Minh City

Viet Nam

Thuy-Chung Kieu-Le

Quoc-Viet Tran

Tran-Nguyen-Sang Truong

Emilie Strady

Ho Chi

Anthropogenic fibres in white clams, Meretrix lyrata, cultivated downstream a developing megacity, Ho Chi

Keywords:

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

Introduction

Microplastics (plastic items from 1 to 5000 μm in size; [START_REF] Arthur | Proceedings of the International Research Workshop on the Occurrence, Effects, and Fate of Microplastic Marine Debris[END_REF] have recently become an emerging environmental pollutant that has received increasing attention from researchers. Microplastics were first observed in the pelagic surface water of the Sargasso Sea in 1972 [START_REF] Carpenter | Plastics on the Sargasso Sea surface[END_REF]. Until now, microplastics have been found in all aquatic environments, in urban areas characterised by dense industrial activities and/or high population density and also in countryside areas characterised by intense aquacultural and agricultural activities [START_REF] Chouchene | Microplastics on Barra beach sediments in Aveiro,Portugal[END_REF][START_REF] Costello | Microplastic pollutants in the coastal dunes of Lake Erie and Lake Ontario[END_REF][START_REF] Lahens | Macroplastic and microplastic contamination assessment of a tropical river (Saigon River, Vietnam) transversed by a developing megacity[END_REF][START_REF] Li | Microplastics in agricultural soils: extraction and characterization after different periods of polythene film mulching in an arid region[END_REF][START_REF] Ma | High levels of microplastic pollution in aquaculture water of fish ponds in the Pearl River estuary of Guangzhou,China[END_REF][START_REF] Pan | Microplastic pollution and ecological risk assessment in an estuarine environment: the Dongshan Bay of China[END_REF][START_REF] Patchaiyappan | Prevalence of microplastics in the sediments of Odisha beaches, southeastern coast of India[END_REF][START_REF] Ríos | Assessing urban microplastic pollution in a benthic habitat of Patagonia Argentina[END_REF][START_REF] Strady | Temporal dynamic of anthropogenic fibers in a tropical river-estuarine system[END_REF][START_REF] Strady | Baseline assessment of microplastic concentrations in marine and freshwater environments of a developing Southeast Asian country,Viet Nam[END_REF][START_REF] Wang | Microplastics profile in a typical urban river in Beijing[END_REF][START_REF] Wu | Accumulation of microplastics in typical commercial aquatic species: a case study at a productive aquaculture site in China[END_REF]. Microplastics have also been observed in rivers and streams in remote areas such as Mount Everest, the Tibetan Plateau and Arctic and Antarctic areas [START_REF] Gonza´lez-Pleiter | Fibers spreading worldwide: microplastics and other anthropogenic litter in an Arctic freshwater lake[END_REF][START_REF] Jiang | Microplastic pollution in the rivers of the Tibet Plateau[END_REF][START_REF] Lusher | Microplastics in Arctic polar waters: the first reported values of particles in surface and sub-surface samples[END_REF][START_REF] Napper | Reaching new heights in plastic pollution-preliminary findings of microplastics on Mount Everest[END_REF][START_REF] Obbard | Microplastics in polar regions: the role of long range transport[END_REF][START_REF] Waller | Microplastics in the Antarctic marine system: an emerging area of research[END_REF]. Microplastics are found in diverse shapes such as fragments, pellets, films, or fibreswith fibres being the most predominant shape [START_REF] Bikker | Microplastics and other anthropogenic particles in the surface waters of the Chesapeake Bay[END_REF][START_REF] Fu | Microplastic pollution research methodologies, abundance, characteristics and risk assessments for aquatic biota in China[END_REF][START_REF] Harris | The fate of microplastic in marine sedimentary environments: a review and synthesis[END_REF][START_REF] Mao | Horizontal and vertical distribution of microplastics in the Wuliangsuhai Lake sediment, northern China[END_REF][START_REF] Napper | The abundance and characteristics of microplastics in surface water in the transboundary Ganges River[END_REF]. Anthropogenic fibres are defined as i) synthetic fibres that have a petrochemical origin (i.e. polyester, polyamide, polypropylene, etc.), ii) artificial fibres originating from artificial cellulose or silk (i.e. viscose and rayon), and iii) natural fibres (i.e. cotton and wool). These fibres are usually used as materials in many applications such as the textile and apparel industries, in production of fishing nets and geotextiles. Anthropogenic fibres are released into the aquatic environments from untreated and treated domestic and industrial wastewater [START_REF] Bui | Microplastics pollution in wastewater: characteristics, occurrence and removal technologies[END_REF][START_REF] Hidayaturrahman | A study on characteristics of microplastic in wastewater of South Korea: identification, quantification, and fate of microplastics during treatment process[END_REF][START_REF] Jiang | Investigation and fate of microplastics in wastewater and sludge filter cake from a wastewater treatment plant in China[END_REF][START_REF] Pico | First evidence of microplastics occurrence in miXed surface and treated wastewater from two major Saudi Arabian cities and assessment of their ecological risk[END_REF], from fishing net degradation used in aquacultural and fishery activities (F. Wu et al., 2020;J . Wu et al., 2020) or from geotextiles used for geotechnical, geo-environmental and civil engineering purposes [START_REF] Elena | Debris of geosynthetic materials on the shore of South-Eastern Baltic (Kaliningrad Oblast, Russian Federation)[END_REF][START_REF] Wiewel | Geotextile composition, application and ecotoXicology-a review[END_REF]. Recently, sludge used as fertiliser or buried in landfills was found to be a source of fibres via leaching into surface water and groundwater [START_REF] Carreras-Colom | A closer look at anthropogenic fiber ingestion in Aristeus antennatus in the NW Mediterranean Sea: differences among years and locations and impact on health condition[END_REF]. The abundance of anthropogenic fibres in aquatic environments may generate negative environmental impacts on the ecosystem and biota. Various aquatic organisms mistake anthropogenic fibres for food and ingest them resulting in negative health impacts from the fibres themselves as well as toXic pollutants (i.e. heavy metals, persistent organic pollutants) in the environment adsorbed on the surface of the (71,526 inhabitants, population density: 102 inhabitants km -2 , HCMC statistical yearbook 2019). The Can Gio mangrove area is subjected to an asymmetric semidiurnal tidal regime and characterised by a tropical monsoon climate. In 2016, the dry season lasted from January to April, while the rainy season continued from May to December with a monthly precipitation of 400-500 mm from August to October [START_REF] Imhen | Weather and climate information[END_REF]. The study site is located at Can Thanh, where long and wide natural sand flats are found near the estuaries of the Long Tau and Soai Rap Rivers. The sea currents also follow the monsoon climate regime, flowing from the southwest to the northeast during the rainy season and vice versa during the dry seasons [START_REF] Fang | A review on the South China Sea western boundary current[END_REF].

fibres [START_REF] Wright | The physical impacts of microplastics on marine organisms: a review[END_REF].

Among various aquatic organisms, bivalves play an important role in both freshwater and marine ecosystems due to their contribution to nutrient recycling, thus creating and modifying the habitat as well as affecting food webs [START_REF] Vaughn | Bivalve impacts in freshwater and marine ecosystems[END_REF]. Moreover, bivalves are an important part of the global food supply. Due to anthropogenic activities, bivalve abundance and species richness are declining [START_REF] Zhang | Direct and indirect effects of microplastics on bivalves, with a focus on edible species: a mini-review[END_REF]; the presence of anthropogenic fibres could be one causative factor. Worldwide, studies have been carried out to assess the accumulation of microplastics or anthropogenic fibres in several marine and freshwater bivalve species. The results have shown that microplastics and fibres were prevalent in all the tested species [START_REF] Berglund | Microplastics in a freshwater mussel (Anodonta anatina) in Northern Europe[END_REF][START_REF] Dowarah | Quantification of microplastics using Nile red in two bivalve species Perna viridis and MeretriX meretriX from three estuaries in Pondicherry, India and microplastic uptake by local communities through bivalve diet[END_REF][START_REF] Klasios | Microplastics and other anthropogenic particles are prevalent in mussels from San Francisco Bay, and show no correlation with PAHs[END_REF][START_REF] Li | Microplastics in commercial bivalves from China[END_REF][START_REF] Narmatha Sathish | Monitoring of microplastics in the clam Donax cuneatus and its habitat in tuticorin coast of gulf of[END_REF][START_REF] Phuong | Factors influencing the microplastic contamination of bivalves from the French Atlantic coast: location, season and/or mode of life?[END_REF][START_REF] Phuong | Contamination of microplastic in bivalve: first evaluation in Vietnam[END_REF][START_REF] Su | Using the Asian clam as an indicator of microplastic pollution in freshwater ecosystems[END_REF][START_REF] Van Cauwenberghe | Microplastics in bivalves cultured for human consumption[END_REF].

Due to plastic waste mismanagement and little public awareness regarding plastic pollution, Vietnam was ranked fourth among the top countries emitting the most plastic waste into the world's oceans [START_REF] Jambeck | Plastic waste inputs from land into the ocean[END_REF]. The abundance of microplastics in the aquatic environments of Vietnam is of concern and varies on a local scale, with the highest concentrations measured in dense urban zone and the lowest ones in remote areas [START_REF] Strady | Baseline assessment of microplastic concentrations in marine and freshwater environments of a developing Southeast Asian country,Viet Nam[END_REF]. Ho Chi Minh City (HCMC, population more than 9 million inhabitants in 2019; GSO, 2019) and its surrounding provinces, form the economic centre of the country and are characterised by a high population density and intensive industrial activities, especially plastic industries, which include food packaging, textile, and apparel industries. The impacts of the urban and industrial activities on the Saigon River can be observed from the spatial and temporal fluctuations in the concentration of anthropogenic fibres from 22 to 519 fibres L -1 [START_REF] Lahens | Macroplastic and microplastic contamination assessment of a tropical river (Saigon River, Vietnam) transversed by a developing megacity[END_REF][START_REF] Strady | Temporal dynamic of anthropogenic fibers in a tropical river-estuarine system[END_REF]. Located downstream of HCMC, the Can Gio Mangrove Biosphere Reserve (Can Gio mangrove) exhibits high biodiversity with more than 200 species of fauna and 52 species of flora [START_REF] Nguyen | The environment in Ho Chi Minh City Harbours[END_REF]. Aquaculture activities such as clam, shrimp, crab, and fish farming as well as fishing provide important livelihoods to local residents [START_REF] Vo | Can Gio Mangrove Biosphere Reserve Evaluation 2012: Current Status, Dynamics, and Ecosystem Services[END_REF]. However, the ecosystem of the Can Gio mangrove is extremely vulnerable. It is impacted by both the upstream urban and industrial activities of HCMC, which release various organic and inorganic pollutants into the mangrove ecosystem [START_REF] Costa-Bo¨ddeker | Ecological risk assessment of a coastal zone in Southern Vietnam: spatial distribution and content of heavy metals in water and surface sediments of the Thi Vai Estuary and Can Gio Mangrove Forest[END_REF], 2020;Nguyen et al., 2019aNguyen et al., , 2019b;;[START_REF] Tran | Bioaccumulation of trace elements in the hard clam, MeretriX lyrata, reared downstream of a developing megacity, the Saigon-Dongnai River Estuary,Vietnam[END_REF], and local issues that directly lead to plastic release, such as solid waste mismanagement (e.g. garbage dumping in the countryside [START_REF] Kuenzer | Assessing the ecosystem services value of can gio mangrove biosphere reserve: combining earth-observation-and household-survey-based analyses[END_REF][START_REF] Pham | Water pollution by intensive brackish shrimp farming in south-east Vietnam: causes and options for control[END_REF]), loss and wear and tear of fishing nets, lines, and floats used in local aquaculture and fishing activities, and the absence of wastewater treatment facilities. A recent study found evidence of the presence of microplastics in the beach sand and seawater of the Can Gio mangrove area (32 to 93 microplastics kg -1 and 6 items L -1 , respectively [START_REF] Vo | Comparison of microplastic pollution in beach sediment and seawater at UNESCO Can Gio mangrove biosphere reserve[END_REF]). However, to date, the accumulation of microplastics (or anthropogenic fibres) in the Can Gio mangrove's biota has not yet been investigated. Therefore, the objective of this study was to characterise the accumulation of anthropogenic fibres in different tissues of white clams (Meretrix lyrata) cultivated at the Can Gio beach by analysing white clams of a similar size range collected monthly. More specifically, the study aimed to examine: (i) the contamination level of anthropogenic fibres in clams, (ii) the mechanisms of anthropogenic fibre accumulation in the different tissues, (iii) the estimation of the annual ingestion of anthropogenic fibres by humans through clam consumption, and (iv) the necessary measures required to reduce accumulation of fibres in clams.

Materials and methods

Study area

The Can Gio Mangrove Biosphere Reserve (10 The white clam Meretrix lyrata (Sowerby, 1851) is a bivalve mollusc commonly found in South East Asia. In Vietnam, white clam farming is an important aquaculture industry for both domestic consumption and export [START_REF] Tcts | Decision 3529/QĐ-BNN-TCTS: Approving the Adjustment of the Mollusk Farming Development Plan to 2020[END_REF]. White clams are distributed in sandy tidal flats (e. g. sand percentage of 60%-90% [START_REF] Ngo | Techniques for Raising Clams, Blood Cockles and Pearl Mussels[END_REF]), mainly in the mid-to lowtidal zone where the slope is relatively flat [START_REF] Nguyen | Some Mollusc Species of Economic Value in Vietnam's Sea Environment[END_REF]. Adult clams are found buried in the substrate at a depth of approXimately 2-6 cm [START_REF] Nguyen | Study on Scientific Basis for Rearing, Conservation and Resource Development of Two Clam Species (MeretriX meretriX Linnaeus 1785 and MeretriX lyrata Sowerby 1851) in the Coastal Area of Nam Dinh[END_REF]. The clams feed on phytoplankton species and other suspended matter in seawater through filter feeding. Among other ecological factors, water temperature and salinity have an important influence on the growth and survival rate of the clams. Therefore, the harvesting duration depends on their living conditions and occurs at the Can Thanh site in the Can Gio beach area after 18-20 months of harvesting [START_REF] Tran | Public Certification Report: Ben Tre Clam Fishery[END_REF]. In clam farming areas, polyethylene nets are often used to enclose the clam culture area [START_REF] Nguyen | Study on Scientific Basis for Rearing, Conservation and Resource Development of Two Clam Species (MeretriX meretriX Linnaeus 1785 and MeretriX lyrata Sowerby 1851) in the Coastal Area of Nam Dinh[END_REF].

Sampling

White clams (Meretrix lyrata) were collected monthly in the Can Thanh cultivation area of Can Gio beach (Fig. 1) from March to August and in October 2016 with approXimately 30-50 medium-sized clam individuals harvested each month. [START_REF] Tran | Bioaccumulation of trace elements in the hard clam, MeretriX lyrata, reared downstream of a developing megacity, the Saigon-Dongnai River Estuary,Vietnam[END_REF] defined three seasonal periods based on monthly precipitation for 2016: a dry season including the March and April sampling months, a transition period including the May-July sampling months, and a rainy season including August and October sampling months. The clams were stored in sterile polyethylene Ziploc bags, immediately transported to the laboratory, and then stored at -18 • C for further analysis.

Sample preparation and digestion

To prevent potential fibre contamination from the bags themselves, the clams were thoroughly rinsed with filtrated tap water and carefully dissected to separate the gill (G), digestive system (D), and remaining tissues (R). The shell length was measured individually. The wet weight (ww) of the G, D, and R of each clam was also recorded individually (Fig. 2). For each sampling month, the G, D, and R of the clams were combined for three individuals of similar size to achieve five pools. The total number of tested (and dissected) individuals was 105 over the seven months of the study, corresponding to a total of 35 pools. The combined samples of the G, D, and R were placed separately in 250 mL Erlenmeyer flasks for digestion using a protocol adapted from [START_REF] Dehaut | Microplastics in seafood: benchmark protocol for their extraction and characterization[END_REF]. ApproXimately 150 mL KOH 10% (Merck®) was poured in each Erlenmeyer flask and the samples were covered with aluminium foil, placed on a hot plate at 40 • C for 24 h, and manually agitated gently. The samples were then filtrated through glass fibre filters (Whatman®, GF/A, 1.6 μm pore size) using a glass filtration unit connected to a vacuum pump. The filters were stored in sterile Petri dishes for stereoscopic observation.

Stereoscopic observation

The anthropogenic fibres on the filters were observed using a Leica stereoscope S6D with the measurement software LAS. The morphology of the fibres, including length and colour, was recorded. As recommended by (GESAMP, 2019), stereomicroscopic identification of microplastics without spectroscopic confirmation can be performed for microplastics of the class size [300-5000 μm]. Due to the absence of μFTIR (Microscopic Fourier Transformed Infrared) or similar equipment allowing systematic identification of particles lower than 300 μm, we selected an observation size range of 300 to 5000 μm for this study. The anthropogenic fibres were recognised according to the following criteria: a fibre should be an elongated line with equal thickness, not tapered towards the ends, presenting three-dimensional bending, no visible cellular or organic structures, and homogeneous colour (Fig. 3). The straight, transparent, or whitish fibres were excluded to avoid biological or organic origin.

Sample contamination control

A specific quality control procedure was established, based on a recommendation from [START_REF] Dehaut | Microplastics in seafood: benchmark protocol for their extraction and characterization[END_REF], to prevent sample contamination from airborne particles, water, reagent, and glassware used during the analysis. First, the work area in the lab was wiped with wet tissues. The technician always wore cotton laboratory clothes and sterile hand gloves. All the steps from sample dissection, digestion, to stereoscopic observation were carried out with little air circulation and using an air conditioner rather than a fan. The exposure time of the sample to the ambient air was reduced to a minimum. The number of people working in the lab was also restricted. Tap water for rinsing clams, glassware, and milli-Q water for preparing KOH solution were filtrated through glass fibre filters (Whatman®, GF/A, 1.6 μm pore size) before use. Glassware and samples were covered with aluminium foil to avoid airborne fibre contamination. Quality controls were performed during the two steps: digestion atmospheric control DAC and observation atmospheric control OAC [START_REF] Strady | Baseline assessment of microplastic concentrations in marine and freshwater environments of a developing Southeast Asian country,Viet Nam[END_REF]. The controls presented very little/no airborne contamination: DAC = 1 to 4 fibres (n = 3 replicates) and OAC = 0 to 1 fibre (n = 3 replicates).

Result analysis

The average fibre concentration over the entire survey and monthly average fibre concentration per individual clam and per gram wet weight of tissues (ww) were determined from observation results. The average fibre concentration over the entire survey and the monthly average fibre concentration in different parts of the clam (per tissue and per gram ww of each tissue) were also calculated. Kruskal Wallis statistical tests were performed to assess temporal variation of fibre accumulation in clams and in different clam tissues by using XLSTAT®.

Results

Abundance of anthropogenic fibres in clams

Over the seven-month survey, anthropogenic fibres were observed in all the clam pools and in all the sampling months (Table 1). Considering each clam individual, the average fibre concentration over the entire survey was 3.6 ± 2.1 fibres individual -1 . The monthly average fibre concentration fluctuated from 2.6 ± 0.6 fibres individual -1 in April, to 5.8 ± 3.1 fibres individual -1 in March (Fig. 4a). This fluctuation did not show temporal differences over the sampling months (Kruskal Wallis test, p > 0.05).

Accounting for each gram of wet tissues, the average fibre concentration over the entire survey was 2.7 ± 2.4 fibres g -1 ww. The monthly average fibre concentration varied from 0.9 ± 0.3 fibres g -1 ww in October to 6.9 ± 3.2 fibres g -1 ww in August (Fig. 4b). This variation presented a statistical difference over the entire survey period (Kruskal Wallis test, p < 0.05).

Abundance of anthropogenic fibres in clam tissues

In clam tissues, anthropogenic fibres were found in 97% of all pools of remaining tissues, 89% of all pools of gills, and 74% of all pools of digestive systems (Table 1). For each clam individual, the average fibre concentration over the entire survey was 1.0 ± 0.8 fibres individual -1 in Data on the number of tested pools, the number of pools with presented fibres and the number of anthropogenic fibres for a whole clam individual and for gills (G), digestive systems (D), and remaining tissues (R) for white clams (Meretrix lyrata) collected in the Can Gio mangrove area.

Month

Number of tested pools Number of pools found fibres in Number of fibres observed in gills, 0.8 ± 0.9 fibres individual -1 in digestive systems, and 1.9 ± 1.4 fibres individual -1 in remaining tissues. Fibre concentration in remaining tissues presented a statistical difference to that in gills and digestive systems (Kruskal Wallis test, p < 0.05). Over the entire survey, the tissue distribution can be summarised by 52% of fibres observed in remaining tissues, 26% observed in gills, and 22% in digestive systems (Fig. 5). The monthly average concentration varied from 0.6 to 1.5 fibres individual -1 in gills, 0.5 to 1.6 fibres individual -1 in digestive systems, and 0.9 to 3.3 fibres individual -1 in remaining tissues (Fig. 4a). The remaining tissues presented the highest monthly fibre counts (47% to 65%) compared to gills (16% to 35%) and digestive systems (15% to 29%), except in August where the highest fibre abundance was observed in gills (47%) rather than in remaining tissues (29%). For each tissue, there was no statistical difference in fibre concentration over the sampling months (Kruskal Wallis test, p > 0.05).

In terms of 1 g wet weight of each tissue, gills presented a higher average fibre concentration over the whole survey with 14.0 ± 19.8 fibres g -1 followed by digestive systems with 7.4 ± 9.7 fibres g -1 and remaining tissues with 1.4 ± 1.1 fibres g -1 , with a significant statistical difference in fibre concentration per gram wet weight between the tissues (Kruskal Wallis test, p < 0.001). Monthly, fibre concentrations in gills varied from a minimum of 3.3 fibres g -1 ww in October to a maximum of 42.7 fibres g -1 ww in August (Fig. 4b). In digestive systems, they varied from 3.7 fibres g -1 ww in April to 20.2 fibres g -1 ww in August. In contrast, fibre concentrations were stable in remaining tissues, from 0.5 to 2.3 fibres g -1 ww. For each tissue, the temporal variation of fibre concentration in 1 g did not present any statistical variation over the sampling months (Kruskal Wallis test, p > 0.05).

Fibre length distribution

Within the observation size range [300-5000 μm], the median fibre length over the survey period was of 627 μm. Fibres shorter than 1000 μm represented nearly 70% of total fibres. Taking into consideration the temporal variation of fibre length, monthly median fibre length varied from 571 μm in April to 667 μm in March, and did not indicate any temporal statistical difference (Kruskal Wallis test, p > 0.05, Fig. 6a).

Fibres longer than 2000 μm or even up to 5000 μm were also observed every month. Notably, beyond the observation size range, seven long fibres up to 11,000 μm and many fibres shorter than 300 μm were also recognised and not recorded in our results.

Over the survey period, considering fibre length in clam tissue, the median fibre length was 561 μm in gills, 611 μm in remaining tissues, and 775 μm in digestive systems. No statistical difference in fibre lengths between tissues was observed (Kruskal Wallis test, p > 0.05, Fig. 6b).

Fibre colour partition

Over the survey period, colour partition of anthropogenic fibres showed a dominance of blue fibres (61%) followed by red (30%) and other colours including transparent, grey, and green (9%). Blue and red fibres were observed in all sampling months, blue fibres were observed in all pools, while red fibres were observed in 94% of all pools. Accordingly, monthly colour partition showed similar colour distribution over the survey period (Fig. 7).

Discussion

Level of contamination: comparison to other bivalve species

The presence of anthropogenic fibres in all clam pools and all sampling months confirms the contamination by anthropogenic fibres of white clams (Meretrix lyrata) reared in the Can Gio beach area. The average concentration of fibres over the whole survey was 3.6 ± 2.1 fibres individual -1 or 2.7 ± 2.4 fibres g -1 ww. This fibre concentration is at the same level of those previously observed in worldwide bivalve species [START_REF] Phuong | Contamination of microplastic in bivalve: first evaluation in Vietnam[END_REF][START_REF] Su | Using the Asian clam as an indicator of microplastic pollution in freshwater ecosystems[END_REF][START_REF] Narmatha Sathish | Monitoring of microplastics in the clam Donax cuneatus and its habitat in tuticorin coast of gulf of[END_REF][START_REF] Gedik | Microplastic pollution profile of Mediterranean mussels (Mytilus galloprovincialis) collected along the Turkish coasts[END_REF][START_REF] Phuong | Factors influencing the microplastic contamination of bivalves from the French Atlantic coast: location, season and/or mode of life?[END_REF]. Microplastic concentrations were of 2.6 items individual -1 or 0.29 items g -1 ww in green mussels (Perna viridis) collected in Central Vietnam [START_REF] Phuong | Contamination of microplastic in bivalve: first evaluation in Vietnam[END_REF], 0.4-5.0 items individual -1 or 0.3-4.9 items g -1 ww in Asian clams (Corbicula fluminea) from Taihu Lake, China [START_REF] Su | Using the Asian clam as an indicator of microplastic pollution in freshwater ecosystems[END_REF], 0.6-1.3 items g -1 ww in clams (Donax cuneatus) from the Tuticorin coast of the Gulf of Mannar, India [START_REF] Narmatha Sathish | Monitoring of microplastics in the clam Donax cuneatus and its habitat in tuticorin coast of gulf of[END_REF], 0.69 items individual -1 or 0.23 items g -1 ww in Mediterranean mussels (Mytilus galloprovincialis) [START_REF] Gedik | Microplastic pollution profile of Mediterranean mussels (Mytilus galloprovincialis) collected along the Turkish coasts[END_REF], 0.61 ± 0.56 items individual -1 in blue mussels (Mytilus edulis), and 2.10 ± 1.71 items individual -1 in the Pacific oyster (Crassostrea gigas) from the French Atlantic coast [START_REF] Phuong | Factors influencing the microplastic contamination of bivalves from the French Atlantic coast: location, season and/or mode of life?[END_REF]. Notably, the differences of laboratory digestion protocols with or without density separation steps between the studies may lead to different extraction efficiency and thus to different concentrations measured in different studies. Moreover, the types and shapes of items considered, i.e. microplastics or anthropogenic fibres, and the differences of observation size range makes these comparisons even trickier.

Accumulation mechanism of anthropogenic fibres in Can Gio white clam tissue

The abundance of anthropogenic fibres in remaining tissues was more prevalent than in digestive systems and gills, over the entire survey as well as in most of the sampling months (Table 1 and Fig. 5). Accordingly, the remaining tissues owed the highest rate of anthropogenic fibres accumulation among all the clams' tissues analysed in this study.

Thus far, the gills and digestive system were observed as the most prevalent organs involved in the accumulation of microplastics by aquatic organisms [START_REF] Su | The occurrence of microplastic in specific organs in commercially caught fishes from coast and estuary area of east China[END_REF]. The respiration, ingestion, digestion, and excretion processes play a crucial role in the accumulation of nondigestible particles including microplastics in the body of the organism. In the present study, anthropogenic fibres were more abundant in remaining tissues implying a supplementary process for fibre accumulation in the organisms. In mussels, the adherence of microplastics to soft tissues was evidenced as a novel way to uptake microplastics beyond ingestion [START_REF] Kolandhasamy | Adherence of microplastics to soft tissue of mussels: a novel way to uptake microplastics beyond ingestion[END_REF]. Based on the adherence mechanism, the fibres in the surrounding waters and benthic sediments adhere to the clam tissues. We presume that the adherence mechanism is also applicable to white clams, depending on the following factors: i) the concentration and vertical distribution of fibres in the sediment, ii) clam mobility in the sediment, and iii) dispersion of the fibres in the surrounding water due to the current and tidal regime, tropical climate conditions (rainfall and wind), and the variation of physico-chemical parameters in the environment. Upstream of HCMC and Saigon River, neither the hydrodynamics, monsoon climate conditions, nor the variation of physico-chemical parameters (pH, T, O2, conductivity, chlorophyll-alpha) affected the concentration of fibres in the surface water of the rivers [START_REF] Strady | Temporal dynamic of anthropogenic fibers in a tropical river-estuarine system[END_REF]. The change in rainfall regime, from dry to rainy season, and the change in wind direction did not influence concentrations measured in the atmospheric fallouts of HCMC [START_REF] Truong | Microplastic in atmospheric fallouts of a developing Southeast Asian megacity under tropical climate[END_REF]. Therefore, we hypothesise that the tropical climate conditions and the tidal hydrodynamics do not influence the adherence of fibres in this environment; no temporal variation was observed affecting the ingestion of accumulated fibres (Figs. 4 and5). A higher accumulation in the gills and digestive systems was observed in August but this did not correspond to any changes in environmental conditions that could support a higher accumulation. We thus hypothesise that a change in the microplastic sources to the surrounding environment could explain this observation. In the beach sediments of Da Nang, Central Vietnam, systematically higher fibre concentrations were measured in the upper sediment layer (0-5 cm) than in the lower one (5-10 cm) [START_REF] Tran | Bioaccumulation of trace elements in the hard clam, MeretriX lyrata, reared downstream of a developing megacity, the Saigon-Dongnai River Estuary,Vietnam[END_REF]. This pattern was also observed in the Bohai beach sediments [START_REF] Yu | Occurrence of microplastics in the beach sand of the Chinese inner sea: the Bohai Sea[END_REF] and in the Amazon macrotidal sandy beach sediments [START_REF] Martinelli Filho | Widespread microplastics distribution at an Amazon macrotidal sandy beach[END_REF]. Therefore, the vertical distribution of fibres in the sediment, coupled with the clam burying mobility in the sediment up to a depth of 4-6 cm [START_REF] Tran | Public Certification Report: Ben Tre Clam Fishery[END_REF], might be important factors influencing the number of fibres that can be both ingested by clams or may adhere to their remaining tissues.

Potential risk of anthropogenic fibres to human health via the ingestion of clams

As with many other bivalves, whole clams are consumed by humans, without the removal of the gastrointestinal tract. Fibres in clams are thus directly transferred to humans by ingestion leading to potential risks to human health.

The fibre ingestion by humans through clam consumption is estimated from the number of individual clams consumed in 2019 by one person and the average fibre concentrations measured in clams during this study. Our study was based on several hypotheses. First, hypothesise that the consumption rate has not changed since 2010, as no data are available from 2011 to 2019, and that the average national clam mass consumption rate is 78%. These values were also derived from a national rate of clam consumption of 66,000 tons and total clam production of 85,000 tons (VNAD, 2011). Thus, with a clam production of 227,000 tons in 2019 (VCFN, 2020), the national clam consumption amount is estimated at 176,152 tons. Second, we hypothesise that the entire national population of all ages (96.48 million inhabitants; (GSO, 2019)) consume clams, which provides an estimated mass of clams consumed per person of 1.8 kg inhabitant -1 yr -1 in 2019. Third, we hypothesise that 1 kg of clams corresponds to roughly 50 individual clams. Fourth, we hypothesise that the average concentration of fibres per clam sold in the markets throughout the country in 2019 is similar to that found in this study, namely 3.6 fibres individual -1 . Accordingly, the number of anthropogenic fibres ingested annually by one person from the ingestion of clams was estimated of about 324 fibres inhabitant -1 yr -1 . Once ingested, these fibres may release toXins into human body before being excreted after a certain time. Although studies on the toXicity of anthropogenic fibres to humans are still limited, the restriction of microplastics and anthropogenic fibres ingested by humans is necessary to beforehand prevent the potential health risks from fibrerelated pollutants.

Necessary measures to reduce accumulation of fibres in clams

Microplastics are mainly released into aquatic environments via wastewater [START_REF] Liu | A review of the removal of microplastics in global wastewater treatment plants: characteristics and mechanisms[END_REF]. Therefore, the reduction of microplastic release from wastewater could be an initiative. In Vietnam, the domestic wastewater treatment rate is quite low and represents approXimately 13% of urban wastewater [START_REF] Monre | Vietnam National Environmental Status Report 2018: Water Environment of River Basins[END_REF]. For industrial wastewaters, the percentage of industrial zones with a centralised wastewater treatment system varies from 16% for the industrial zones with small and medium sized enterprises to 88% for those with large-sized enterprises [START_REF] Monre | Vietnam National Environmental Status Report 2018: Water Environment of River Basins[END_REF]. The treatments used in Vietnamese wastewater treatment plants (WWTPs) are: primary treatments (e.g. grit chamber and primary sedimentation tank) and secondary treatments (e.g. activated sludge, trickling filters, moving bed bioreactors and aerated lagoons). Thus far, the retention of microplastics in Vietnamese WWTPs has not been studied. At a global level, microplastic retention was observed as 40%-99% after the grit chamber and primary sedimentation tank, from 29%-97% after sand filtration, from 8%-99% after the coagulation step, and from 17%-98% after the conventional activated sludge treatment (i. e. secondary sedimentation tank) [START_REF] Bui | Microplastics pollution in wastewater: characteristics, occurrence and removal technologies[END_REF]. The variables measured by the researchers depend on internal parameters (duration, chemical parameters) set up at the WWTP, the type and size of microplastics observed to evaluate their retention, and the management of the WWTP. Thus, the increase of the wastewater treatment rate coupled with supporting research to evaluate the microplastic retention and adapt the technology to achieve the highest retention evaluation could help reduce the release of anthropogenic fibres into the environment.

Another major source of fibres is from fishing equipment (F. Wu et al., 2020; J . Wu et al., 2020). Since their first introduction to the Plastic Waste Management in Ho Chi Minh City (2020-2030). Among various tasks, the reduction of anthropogenic fibre pollution and its release into the aquatic environment is addressed by focusing on reducing microplastic discharge from wastewater, reducing plastic waste, and on recovering lost fishing equipment.

Conclusions

This study confirmed the contamination of white clams (Meretrix lyrata) cultivated in the Can Gio sand beach with anthropogenic fibres with an average concentration over a seven-month sampling campaign of 3.6 ± 2.1 fibres individual -1 or 2.7 ± 2.4 fibres g -1 . The accumulation of the fibres in clams does not seem through ingestion and respiration processes only but may also be from the adherence of the fibres to the clam tissues. Moreover, fibres shorter than 1000 μm were predominantly found in the clams. An estimation of the number of fibres ingested by each person per year from clam consumption was approXimately 324 fibres inhabitant -1 yr -1 . To reduce fibre ingestion by humans, measures for reducing the release of fibres into the environment, especially from wastewater and fishing equipment.

Declaration of competing interest

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. fishing equipment industry, synthetic or semisynthetic fibres are now the main materials used to produce fishing nets and ropes [START_REF] Lusher | Microplastics in fisheries and aquaculture: status of knowledge on their occurrence and implications for aquatic organisms and food safety[END_REF]. Owing to aquaculture and fishery activities, a large quantity of fishing equipment are left or lost in aquatic environments. [START_REF] Jang | Estimation of the annual flow and stock of marine debris in South Korea for management purposes[END_REF] estimated that more than 44,000 tons of fishing equipment is lost annually in the Korean Sea. In European waters, derelict fishing equipment was found to contribute to 33% of the total litter items (Pham et al., 2014). [START_REF] Zhang | Neglected microplastics pollution in the nearshore surface waters derived from coastal fishery activities in Weihai,China[END_REF] reported a higher concentration of microplastic fibres in the surface waters of the marine culture areas as compared to other areas in Weihai, China. Fibres were also recognised as the predominant type of microplastic ingested by commercial oysters (Ostrea denselamellosa) and razor clams (Sinonovacula constricta) collected from the aquaculture sites at Xiangshan Bay, China (F. Wu et al., 2020). A laboratory analysis of microplastic release by the degradation of fishing nets recovered from the Venetian Lagoon was carried out by [START_REF] Montarsolo | Study on the microplastics release from fishing nets[END_REF]. The results showed that under the simulated marine environment, textile filament fishing nets released 64-750 fibres g -1 . In the Can Gio mangrove area, aquaculture and fishing activities are important for the livelihood of residents. In other words, the existence of fishing equipment in the environment, which is being used or lost, is guaranteed. In particular, in the Can Thanh sand flats, the use of fishing nets to enclose the clam culture areas has been observed. This leads to the release of anthropogenic fibres into the aquatic environment due to the aquaculture and fishing activities in this area. This source of fibres could be reduced by the recovery of the lost fishing equipment.

Recently, the Ho Chi Minh City council promulgated the Decision No. 2626/QĐ-UBND dated the 7th July 2020 on the Action Plan for Marine

Fig. 2 .

 2 Fig. 2. Monthly average shell length (cm) and monthly average wet weight (g) of gills (G), digestive systems (D) and remaining tissues (R) of white clam (Meretrix lyrata) collected in the Can Gio mangrove area over the sampling period.

Fig. 1 .

 1 Fig. 1. Study area map with sampling site for white clams (Meretrix lyrata) in the Can Gio mangrove area.

Fig. 3 .

 3 Fig. 3. Anthropogenic fibres observed in white clams (Meretrix lyrata) collected in the Can Gio mangrove area.

Fig. 4 .

 4 Fig. 4. Average concentration over the entire survey and the monthly average concentration of anthropogenic fibre in white clams (Meretrix lyrata) collected in the Can Gio mangrove area for the entire clam tissues and for gills (G), digestive systems (D) and remaining tissues (R) calculated for (a) one clam individual, and (b) 1 g wet weight of tissues.

Fig. 5 .

 5 Fig. 5. Partitioning of anthropogenic fibre abundance in gills (G), digestive systems (D) and remaining tissues (R) in white clam (Meretrix lyrata) collected in the Can Gio mangrove area for every month and for the entire survey.

Fig. 6 .

 6 Fig. 6. BoXplots of anthropogenic fibre length observed in white clam (Meretrix lyrata) collected in the Can Gio mangrove area for (a) every month and the entire survey, (b) the clams' tissues including gills (G), digestive systems (D) and remaining tissues (R).

Fig. 7 .

 7 Fig. 7. Colour partition of anthropogenic fibres in white clam (Meretrix lyrata) collected in the Can Gio mangrove area for every month and for the entire survey.

  Kieu-Le: Conceptualization, Investigation, Writingoriginal draft, Writingreview & editing. Quoc-Viet Tran: Investigation, Writingreview & editing. Tran-Nguyen-Sang Truong: Investigation, Writingreview & editing. Emilie Strady: Conceptualization, Writingreview & editing, Supervision.

  • 22 ′ -10 • 44 ′ N and 106 • 46 ′ -107 • 01 ′ E) is a part of HCMC with a low population density

	2.2. White clam (Meretrix lyrata)

Table 1

 1

			G	D	R	Whole clam	G	D	R	Whole clam
	Mar	5	5	4	5	5	14	24	49	87
	Apr	5	4	3	5	5	11	8	25	44
	May	5	5	4	5	5	22	11	29	62
	J u n	5	5	3	5	5	15	8	29	52
	J u l	5	4	3	5	5	9	8	32	49
	Aug	5	4	4	4	5	21	11	13	45
	Oct	5	4	5	5	5	9	12	21	42
	All	35	31	26	34	35	101	82	198	381
	(%)		88.6%	74.3%	97.1%	100.0%	26.5%	21.5%	52.0%

France and the Vietnam National University, Ho Chi Minh City (VNU-HCM) under grant number C2018-20-17. This study was undertaken under the framework of the JEAI PLASTIC research team supported by the French National Research Institute for Development (IRD). We acknowledge the time and facilities provided by the Ho Chi Minh City University of Technology (HCMUT), VNU-HCM for this study.

Acknowledgements

This study was partly funded by CNRS-INSU EC2CO-Ecodyn grants,

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.marpolbul.2021.113302.


Mouhannad Alattar

Francoise Sailhan

Julien Bourgeois

Modeling and Detecting Intrusions in ad hoc Network Routing Protocols

Ad hoc networks mostly operate over open and unprotected environments and are as such vulnerable to a wide range of attacks. Preventive techniques e.g., firewall and encryption, are no longer sufficient and should be coupled with advanced intrusion detection. We propose a distributed intrusion detection system that analyses activity logs so as to generate the rules which are used to detect intrusion. In order to deal with the distributed nature of an ad hoc network, the proposed system correlates information found in the multiple traces provided by surrounding devices. Performance is further evaluated, in terms of e.g., intruder detection rate and false positive.

I. INTRODUCTION

Securing ad hoc networks is not a trivial task because these networks rely on open radio-based medium of communication. In addition, the lack of centralized points e.g., switches and routers, complicates the deployment of preventive strategies. Thus, traditional ways of securing networks e.g., firewall, should be enriched with reactive mechanisms including intrusion detection system. As a first step upon detecting intrusions, we first categorize the attacks threatening routing protocols; their central role, i.e., determining multi-hops paths among the devices, designates these latter as the favourite target of attackers. Then, we propose a Distributed Intrusion Detection System (DIDS), which analyses the activity logs so as to discover a sequence of events that characterizes an intrusion attempt. Then, logs are correlated so that the DIDS correctly identifies more intrusions and reduces the number of false positive. We further examplify and experimentally evaluate the performance of our DIDS focusing on a specific attacks, the so-called spoofing link attack, which aims at undermining the Optimized Link State Routing (OLSR) protocol [START_REF] Clausen | Optimized link state routing protocol (olsr)[END_REF].

The remainder of this paper is organized as follows. We first present the attacks on ad hoc routing protocols ( §II). Then, we envisage the development of the proposed DIDS ( §III) and its evaluation ( §IV). Finally, we conclude this article presenting future research directions ( §V).

II. ATTACKS ON OLSR

In ad hoc network, routing protocols constitute one of the favourite target of intruders; the reason is threefold. First, no security countermeasure is specified as a part of the RFCs proposed by IETF working group 1 . Second, the absence of the centralized infrastructure complicates the deployment of preventive measures e.g., firewalls. Third, any device may operate as router, which facilitates the manipulation of multihops messages as well as the compromising of the routing 1 http://www.ietf.org/dyn/wg/charter/manet-charter.html functionality. Attacks targeting ad hoc routing protocol fall into two main categories, passive (i.e., observing the traffic) versus active (i.e., an authorized change is attempted). Active attacks are further sub-classified according to the undertaken action on the routing messages [START_REF] Peng | How to misuse aodv:a case study of insider attacks against mobile ad-hoc routing protocols[END_REF]:

• Drop attacks consist in dropping one (or further) routing message(s). • Modify and forward attacks modify received routing message(s) before forwarding it. • Forge reply attacks aim at sending false response(s) to routing message(s). • Active forge attacks proactively generate novel routing message(s). In this paper, we focus on a particular active forge attack corresponding to a spoofing link attack. We exemplify this attack using the OLSR protocol [START_REF] Clausen | Optimized link state routing protocol (olsr)[END_REF]. In a nutshell, OLSR aims at maintaining a constantly updated view of the network topology on each device. One fundamental of OLSR is the notion of multipoint relay (MPR): a subset of 1-hop neighbors that covers all the 2-hops neighboors and forwards the control traffic in the entire network. In practice, a node recognizes the 1-hop neighboors through periodic heartbeat messages, termed Hello messages. A MPR declares the nodes that selected itself to act as MPR via Topology Control (TC) message, which is intended to be diffused in the entire network. Thanks to TC messages, any device computes the shortest path (in term of number of hops) to any destination, such path being represented as a sequence of MPRs. Overall, OLSR is subject to a variety of attacks, including spoofing link attack, that majoritly target MPRs ; these latter constituing an attractive post for launching further attacks (e.g., deleting messages). In practice, one possible strategy consists in corrupting the MPR selection.

A. Spoofing link attack

An attacker aspiring being selected as an MPR may corrupt the MPR selection (equation 1). Towards this goal, the intruder advertises a falsified local topology: I sends a Hello message (V Hello(N S I ) ←-------I) to a victim V so as to declare a neighboor set N S I differing from the real set N S I . The difference results from inserting 2 (i) a non-existing node N (N / ∈ N with N defining the set of nodes composing the network) and/or (ii) an existing, but non-neighbooring, node E (E ∈ N S I ∩N : E / ∈ N S I ). Thus, the connectivity of I is increased Card(N S I \N S I ∩ N S I ) > 0. Recall that the set of MPRs is selected so that all the 2-hop neighboors are covered, I is hence selected as a MPRs of V . This affirmation is verified as long as no other intruder I (respectively legitimate node M ) advertises the same neighboor N (respectively E) .

I Hello(N S V ) ← -----------V, V Hello(N S I ) ← ----------I, (∃N ∈ N S I : N / ∈ N ∩ N S I ) ∨ (∃E ∈ N S I ∩ N : E / ∈ N S I ) ⇓ ⇓ Card(N S I \N S I ∩ N S I ) > 0 Card((N S I \[N S I ∩ N S I ]) ∩ N ) > 0 ∃M ∈ N \I : M ∈ N S V ∧ E ∈ N S M ∃I ∈ I : I ∈ N S V ∧ N ∈ N S I ⇓ ⇓ I ∨ M ∈ M P R V , I ∨ I ∈ M P R V , ⇓ I ∈ I. (1)
Such an attack can be detected relying on a intrusion detection system.

III. INTRUSION DETECTION

We proposed a DIDS that traces and detects the source of a network-based intrusion. This DIDS includes a host-based tracing mechanism that keeps track of the OLSR activities (routing logs) and analyses these latter to detect evidences. Each device further cooperates with one another so as to correlate evidences and match it against predefined intrusion signatures. In spoofing link attack, recall that the intruder increases artificially its connectivity so as to be chosen as MPR, we define three evidences that render a MPR suspect:

• a MPR relays packets in an abnormal way e.g., packets are dropped and do not reach their destination, • a MPR is replaced suspiciously by a new MPR, • a MPR is the only one covering one node. If one of the above evidence is discovered, advanced correlation and investigation is performed: messages are exchanged with the 2-hop neighboors that are covered by the suspicious MPR.

IV. PERFORMANCE EVALUATION

In order to evaluate our DIDS, we couple a network simulator (NS3) 3 [START_REF] Riley | The ns-3 network simulator[END_REF] with LinuX Containers virtual machines (LXC)4 [START_REF] Bhattiprolu | Virtual servers and checkpoint/restart in mainstream linux[END_REF]. NS3 simulates a MANET (and hence owns an implementation of the OLSR protocol) while each simulated devices owns a LXC container embeding a DIDSs. While offering the capability of monitoring the memory consumption of each node in the virtual machine, this platform permits to easily experiment a MANET (herein 25 mobile nodes including 5 intruders are simulated). Performance is further evaluated in terms of intruder detection rate, resource consumption and false positive (Figure ??). The detection rate rises up to 95% with a node's speed equal to 10 m/s. Increasing the speed leads to a decrease of the detection rate that reaches 55% with a node speed of 20 m/s (i.e., 180 km/h). This decrease results from the difficulty to obtain investigation correspondences when the devices mobility rises. In counterpart, the number of false positive observed is limited (ranging between 0 up to 3) and has almost no relation with the mobility. The network overhead resulting from DIDS detection messages is 10 times smaller than the overhead attributed to the OLSR protocol. Furthermore, our system causes an increment in the container used memory ranging from 16 to 52 MB, meaning that such a DIDS can be deployed on resource-constrained devices. Overall, our system has an accepted (respectively high) intruder detection rate in high (respectively moderate) mobility network.

V. CONCLUSION AND FUTURE WORK

In this paper, we present a distributed intrusion detection system that is focusing on the network intrusions targeting the routing protocol in MANET. The proposed DIDS reffers to a host-based detection system that matches logs against intrusion signatures. It requires no prior knowledge about the ad hoc network. Therefore, our system has an advantage over anomaly-based IDSs, which search for deviations from a normal expected behavior (and hence is generated from training data) so as to detect the intrusions. In counterpart, our system cannot detect a not-defined intrusion. DIDS performance is investigated against spoofing link attack. The detection rate reaches up to (95%) with a mobility speed more than the running average speed of a human being. The number of false positive is limited. Even though the amount of logs to be analyzed increases exponentially with network size, resource consumption is still suitable for the resourceconstrained devices.

A falsification of the local topology may also consists in suppressing existing neighboor(s) in the advertised neighbooring set. Nevertheless, rather than facilitating the selection of the intruder as MPR, this alteration reduces the connectivity of the intruder perceived by other and hence mortgages the probability of being selected as MPR.

http://www.nsnam.org

http://lxc.sourceforge.net


Sarra Zid

Jean-Patrick Bazile

Jean-Luc Daridon

Jean-Noël Jaubert

Jean-Louis Havet

Marie Debacq

Stéphane Vitu

High-Pressure Phase Equilibria Measurements of the Carbon Dioxide + Cycloheptane Binary System

Keywords: carbon dioxide, cycloheptane, phase equilibria, high pressure, equation of state, saturation pressure

The phase behavior of the carbon dioxide + cycloheptane binary system for which no literature data are available has been examined at temperatures ranging from (292.6 to 372.8) K. Saturation pressures, ranging from (23.9 to 154.8) bar, were acquired by a synthetic and visual method. In concrete terms, an adjustable volume high-pressure cell was utilized to measure bubble and dew point pressures by visual detection of phase transitions at constant overall composition. A total of 11 different mixtures with mole fractions of carbon dioxide ranging from (0.20 to 0.96) were prepared and a total of 99 experimental points were reported. The experimental results obtained in this work reveal the absence of liquid-liquid immiscibility in the studied temperature range and make it possible to conclude that the vapor-liquid critical line of the binary system is continuous and uninterrupted between the two pure compounds. Experimental data were correlated with the Peng-Robinson equation of state and classical Van der Waals mixing rules with temperaturedependent binary interaction parameters. They were also compared to the PPR78 model, an entirely predictive group contribution method in which the kij depends on temperature.

Introduction

Considering the vast amount of industrially used chemicals, the number of possible mixtures encountered in the different branches of industry is practically unlimited. It is thus unthinkable to measure thermodynamic and transport properties for all the possible industrial mixtures in a wide range of temperature and pressure. Nowadays, in various industrial sectors, several thermodynamic models can be successfully employed to avoid doing systematic and timeconsuming experimental measurements. However, experimental data are still needed for many application areas. 1,[START_REF] Sima | High-Pressure Phase Equilibrium for Carbon Dioxide + Ethyl n -Butyrate Binary System[END_REF] Even in the oil and gas sector, high-pressure phase equilibrium data as well as thermodynamic data regarding to uncommon molecules (such as naphthenic components with more than six carbon atoms in ring or with multiple aromatic ring) are welcomed. 1,[START_REF] Vitu | Experimental Determination and Modelling of High-Pressure Phase Behavior for the Binary System CO2 + Cyclooctane[END_REF] Due to the important role of carbon dioxide in the chemical, pharmaceutical and petrochemical industries, phase equilibrium data of mixtures containing CO2 remain crucial. [START_REF] Peper | High-Pressure Fluid-Phase Equilibria: Trends, Recent Developments, and Systems Investigated (2009-2012[END_REF][START_REF] He | New Vapor-Liquid Phase Equilibrium Data of CO2 in Several Heavy n-Alkanes at High Pressures[END_REF][START_REF] Moorcroft | Influence of Unsaturation on the High-Pressure Phase Behavior of C18 Fatty Acid Methyl Esters + CO2[END_REF][START_REF] Sima | High-Pressure Phase Equilibria of Carbon Dioxide + 1,4-Dioxane Binary System[END_REF][START_REF] Gao | The Solubility of CO2 in (Hexane + Cyclohexane) and (Cyclopentane + Ethylbenzene) and (Toluene + Undecane) Systems at High Pressures[END_REF] In our previous works [START_REF] Vitu | Experimental Determination and Modelling of High-Pressure Phase Behavior for the Binary System CO2 + Cyclooctane[END_REF][START_REF] Vitu | High-Pressure Phase Behaviour of the Binary System CO2 + Cis-Decalin from[END_REF] , a lack of data for two kinds of binary systems containing carbon dioxide + a naphthenic compound was noticed; they are CO2 + branched naphthenes and CO2 + naphthenes that contains more than 6 carbon atoms in ring (cycloheptane, cyclooctane, etc.). Indeed, the binary systems CO2 + cyclopentane and CO2 + cyclohexane were widely investigated in the past and more details about the different studies related to these two systems can be found elsewhere. [START_REF] Sima | The Effect of the Naphthenic Ring on the VLE of (Carbon Dioxide + Alkane) Mixtures[END_REF][START_REF] Vitu | Predicting the Phase Equilibria of CO2 + Hydrocarbon Systems with the PPR78 Model (PR EoS and kij Calculated through a Group Contribution Method)[END_REF] No literature data were available for the binary mixture CO2 + cyclooctane under pressure and we thus recently published [START_REF] Vitu | Experimental Determination and Modelling of High-Pressure Phase Behavior for the Binary System CO2 + Cyclooctane[END_REF] bubble and dew points for this system. The situation is almost similar for the binary system CO2 + cycloheptane. Only five solubilities of CO2 in cycloheptane at atmospheric pressure have been published. [START_REF] Gironi | A Simple Method for Determining the Solubility of Gases in Liquids: Application to CO2-Cycloparaffin Systems[END_REF] The purpose of this study is to disseminate new VLE measurements for the CO2 + cycloheptane system under pressure, on the full composition range and therefore to fill in the data gaps concerning such systems. A high-pressure device was employed to measure phase transitions. The experimental data were correlated using the Peng-Robinson equation of state. This work is the continuation of a series of paper providing missing experimental data with regard to basic binary mixtures [START_REF] Zid | Fluid Phase Equilibria for the CO2 + 2,3-Dimethylbutane Binary System from 291[END_REF][START_REF] Vitu | Phase Equilibria Measurements of CO2 + Methyl Cyclopentane and CO2 + Isopropyl Cyclohexane Binary Mixtures at Elevated Pressures[END_REF] or to more uncommon systems. [START_REF] Vitu | Experimental Determination and Modelling of High-Pressure Phase Behavior for the Binary System CO2 + Cyclooctane[END_REF][START_REF] Vitu | High-Pressure Phase Behaviour of the Binary System CO2 + Cis-Decalin from[END_REF][START_REF] Vitu | Bubble and Dew Points of Carbon Dioxide + a Five-Component Synthetic Mixture: Experimental Data and Modeling with the PPR78 Model[END_REF]

Experimental section

Materials

The description of materials used in this work is reported in Table 1. The chemicals were utilized as we received them. Purities given in Table 1 are those provided by the manufacturers.

Apparatus and method

The apparatus employed and the experimental strategy were widely described in our previous papers. [START_REF] Vitu | Experimental Determination and Modelling of High-Pressure Phase Behavior for the Binary System CO2 + Cyclooctane[END_REF][START_REF] Zid | Fluid Phase Equilibria for the CO2 + 2,3-Dimethylbutane Binary System from 291[END_REF][START_REF] Daridon | Fluid-Fluid and Fluid-Solid Phase Equilibria in Carbon Dioxide + Waxy Systems 1. CO2 + n-C17[END_REF] The technique used to perform saturation pressure measurements was a synthetic method, without sampling and analyses. This method is called synthetic-visual method with variable-volume cell (SynVisVar) as defined by Dohrn and co-workers. [START_REF] Peper | High-Pressure Fluid-Phase Equilibria: Trends, Recent Developments, and Systems Investigated (2009-2012[END_REF] The main equipment was an adjustable-volume high pressure cell. The device includes a mobile piston and two sapphire windows: one for visual observation of the fluid contained in the cell thanks to an endoscope and the other to light up the interior of the cell. The mixture was continuously mixed by means of a small bar magnet. Internal volume of the cell ranged from 8 to 30 cm 3 and the internal temperature was maintained by circulating a coolant distributed through three lines arranged in the cell wall.

The mixture temperature enclosed in the cell was measured with an uncertainty of 0.1 K using a Pt100 probe inserted in the cell. The pressure was acquired by a Kulite pressure gauge installed inside the cell to restrict dead volumes. The pressure gauge was calibrated, between 1 and 1000 bar, with an uncertainty better than 0.02%.

In a first step, the device was partly filled with a known quantity of liquid cycloheptane by vacuum extraction. The mass of cycloheptane loaded (𝑚 𝑐𝑦𝑐𝑙𝑜ℎ𝑒𝑝𝑡𝑎𝑛𝑒 ) was measured with a precision balance (Ohaus). In a second step, the CO2 mass (𝑚 𝐶𝑂 2 ) was added under pressure in the cell. To do so, the carbon dioxide was firstly loaded in an aluminum pressure vessel. The vessel was weighed by means of a Metler Toledo high-capacity precision balance. So as CO2 weighing not to be disturbed by the connecting line, the CO2 vessel was linked to the cell through a flexible lightweight tubing. This flexible high-pressure capillary does not affect the stability of the balance during the weighing. Moreover, the capillary tube has a small diameter (1/32") and the CO2 was stored in liquid-vapor equilibrium in the tank in order to keep the pressure constant during CO2 injection and therefore prevent gas mass changes in the connecting line.

The expanded uncertainties in mass loaded in the apparatus were deemed to be better than 0.005 g for cycloheptane and 0.005 g for CO2. The composition (𝑧 𝐶𝑂 2 = 𝑧 1 ) of the CO2 + cycloheptane binary system contained in the cell can be calculated and the standard uncertainty in composition is obtained through the following expression

𝑢(𝑧 1 ) = 𝑢(𝑧 𝐶𝑂 2 ) = 𝑧 𝐶𝑂 2 (1 -𝑧 𝐶𝑂 2 ) √ ( 𝑢(𝑚 𝐶𝑂 2 ) 𝑚 𝐶𝑂 2 ) 2 + ( 𝑢(𝑚 𝑐𝑦𝑐𝑙𝑜ℎ𝑒𝑝𝑡𝑎𝑛𝑒 ) 𝑚 𝑐𝑦𝑐𝑙𝑜ℎ𝑒𝑝𝑡𝑎𝑛𝑒 ) 2 (1)
Saturation pressures were obtained visually. The phase boundaries were assessed by determining the disappearance conditions of one phase of a two phase equilibrium system.

The experiments were conducted, at constant temperature, by examining the disappearance of the vapor phase for a bubble-point pressure measurement. For a dew point, disappearance of the liquid phase was examined. The principle of the procedure is, in a first step, to reach a two-phase equilibrium by modifying the pressure and, in a second step, to eliminate the last phase formed by slowly inverting the pressure variation. Experiments were replicated to refine the measurements.

Bubble pressures and dew pressures were determined with a standard uncertainty of 0.2 bar and 0.4 bar, respectively. 13,16

Experimental results

For the CO2 + cycloheptane binary system, 83 bubble points and 16 dew points were acquired, by isoplethic measurements, for 11 different compositions ranging from 0.1996 to 0.9600 CO2 mole fraction so as to achieve a detailed description of the phase diagram. The obtained data are presented in Table 2 with uncertainties and are partly depicted in Figure 1. Measurements were carried out with a particular care for the CO2 rich mixtures at low temperature, by doing intermediate temperature steps, in order to detect a possible liquid-liquid phase splitting.

However, despite all this attention, no liquid-liquid immiscibility was encountered in the investigated temperature range. From the experimental data obtained in this work, it can be concluded that the vapor-liquid critical line of the CO2 + cycloheptane system is most likely uninterrupted between the pure compounds critical point so that the system is either a type I or a type II binary system in the nomenclature of van Konynenburg and Scott. [START_REF] Van Konynenburg | Critical Lines and Phase Equilibria in Binary Van Der Waals Mixtures[END_REF][START_REF] Privat | Classification of Global Fluid-Phase Equilibrium Behaviors in Binary Systems[END_REF] To complete the analysis of the system CO2 + cycloheptane, it is more convenient to represent data in an isothermal (P,x1,y1) diagram. However, phase transition measurements were not carried out at exactly the same temperatures for the 11 different mixtures prepared. The isothermal curves (P, 𝑥 𝐶𝑂 2 ) thus cannot be graphed directly from the measured data presented in Table 2. Interpolated values were calculated using second order polynomials to describe the pressuretemperature relationship of the raw experimental data at constant composition, following the reliable method previously described in detail by Schwarz et al. [START_REF] Moorcroft | Influence of Unsaturation on the High-Pressure Phase Behavior of C18 Fatty Acid Methyl Esters + CO2[END_REF][START_REF] Schwarz | Phase Equilibrium of Propane and Alkanes[END_REF][START_REF] Schwarz | High Pressure Phase Behavior of the Homologous Series CO2 + 1-Alcohols[END_REF][START_REF] Latsky | High Pressure Bubble-and Dew-Point Data for Systems Containing CO2 with 1-Decanol and n-Hexadecane[END_REF] The following second order polynomial was used

𝑃 = 𝐴𝑇 2 + 𝐵𝑇 + 𝐶 (2)
The coefficients of the polynomials (see Table 3) were calculated by fitting the raw experimental data reported in Table 2. These polynomial functions were then employed to generate isothermal data. Fitted coefficients are presented in Table 3 with R 2 values for each composition studied. It is obvious that the functions and the coefficients detailed in Table 3 can be safely used to describe the studied binary system in the strict temperature range of 293.15 to 373.15 K. The (P,x1,y1) phase diagrams resulting from these interpolations were displayed in Figure 2 for different isotherms.

Table 3. Coefficients of the P-T polynomial function (P) used to fit the experimental data reported in

Modeling and discussion

Initially, the PPR78 model was used to predict the experimental points. This model is today routinely employed [START_REF] Chiko | Comparison of CP-PC-SAFT and PC-SAFT with k12 = 0 and PPR78 in Predicting Binary Systems of Hydrocarbons with Squalane, n-Dodecylbenzene, Cis-Decalin, Tetralin, and Naphthalene at High Pressures[END_REF] and is fully described elsewhere. [START_REF] Jaubert | VLE Predictions with the Peng-Robinson Equation of State and Temperature-Dependent kij Calculated through a Group Contribution Method[END_REF][START_REF] Jaubert | Thermodynamic Models for Chemical Processes[END_REF] Only most important points of the model are thus recalled below.

For a pure component, the equation of state proposed by Peng and Robinson 25 is

=- - + + - i i i i i a (T ) RT P v b v( v b ) b ( v b ) (3)
And

-1 -1 , , 2 22 , ,, 2 2 8
=   =   = + -        = + -  = + - + ci i ci ci ii c i c i i i i i i i i i R RT b P RT T am PT m m       3              i  (4)
P is the pressure, R the ideal gas constant, T the temperature, v the molar volume, Tc the critical temperature, Pc the critical pressure and  the acentric factor.

In the PPR78 model, classical mixing rules are employed

) NN i j i j ij ij N ii i a z z a a k T b z b == =  =-      =      (5) 11 1 (1 ( )
zi is the mole fraction of component i in the mixture, N is the number of components in the mixture and kij(T) is the binary interaction parameter. In the PPR78 model, kij is temperature-dependent and determined by group contribution:

2 1 11 () () 1 298.15 ( )( ) 2 () ( ) ( ) 2 kl gg kl B NN j A i ik jk il jl kl ij kl ij ij ij aT aT A T b b kT a T a T bb      -   ==    - - -  - -     =    (6)
In eq 6, ai and bi are calculated by eq 4, Ng is the number of different groups presents in the mixture and ik is the fraction of molecule i occupied by group k. Akl and Bkl are constant group interaction parameters between group k and group l. The critical properties (Tc and Pc) and the acentric factor () of the two components involved in this work come from the DIPPR database.

In Figure 2, dashed isothermal curves (bubble and dew curves) foreseen by the PPR78 approach are drawn for comparison with interpolated data obtained from the original measurements at six temperatures from 293.15 K to 373.15 K. Figure 2 illustrates that the binary system is qualitatively and to a lesser extent quantitatively predicted accurately by the PPR78 model. With such a model, an average overall deviation on the liquid phase composition of 0.054 and a deviation of 0.030 on the gas phase composition were obtained, respectively. Such deviations are not surprising since (i) no parameters were fitted to represent the experimental data and (ii) this binary system was completely absent from the database used to adjust the interaction parameters of the model (the system CO2 + cycloheptane is indeed studied for the first time in this paper).

The PPR78 model foresees a type IV phase diagram in the van Konynenburg and Scott nomenclature. The predicted vapor-liquid critical locus is thus interrupted, with the presence of a lower critical end point (at 320.4 K), an upper critical end point (at 324.9 K) and a very narrow liquid-liquid-vapor three-phase line. Such a 3-phase domain was not found experimentally despite a careful examination of the mixture inside the cell in the CO2 rich region. So as to improve the description of the mixture behavior, it was decided to consider each molecule of the binary system as a single group. By doing so, eq 6 becomes

2 1 () () 298.15 () ( ) ( ) 2 ij ij B j A i ij ij ij ij ij aT aT A T b b kT a T a T bb  -        - -     =   (7)
The parameters Aij and Bij were fitted to smoothed experimental data to minimize, at specified temperature and pressure, the mean absolute deviations between calculated and experimental liquid phase and gas phase compositions. A12 = 122.1 MPa and B12 = 281.1 MPa were found to be the optimal values. Isothermal bubble and dew curves calculated by this method are solid lines in Figure 2. This figure shows that a slightly better representation of the data is obtained in the mixture critical area. By using these adjusted parameters, a continuous vapor-liquid critical locus is now calculated between the two pure compounds, which is in accordance with the experimental observation. The binary interaction parameter is high and slightly decreases with temperature: from 0.1155 at 293.15 K to 0.1007 at 373.15 K, which is in accordance with kij values reported in the literature for comparable systems.

8,11,14

Finally, Figures 1 and2 highlight that the Peng-Robinson equation of state is not able to perfectly correlate simultaneously the bubble curve (for low CO2 compositions) and the critical region. This is a well-documented weakness of cubic equations of state when size-asymmetric systems are correlated. As shown in Figure 2, the PPR78 model (dashed lines) satisfactorily correlates the bubble curve but the prize to pay is an overestimation of the critical pressures. Conversely, the Peng-Robinson equation of state with temperature-dependent kij fitted against the measured experimental data better correlates the critical region than the PPR78 prediction but at the expense of less accurate bubble pressures at lower CO2 mole fractions.

Conclusions

The synthetic method was applied to measure new saturation pressures of the CO2 + cycloheptane binary system between 292.6 K and 372.8 K. To do so, 11 mixtures from (0.20 to 0.96) CO2 mole fractions were prepared and studied. A total of 83 bubble points and 16 dew points This experimental work, and a previous one 3 devoted to the binary system CO2 + cyclooctane, can be used to discuss the phase behavior evolution regarding to the homologous series of CO2 + naphtenes binary mixtures. Mixtures of CO2 with cyclopentane and cyclohexane exhibit type I or II phase behavior. [START_REF] Sima | The Effect of the Naphthenic Ring on the VLE of (Carbon Dioxide + Alkane) Mixtures[END_REF][START_REF] Vitu | Predicting the Phase Equilibria of CO2 + Hydrocarbon Systems with the PPR78 Model (PR EoS and kij Calculated through a Group Contribution Method)[END_REF] From the results reported in this paper, it can be concluded that the binary system CO2 + cycloheptane also belongs to type I or II since the vapor-liquid critical curve is, highly likely, continuous. Finally, the binary mixture CO2 + cyclooctane probably exhibits type V phase behavior [START_REF] Vitu | Experimental Determination and Modelling of High-Pressure Phase Behavior for the Binary System CO2 + Cyclooctane[END_REF] but more experiments are necessary to validate this assumption.

In the future, additional measurements of the system CO2 + cyclooctane are planned, with small steps in temperature and a particular focus on the CO2 rich region of the binary diagram to confirm, or not, our supposition. Measurement of the binaries CO2 + cyclodecane and CO2 + cyclododecane are also envisaged.

For Table of Contents Only

  b Standard uncertainties are u(T) = 0.1 K, u(P) = 0.2 bar for bubble points and u(P) = 0.4 bar for dew points. c Each composition z1 is reported with its standard uncertainty u(z1). d z1 + z2 = 1.

Figure 1 .

 1 Figure 1. (P, T) phase envelopes of the binary system CO2 (1) + cycloheptane (2). CO2 mole fractions are: z1 = 0.2999 (green envelope and green points); z1 = 0.5000 (black envelope and black points); z1 = 0.7000 (red envelope and red points); z1 = 0.3753 (pink envelope and pink points); z1 = 0.8499 (yellow envelope and yellow points); z1 = 0.9600 (blue envelope and blue points). ○: experimental bubble points measured in this work. •: experimental dew points measured in this work. Solid lines: calculated phase envelopes with the Peng-Robinson equation of state and a temperature-dependent kij. : calculated mixture critical point.

Figure 2 .

 2 Figure 2. Isothermal phase equilibria of the binary system CO2 + cycloheptane. ○: interpolated experimental phase transition points. Dashed lines: bubble and dew curves predicted with the PPR78 model. Solid lines: bubble and dew curves calculated with the PR equation of state and a temperature-dependent kij fitted against interpolated experimental data.

  were measured with pressures up to 154.8 bar. Two approaches were employed to model the experimental data: the fully predictive PPR78 model and the equation of state proposed by Peng and Robinson combined with a temperature-dependent binary interaction parameter fitted to experimental data.

Table 1 . Description and specification of the pure compounds used in this study

 1

	compound	linear formula	CAS number	supplier	mass fraction purity b
	carbon dioxide	CO2	124-38-9	Linde	0.99995
	cycloheptane	C7H14	291-64-5	Acros Organics a	0.995

a Thermo Fisher Scientific. b information provided by the manufacturers.

Table 2 . Experimental bubble-point and dew-point pressures P, at temperature T, for the CO2 (1) + cycloheptane (2) binary system at 11 different CO2 mole fractions z1 a,b,c,d

 2

	z1 = 0.1996  0.0006 z1 = 0.2999  0.0006 z1 = 0.3753  0.0005 z1 = 0.5000  0.0004 z1 = 0.6002  0.0003 z1 = 0.7000  0.0002
	T/K	P/bar	T/K	P/bar	T/K	P/bar	T/K	P/bar	T/K	P/bar	T/K	P/bar
	293.5	23.9	293.1	32.8	293.9	38.3	293.8	44.0	292.6	46.1	292.7	47.6
	302.4	27.3	303.4	37.4	302.6	44.1	302.6	52.0	302.9	56.2	302.7	58.5
	311.6	30.5	311.9	42.4	313.4	51.2	311.7	60.6	312.3	66.5	311.6	68.9
	321.5	33.9	321.5	47.3	322.7	57.7	321.3	69.5	321.3	76.6	321.2	81.1
	331.1	37.6	332.8	52.0	332.3	64.1	330.9	78.5	331.1	88.3	330.8	94.7
	341.4	40.7	342.8	57.5	342.2	70.7	341.5	88.4	341.5	100.4	340.9	107.9
	349.1	42.9	351.0	62.0	350.5	76.0	350.9	97.3	350.9	111.0	350.8	121.2
	358.9	46.5	359.5	66.6	360.4	82.5	361.2	106.4	360.7	122.2	361.4	134.8
	371.7	49.9	371.3	72.0	371.7	89.7	372.8	115.5	371.4	132.6	371.7	146.2
	z1 = 0.8000  0.0002 z1 = 0.8499  0.0001 z1 = 0.9000  0.0001 z1 = 0.9300  0.0001 z1 = 0.9600  0.0002
	T/K	P/bar	T/K	P/bar	T/K	P/bar	T/K	P/bar	T/K	P/bar
	293.3	50.1	293.9	51.1	293.1	51.1	293.7	52.2	293.5	53.5
	304.4	63.3	303.5	62.4	303.1	62.7	302.6	63.4	303.0	65.7
	312.5	73.0	312.6	74.1	312.6	75.6	312.2	76.6	312.0	79.5
	322.5	86.9	322.0	87.4	321.5	89.2	322.5	91.5	321.2	91.1
	332.0	101.0	332.6	103.4	331.4	103.5	331.5	104.0	330.0	100.7
	341.6	115.5	341.4	117.8	341.2	117.2	340.9	115.8	341.3	112.6
	350.6	128.6	351.1	130.9	351.4	130.7	351.4	127.7	351.3	119.0
	360.7	141.7	359.8	141.9	361.2	141.8	361.1	137.3	360.9	122.0
	371.8	154.4	372.4	154.8	370.6	150.0	371.6	143.5	371.6	123.4

a Bold data are dew points, the rest are bubble points.

Table 2 in order to generate isothermal data for the CO2 (1) + cycloheptane (2) binary system between 293.15 K and 373.15 K a,b

  The polynomial function is 𝑃 = 𝐴𝑇 2 + 𝐵𝑇 + 𝐶. b Using these coefficients, original experimental data (Table2) are correlated with AAD of 0.78 bar (0.95%).

	z1/mol•mol -1	A/bar•K -2	B/bar•K -1	C/bar	R 2
	0.1996	-5.59793 × 10 -04	0.70530	-134.850	0.9995
	0.2999	1.01568 × 10 -04	0.43853	-104.594	0.9992
	0.3753	-2.83741 × 10 -04	0.85081	-187.328	1.0000
	0.5000	-6.72802 × 10 -04	1.36499	-299.327	0.9998
	0.6002	1.02935 × 10 -04	1.05265	-271.631	0.9995
	0.7000	4.95344 × 10 -04	0.94864	-273.623	0.9992
	0.8000	2.45485 × 10 -04	1.20661	-326.420	0.9986
	0.8499	-1.13277 × 10 -03	2.12810	-478.551	0.9976
	0.9000	-2.32288 × 10 -03	2.86794	-591.984	0.9982
	0.9300	-4.89489 × 10 -03	4.47474	-841.614	0.9982
	0.9600	-1.02564 × 10 -02	7.75416	-1340.672	0.9977
	a

Corresponding Author

* stephane.vitu@lecnam.net

Notes

The authors declare no competing financial interest.