it should also be considered as an important microplastics source in the Mediterranean basin. In most studies, whether in the surface water, sediments or biota, fibers were generally the dominant microplastic shape found [START_REF] Lefebvre | Microplastics FTIR characterisation and distribution in the water column and digestive tracts of small pelagic fish in the Gulf of Lions[END_REF]Renzi et al., 2019b;[START_REF] Compa | Ingestion of microplastics and natural fibres in Sardina pilchardus (Walbaum, 1792) and Engraulis encrasicolus (Linnaeus, 1758) along the Spanish Mediterranean coast[END_REF]. In the present study, fragments constituted more than 77%, 90% and 50% of items found in surface water, sediments and biota samples, respectively. The same pattern was found in a study done near an abandoned coastal landfill, where fragments constituted the majority of found items [START_REF] Kazour | Sources of microplastics pollution in the marine environment: Importance of wastewater treatment plant and coastal landfill[END_REF]. Such dominance of fragments in the analyzed samples highlights on the potential role of Lebanese coastal landfills in fragments input into the Mediterranean Basin. Other factors can also lead to this severe microplastics contamination and, most precisely, on fragments abundance on the Lebanese beaches. [START_REF] Munari | Plastic debris in the Mediterranean Sea: Types, occurrence and distribution along Adriatic shorelines[END_REF] indicated a high number of fragments (approximately 90% of the samples) was observed near beaches affected by riverine run-off. All of the sampled Lebanese beaches are affected by riverine runoff as well as a significant anthropogenic pressure leading to a major microplastics pollution along the beaches (100 times higher than other reported studies, see Supplementary Table 3). These microplastics are prone to re-enter the surface water with waves hitting the shoreline (directed by wind speed and direction) increasing their chance to end up in nearby regions via the northward general water circulation pattern.

The above stated factors increase microplastics availabilityto marine organisms leading to potential human health risks. In Lebanon, an average of 19.7g of fish and seafood per person is consumed [START_REF] Nasreddine | Food consumption patterns in an adult urban population in Beirut, Lebanon[END_REF] making it around 7 kg per caput per annum. Converting the average of ingested MPs/g in oysters to the number of seafood ingested by a Lebanese person, we would have approximatively 31, 500 MPs ingested per person (4500 MPs/kg of oysters). This is similar to that obtained by Catarino et al. (2018) (13,731 and 68,415 particles/y/person) yet lower than that obtained by Van Cauwenberghe & Janssen (2014) (11, 000 MPs/y/person).

European anchovies are eaten wholly by the Lebanese population as Bizree (juvenile pelagic fish caught by purse seiners) as well as spiny oysters that are served in various Lebanese restaurants raising the concern on the human consumption of these contaminated seafood.

Conclusion

These results should be considered as a preliminary analysis on MPs levels recorded off the Lebanese coast in three different matrices. This is the first contribution to characterize the area and to understand the degree of its MPs pollution. More studies are necessary to evaluate the ecological impacts of these MPs on the marine biodiversity in the Levantine Basin. The levels of MPs found were alarmingly high in comparison to other Mediterranean regions. The high number of ingested items by both anchovies and oysters could suggest a potential health concern since these two species are wholly consumed (without being degutted). This study highlighted the role of landfills in MPs entry into the coastal marine environment. Coastal landfills and mismanaged wastes are important issues existing in 80% of the Mediterranean Basin explaining the elevated number of MPs found in this semi-enclosed basin. The whole Mediterranean should be regarded as an important case study for microplastics pollution where more initiative should be taken to insure an appropriate waste management and, hence, decreasing plastics pollution.

A) B) C) D)

1

Figure 1 :

 1 Figure 1: Localization of the sampling sites (Tripoli, Beirut and Sidon) along the Lebanese coast.

Figure 2 :Figure 3 :

 23 Figure 2: A) Concentration of suspected microplastics (MPs/m3) according to their shapes found in the surface water; B) Suspected microplastics (MPs) and macroplastics (MaPs) concentration (items/g) found in one gram of sublittoral sediments; C) Average number (±SD) of ingested suspected microplastics by shape categories in the digestive tract of (E) European anchovies (MP/ind) and in the soft tissues of (O) spiny oysters (MP/gram) at the three sampling sites (* indicate statistically significant difference).

Figure 4 :Figure 5 :

 45 Figure 4: Proportion of size class composition of suspected microplastics in the A) Water surface, B) anchovies and C) spiny oysters from the different sampling sites A)

Table 1 :

 1 Literature data on microplastics abundance in Mediterranean surface waters and instrumental analysis methods used for detection of MPs. To compare with our results, only studies expressing concentrations in m 3 are presented.

			Mesh size
	Location	Sampling nets		Identification method	MPs abundances	Reference
			(µm)
	NW Mediterranean (the Rhône					Constant et al.,
		Manta trawl net	333	Stereomicroscope/FTIR 0.18-0.19 items/m 3
	River and the Têt River)					(2018)
	NW Mediterranean Sea (Ligurian
		WP2 standard net	200	Stereomicroscope	0.62 ± 2 items/m 3	Fossi et al., (2012)
	Sea and Sardinian Sea)
	NW Mediterranean Sea (Gulf of				0.23 ± 0.20	Lefebvre et al.,
		WP2 plankton net	200	Stereomicroscope/FTIR
	Lion)				items/m 3	(2019)
	NW Mediterranean Sea (Gulf of				0.31 ± 1.17
		Neuston net	200	Stereomicroscope		Fossi et al., (2016)
	Asinara and the Sardinian Sea)				items/m 3
	NW Mediterranean Sea (North	WP2 standard ring net	200	Stereomicroscope	0.17 ± 0.32	Panti et al., (2015)

Table 2 :

 2 Average (±SD) of ingested items found in the digestive track of European anchovies (Engraulis encrasicolus) collected in different areas of the Mediterranean Sea.

	Sites	Digestion method	Concentration (items/ind)	Occurrence	Polymers Identification method	Main polymer	Reference
			0.07
	Spanish Western Mediterranean coasts	None. Visual observation	(±0.35) to 0.33	15%	FTIR		PET	Compa et al., (2018)
			(±0.26)
	Western Mediterranean
	Sea (Iberian Peninsula coast and the Balearic	None Visual observation	0.07 ± 0.26	2.56%	None		-	Rios-Fuster et al., (2019)
	Islands)
	North Western of the Mediterranean Sea (Gulf of Lions)	None. Visual observation	0.11 ± 0.31	11%	ATR-FTIR		PET	Lefebvre et al., (2019)
	Northwestern Mediterranean Sea	NaClO	0.85	40%	Raman spectroscopy	PE, PP, and PET	Collard et al., (2015)
	Northern Mediterranean (Adriatic Sea)	H2O2	1.25	91%	μ-FT-IR		PVC	Renzi et al., (2019a)
	Eastern Mediterranean Sea (Lebanese coast)	KOH 10%	2.5 ± 0.3	83.4%	Raman Spectroscopy		PS	Present study

Acknowledgments

The authors would like to thank G. Duflos, A. Dehaut and L. Hermabessiere from the Agence Nationale de Sécurité Sanitaire de L'alimentation, de L'environnement et du Travail (ANSES) -Boulogne-sur-mer for their help and advices during the sampling preparation. The authors would also like to thank Elie Tarek from the National Center of Marine Sciences -Batroun for helping us acquiring our field samples.

Funding

Maria Kazour is financially supported by a PhD fellowship from the National Council for Scientific Research (Lebanon) and Université du Littoral Côte d'Opale (France). This work has been partially financially supported by the European Union (ERDF), the French Government, the Région Hauts-de-France and IFREMER, in the framework of the project CPER MARCO 2015-2020.

Declaration of interests:


Nathalie Lécrivain
email: nathalie.lecrivain10@gmail.com

Antoine Duparc

Bernard Clément

Emmanuel Naffrechoux

Victor Frossard

Tracking sources and transfer of contamination according to pollutants variety at the sediment-biota interface using a clam as bioindicator in peri-alpine lakes

Keywords: Trace metal, polycyclic aromatic hydrocarbon, bioindicator, bioconcentration, Corbicula fluminea, littoral

Point pollution sources may differently impact lakes littoral, possibly leading to local ecological risks.

The concomitant chemical analysis of littoral-benthic organisms and sediment can provide insights into the bioavailability and thus the ecological risk of contaminants. In this study, the autochthonous Corbicula fluminea was used to assess the sources and transfer of six trace metals (TMs) and fourteen Polycyclic Aromatic Hydrocarbons (PAHs) to the littoral-benthic biota of a large lake. The contaminant concentrations spatially varied with a value scale from 1 to 280 000 times along the lake littoral in both the sediment and clams. Multiple linear regressions were performed to explain the spatial variability of Corbicula fluminea contamination by considering both watershed and in-lake sources. The concentration of the sum of PAHs in clams was significantly correlated with sediment contamination, suggesting that PAHs contamination of the benthic biota mainly occur from the sediment. Most of the internal TM concentrations of clams were significantly correlated with stormwater drainage areas in the lake watershed, highlighting the importance of stormwater runoffs in the littoral biota contamination. The transfer of TMs and PAHs was assessed through the bioconcentration factor defined as the ratio of internal and sediment concentrations. As, Cd, Cu, Zn and light molecular weight PAHs were more bioconcentrated in C. fluminea than Pb, Sn and heavy molecular weight PAHs, suggesting differences in their bioavailability. This study underlines the relevance of using autochthonous organisms as bioindicators of lake littoral biota contamination concomitantly with sediment matrices, and illustrates the challenge of tracking pollution sources in lakes.

1. Introduction

The sediment compartment is of great importance when assessing the ecological risk of contaminants for biological organisms (Atkinson et al., 2007;[START_REF] Bryan | Bioavailability, accumulation and effects of heavy metals in sediments with special reference to United Kingdom estuaries: a review[END_REF][START_REF] Burton | Sediment Toxicity Assessment: 0[END_REF]. Chemical analyses of sediments can be compared to ecotoxicological thresholds from the literature (e.g. Threshold Effect Concentration [START_REF] Macdonald | Development and Evaluation of Consensus-Based Sediment Quality Guidelines for Freshwater Ecosystems[END_REF]). Sediment can also be used in bioassays as a contaminated matrix to which study laboratory organisms are exposed to assess their response [START_REF] Triffault-Bouchet | Assessment of contaminated sediments with an indoor freshwater/sediment microcosm assay[END_REF]. In the literature, the sediment is considered as a more stable and integrative compartment compared to water [START_REF] Payne | Acetylcholinesterase, an old biomarker with a new future? Field trials in association with two urban rivers and a paper mill in Newfoundland[END_REF][START_REF] Sutherland | Bed sediment-associated trace metals in an urban stream, Oahu, Hawaii[END_REF] and can exhibit significantly higher contaminant concentrations leading to a higher ecological risk (Atkinson et al., 2007;[START_REF] Bryan | Bioavailability, accumulation and effects of heavy metals in sediments with special reference to United Kingdom estuaries: a review[END_REF]. However, the binding capacity of pollutants to the sediment greatly varies according to their molecular structure. As an example, PAHs with low octanol / water partitioning coefficient (Kow) tend to bind to the sediment organic matter in a lesser extent than those with higher Kow [START_REF] Harner | Using passive air samplers to assess urban-rural trends for persistent organic pollutants. 1. Polychlorinated biphenyls and organochlorine pesticides[END_REF][START_REF] Naffrechoux | Historical Profiles of PCB in Dated Sediment Cores Suggest Recent Lake Contamination through the "Halo Effect[END_REF][START_REF] Shoeib | Using measured octanol-air partition coefficients to explain environmental partitioning of organochlorine pesticides[END_REF]. Secondly, many abiotic (e.g. temperature, dissolved oxygen, hydrodynamic regime) and biotic (e.g. trophic interactions, bioturbation) factors can influence the bioavailability of sediment-borne contaminants [START_REF] Eggleton | A review of factors affecting the release and bioavailability of contaminants during sediment disturbance events[END_REF]. It is therefore essential to perform concomitant measurements in biological matrices, in addition to sediment, to provide better insights regarding the bioavailability of sediment-borne contaminants [START_REF] Guo | Biological toxicity response of Asian Clam (Corbicula fluminea) to pollutants in surface water and sediment[END_REF] and their transfer throughout lake food webs.

The Asian clam Corbicula fluminea is a benthic filter-feeder [START_REF] Cataldo | Condition index and growth rates of field caged Corbicula fluminea (Bivalvia) as biomarkers of pollution gradients in the Paraná river delta (Argentina)[END_REF] commonly used as a bioindicator of the ecological status of stream and coastal environments over large time [START_REF] Riva | The case of pollution of Lake Maggiore: a 12year study with the bioindicator mussel Dreissena polymorpha[END_REF] and spatial [START_REF] Goldberg | The mussel watch concept[END_REF] scales. C. fluminea is a widespread bivalve mollusk living in the upper part of sediment and filtering water at the sediment surface. These characteristics make C. fluminea a great biological model to test for the bioavailability of contamination at the sediment-water interface.

Lake Annecy is a large French peri-alpine lake with an anthropized watershed. Although the physicochemical and biological parameters of the water column have been monitored for several decades [START_REF] Frossard | Suivi de la qualité des eaux du lac d'Annecy[END_REF], only a few studies have focused on anthropogenic contamination [START_REF] Grebe | Sédiments du lac d'Annecy : Etude des concentrations en HAP et PCB, indices oligochètes[END_REF][START_REF] Lallée | Etude bibliographique de la contamination des milieux lacustres par les micropolluants (PCB HAP) -Application au lac d'Annecy[END_REF][START_REF] Naffrechoux | Historical Profiles of PCB in Dated Sediment Cores Suggest Recent Lake Contamination through the "Halo Effect[END_REF] and mostly concern contamination in the deepest part of the lake.

Using Lake Annecy as case study, we aim at assessing whether the contamination of C. fluminea provides insights (1) into the transfer and bioavailability of sediment-borne pollutants to benthic organisms in the lake littoral zone, and (2) into the identification of sites and point sources that may exert ecological risks. In particular, this study attempts to assess the contribution of potential in-lake versus watershed sources in the contamination of the littoral biota by studying C. fluminea multicontamination.

Two contaminant families were investigated; Trace Metals and Metalloids (TMs) and Polycyclic Aromatic Hydrocarbons (PAHs). These families are widespread in the lacustrine environment [START_REF] Järup | Hazards of heavy metal contamination[END_REF][START_REF] Pacyna | Sources and Fluxes of Harmful Metals[END_REF][START_REF] Schwarzenbach | Global water pollution and human health[END_REF] and mainly reflect domestic, industrial and transport activities (e.g. wood and fossil fuel combustion, fuel leakage, corrosion of roofs and vehicle bodies, road, brake and tire wear abrasions (Abdel-Shafy and Mansour, 2016;[START_REF] Shen | Global atmospheric emissions of polycyclic aromatic hydrocarbons from 1960 to 2008 and future predictions[END_REF]).

Among these anthropogenic activities, the pollutants emitted from transport activities can both originate from in-lake (e.g. boats emissions [START_REF] Hoch | Organotin compounds in the environment -an overview[END_REF]) and lake watershed. The objective was to identify a large array of potential pollution sources with these two contaminant families.

In addition, TMs and PAHs differ by their mobility, persistence, bioavailability and toxicity [START_REF] Naimo | A review of the effects of heavy metals on freshwater mussels[END_REF]UNEP, 2003), thereby justifying a large-scale assessment of C. fluminea's capacity to reflect biota contamination in the littoral zone.

We first expected that C. fluminea contamination would exhibit significant spatial variability in the littoral zone due to the influence of local point sources of pollution. In one side, harbors were expected to exert significant in-lake influence on littoral biota contamination, with a greater concentration of TMs in organism [START_REF] Guevara-Riba | Assessment of metal mobility in dredged harbour sediments from Barcelona, Spain[END_REF]. In the other side, external sources, such as surface and stormwater runoffs, were expected to play a role in the contamination by PAHs of the littoral biota [START_REF] Brown | Sources of heavy metals and polycyclic aromatic hydrocarbons in urban stormwater runoff[END_REF]. In addition, two buffer zones around the study sites of respectively 200 and 600 m were assessed in order to account for small and medium-scale sources that may influence C. fluminea contamination. We therefore expected that point sources included in the small buffer zone (i.e. 200 m) would more precisely explain the clams contamination than higher scales. The last assumption was that C. fluminea contamination would differ from sediment contamination according to the bioavailability of the contaminants. Particularly, differences in PAH contaminations in the sediment and biological matrices should be greater (i.e. by bioconcentration effect) than for TMs due to their greater reactivity (i.e. lipophilicity, molecular degradation through biological, chemical and physical processes; [START_REF] Moza | Photooxidation of naphthalene and phenanthrene in hexane as an oil film on water[END_REF]. However, some differences should be expected according to light and heavy molecular weight PAHs that exhibit different molecular properties (e.g. water / octanol partitioning coefficient Kow) [START_REF] Ha | QSAR study using acute toxicity of Daphnia magna and Hyalella azteca through exposure to polycyclic aromatic hydrocarbons (PAHs)[END_REF][START_REF] Noh | Bioaccumulation of Polycyclic Aromatic Hydrocarbons (PAHs) by the Marine Clam, Mactra veneriformis, Chronically Exposed to Oil-Suspended Particulate Matter Aggregates[END_REF].

Materials and Methods

The study lake

Lake Annecy is a large monomictic and oligotrophic lake located in Savoie (France), on the northwest edge of the French Alps (45°51'N, 6°10'E, Figure 1A). It has a surface area of 27 km 2 and mean and maximum depths of 41 and 65 m, respectively. The 250 km 2 -catchment area of Lake Annecy is covered by forests (56 %), agricultural areas (20 %), urban areas (17 %) and water bodies (6 %) (Corinne Land Cover, 2012). Its watershed has been subject to rapid anthropization over recent decades [START_REF] Rulliere | assainissement des eaux du Lac d'Annecy[END_REF]. The total population in the watershed, about 125 000 inhabitants, doubles in summer during the tourist season. Red circles indicate main sampling sites and green triangles replicates for assessment of local contamination variability.

Sampling sites

Twenty littoral sites were selected to cover the overall lake littoral and included different land-use contexts (i.e. beaches, reed beds, roadsides, harbors) (Figure 1-B). At all sites, surface sediments (i.e. the first 5-cm sediments) and C. fluminea were sampled using an Ekman grab at a water depth of 1.5 to 2.5 m, depending on the topographic constraints. Sediment samples were homogenized, sieved through a 2-mm mesh to remove debris and stored at 4°C. The sampled C. fluminea were selected according to similar ranges of shell size (i.e. between 1.5 and 2.5 cm) to limit possible ontogenic differences in contamination. We checked for the representativeness of each sample by sampling 3 replicates close to each other (<12m) in the site S2 (Figure 1). No significant differences in C. fluminea contamination for PAHs and TMs were found (see details in Figure A1 in the Appendix).

Contamination quantification

The glassware used for sampling and measurements was prior washed in a detergent bath (5% Gigapur® 05 (Shimitek, France) for 12 h, followed by an acid bath (5% nitric acid) during 6 hours and rinsed with deionized water.

TM concentrations in sediment and organism samples were assessed by inductively coupled plasma mass spectrometry (ICP-MS) according to the European guideline NF EN ISO17294-2 (AFNOR, 2016). Incertitude measurements were always under 25 % and detection limit for these TMs was about 0.1 μg L -1 . PAH concentrations in sediment and organism samples were measured by LCME Laboratory using high performance liquid chromatography (HPLC) with fluorescence detection. The employed methodology was similar as the one described in [START_REF] Lécrivain | Multi-contamination (heavy metals, polychlorinated biphenyls and polycyclic aromatic hydrocarbons) of littoral sediments and the associated ecological risk assessment in a large lake in France (Lake Bourget)[END_REF]. The limit of detection and quantification corresponded to 6 and 12 times the blank standard deviation, respectively. A3). The sum of the 14 PAHs was thus expressed in μg kg -1 OM for the sediments and in μg kg -1 lipids for C. fluminea. Grain size distribution of sediment was measured by laser diffractometry (Malvern Mastersizer 2000G) and presented a homogeneous sandy size group (Figure A3). The grain size distribution of sampled sediment could not consequently exert a major bias in the contaminant bioavailability [START_REF] Eggleton | A review of factors affecting the release and bioavailability of contaminants during sediment disturbance events[END_REF][START_REF] Reid | Bioavailability of persistent organic pollutants in soils and sediments-a perspective on mechanisms, consequences and assessment[END_REF][START_REF] Zhang | Effects of sediment geochemical properties on heavy metal bioavailability[END_REF]. Concentrations below the detection limits were estimated at a value equal to the detection limit divided by √2 according to [START_REF] Croghan | Methods of dealing with values below the limit of detection using SAS[END_REF]. The raw data of sediment and C. fluminea contaminant concentrations are presented in Table A1 in the Appendix.

Bioconcentration factor (BCF)

It was assumed that the sampled organisms had already reached an equilibrium regarding the ambient contamination since they were autochthonous and adult clams [START_REF] Jacomini | Bioaccumulation of atrazine in freshwater bivalves Anodontites trapesialis (Lamarck, 1819) and Corbicula fluminea (Müller, 1774)[END_REF][START_REF] Thorsen | Toxicokinetics of environmental contaminants in freshwater bivalves[END_REF]. The transfer from sediment borne contaminants to the clams could so be assessed through the ratio of the non-normalized contaminant concentrations between C. fluminea and the sediments designated as the bioconcentration factor (BCF) [START_REF] Jia | Distribution, contamination and accumulation of heavy metals in water, sediments, and freshwater shellfish from Liuyang River, Southern China[END_REF][START_REF] Mendoza-Carranza | Distribution and bioconcentration of heavy metals in a tropical aquatic food web: A case study of a tropical estuarine lagoon in SE Mexico[END_REF][START_REF] Mountouris | Bioconcentration of heavy metals in aquatic environments: the importance of bioavailability[END_REF]. A contaminant was considered as bioconcentrated when the BCF was equal to or greater than 1. The relationship between the BCFs and the sediment contamination was studied to assess whether potential biological regulation mechanisms may occur in the organisms exposed to an increasing sediment contamination [START_REF] Mcgeer | Inverse relationship between bioconcentration factor and exposure concentration for metals: implications for hazard assessment of metals in the aquatic environment[END_REF].

Explanatory variables of C. fluminea contamination

We defined explanatory variables which represented in-lake and external contamination sources to identify which one contributed to contaminant concentration in clams. First, potential in-lake sources of pollution were considered using the total engine power of boats (hp) in the harbors, to provide a realistic pollution capacity of the boats (Figure A4 2008) and [START_REF] Marchand | waver: Calculate Fetch and Wave Energy. R package version 0[END_REF], by coupling bathymetric data from the BRGM / SILA lake [START_REF] Bisson | Arsenic et ses dérivés inorganiques[END_REF] and the direction and wind speed from the Météo-France

Meythet station (i.e. hourly data over 4 annual cycles). The average wave energy within a 200 m diameter circle around the site was calculated over the entire meteorological time range (Figure A6 in the Appendix). The mean slope (%) within a 50 m radius around the site was calculated from BRGM / SILA (1990) bathymetric lake data. The sediment contamination was also considered to explain the contamination of C. fluminea.

Data analysis

We assessed the contamination in C. fluminea performing multiple linear regressions with backward selection, using C. fluminea contamination (for each TMs separately and for the sum of the PAHs) as response variable against the potential explanatory variables (i.e. section 2.5). Site S11 was removed prior to modeling for the PAHs because of its concentration several orders of magnitude higher than the other sites that can be explained by the vicinity of a main road with huge daily traffic. For the TMs, sites S7, S8, S10, S13, S16, S17, S18, S20 could not be considered since the biological masses sampled were not sufficient to carry out the analyses. Results of the models were illustrated in two heatmaps, one for each study scale (i.e. radii of 200 and 600 m). All the statistical analyses and models as well as the graphical displays were carried out using R [START_REF] Team | R: A language and environment for statistical computing[END_REF].

Results

Sediment and C. fluminea contaminations

The TMs contamination of the sediment exhibited a high spatial heterogeneity along lake littoral with a value scale from 1 to 8 (As), 9 (Pb), 12 (Cu), 31 (Zn) and 42 (Sn) times (Figure 2). Similarly, internal concentrations in clams were also highly variable with a value scale from 1 to 2 (As, Cd), 3

(Sn) and 4 (Cu, Pb) times (Figure 3). However, the pattern of TMs contamination was significantly different between the sediment and the organisms along the lake littoral (Chi 2 tests, p-value < 0.05).

Contamination transfer

BCFs differed significantly among contaminants for both PAHs and TMs and among sites for PAHs (ANOVA, p-value < 0.01; Figure 6). For TMs, the BCFs for As, Cu and Zn were > 1 while they were < 1 for Pb and Sn (Figure 6A). The BCF for Cd could not be calculated, as it was not detected in the sediment.

BCFs were higher for light PAHs compared to heavy ones, especially Flu for which values exceeded 400 (Figure 6B). Regarding the spatial distribution of BCFs along the lake littoral, S2 and S3 had the highest BCFs (Table A2 in the Appendix).

Contamination sources

All the candidate variables were significantly related to C. fluminea contamination for at least one contaminant for a given buffer radius, and the R-squared were relatively high and varied from 0.49 to 0.9 (except 0.28 for Pb at 200 m radius, Table 1). Adjusted-R² 0.54 0.63 NA 0.53 0.9 0.83 0.28 0.58 0.49 0.57 0.78 0.6 0.59 0.59

For the TMs, the sediment contamination was not correlated with that of C. fluminea, meaning that the concentrations in the clams were likely not directly impacted by the sediment contamination (Figure 7). All other variables were at least significant for one TM and, most of them were positively correlated with C. fluminea contamination, suggesting that these variables may favor their contamination. However, at a large scale (i.e. radius = 600 m), some significant negative relationships indicate that the influence of the explanatory variables may differ from one TM to another (Figure 7-B). For example, the number of stormwater outlets exerted a significant positive influence on the contamination for As and Cd but a negative one for Cu, Pb and Sn.

We also noted that many more variable were detected to have an effect on clams contamination in TMs at the larger scale (600 m radius, Figure 7-B) than at the local scale (200 m radius, Figure 7-A), except for the proportion of organic matter in sediment. R-square were also similar or higher for the 600 m radius confirming the better prediction power at this scale (Table 1).

Considering the sum of PAHs, the main explanatory variable was the sediment contamination while the slope of the site had a marginally negative influence on internal PAH concentrations in C. fluminea whatever the scale of the study (Figure 7). This result was similar for both radii. compared to polluted sites such as Lake Kentucky (171.5 μg kg -1 DM; [START_REF] Lapviboonsuk | Polynuclear Aromatic Hydrocarbons in Sediments and Mussel Tissue from the Lower Tennessee River and Kentucky Lake[END_REF]). In contrast, mean TM contaminations of C. fluminea were very similar to those measured in the C. fluminea from the littoral zone of Lake Bourget, located 20 kilometers from Lake Annecy (Table A3 in the Appendix). Similar concentrations were also reported by [START_REF] Shoults-Wilson | Comparison of metal concentrations in Corbicula fluminea and Elliptio hopetonensis in the Altamaha River system, Georgia, USA[END_REF] from the Altamaha River (Georgia, USA), except for Cd for which the contamination in Lake Annecy was on average 10 times lower than in the Altamaha River and even lower than the detection threshold in the sediment. In this study, the magnitude of both TM and PAH contaminations of C. fluminea varied from 5 to 10 times among sites and the patterns of contamination were related to different contamination sources. These benthic organisms may therefore efficiently reflect the spatial differences in the magnitude of contamination that the littoral faces.

In-lake versus watershed sources of contamination

In this study, both the 200 m and 600 m buffers were expected to account for small and medium-scale constraints that may influence C. fluminea contaminations. The explanatory powers of the multiple linear regression models performed with the 600 m buffer radii were higher in term of value and in number of significant variables, emphasizing the importance of selecting an appropriate buffer zone in such studies. The explanatory powers of the multiple linear regression models used to explain C. fluminea contamination were rather high although variable, suggesting that other non-considered parameters could also contribute to (i.e. sources) or influence (i.e. environmental variables) the contamination of organisms, such as bioturbation [START_REF] Remaili | Effects of enhanced bioturbation intensities on the toxicity assessment of legacy-contaminated sediments[END_REF][START_REF] Schaffner | Effects of Physical Chemistry and Bioturbation by Estuarine Macrofauna on the Transport of Hydrophobic Organic Contaminants in the Benthos[END_REF].

The significant positive correlations of C. fluminea contamination by Cu, Pb, Sn and Zn with the drainage area in the lake watershed highlighted the potential role of stormwater as a major source of littoral biota contamination by TMs. This result is consistent with other studies that highlighted the influence of stormwater in lake contamination [START_REF] Mclellan | Distribution and Fate of Escherichia coli in Lake Michigan Following Contamination with Urban Stormwater and Combined Sewer Overflows[END_REF][START_REF] Zhang | Antibiotic concentration and antibiotic-resistant bacteria in two shallow urban lakes after stormwater event[END_REF]. Stormwater was also expected to explain the PAH contamination of C. fluminea (Brown and Peake 2005), although no significant correlation have been identified in this study. The influence of the total number of stormwater outlets was not consistent between the different TMs (i.e. positive and negative correlations). This result could have been influenced by the drainage area and watershed land cover, which could vary considerably between the outlets.

As expected, C. fluminea contamination was also positively correlated with the total engine power of boats in harbors for Pb and Zn, elements generally measured in high abundance in harbors [START_REF] Guevara-Riba | Assessment of metal mobility in dredged harbour sediments from Barcelona, Spain[END_REF]. Clam As and Cd contaminations exhibited less significant correlations with the candidate variables. This result may be partly due to the fact that these elements are highly mobile between the different compartments (i.e. sediment, water and biota) [START_REF] Frémion | Key parameters influencing metallic element mobility associated with sediments in a daily-managed reservoir[END_REF] and therefore possibly less directly related to point sources.

C. fluminea as an indicator of contaminant bioavailability

The lack of significant correlation between the TM concentrations in C. fluminea and those in the sediment, as well as the differences in BCF between the different contaminants, suggest substantial variability in contaminant bioavailability. These results demonstrated that chemical analyses performed on solely abiotic matrices do not provide robust evidence of the bioavailability of the contaminants and consequently their effective ecological risk [START_REF] Guo | Biological toxicity response of Asian Clam (Corbicula fluminea) to pollutants in surface water and sediment[END_REF]. The molecular characteristics of contaminants can induce significant differences in their uptake (e.g. passive or active transfers), storage and excretion by the organism, as illustrated in our study by the higher bioconcentration of light PAHs compared to heavy ones. Such differential bioconcentration was already highlighted by previous studies [START_REF] Meador | Comparative bioaccumulation of polycyclic aromatic hydrocarbons from sediment by two infaunal invertebrates[END_REF][START_REF] Noh | Bioaccumulation of Polycyclic Aromatic Hydrocarbons (PAHs) by the Marine Clam, Mactra veneriformis, Chronically Exposed to Oil-Suspended Particulate Matter Aggregates[END_REF]). An explanation of such variability is that environmental variables, such as slope and wave energy in this study, influence the benthic organism exposure to littoral contamination. Indeed, these two variables could reflect the disturbance level at the sampling location leading to a transport and re-suspension of the sediment and so an increase in the exposure of benthic organisms to littoral contamination [START_REF] Eggleton | A review of factors affecting the release and bioavailability of contaminants during sediment disturbance events[END_REF][START_REF] Frémion | Impact of sediments resuspension on metal solubilization and water quality during recurrent reservoir sluicing management[END_REF][START_REF] Phelps | Active biomonitoring with Corbicula for USEPA priority pollutant and metal sources in the Anacostia River (DC, Maryland, USA)[END_REF]. In addition, Cu contamination in C. fluminea was negatively correlated with the abundance of organic matter in the sediment, which was in agreement with the fact that Cu is highly bound to the organic matter fraction of the sediment [START_REF] Frémion | Dynamique des flux sédimentaires et des éléments métalliques en lien avec l'exploitation courante et exceptionnelle d'un barrage hydroélectrique[END_REF][START_REF] Tiquio | Sediment-bound trace metals in Golfe-Juan Bay, northwestern Mediterranean: Distribution, availability and toxicity[END_REF], which may reduce its bioavailability. These environmental variables can therefore be considered in the ecological risk assessment.

Conclusion

C. fluminea appeared to appropriately reflect the spatial heterogeneity of lake littoral contamination (both in magnitude and profile) and the target contaminated "hot-spots" locally. The identification of stormwater runoffs as potentially important sources of TM contamination for the littoral biota provided insights into the assessment of the ecological risk of the pollution in the littoral zone of Lake Annecy and useful information for identifying and suggesting remedial actions (e.g. stormwater management). However, regarding persistent organic pollutants such as PAHs, linking organisms and potential watershed sources was not possible in this study. This result underlines possible changes in the contamination pattern from the source to the biota for persistent organic pollutants. This study supports the need to consider littoral contamination as complementary to that at a single site in the deepest zone (i.e. management monitoring) to fully appreciate the pressure of contamination on the lake. In view to continuing this work, it would be interesting to couple the variability of BCF between the contaminants with foodweb models to predict the preferential transfer of contaminants to patrimonial fishes such as whitefish and arctic char in Lake Annecy. In light of the bioconcentrated TMs and PAHs in C. fluminea in the current study, enzymatic biomarkers in these autochthonous organisms could be performed to obtain bioindicators of the detrimental effects caused by their contamination and thus go further in the ecological risk assessment. In addition, performing risk assessment at higher trophic levels would be relevant since C. fluminea is an important source of

Figure 1 .

 1 Figure 1. Map of Lake Annecy in the east of France (A) and the locations of the sampling sites (B).

Among the 16

 16 PAHs prioritized by the US Environmental Protection Agency (US-EPA), 14 were quantified; acenaphthene (Ace), fluorene (Flu), phenanthrene (Phe), anthracene (Ant), fluoranthene (Fla), pyrene (Pyr), benzo[a]anthracene (BaA), chrysene (Chr), benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), dibenzo[ah]anthracene (DbahA), benzo[g,h,i]perylene (BghiP) and indeno[1,2,3-c,d]pyrene (IP). Light molecular weight PAHs, also called light PAHs are Ace, Flu, Phe, Ant and Pyr, while the others are considered as heavy PAHs. To limit biases in the analysis of contamination data related to physicochemical properties, PAH concentrations in sediments and C. fluminea were normalized to organic matter (OM) concentration and lipid content, respectively. The average percentage of lipids for all the C. fluminea sampled was estimated at 12.9% based on C:N ratios (Figure A2 in the Appendix). The organic matter content of the sediments was estimated by measuring loss on ignition according to Heiri et al. (2001) (Figure

  in the Appendix). A total of 1635 boats were counted throughout the lake littoral, moored at private pontoons and in large public harbors. The individual power of each engine was read directly on the engine for 70% of the boats and estimated for the remaining 30%. Second potential watershed sources consisted of the number of stormwater outlets and their associated drainage areas (FigureA4in the Appendix). To test the influence of the spatial scale in C. fluminea contamination by these candidate variables, two buffer sizes around the sampling sites were considered, i.e. 200 and 600 m (FigureA5in the Appendix). The mean wave energy and slope and the sediment organic content of the sampling sites were also considered to influence the contamination of C. fluminea. The mean wave energy was calculated as a proxy of the hydrodynamic regime within a 200 m diameter circle around each site, based on the hydrodynamic models described inRohweder et al. (

Figure 2 .

 2 Figure 2. Mapping the TMs sediment contamination (mg kg -1 DM) along Lake Annecy littoral.

Figure 3 .

 3 Figure 3. Mapping the TMs C. fluminea contamination (mg kg -1 DM) along Lake Annecy littoral.

Figure 4 .

 4 Figure 4. Map of the sum of PAHs in the sediment (µg kg -1 OM) on the left side and in C. fluminea (µg kg -1 lipids) on the right side.

Figure 5 .

 5 Figure 5. Barplots representing the composition in light and heavy PAHs in A) the sediments (µg kg -1 OM) and B) C. fluminea (µg kg -1 lipids).

Figure 6 .

 6 Figure 6. Boxplots of the bioconcentration factors (BCF) for A) TMs and B) PAHs. The horizontal

Figure 7 .

 7 Figure 7. Heatmaps illustrating the results from the multiple linear regressions relating C. fluminea

Table 1 .

 1 Adjusted R-squared (Adjusted-R²) values for each variable and radius that indicate the proportion of variations in C. fluminea contamination explained in the final models.

	Contaminants	As	Cd	Cu	Pb	Sn	Zn	Sum of PAHs

Acknowledgment

The authors would like to thank the SILA (Syndicat Mixte du Lac d'Annecy) for sharing the data about stormwater outlets and their associated drainage areas on Lake Annecy watershed as well as the Association Agréée de Pêche et de Protection des Milieux Aquatiques (APPMA) "Annecy Lac Pêche" for sharing the data of their survey of 1636 boats in Lake Annecy during the summer 2017. The authors are specifically indebted to Yann Magnani, Pierre Boutillon and Céline Chasserieau for their support in sampling design and constructive comments along the course of this study. The authors also would like to thank the anonymous reviewers for their constructive comments on the early version of this article.

Financial support

This work has been financially supported by Association Agréée de Pêche et de Protection des Milieux Aquatiques (APPMA) "Annecy Lac Pêche" and the Fédération Départementale de Pêche et Protection des Milieux Aquatiques de Haute-Savoie.

Appendix

In addition to the details of TMs and PAHs contents in clams and sediments in study sites, the Appendix file provides information about intra-site variability of C. fluminea contamination, the method used for estimating lipid contents in the organisms, sediment characterization, spatial distribution of potential in-lake and watershed sources of contamination, relationships between the results of linear regressions and different buffer sizes for the buffer sizes selection and hydrodynamics details on Lake Annecy shoreline.