Guilherme V B Ferreira
email: guilherme.vbf@gmail.com

Anne K S Justino

Leandro Nolé

Véronique Lenoble

Vincent Fauvelle

Natascha Schmidt

Teodoro Vaske Junior

Thierry Frédou

Flávia Lucena-Frédou

Plastic in the inferno: Microplastic contamination in deep-sea cephalopods (Vampyroteuthis infernalis and Abralia veranyi) from the southwestern Atlantic

Keywords: Plastics, Mesopelagic zone, Marine Litter, Deep-sea squids, Oceanic islands

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

Plastics have a crucial role in the global economy as raw materials, positively influencing the market dynamics due to the low production costs, high durability, and versatility [START_REF] Andrady | Applications and societal benefits of plastics[END_REF][START_REF] Derraik | The pollution of the marine environment by plastic debris: A review[END_REF]. However, during the last decades, the great demand and further production of plastics have rapidly increased [START_REF] Geyer | Production, use, and fate of all plastics ever made[END_REF], arising major concerns regarding their final fate and hazardous potential. Microplastics (<5 mm; MPs) [START_REF] Arthur | Proceedings of the International Research Workshop on the Occurrence, Effects, and Fate of Microplastic Marine Debris[END_REF] have gained attention due to their toxicity and the widespread occurrence as a contaminant in the aquatic environment [START_REF] Bhagat | Zebrafish: An emerging model to study microplastic and nanoplastic toxicity[END_REF][START_REF] Eriksen | Plastic Pollution in the World's Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Afloat at Sea[END_REF].

Inshore economic activities yield the major waste production. Continental areas are the main plastic reservoir in the biosphere [START_REF] Gesamp | Sources, fate and effects of microplastics in the marine environment: part two of a global assessment[END_REF][START_REF] Van Sebille | A global inventory of small floating plastic debris[END_REF], where a considerable portion of these products are mismanaged along the production chain or disposed into landfills, then later flushed by rainfall into waterways [START_REF] Jensen | Sources, distribution and fate of microfibres on the Great Barrier Reef[END_REF]. Improper sewage disposal, inefficient wastewater treatment plants, and atmospheric transport are additional sources of MPs to the aquatic ecosystems [START_REF] Allen | Atmospheric transport and deposition of microplastics in a remote mountain catchment[END_REF][START_REF] De Falco | The contribution of washing processes of synthetic clothes to microplastic pollution[END_REF]. Once in the drainage basin, advection flow will transport MPs downstream to the estuary [START_REF] Lima | Distribution patterns of microplastics within the plankton of a tropical estuary[END_REF].

Estuaries can supply many MPs to the adjacent coastal waters and are acknowledged as the main source to the ocean [START_REF] Lebreton | River plastic emissions to the world's oceans[END_REF], mostly during increased river flow periods [START_REF] Cheung | Seasonal variation in the abundance of marine plastic debris in the estuary of a subtropical macro-scale drainage basin in South China[END_REF].

Microplastics are ubiquitous at the ocean surface [START_REF] Hardesty | Using numerical model simulations to improve the understanding of micro-plastic distribution and pathways in the marine environment[END_REF], drifting according to the wind, geostrophic currents, stokes drift, and surface currents that will lead to convergence areas [START_REF] Jiang | Greenland Sea Gyre increases microplastic pollution in the surface waters of the Nordic Seas[END_REF][START_REF] Mountford | Eulerian Modeling of the Three-Dimensional Distribution of Seven Popular Microplastic Types in the Global Ocean[END_REF][START_REF] Van Sebille | A global inventory of small floating plastic debris[END_REF]. The so-called hot spots of MP pollution tend to be correlated with ocean gyres and polar zones [START_REF] Jiang | Greenland Sea Gyre increases microplastic pollution in the surface waters of the Nordic Seas[END_REF][START_REF] Lima | Global patterns for the spatial distribution of floating microfibers: Arctic Ocean as a potential accumulation zone[END_REF].

Peaks in the density (particles/m 3 ) of this contaminant are also associated with the proximity of the main sources, mostly enclosed seas surrounded by densely inhabited areas [START_REF] Van Sebille | A global inventory of small floating plastic debris[END_REF]. However, several aspects related to the occurrence, density and dispersion of MPs into deeper ocean layers are still poorly understood.

The density of MPs in the deep sea relies on thermohaline currents [START_REF] Kane | Seafloor microplastic hotspots controlled by deep-sea circulation[END_REF] and a complex sinking pattern, which encompasses oceanographic parameters, specific features of particles (e.g., the density of polymers, surface area, and fragmentation rates), and incorporation by marine snow and faecal pellets [START_REF] Onink | The Role of Ekman Currents, Geostrophy, and Stokes Drift in the Accumulation of Floating Microplastic[END_REF][START_REF] Kvale | The global biological microplastic particle sink[END_REF]. Microplastics comprise a heterogeneous assemblage of particles that vary in shape, colour, size and chemical composition, including several polymer derivatives of the petrochemical industry [START_REF] Barnes | Accumulation and fragmentation of plastic debris in global environments[END_REF]. Most polymers have a molecular density higher than seawater and are thus negatively buoyant [START_REF] Mountford | Eulerian Modeling of the Three-Dimensional Distribution of Seven Popular Microplastic Types in the Global Ocean[END_REF]. Advection flow might play a crucial role in their sinking rates [START_REF] Kvale | The global biological microplastic particle sink[END_REF]. However, some polymers (e.g., polyethylene, polystyrene, and polypropylene) [START_REF] Geyer | Production, use, and fate of all plastics ever made[END_REF] are positively buoyant and likely require additional mechanisms and/or greater residence time to finally sink through the water column [START_REF] Koelmans | All is not lost: Deriving a top-down mass budget of plastic at sea[END_REF][START_REF] Long | Interactions between microplastics and phytoplankton aggregates: Impact on their respective fates[END_REF].

Due to their prevalence, persistence, diminutive size, and colonisation by microorganisms, MPs are highly susceptible to be ingested throughout the food web [START_REF] Everaert | Risk assessment of microplastics in the ocean: Modelling approach and first conclusions[END_REF]Ferreira et al., 2019b;[START_REF] Roch | Uptake routes of microplastics in fishes: practical and theoretical approaches to test existing theories[END_REF]. Moreover, marine organisms play an important role in vertical MP transportation [START_REF] Kvale | The global biological microplastic particle sink[END_REF]. Zooplankton modifies sinking rates of egested MPs through their incorporation within faecal pellets [START_REF] Cole | Microplastics Alter the Properties and Sinking Rates of Zooplankton Faecal Pellets[END_REF]. Vertical migratory species from the mesopelagic realm induce important vertical biomass fluxes [START_REF] Davison | Carbon export mediated by mesopelagic fishes in the northeast Pacific Ocean[END_REF][START_REF] Eduardo | Hatchetfishes (Stomiiformes: Sternoptychidae) biodiversity, trophic ecology, vertical niche partitioning and functional roles in the western Tropical Atlantic[END_REF]. Thereby, the diel migration pattern of preying upon contaminated resources (contaminated habitat and food) in the uppermost ocean layers and returning to the deep layers suggests that vertical migration contributes to MP transportation.

Despite providing key ecological links between different depth strata [START_REF] Robinson | Mesopelagic zone ecology and biogeochemistry -A synthesis[END_REF], cephalopods are among the least studied groups regarding MP contamination [START_REF] Alejo-Plata | Description of buoyant fibers adhering to Argonauta nouryi (Cephalopoda: Argonautidae) collected from the stomach contents of three top predators in the Mexican South Pacific[END_REF][START_REF] Oliveira | Microplastics presence in cultured and wildcaught cuttlefish, Sepia officinalis[END_REF]. Yet, surveys have pointed to microplastic contamination among the most common preys (copepods, euphausiids, crabs, tunicates, and fishes) and predators (cetaceans) of deep-sea cephalopods [START_REF] Choy | The vertical distribution and biological transport of marine microplastics across the epipelagic and mesopelagic water column[END_REF][START_REF] Davison | Plastic ingestion by mesopelagic fishes in the North Pacific Subtropical Gyre[END_REF][START_REF] Desforges | Ingestion of Microplastics by Zooplankton in the Northeast Pacific Ocean[END_REF]Lusher et al., 2015;[START_REF] Wieczorek | Frequency of microplastics in mesopelagic fishes from the Northwest Atlantic[END_REF]. Cephalopods are exposed to at least two main contamination pathways; the contaminants present in the water column and those in the prey's digestive tract. Vampyroteuthis infernalis (Chun, 1903) is the only living species from the Vampyroteuthidae family. It usually inhabits meso-and bathypelagic zones throughout the oceans [START_REF] Hoving | Vampire squid: Detritivores in the oxygen minimum zone[END_REF] with a slow-swimming and opportunistic behaviour foraging on copepods, but mostly on marine snow [START_REF] Golikov | The first global deep-sea stable isotope assessment reveals the unique trophic ecology of Vampire Squid Vampyroteuthis infernalis (Cephalopoda)[END_REF]. The Enoploteuthidae Abralia veranyi (Rüppel, 1844) inhabits the upper layers of the mesopelagic zone in the Atlantic Ocean, migrating towards the epipelagic zone to prey on copepods, decapods, and fish [START_REF] Guerra-Marrero | Migratory patterns, vertical distributions and diets of Abralia veranyi and Abraliopsis morisii (Cephalopoda: Enoploteuthidae) in the eastern North Atlantic[END_REF].

The objectives of this study were to (i) evaluate if cephalopods from the southwestern tropical Atlantic (V. infernalis and A. veranyi) are contaminated by microplastic particles and (ii) investigate whether cephalopods with different ecological behaviour exhibit different patterns of contamination. To our knowledge, this is one of the first studies to evaluate microplastic contamination in deep-sea cephalopods from the South Atlantic and the first to evaluate contamination in V. infernalis and A. veranyi.

Mesopelagic cephalopods were collected in the southwestern tropical Atlantic during the multidisciplinary research cruise ABRACOS2 (Acoustics along the BRAzilian COaSt 2), onboard the French R/V Antea from 9 th April to 6 th May 2017 [START_REF] Bertrand | ABRACOS 2 cruise, RV Antea[END_REF] (Fig. 1). Samplings were carried out off northeastern Brazil using a micronekton trawl net with different mesh sizes (body mesh: 40 mm, cod-end mesh: 10 mm, estimated opening area: 120 m 2 ). Individuals were selected from 5 sampling stations, and vertical trawls were conducted both day and night, between 25 and 1,113 m depth at 2-3 kt [START_REF] Eduardo | Hatchetfishes (Stomiiformes: Sternoptychidae) biodiversity, trophic ecology, vertical niche partitioning and functional roles in the western Tropical Atlantic[END_REF].

The study area encompasses the Fernando de Noronha Archipelago, Rocas Atoll, and adjacent seamounts (Fig. 1), which together are considered an Ecologically and Biologically Significant Marine Area and include Marine Protected Areas (CBD, 2014).

Additionally, Fernando de Noronha Archipelago is classified as an UNESCO Natural Heritage of Humanity, and the number of tourists reaches >50,000 in the peak season (austral summer season) (IBGE, 2010). The surface waters of the study area are not estimated to be an accumulation zone for MPs [START_REF] Jiang | Greenland Sea Gyre increases microplastic pollution in the surface waters of the Nordic Seas[END_REF][START_REF] Lima | Global patterns for the spatial distribution of floating microfibers: Arctic Ocean as a potential accumulation zone[END_REF]; the area is dominated by the South Equatorial Current (SEC) and South Equatorial Undercurrent (SEUC) and is characterised by a thin thermocline and high stratification [START_REF] Assunção | 3D characterisation of the thermohaline structure in the southwestern tropical Atlantic derived from functional data analysis of in situ profiles[END_REF].

Promptly upon collection at each sampling station, specimens (V. infernalis n=19 and A. veranyi n=8) were fixed in a 4% formalin solution and then preserved in a 70% ethanol solution in the laboratory. Organisms were identified [START_REF] Nesis | Cephalopods of the world[END_REF][START_REF] Roper | Cephalopods of the world[END_REF], measured (nearest 0.001 cm of total length and dorsal mantle length) and weighted (nearest 0.0001 g of total weight). Since precautionary measures to avoid contamination were not implemented during sampling and storage, individuals were thoroughly washed before analysis with filtered (glass fibre filter) distilled water to remove any particles attached to the outermost tissue. Then, beaks were removed, and the entire animal was submitted to chemical digestion through sodium hydroxide (NaOH 1 mol/L; PA 97%).

We implemented chemical digestion through base reaction (NaOH), due to greater logistical performance (short incubation period and cost-effective method) and for preserving the integrity of polymers after chemical digestion [START_REF] Budimir | Effective and easy to use extraction method shows low numbers of microplastics in offshore planktivorous fish from the northern Baltic Sea[END_REF].

Specimens were individually stored in beakers covered by glass lids. Each beaker was filled with NaOH solution (100 mL of solution per gram of organic sample) and ovendried at 60ºC for 24 h [START_REF] Lusher | Sampling, isolating and identifying microplastics ingested by fish and invertebrates[END_REF][START_REF] Wieczorek | Frequency of microplastics in mesopelagic fishes from the Northwest Atlantic[END_REF]. Samples were mixed with a glass stick two times during the incubation period to homogenise the solution.

Samples (output of digestion process) were filtered through a glass fibre filter (GF/F 0.7 µm pore size Whatman) using a membrane filtration system with a vacuum set up (equipped with laboratory glassware). The glass fibre filters with the digestion residues were then placed in covered Petri dishes and oven-dried at 60ºC for 24 h (for the complete protocol, see [START_REF] Justino | Microplastic contamination in tropical fishes: An assessment of different feeding habits[END_REF]. Two observers visually examined samples to reduce identification bias (overestimation and/or underestimation of contaminants) on a stereomicroscope (Zeiss Stemi 508, using 40-50x magnification with a detection limit of 20 µm) coupled with a device camera (Axiocam 105 Color). Items suspected to be microplastics were photographed, counted, measured (Zeiss Zen 3.2), and categorised according to morphological and optical characteristics into (i) fibres (filamentous shape), fragments (thick with an irregular shape), film (flat with an irregular shape), foam (soft with an irregular shape) and beads (spherical shape), and (ii) black, blue, green, red and white [START_REF] Lusher | Sampling, isolating and identifying microplastics ingested by fish and invertebrates[END_REF]. Petri dishes were kept closed during the entire identification process to avoid airborne contamination of samples.

As chemical digestion is not an efficient process to distinguish between plastic fibres and cellulose fibres, a sub-sample of particles extracted from the specimens were identified through Laser Directed Infra-Red analysis (LDIR). The selection of particles composing the sub-sample followed their shape as criteria rather than differences between the species. The absorbance of polymers was obtained using the Agilent 8700 LDIR Chemical Imaging System with the Microplastics Starter 1.0 library. Each spectral curve resulted from scans performed in the wavelength ranging from 1800 to 975 cm -1 (Ourgaud et al., In prep.). The specific polymer was asserted when a given particle registered above 70% of similarity with the reference spectrum.

Several procedures were taken to avoid airborne contamination of samples. All analyses were conducted under a laminar flow cabinet in a subsection of the laboratory designed to limit the flow of people [START_REF] Wieczorek | Frequency of microplastics in mesopelagic fishes from the Northwest Atlantic[END_REF]. As clothing made from synthetic fibres represents a potential source of contamination [START_REF] Su | Microplastics biomonitoring in Australian urban wetlands using a common noxious fish (Gambusia holbrooki)[END_REF], latex gloves, 100% cotton lab coats, and facemasks were worn during laboratory analysis.

All solutions used during the MP extraction were made using distilled water and filtered over a 47 mm GF/F filter (0.7 µm pore size). Before the analysis, work surfaces, equipment, and manipulation instruments were thoroughly cleaned with 70% alcohol. All manipulation instruments were rinsed with filtered distilled water and double-checked under a stereomicroscope for contamination before use.

A procedural blank was run in conjunction with each sample batch (~5 samples) to determine airborne contamination during the analysis. For blanks, a beaker was filled with 300 ml of NaOH solution and submitted to the same procedures applied to the samples [START_REF] Justino | Microplastic contamination in tropical fishes: An assessment of different feeding habits[END_REF]. Out of the five procedural blanks implemented during the analysis, two registered contamination, a single blue fibre. Any contamination evinced from the procedural blanks was subtracted from the corresponding sample batch (1 blank for each ~5 samples). Thereby, particles matching these characteristics (shape and colour) were omitted from the dataset and analysis.

The Percentage of the Frequency of Occurrence (FO%) was calculated to assess the overall contamination status of the species. The FO% was calculated as the percentage of individuals in a given species in which MP particles were recorded. As the data on MPs extracted did not meet parametric assumptions, Mann-Whitney tests were applied to determine whether there were any differences in the number and size (shape, colour and the overall particles, regardless of shape and colour) of MPs between the species. The number of extracted MPs was expressed as abundance (particles individual -1 ), containing all analysed individuals, including those that did not ingest MPs [START_REF] Provencher | Quantifying ingested debris in marine megafauna: A review and recommendations for standardisation[END_REF]. All analyses were carried out using R 3.6 (R Core Team, 2020) with a 5% significance level.

In this study, two species of deep-sea cephalopods were analysed. Both species registered a high prevalence (FO%) of MPs (V. infernalis 100% and A. veranyi 87.5%) (Table 1). Contamination levels (number of particles) varied between the species, ranging from 1 to 33 particles for V. infernalis (a total of 182 particles) and from 0 to 7 particles for A. veranyi (a total of 19 particles). Vampyroteuthis infernalis (9.58 ±8.25 part. ind. -1 ; Mean ± Standard deviation) (W = 134.5, p≤0.05) was the more contaminated species (A. veranyi 2.37 ±2.13 part. ind. -1 ) (Fig. 2 and Supplementary material).

Plastic particles extracted from cephalopods ranged from 0.06 to 3.91 mm in size. Most particles were categorised within the MP size range (99% of particles < 5 mm). Independent of shape and colour, the average length of particles extracted from A. veranyi (0.63 ±1.19 mm) (W = 116, p≤0.05) was longer than that of V. infernalis (0.61 ±0.34 mm) (Fig. 2 and Supplementary material).

In total, 201 MP particles corresponding to five main groups were recorded (Fig. 3). Fragments were the most common shape of MP extracted for both species (V. infernalis 3.36 ±3.67 part. Ind. -1 and A. veranyi 1.5 ±1.69 part. ind. -1 ), followed by fibres [3.05 ±2.34 part. Ind. -1 (W = 120.5, p≤0.05) and 0.5 ±0.75 part. ind. -1 , respectively] and beads (1.84 ±6.6 part. Ind. -1 and 0.37 ±0.52 part. ind. -1 , respectively) (Table 1). Microplastics categorised as foam (1.21 ±2.01 part. ind. -1 ) and films (0.1 ±0.31 part. ind. -1 ) were only recorded in V. infernalis (Table 1 and Supplementary material).

The MP shapes did not significantly differ in size according to species. Among the different shapes, fibres showed the broadest size range for both species (V. infernalis 0.91 ±0.92 mm and A. veranyi 0.64 ±1.23 mm) (Fig. 2). Fragments (V. infernalis 0.33 ±0.37 mm and A. veranyi 0.13 ±0.14 mm) and foam (V. infernalis 0.16 ±0.22 mm) were mostly ingested as small particles, whereas film (V. infernalis 0.01 ±0.04 mm) and beads (V. infernalis 0.04 ±0.06 mm and A. veranyi 0.03 ±0.04 mm) were the smallest fractions of ingested MPs (Fig. 2 and Supplementary material).

Concerning the colour of extracted MPs, white particles (6 ±7.17 part. ind. -1 ) prevailed in V. infernalis (W = 84.5, p≤0.05), followed by blue (1.26 ±2.15 part. ind. - 1 ) and red (1.15 ±1.64 part. ind. -1 ), whereas A. veranyi was most contaminated by blue (1.12 ±1.35 part. ind. -1 ), white (0.87 ±0.64 part. ind. -1 ) and black particles (0.25 ±0.7 part. ind. -1 ) (Table 1 and Supplementary material).

The subsample of particles analysed by LDIR comprehended 5.5% of the total particles extracted from the cephalopods. LDIR analysis successfully identified 78% of the subset as plastic polymers, including seven different polymers (polyethylene, polyethylene terephthalate, polyvinyl chloride, polyamide, styrene-butadiene rubber, chlorinated polyisoprene and polyurethane) (Fig. 4). Cellulose and wool particles were not registered in the subsample tested, but 22% of particles could not be identified through the assessed library. Thereby, those particles can be marine debris of natural origin or highly weathered plastic polymers.

Plastic pollution has become a major environmental issue [START_REF] Eriksen | Plastic Pollution in the World's Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Afloat at Sea[END_REF][START_REF] Raubenheimer | Can the Basel and Stockholm Conventions provide a global framework to reduce the impact of marine plastic litter?[END_REF]. Microplastics are asserted to contaminate the soil, air, and water bodies, which are the main pathway for their dispersion and accumulation [START_REF] Allen | Atmospheric transport and deposition of microplastics in a remote mountain catchment[END_REF][START_REF] Corradini | Evidence of microplastic accumulation in agricultural soils from sewage sludge disposal[END_REF][START_REF] Lebreton | River plastic emissions to the world's oceans[END_REF]. Despite the recent awareness of the ubiquity of MPs, little is known regarding the general patterns of dispersion, fragmentation, and interaction with marine biota in the deepest ocean layers [START_REF] Kane | Seafloor microplastic hotspots controlled by deep-sea circulation[END_REF][START_REF] Liu | Elucidating the vertical transport of microplastics in the water column: A review of sampling methodologies and distributions[END_REF][START_REF] Porter | Role of Marine Snows in Microplastic Fate and Bioavailability[END_REF]. Indeed, plastics (> 330 µm) contaminating the upper ocean layers represent less than 1% of the estimated global annual plastic inputs to the ocean [START_REF] Jambeck | Plastic waste inputs from land into the ocean[END_REF][START_REF] Van Sebille | A global inventory of small floating plastic debris[END_REF]. The remaining missing plastics are likely fragmenting into smaller pieces, diffusing into deeper layers, sediments [START_REF] Dai | Occurrence of microplastics in the water column and sediment in an inland sea affected by intensive anthropogenic activities[END_REF][START_REF] Courtene-Jones | Microplastic pollution identified in deep-sea water and ingested by benthic invertebrates in the Rockall Trough, North Atlantic Ocean[END_REF][START_REF] Gerigny | Seafloor litter from the continental shelf and canyons in French Mediterranean Water: Distribution, typologies and trends[END_REF][START_REF] Kane | Seafloor microplastic hotspots controlled by deep-sea circulation[END_REF][START_REF] Kanhai | Microplastics in sub-surface waters of the Arctic Central Basin[END_REF] and being assimilated by deep-sea biota [START_REF] Choy | The vertical distribution and biological transport of marine microplastics across the epipelagic and mesopelagic water column[END_REF][START_REF] Sathish | Occurrence of microplastics in epipelagic and mesopelagic fishes from Tuticorin, Southeast coast of India[END_REF].

Cephalopods inhabiting coastal areas have been asserted to ingest MPs [START_REF] Daniel | Microplastics in the edible tissues of shellfishes sold for human consumption[END_REF][START_REF] Oliveira | Microplastics presence in cultured and wildcaught cuttlefish, Sepia officinalis[END_REF]. [START_REF] Oliveira | Microplastics presence in cultured and wildcaught cuttlefish, Sepia officinalis[END_REF] identified higher contamination levels (number of particles) in wild cuttlefish (Sepia officinalis) than in those from a cultured marine station on the coast of the Iberian Peninsula, likely due to the retention of particles in the filtration system of culture tanks. [START_REF] Daniel | Microplastics in the edible tissues of shellfishes sold for human consumption[END_REF] investigated MPs in the edible tissues of four shellfish species on the Arabian Sea coast and detected the highest levels of contamination in the squids (Uroteuthis duvaucelii). This is the first study to investigate MPs in deep-sea cephalopods from the southwestern Atlantic and to provide information on V. infernalis and A. veranyi. Surveys on the deep-sea are logistically challenging, and literature in this field is scarce. To our knowledge, only two studies, both regarding macroplastics (>5 mm), have been published on deepsea representants of this group [START_REF] Braid | Preying on commercial fisheries and accumulating paralytic shellfish toxins: A dietary analysis of invasive Dosidicus gigas (Cephalopoda Ommastrephidae) stranded in Pacific Canada[END_REF][START_REF] Rosas-Luis | Description of plastic remains found in the stomach contents of the jumbo squid Dosidicus gigas landed in Ecuador during 2014[END_REF]. Both studies focused on the jumbo squid Dosidicus gigas, describing ingestion of large particles, mainly fishing lines. [START_REF] Braid | Preying on commercial fisheries and accumulating paralytic shellfish toxins: A dietary analysis of invasive Dosidicus gigas (Cephalopoda Ommastrephidae) stranded in Pacific Canada[END_REF] also observed the ingestion of plastic beads, but the dimension of particles was not assessed. Thereby, comparisons between our findings and other studies on deep-sea cephalopods are limited.

Microplastic ingestion is correlated with the density (particles m -3 ) of contaminants in the environment [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], which can significantly differ according to depth, turbulence, and specific areas within the ocean basin [START_REF] Kooi | Ups and Downs in the Ocean: Effects of Biofouling on Vertical Transport of Microplastics[END_REF][START_REF] Lima | Global patterns for the spatial distribution of floating microfibers: Arctic Ocean as a potential accumulation zone[END_REF][START_REF] Reisser | The vertical distribution of buoyant plastics at sea: An observational study in the North Atlantic Gyre[END_REF]. The feeding strategy of species is also influenced by the levels of contamination (Ferreira et al., 2019a;[START_REF] Mcneish | Microplastic in riverine fish is connected to species traits[END_REF][START_REF] Messinetti | Effects of polystyrene microplastics on early stages of two marine invertebrates with different feeding strategies[END_REF][START_REF] Miller | Bioaccumulation and biomagnification of microplastics in marine organisms: A review and meta-analysis of current data[END_REF][START_REF] Mizraji | Is the feeding type related with the content of microplastics in intertidal fish gut?[END_REF][START_REF] Setälä | Ingestion and transfer of microplastics in the planktonic food web[END_REF].

Marine biota can ingest MPs through three main pathways [START_REF] Moore | Synthetic polymers in the marine environment: A rapidly increasing, long-term threat[END_REF]: (i) actively when they are mistaken for actual prey [START_REF] Moore | Synthetic polymers in the marine environment: A rapidly increasing, long-term threat[END_REF], which in this case are very similar in size (copepods, mysids, and early-stage decapods) [START_REF] Figueiredo | Body size and stable isotope composition of zooplankton in the western tropical Atlantic[END_REF] or (ii) passively, trough particles adhered and in the surroundings of prey during the feeding process, such as suggested for cephalopods in field studies [START_REF] Alejo-Plata | Description of buoyant fibers adhering to Argonauta nouryi (Cephalopoda: Argonautidae) collected from the stomach contents of three top predators in the Mexican South Pacific[END_REF][START_REF] Rosas-Luis | Description of plastic remains found in the stomach contents of the jumbo squid Dosidicus gigas landed in Ecuador during 2014[END_REF]. Additionally, MPs can also be (iii) transferred within the trophic web by ingesting contaminated prey (Ferreira et al., 2019b;[START_REF] Nelms | Investigating microplastic trophic transfer in marine top predators[END_REF][START_REF] Setälä | Ingestion and transfer of microplastics in the planktonic food web[END_REF]. Indeed, the trophic transfer has been acknowledged as the core pathway for MP ingestion in cephalopods [START_REF] Daniel | Microplastics in the edible tissues of shellfishes sold for human consumption[END_REF][START_REF] Braid | Preying on commercial fisheries and accumulating paralytic shellfish toxins: A dietary analysis of invasive Dosidicus gigas (Cephalopoda Ommastrephidae) stranded in Pacific Canada[END_REF]; this group, in turn, may also act as vectors transferring MPs to higher trophic levels [START_REF] Alejo-Plata | Description of buoyant fibers adhering to Argonauta nouryi (Cephalopoda: Argonautidae) collected from the stomach contents of three top predators in the Mexican South Pacific[END_REF] and potentially to humans as seafood [START_REF] Daniel | Microplastics in the edible tissues of shellfishes sold for human consumption[END_REF].

Both species analysed here (A. veranyi and V. infernalis) exhibited a high prevalence of MPs (FO% and part. ind -1 ) when compared to other deep-sea biota [START_REF] Braid | Preying on commercial fisheries and accumulating paralytic shellfish toxins: A dietary analysis of invasive Dosidicus gigas (Cephalopoda Ommastrephidae) stranded in Pacific Canada[END_REF][START_REF] Daniel | Microplastics in the edible tissues of shellfishes sold for human consumption[END_REF][START_REF] Davison | Plastic ingestion by mesopelagic fishes in the North Pacific Subtropical Gyre[END_REF][START_REF] Rosas-Luis | Description of plastic remains found in the stomach contents of the jumbo squid Dosidicus gigas landed in Ecuador during 2014[END_REF][START_REF] Wieczorek | Frequency of microplastics in mesopelagic fishes from the Northwest Atlantic[END_REF]. MP ingestion was observed among marine copepods, euphausiids, crabs, tunicates, and mesopelagic fishes that are common prey for the species analysed in this study [START_REF] Bernal | Mesopelagic fish composition and diets of three myctophid species with potential incidence of microplastics, across the southern tropical gyre[END_REF][START_REF] Choy | The vertical distribution and biological transport of marine microplastics across the epipelagic and mesopelagic water column[END_REF][START_REF] Desforges | Ingestion of Microplastics by Zooplankton in the Northeast Pacific Ocean[END_REF][START_REF] Lusher | Microplastic interactions with North Atlantic mesopelagic fish[END_REF][START_REF] Sathish | Occurrence of microplastics in epipelagic and mesopelagic fishes from Tuticorin, Southeast coast of India[END_REF][START_REF] Wieczorek | Frequency of microplastics in mesopelagic fishes from the Northwest Atlantic[END_REF], which indicates the possibility of this contamination pathway.

The species analysed in this study have different ecological behaviour. Abralia veranyi inhabits the upper mesopelagic zone (200-500 m depth) during daytime and migrates towards the epipelagic zone to forage during the night-time [START_REF] Roper | Cephalopods of the world[END_REF] as part of the DSL (Deep Scattering Layer) migrants. According to the size range of captured A. veranyi, most of the individuals were under ontogenetic shift, reaching the adult phase (dorsal length > 30.9 mm), when they prey mostly on zooplankton and fish [START_REF] Guerra-Marrero | Migratory patterns, vertical distributions and diets of Abralia veranyi and Abraliopsis morisii (Cephalopoda: Enoploteuthidae) in the eastern North Atlantic[END_REF]. In general, MP occurrence is greater in surface and subsurface waters than in intermediate layers [START_REF] Bagaev | Anthropogenic microlitter in the Baltic Sea water column[END_REF][START_REF] Kvale | The global biological microplastic particle sink[END_REF][START_REF] Zobkov | Microplastic content variation in water column: The observations employing a novel sampling tool in stratified Baltic Sea[END_REF]. Abralia veranyi forage on the ocean layer with the higher density of MPs and feeds on copepods and fishes, both frequently reported to ingest MPs [START_REF] Bernal | Mesopelagic fish composition and diets of three myctophid species with potential incidence of microplastics, across the southern tropical gyre[END_REF][START_REF] Botterell | Bioavailability and effects of microplastics on marine zooplankton: A review[END_REF][START_REF] Wieczorek | Frequency of microplastics in mesopelagic fishes from the Northwest Atlantic[END_REF]; hence the recurrent and relatively high contamination levels observed are not surprising. Contamination levels were higher than those observed for the large predatory squid D. gigas [START_REF] Braid | Preying on commercial fisheries and accumulating paralytic shellfish toxins: A dietary analysis of invasive Dosidicus gigas (Cephalopoda Ommastrephidae) stranded in Pacific Canada[END_REF][START_REF] Rosas-Luis | Description of plastic remains found in the stomach contents of the jumbo squid Dosidicus gigas landed in Ecuador during 2014[END_REF], which forages in deeper layers, but lower than those for S. officinalis that feeds in shallower coastal waters [START_REF] Oliveira | Microplastics presence in cultured and wildcaught cuttlefish, Sepia officinalis[END_REF]. The feeding habitat (epipelagic zone) might also have influenced the size of MPs ingested by A.

veranyi. This species showed the longest MPs, and the size of these particles in the water column is negatively correlated with depth [START_REF] Dai | Occurrence of microplastics in the water column and sediment in an inland sea affected by intensive anthropogenic activities[END_REF][START_REF] Zobkov | Microplastic content variation in water column: The observations employing a novel sampling tool in stratified Baltic Sea[END_REF].

Vampyroteuthis infernalis inhabits deeper layers from the lower mesopelagic zone (500-1000 m depth) [START_REF] Hoving | Vampire squid: Detritivores in the oxygen minimum zone[END_REF]. Individuals analysed in this study corresponded to the juvenile phase (dorsal length < 66 mm) [START_REF] Hoving | Vampire squid reproductive strategy is unique among coleoid cephalopods[END_REF][START_REF] Schwarz | Life history traits of the deep-sea pelagic cephalopods Japetella diaphana and Vampyroteuthis infernalis[END_REF], when they display a transition in the locomotion pattern towards a more passive predatory behaviour, feeding on aggregated marine snow and POM (Particulate Organic Matter) associated feeder invertebrates [START_REF] Golikov | The first global deep-sea stable isotope assessment reveals the unique trophic ecology of Vampire Squid Vampyroteuthis infernalis (Cephalopoda)[END_REF][START_REF] Hoving | Vampire squid: Detritivores in the oxygen minimum zone[END_REF]. To a smaller degree, V. infernalis juveniles also actively prey upon zooplankton [START_REF] Seibel | Flight of the vampire: Ontogenetic gait-transition in Vampyroteuthis infernalis (Cephalopoda: Vampyromorpha)[END_REF].

Despite MPs being less available in the foraging habitat of V. infernalis, this species registered higher contamination levels than A. veranyi, other cephalopod species [START_REF] Braid | Preying on commercial fisheries and accumulating paralytic shellfish toxins: A dietary analysis of invasive Dosidicus gigas (Cephalopoda Ommastrephidae) stranded in Pacific Canada[END_REF][START_REF] Daniel | Microplastics in the edible tissues of shellfishes sold for human consumption[END_REF][START_REF] Rosas-Luis | Description of plastic remains found in the stomach contents of the jumbo squid Dosidicus gigas landed in Ecuador during 2014[END_REF] and fishes [START_REF] Boerger | Plastic ingestion by planktivorous fishes in the North Pacific Central Gyre[END_REF][START_REF] Lusher | Microplastic interactions with North Atlantic mesopelagic fish[END_REF]Wieczorek et al., 201), which specific ecological traits might explain. Marine aggregates (e.g., marine snow and faecal pellets) play an essential role in transporting MPs from the surface to the deep ocean [START_REF] Cole | Microplastics Alter the Properties and Sinking Rates of Zooplankton Faecal Pellets[END_REF][START_REF] Kvale | The global biological microplastic particle sink[END_REF][START_REF] Möhlenkamp | Plastic microbeads from cosmetic products: An experimental study of their hydrodynamic behaviour, vertical transport and resuspension in phytoplankton and sediment aggregates[END_REF]. Thereby, V. infernalis, feeding on faecal pellets and marine snow [START_REF] Golikov | The first global deep-sea stable isotope assessment reveals the unique trophic ecology of Vampire Squid Vampyroteuthis infernalis (Cephalopoda)[END_REF][START_REF] Hoving | Vampire squid: Detritivores in the oxygen minimum zone[END_REF], is likely more vulnerable to contamination. Additionally, V. infernalis is asserted to have the lowest metabolic rates among all cephalopods, as an adaptative trait to live within the oxygen minimum zones (OMZs) [START_REF] Seibel | Decline in Pelagic Cephalopod Metabolism With Habitat Depth Reflects Differences in Locomotory Efficiency[END_REF], contributing to low egestion rates, which may result in a momentary build-up of MP particles in the body.

The MPs extracted from A. veranyi and V. infernalis were heterogeneous, varying in shape and colour. However, no differences were observed between species, suggesting the absence of active ingestion of particles or, at least, a similar "preference" for the same colours and shapes. Nevertheless, according to the size of the specimens and the tiny dimensions of the particles, active predation (confusion with actual prey) is unlikely to occur. The colour composition of MPs extracted from cephalopods resembled those observed in epipelagic and deep-sea water samples elsewhere (Artic and Atlantic Ocean) [START_REF] Campos Da Rocha | Microplastic pollution in Southern Atlantic marine waters: Review of current trends, sources, and perspectives[END_REF][START_REF] Courtene-Jones | Microplastic pollution identified in deep-sea water and ingested by benthic invertebrates in the Rockall Trough, North Atlantic Ocean[END_REF][START_REF] Kanhai | Microplastics in sub-surface waters of the Arctic Central Basin[END_REF].

Fragments were the most common shape of MP ingested for both species, as observed for U. duvaucelii and deep-sea fishes from the North Pacific Central Gyre, Northeast Atlantic and Eastern Mediterranean [START_REF] Anastasopoulou | Plastic debris ingested by deep-water fish of the Ionian Sea (Eastern Mediterranean)[END_REF][START_REF] Boerger | Plastic ingestion by planktivorous fishes in the North Pacific Central Gyre[END_REF][START_REF] Daniel | Microplastics in the edible tissues of shellfishes sold for human consumption[END_REF][START_REF] Pereira | Microplastic in the stomachs of open-ocean and deep-sea fishes of the North-East Atlantic[END_REF]. The contamination by fibres was very relevant, being the second most common shape extracted and prevailing among the larger particles (>1 mm), likely due to their high density in the environment. Fibres comprehend more than 90% of MPs in the water column [START_REF] Kanhai | Microplastics in sub-surface waters of the Arctic Central Basin[END_REF][START_REF] Lima | Global patterns for the spatial distribution of floating microfibers: Arctic Ocean as a potential accumulation zone[END_REF] and generally is the most common shape ingested by deep-sea organisms [START_REF] Gago | Ingestion of plastic debris (macro and micro) by longnose lancetfish (Alepisaurus ferox) in the North Atlantic Ocean[END_REF][START_REF] Oliveira | Microplastics presence in cultured and wildcaught cuttlefish, Sepia officinalis[END_REF][START_REF] Sathish | Occurrence of microplastics in epipelagic and mesopelagic fishes from Tuticorin, Southeast coast of India[END_REF][START_REF] Wieczorek | Frequency of microplastics in mesopelagic fishes from the Northwest Atlantic[END_REF].

Despite beads being seemingly less common in water samples [START_REF] Barrows | Marine environment microfiber contamination: Global patterns and the diversity of microparticle origins[END_REF], the spherical MPs were also observed in D. gigas [START_REF] Braid | Preying on commercial fisheries and accumulating paralytic shellfish toxins: A dietary analysis of invasive Dosidicus gigas (Cephalopoda Ommastrephidae) stranded in Pacific Canada[END_REF]. Beads have the highest sinking rate among the different MP shapes [START_REF] Khatmullina | Settling velocity of microplastic particles of regular shapes[END_REF], positively influencing their density (particles m -3 ) in the deep sea.

Given the size range of extracted MPs, they are likely not bioaccumulated, since only the smaller fraction of microplastics (< 5 μm) and nanoplastics (< 1μm) are prone to be assimilated into tissues and organs [START_REF] Lee | Bioaccumulation of polystyrene nanoplastics and their effect on the toxicity of Au ions in zebrafish embryos[END_REF][START_REF] Lu | Uptake and Accumulation of Polystyrene Microplastics in Zebrafish (Danio rerio) and Toxic Effects in Liver[END_REF][START_REF] Sökmen | Polystyrene nanoplastics (20 nm) are able to bioaccumulate and cause oxidative DNA damages in the brain tissue of zebrafish embryo (Danio rerio)[END_REF]. However, particles within this size fraction could not be assessed using the methodology implemented herein.

The assimilation of plastics is associated with the uptake of several chemical additives (e.g., plasticisers, pigments, flame retardants, and UV stabilisers) introduced during the manufacturing and pollutants adsorbed from the environment [START_REF] Batel | Transfer of benzo[a]pyrene from microplastics to Artemia nauplii and further to zebrafish via a trophic food web experiment: CYP1A induction and visual tracking of persistent organic pollutants[END_REF][START_REF] Galloway | Interactions of microplastic debris throughout the marine ecosystem[END_REF]. The large surface area and the hydrophobic nature of MPs increase the sorption capability of several organic pollutants (e.g., DDT, PCBs and PAHs) [START_REF] Bakir | Competitive sorption of persistent organic pollutants onto microplastics in the marine environment[END_REF][START_REF] Rios | Persistent organic pollutants carried by synthetic polymers in the ocean environment[END_REF][START_REF] Rochman | Policy: Classify plastic waste as hazardous[END_REF], whereas the biofouling of MPs enhances the sorption of heavy metals and antibiotics [START_REF] Richard | Biofilm facilitates metal accumulation onto microplastics in estuarine waters[END_REF]Wang et al., 2020). The sorption of pollutants is influenced by environmental conditions, polymer composition, and residence time; plastics with longer stays in the ocean accumulate higher concentrations of pollutants [START_REF] Rochman | Long-term sorption of metals is similar among plastic types: Implications for plastic debris in aquatic environments[END_REF]. Polyethylene, for instance, the most common polymer extracted from the cephalopods in this study, is asserted to have a greater sorption capability than other polymers [START_REF] Alimi | Microplastics and Nanoplastics in Aquatic Environments: Aggregation, Deposition, and Enhanced Contaminant Transport[END_REF].

The role of MPs as vectors for pollutants and their adverse effects on cephalopods has not been evaluated to date. However, experimental research on MP ingestion by fish indicates several adverse effects (e.g., intestinal damage, oxidative stress, immunotoxicity, genotoxicity, developmental, reproductive and locomotor toxicity) [START_REF] Bhagat | Zebrafish: An emerging model to study microplastic and nanoplastic toxicity[END_REF]. Further research is necessary to investigate if MP contamination can reduce the swimming and feeding activity of cephalopods, as was observed for fish [START_REF] De Sá | Effects of microplastics on juveniles of the common goby (Pomatoschistus microps): Confusion with prey, reduction of the predatory performance and efficiency, and possible influence of developmental conditions[END_REF][START_REF] Qiang | Exposure to microplastics decreases swimming competence in larval zebrafish (Danio rerio)[END_REF]. If so, deep-sea cephalopods could be more vulnerable to the adverse effects given the extended diel vertical migration to forage. Cephalopods are widely recognised as playing a crucial role in many marine ecosystems, both as predators and prey. The vampire and midwater squids are important prey for harvestable fish stocks and highly threatened species of the southwestern Atlantic [START_REF] Vaske Júnior | Feeding strategy of the night shark (Carcharhinus signatus) and scalloped hammerhead shark (Sphyrna lewini) near seamounts off northeastern Brazil[END_REF][START_REF] Vaske Júnior | Forage fauna in the diet of bigeye tuna (Thunnus obesus) in the western tropical Atlantic Ocean[END_REF]). Yet, they remain poorly known worldwide while increasingly at risk in several ways, such as climatic change (Levin et al., 2019) and deep-sea exploitation [START_REF] Drazen | Midwater ecosystems must be considered when evaluating environmental risks of deep-sea mining[END_REF]. Additionally, we show that both A. veranyi and V. infernalis are highly contaminated by MPs, which threatens their survival and may enhance the plastic transportation between oceanic layers.

With the observed pervasion of plastic into the ocean, we reaffirm that the structure and function of deep-sea ecosystems could undergo changes that, given the current state of knowledge, may go mostly unnoticed by scientists and marine resource managers. More studies encompassing a greater sample size, various sampling depths, oceanographic parameters, and different taxonomic and ecological groups are required to understand the interaction with MPs in these habitats and investigate if the driving force responsible for the biological pump could also "sequestrate" microplastics into deeper ocean layers, as a plastic pump. 5.06/20 and BFP-0107-5.06/21) to Guilherme Ferreira, CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), which provided research grants to Flávia Lucena Frédou (CNPq n°305014/2016-1); Thierry Frédou (CNPq n°312211/2017-1); and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for granting of a scholarship at doctoral level to Anne Justino. All the authors thanks to the members of the laboratory BIOIMPACT for their support. 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.

FIGURE CAPTIONS

  Understanding general MP dispersion into the deep ocean and their final fate is one of the most relevant open questions in the field. The broad range of shapes and identified polymers indicate multiple sources of contamination. The biological activity influences the MP sinking rates through biofouling, aggregation in marine snow and faecal pellets. Additionally, vertical migratory species may play a significant role. Cephalopods and other vertical migrants ingest MPs in shallower waters with high MP density and likely egest those contaminants into deeper layers during resting periods.

Figure 1 -

 1 Figure1-Study area in the southwestern tropical Atlantic, sampling stations where V. infernalis and A. veranyi were captured (5 stations).

Figure 2 -

 2 Figure 2 -Mean (±standard error) (a) number, (b) length, and (c) (%) shape according to the size of ingested microplastics by V. infernalis and A. veranyi from the southwestern tropical Atlantic.

Figure 3 -

 3 Figure 3 -Microplastic particles extracted from deep-sea cephalopods (V. infernalis and A. veranyi): (a) fibre, (b) tangled fibres, (c) fragment, (d) foam, (e) film and (f -g) beads.

Figure 4 -

 4 Figure 4 -Microplastic polymers identified by the LDIR analysis: (a) particle composition, (b) types of plastic polymers, (c) PVC (polyvinyl chloride) MP fragment (solid line: particle spectrum; dotted line: reference spectrum), (d) PET (polyethylene terephthalate) MP fibre (solid line: particle spectrum; dotted line: reference spectrum), (e) PE (polyethylene) MP foam and (f) PU (polyurethane) MP fragment.

ACKNOWLEDGEMENTS

This study was supported by the LMI TAPIOCA program CAPES/COFECUB (88881.142689/2017-01), MicroplastiX project/JPI-Oceans/CONFAP/FACEPE (n°APQ-0035-1.08/19) and FUNBIO/Humanize under the grant "Programa Bolsas Funbio -Conservando o Futuro 2019 (02/2019)". We thank the FACEPE (Fundação de Amparo à Ciência e Tecnologia de Pernambuco) trough research grant (BFP-0049-

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 study.


Anne K S Justino

Véronique Lenoble

Latifa Pelage

Guilherme V B Ferreira

Rafaela Passarone

Thierry Frédou

Flávia Lucena Frédou

Microplastic contamination in tropical fishes: an assessment of different feeding habits

Keywords: Trophic transfer, Plastic extraction, Trophic guild, Plastic pollution, Tropical Atlantic

Marine ecosystems are reported to be contaminated by microplastics (MPs) (< 5 mm), however the ecological mechanisms involved in the ingestion of debris by marine organisms are relatively unknown. By developing and optimising an appropriate protocol of gut digestion for fish species, this study explores a tropical estuarine environment to unriddle the processes responsible for the different ingestion rates of plastic debris. A total of 82 fishes with different feeding habits were analysed, Centropomus undecimalis (n = 30; Piscivore), Bairdiella ronchus (n = 21; Zoobenthivore) and Gobionellus stomatus (n = 31; Detritivore). The microplastic ingestion varied with the feeding strategy; C. undecimalis, the predator, was the most contaminated species. Overall, most MPs were fibres (47%), followed by pellets (40%) and fragments (13%), although these proportions varied among species. A high level of contamination was found in the Estuarine Complex of Santa Cruz Channel, Northeast of Brazil, with many sources of MPs contaminants, as for many other tropical estuaries, which probably accumulate in the sediment and water column, with unknown consequences for human health.

Introduction

Estuaries are well known for providing ecosystem services by supplying essential goods (raw materials and food) and offering an attractive environment for population growth and cultural activities [START_REF] Atkins | Management of the marine environment: Integrating ecosystem services and societal benefits with the DPSIR framework in a systems approach[END_REF]. In addition, they carry out an important ecological function since fishes use these environments for protection, feeding, reproduction, settlement, and nursery [START_REF] Krumme | Spring-neap cycle as a major driver of temporal variations in the feeding of intertidal fishes: evidence from the sea catfish Sciades herzbergii (Ariidae) of equatorial West Atlantic mangrove creeks[END_REF][START_REF] Potter | The ways in which fish use estuaries: a refinement and expansion of the guild approach[END_REF]Lima et al., 2016b;[START_REF] Ramos | Seasonal and spatial ontogenetic movement of Gerreidae in a Brazilian tropical estuarine ecocline and its application for nursery habitat conservation[END_REF]Ferreira et al., 2019). Worldwide, estuaries are usually surrounded by large metropolises, and the expansion of the urban population is directly associated with impacts on the coastal ecosystems [START_REF] Freeman | Impacts of urbanisation and development on estuarine ecosystems and water quality[END_REF]. Plastic pollution is one of the most significant environmental problems of the 21st century since large quantities of these materials are being mismanaged and/or illegally disposed of in marine ecosystems [START_REF] Dauvergne | Why is the global governance of plastic failing the oceans?[END_REF][START_REF] Ostle | The rise in ocean plastics evidenced from a 60-year time series[END_REF]. Once in the environment, these plastic materials are weakened by natural processes (e.g., hydrodynamic forces, solar radiation, and biological actions) [START_REF] Thompson | Lost at sea: where is all the plastic?[END_REF][START_REF] Jambeck | Plastic waste inputs from land into the ocean[END_REF] and fragmented into smaller parts known as microplastics (< 5 mm).

Plastic debris poses several risks to marine biota [START_REF] Galloway | Interactions of microplastic debris throughout the marine ecosystem[END_REF]. Ingestion can be hazardous, causing digestive injuries, decreasing predatory efficiency, or inducing toxic effects [START_REF] De Sá | Effects of microplastics on juveniles of the common goby (Pomatoschistus microps): confusion with prey, reduction of the predatory performance and efficiency, and possible influence of developmental conditions[END_REF][START_REF] Moore | Synthetic polymers in the marine environment: a rapidly increasing, longterm threat[END_REF][START_REF] Teuten | Potential for plastics to transport hydrophobic contaminants[END_REF][START_REF] Barboza | Single and combined effects of microplastics and mercury on juveniles of the European seabass (Dicentrarchus labrax): Changes in behavioural responses and reduction of swimming velocity and resistance time[END_REF]. Microplastics can adsorb pollutants available in the water column, such as persistent organic pollutants (POPs) [START_REF] Oehlmann | A critical analysis of the biological impacts of plasticisers on wildlife[END_REF][START_REF] Frias | Organic pollutants in microplastics from two beaches of the Portuguese coast[END_REF][START_REF] Rochman | Ingested plastic transfers hazardous chemicals to fish and induces hepatic stress[END_REF] or heavy metals [START_REF] Ashton | Association of metals with plastic production pellets in the marine environment[END_REF][START_REF] Holmes | Adsorption of trace metals to plastic resin pellets in the marine environment[END_REF], which may be further bioaccumulated and biomagnified in the food web [START_REF] Batel | Transfer of benzo [a] pyrene from microplastics to Artemia nauplii and further to zebrafish via a trophic food[END_REF][START_REF] Teuten | Transport and release of chemicals from plastics to the environment and to wildlife[END_REF]. Furthermore, microplastics may be transferred in the trophic chain by predating contaminated prey [START_REF] Ferreira | Plastic debris contamination in the life cycle of Acoupa weakfish (Cynoscion acoupa) in a tropical estuary[END_REF]2019;[START_REF] Chagnon | Plastic ingestion and trophic transfer between Easter Island flying fish (Cheilopogon rapanouiensis) and yellowfin tuna (Thunnus albacares) from Rapa Nui (Easter Island)[END_REF]. The trophic transfer has already been pointed out as a relevant contamination mechanism for estuarine species [START_REF] Athey | Trophic transfer of microplastics in an estuarine food chain and the effects of a sorbed legacy pollutant[END_REF].

Although the long-term effects of microplastic contamination are still unknown, the scientific community continually emphasises the importance of using reliable and replicable methods of investigations [START_REF] Hermsen | Quality criteria for the analysis of microplastic in biota samples: a critical review[END_REF][START_REF] Markic | Plastic ingestion by marine fish in the wild[END_REF]. A useful approach to obtain plastic in marine wildlife is based on chemical digestion protocols, which are efficient and low cost to work with [START_REF] Karami | A high-performance protocol for extraction of microplastics in fish[END_REF][START_REF] Kühn | The use of potassium hydroxide (KOH) solution as a suitable approach to isolate plastics ingested by marine organisms[END_REF]. Digestion protocols are a practical and secure way to extract and isolate microplastics in organisms, being widely used in the investigation of fish contamination by plastics [START_REF] Foekema | Plastic in North Sea fish[END_REF][START_REF] Tanaka | Microplastic fragments and microbeads in digestive tracts of planktivorous fish from urban coastal waters[END_REF]Bellas et al., 2016;[START_REF] Hermsen | Detection of low numbers of microplastics in North Sea fish using strict quality assurance criteria[END_REF][START_REF] Bessa | Occurrence of microplastics in commercial fish from a natural estuarine environment[END_REF][START_REF] Pellini | Characterisation of microplastic litter in the gastrointestinal tract of Solea solea from the Adriatic Sea[END_REF][START_REF] Herrera | Microplastic ingestion by Atlantic chub mackerel (Scomber colias) in the Canary Islands coast[END_REF][START_REF] Su | Microplastics biomonitoring in Australian urban wetlands using a common noxious fish (Gambusia holbrooki)[END_REF]. However, the lack of security procedures (e.g., cleaned workroom and blanks procedures) during the implementation of digestion protocols may lead to the overestimation of contaminants since samples are more prone to airborne and crosscontamination [START_REF] Torre | Anthropogenic microfibres pollution in marine biota. A new and simple methodology to minimise airborne contamination[END_REF][START_REF] Hermsen | Detection of low numbers of microplastics in North Sea fish using strict quality assurance criteria[END_REF][START_REF] Hermsen | Quality criteria for the analysis of microplastic in biota samples: a critical review[END_REF]. Moreover, the procedure must ensure data reliability through an effective and careful extraction of the microplastics and the implementation of a robust sample size with a minimum of 10 samples [START_REF] Markic | Plastic ingestion by marine fish in the wild[END_REF].

Several studies conducted in South Atlantic estuaries evaluated the ingestion of plastics by marine organisms, mammals [START_REF] Attademo | Debris ingestion by the Antillean Manatee (Trichechus manatus manatus)[END_REF], mussels [START_REF] Santana | Microplastic contamination in natural mussel beds from a Brazilian urbanised coastal region: Rapid evaluation through bioassessment[END_REF][START_REF] Birnstiel | Depuration reduces microplastic content in wild and farmed mussels[END_REF], turtles [START_REF] Guebert-Bartholo | Using gut contents to assess foraging patterns of juvenile green turtles Chelonia mydas in the Paranaguá Estuary, Brazil[END_REF], and microplastics interactions with ichthyoplankton (Lima et al., 2016a), and fish assemblages [START_REF] Vendel | Widespread microplastic ingestion by fish assemblages in tropical estuaries subjected to anthropogenic pressures[END_REF]. However, few studies investigated the ecological and biological dynamics associated with microplastic intakes on wild fishes [START_REF] Ferreira | Plastic debris contamination in the life cycle of Acoupa weakfish (Cynoscion acoupa) in a tropical estuary[END_REF][START_REF] Dauvergne | Why is the global governance of plastic failing the oceans?[END_REF][START_REF] Amorim | Ingestion of microplastic by ontogenetic phases of Stellifer brasiliensis (Perciformes, Sciaenidae) from the surf zone of tropical beaches[END_REF][START_REF] Dantas | Plastic intake does not depend on fish eating habits: Identification of microplastics in the stomach contents of fish on an urban beach in Brazil[END_REF].

Furthermore, the bioaccumulation of microplastics in marine species is highly influenced by feeding strategies [START_REF] Miller | Bioaccumulation and biomagnification of microplastics in marine organisms: A review and meta-analysis of current data[END_REF]. However, the possible correlation between feeding habits and MPs ingestion is not yet well known.

Three estuarine species were chosen to test our hypothesis that microplastic ingestion varies according to the feeding strategies: (1) Centropomus undecimalis (Bloch, 1792); (2) Bairdiella ronchus (Cuvier, 1830); and (3) Gobionellus stomatus (Starks, 1913). C. undecimalis is an essential economic living resource for the commercial and subsistence fisheries in South America [START_REF] Carpenter | The living marine resources of the Western Central Atlantic[END_REF]. While adults of C. undecimalis inhabit coastal areas and migrate towards the estuary, their juvenile stage uses the estuarine areas as a nursery ground (Ferreira et al., 2019). C. undecimalis is classified as opportunistic predator, feeding on a large variety of available preys in the environment, with a piscivorous tendency [START_REF] Lira | Feeding ecology of Centropomus undecimalis (Bloch, 1792) and Centropomus parallelus (Poey, 1860) in two tropical estuaries in Northeastern Brazil[END_REF]Ferreira et al., 2019). B. ronchus is classified as zoobenthivore (Ferreira et al., 2019), which preys on invertebrates associated with the sediment [START_REF] Elliott | The guild approach to categorising estuarine fish assemblages: a global review[END_REF]. G. stomatus is a detritivore fish (Ferreira et al., 2019) consuming detritus and microphytobenthos [START_REF] Elliott | The guild approach to categorising estuarine fish assemblages: a global review[END_REF]. Although B. ronchus and G. stomatus were not of economic importance, they play a significant ecological role within the estuarine ecosystem and are the main energy source for the C. undecimalis [START_REF] Lira | Trophic flow structure of a neotropical estuary in northeastern Brazil and the comparison of ecosystem model indicators of estuaries[END_REF][START_REF] Gonzalez | Trophic resource partitioning of two snook fish species (Centropomidae) in tropical estuaries in Brazil as evidenced by stable isotope analysis[END_REF].

Understanding the role of microplastics as a component of anthropic pollution in this ecosystem is crucial to assess adverse impacts on regional biodiversity and the quality of fisheries resources that are being traded and consumed. In this context, the present study aims to (i) apply an adapted extraction protocol to assess MPs in the digestive tract of fishes, assuring the integrated quality control and appropriate sampling size, (ii) describe microplastics contamination in fishes in estuarine waters, and (iii) identify the main types of microplastics considering the different feeding strategies.

Materials and Methods

Study area

The Estuarine Complex of the Santa Cruz Channel (ECSC) (Fig. 1) is located along the northeast Brazilian coast. The climate is classified as tropical, hot and humid, with an average of 26ºC (±2.8ºC) annual air temperature and two seasons defined according to the level of precipitation (rainy and dry seasons) [START_REF] Medeiros | The itamaracá estuarine ecosystem, Brazil[END_REF].

The ECSC is a tidal channel that surrounds the Itamaracá Island, separating it from the mainland, with a total area of 22 km, a maximum width of 1.5 km and a depth between 4 and 5 m [START_REF] Lira | Feeding ecology of Centropomus undecimalis (Bloch, 1792) and Centropomus parallelus (Poey, 1860) in two tropical estuaries in Northeastern Brazil[END_REF]. The main tributaries are formed by the Arataca, Botafogo and Igarassu Rivers, and the predominant vegetation is the mangrove forest [START_REF] Medeiros | The itamaracá estuarine ecosystem, Brazil[END_REF]. The ECSC is surrounded by two cities (Itapissuma and Itamaracá), which have their economies mainly focused on the industrial and agricultural sectors (IBGE, 2011), whereas the local economy is primarily supported by artisanal fisheries, aquaculture, and tourism [START_REF] De Moura | A multi-temporal remote sensing and gis based inventory of the mangroves at itamaracá estuarine system, Northeastern Brazil[END_REF].

Sampling and Laboratory procedures

Three demersal species were selected for this study, given their commercial and or subsistence importance for riverine populations and their ecological interactions in the trophic chain [START_REF] Lira | Feeding ecology of Centropomus undecimalis (Bloch, 1792) and Centropomus parallelus (Poey, 1860) in two tropical estuaries in Northeastern Brazil[END_REF][START_REF] Dauvergne | Why is the global governance of plastic failing the oceans?[END_REF]: Gobionellus stomatus, Bairdiella ronchus, and Centropomus undecimalis, classified as Detritivores, Zoobenthivores and Piscivores respectively, according to Ferreira et al. (2019) for the area. For the common Snook C. undecimalis, individuals were collected in the juvenile stage (Total Length < 26.3 cm) (Ferreira et al., 2019), ensuring that the obtained specimens had not left the estuarine ecosystem. This procedure was carried out to ensure that contaminants' intake occurred within the estuarine boundary since adults of C. undecimalis perform migrations toward coastal areas. As B. ronchus and G. stomatus are not caught outside the estuary of ECSC (Ferreira et al., 2019), we did not fix these species' ontogeny, and juveniles and adults were analysed. A total of 82 individuals were obtained by local fishermen. After each sampling, the specimens were labelled, and the individuals were frozen. In the laboratory, individuals were identified [START_REF] Menezes | Manual de peixes marinhos do sudeste do Brasil. Teleostei (5)[END_REF], measured and weighted. Each individual had their organs (stomach and intestine) carefully removed, weighed and stored again for the digestion analysis. The minimum sample size for each species was 20 individuals to avoid the bias of a low sample size, which is twice the number suggested by [START_REF] Markic | Plastic ingestion by marine fish in the wild[END_REF].

Quality control and Extraction protocol

Firstly, to guarantee quality control and avoid potential airborne contamination, several steps were implemented. The entire process of digestion, filtration, identification and storage of the microplastic samples was carried out in a cleaned and reserved room within the main laboratory, reserved only for microplastic analysis. The flow of people was limited; cotton lab coats and disposable latex gloves were worn during the entire process. Also, all used tools were previously cleaned with alcohol 70% and rinsed with filtered distilled water. The solutions utilised in the various procedures were filtered using a vacuum pump system (equipped with laboratory glassware) through a 47 mm GF/F 0.7 µm glass fibre filter (Whatman).

The digestive tracts were rinsed with distilled water before being placed in a beaker, submerged in NaOH (1 mol/L; PA 97%) solution, and covered by a glass lid (Fig. 2). The entire digestive tracts were submitted to NaOH without further dissection of those organs to avoid airborne contamination during handling. The proportion used was 1:100 w/v for 1 g of digestive tract weight, 100 ml of NaOH (1 mol/L) solution. The mixture was oven-dried at 60° C for 24 h, mixed from time to time with a glass stick. The samples digested in the previous step, and the procedural blanks were filtered using a vacuum pump system through a 47 mm glass fibre filter (GF/F 0.7 µm Whatman). After filtration, filters were carefully set in a Petri dish (47 mm diameter) and covered. These filters were oven-dried at 60° C for 24 h. The microplastics were identified using a stereomicroscope (Zeiss Stemi 508) with 6.3 -50 times magnification with a detection limit of 20 µm, photographed (Axiocam 105 Color), measured (Zeiss Zen 3.2) from the filter and stored in covered Petri dishes (Fig. 2). They were then categorised by type: (i) fibres (filamentous shape), (ii) fragments (irregular shape) or (iii) pellets (spherical shape). The digestion protocol is a useful tool to separate the organic materials and facilitates visual identification, although it is not sufficient for identifying the polymers. Thereby, we also applied the method described by Ferreira et al. (2019) to confirm plastic debris by drying the samples in an oven to verify whether their physical characteristics changed or not.

Procedural blanks were made for each day of analysis before beginning the sample digestion. For blanks, a beaker was filled with 50 ml of NaOH (1 mol/L) solution and covered with a glass lid, and these blanks were exposed to the same protocol applied to the samples. A total of 10 blanks were made; among them, four blanks were observed with eight tiny particles (<100 µm) considered as paint fragments. Thereby, all particles further identified with any resemblance to those observed in the blanks were excluded from posterior analyses.

Data Analysis

Kruskal-Wallis tests were used to verify if ingested microplastics (total MPs and different types) presented significant differences in number and length in relation to species (C. undecimalis, B. ronchus and G. stomatus). When the Kruskal-Wallis showed significant differences, the post hoc pairwise comparisons Dunn's test was performed to investigate the sources of variance [START_REF] Dunn | Multiple comparisons using rank sums[END_REF]. All statistical analyses were carried out with the software R version 3.6.3 (R Core Team, 2020) and were conducted considering a level of significance of 5%.

Results

A total of 30 individuals of Centropomus undecimalis, 31 Gobionellus stomatus, 21

Bairdiella ronchus were analysed. Microplastics were present in 77% of C. undecimalis, 74% of G. stomatus, and 67% of B. ronchus individuals. A total of 176 particles of MPs were recovered from 82 fishes (Table I).

According to the number of MPs, ingestion significantly differed between species (pvalue ≤ 0.05), C. undecimalis being the most contaminated (3.3 ± 2.9 MPs fish -1 ), followed by G. stomatus (1.7 ± 1.5 MPs fish -1 ) and B. ronchus (1.2 ± 1.3 MPs fish -1 ) (Fig. 3). Significant differences were recorded between C. undecimalis and B. ronchus (chi-squared = 7.873, df = 2, p-value ≤ 0.05). Concerning the length of ingested MPs, no significant differences among the same type of MPs were observed when comparing MPs' size between species.

Regarding the types of MPs ingested by fishes, most were fibres (47%), followed by pellets (40%) and fragments (13%), and proportions varied between the species. C. undecimalis registered 68% of pellets, 28% of fibres, and 4% of fragments, B. ronchus and G. stomatus registered 23% and 4% of pellets, 62% and 71% of fibres and 15% and 25% of fragments, respectively (Fig. 4 and5). I. The ecological parameters and biological aspects of the analysed species. Feeding habit: DV (Detritivores), ZB (Zoobenthivores) and PV (Piscivores) obtained for the area by Ferreira et al. (2019). TL (Total Length); SL (Standard Length); TW (Total Weight); FO% (Frequency of occurrence).

Discussion

Quality Assurance and Quality Control

In our study, we applied an analytical method to extract microplastics (MPs) in the digestive tract of fishes, involving a careful procedure of quality control, using sodium hydroxide (NaOH 1 M), and a sampling size with a minimum of 20 individuals for each species, following the recent recommendations of [START_REF] Markic | Plastic ingestion by marine fish in the wild[END_REF] and [START_REF] Hermsen | Quality criteria for the analysis of microplastic in biota samples: a critical review[END_REF].

The application of digestion protocols to extract microplastics in marine biota has been growing worldwide [START_REF] Lusher | Sampling, isolating, and identifying microplastics ingested by fish and invertebrates[END_REF]. Digestion protocol is a reliable method for isolating microplastics in the biota, facilitating the observation of non-organic materials. For fish, the most common method used is alkaline digestion. Indeed, for many authors, this is considered the most suitable method to remove organic material and isolate plastic debris [START_REF] Karami | A high-performance protocol for extraction of microplastics in fish[END_REF][START_REF] Kühn | The use of potassium hydroxide (KOH) solution as a suitable approach to isolate plastics ingested by marine organisms[END_REF][START_REF] Schirinzi | A new digestion approach for the extraction of microplastics from gastrointestinal tracts (GITs) of the common dolphinfish (Coryphaena hippurus) from the western Mediterranean Sea[END_REF]. Although the efficiency of potassium hydroxide (KOH 10%) has been more often tested, the use of sodium hydroxide (NaOH 1M) has also been widely tested and demonstrated to be very useful (Bellas et al., 2016;[START_REF] Baalkhuyur | Microplastic in the gastrointestinal tract of fishes along the Saudi Arabian Red Sea coast[END_REF][START_REF] Budimir | Effective and easy to use extraction method shows low numbers of microplastics in offshore planktivorous fish from the northern Baltic Sea[END_REF][START_REF] Morgana | Microplastics in the Arctic: A case study with sub-surface water and fish samples off Northeast Greenland[END_REF][START_REF] Wieczorek | Frequency of microplastics in mesopelagic fishes from the Northwest Atlantic[END_REF][START_REF] Su | Microplastics biomonitoring in Australian urban wetlands using a common noxious fish (Gambusia holbrooki)[END_REF], and ensures polymer integrity after chemical digestion [START_REF] Budimir | Effective and easy to use extraction method shows low numbers of microplastics in offshore planktivorous fish from the northern Baltic Sea[END_REF].

Although adaptations of digestion protocol have been carried out around the world [START_REF] Kühn | The use of potassium hydroxide (KOH) solution as a suitable approach to isolate plastics ingested by marine organisms[END_REF][START_REF] Karami | A high-performance protocol for extraction of microplastics in fish[END_REF], so far, only three studies in estuaries of South America have used a chemical digestion protocol for microplastics extraction in the digestive tract of fishes, with the implementation of adequate quality assurance and quality control (QA/QC) [START_REF] Arias | Evidence of Microplastic Ingestion by Fish from the Bahía Blanca Estuary in Argentina, South America[END_REF][START_REF] Ribeiro-Brasil | Contamination of stream fish by plastic waste in the Brazilian Amazon[END_REF][START_REF] Garcés-Ordóñez | Prevalence of microplastic contamination in the digestive tract of fishes from mangrove ecosystem in Cispata, Colombian Caribbean[END_REF]. Implementing QA/QC procedures is essential to avoid cross-contamination of microplastic samples [START_REF] Lusher | Sampling, isolating, and identifying microplastics ingested by fish and invertebrates[END_REF][START_REF] Hermsen | Detection of low numbers of microplastics in North Sea fish using strict quality assurance criteria[END_REF][START_REF] O'connor | Microplastics in Freshwater Biota: A Critical Review of Isolation, Characterisation, and Assessment Methods[END_REF]. This treatment is necessary to minimise over/underestimation of microplastics due to airborne contamination and or loss of particles during sample handling. Moreover, the sample size is also an essential factor in the analysis. Among the studies in South America, only [START_REF] Arias | Evidence of Microplastic Ingestion by Fish from the Bahía Blanca Estuary in Argentina, South America[END_REF] have chosen a sample size n > 10. Such bias is unlikely to occur in our study, where the sample size was the highest in South America. Therefore, our results are expected to adequately reflect the contamination in the Estuarine Complex of Santa Cruz Channel (ECSC), and the protocol here used can safely be replicated in other studies.

Microplastic ingestion by fishes

In our study, microplastic (MP) contamination rates were high (frequency of occurrence of 73%) for the three analysed demersal estuarine species. Overall, demersal estuarine species are frequently reported to have a high ingestion rate of MPs [START_REF] Ferreira | Plastic debris contamination in the life cycle of Acoupa weakfish (Cynoscion acoupa) in a tropical estuary[END_REF]2019;[START_REF] Arias | Evidence of Microplastic Ingestion by Fish from the Bahía Blanca Estuary in Argentina, South America[END_REF]. Demersal species inhabit and feed on the fauna associated with the substrate (Elliot et al., 2017), which might be in direct contact with contaminated sediment. Indeed, the estuarine sediment is a significant accumulation zone for MPs [START_REF] Zhang | Transport of microplastics in coastal seas[END_REF]. As estuaries are a transitional ecosystem, often surrounded by urban areas and exposed to domestic sewage discharge, they receive contaminants of both riverine and tidal inputs [START_REF] Lebreton | River plastic emissions to the world's oceans[END_REF].

Thereby, these ecosystems are more prone to be contaminated by MPs, especially fibres [START_REF] Browne | Accumulation of Microplastic on Shorelines Woldwide: Sources and Sinks[END_REF][START_REF] Bessa | Occurrence of microplastics in commercial fish from a natural estuarine environment[END_REF]. In addition to the sewage discharge, fibres can also be originated by the use, maintenance, discarding and loss of fisheries gear [START_REF] Lima | Distribution patterns of microplastics within the plankton of a tropical estuary[END_REF].

MPs fibres occurred the most in the estuarine bottom, possibly due to the rapid sinking of these types of MPs [START_REF] Lima | Distribution patterns of microplastics within the plankton of a tropical estuary[END_REF].

Overall, the most ingested MP type were fibres, as observed in most of the studies worldwide [START_REF] Wright | Microplastic ingestion decreases energy reserves in marine worms[END_REF][START_REF] Foekema | Plastic in North Sea fish[END_REF]Bellas et al., 2016;[START_REF] Herrera | Microplastic ingestion by Atlantic chub mackerel (Scomber colias) in the Canary Islands coast[END_REF][START_REF] Bessa | Occurrence of microplastics in commercial fish from a natural estuarine environment[END_REF]. Although all types of plastics were ingested by the species analysed in this study, ingestion rates varied. Fibres were the most frequent type in G. stomatus (representing 71% of the ingested MPs) and B. ronchus (68%). G. stomatus and B. ronchus are detritivore and zoobenthivore species, respectively, depending on the organisms associated with the substrate or the organic matter available. Consequently, they are more vulnerable to the MPs fibres contaminating estuarine sediments. However, pellets were the most frequent type in C. undecimalis (68%), differently from the results observed by Ferreira et al. (2019) for the same species, which registered mostly ingestion of filaments. Pellets also dominated the diet of fish along the coast of Salvador (Brazil) (Miranda & Carvalho-Souza,2016) and from the Amazon estuary [START_REF] Pegado | First evidence of microplastic ingestion by fishes from the Amazon River estuary[END_REF]. Different from the other types of MP ingested, pellets are primary microplastics [START_REF] Fendall | Contributing to marine pollution by washing your face: Microplastics in facial cleansers[END_REF], which are manufactured as MPs mainly for the cosmetics industry (e.g., microbeads). This type of MP can be accidentally discharged into the environment during the transport of this raw material [START_REF] Ogata | International Pellet Watch: global monitoring of persistent organic pollutants (POPs) in coastal waters. 1. Initial phase data on PCBs, DDTs, and HCHs[END_REF] or by the release of domestic sewage [START_REF] Tanaka | Microplastic fragments and microbeads in digestive tracts of planktivorous fish from urban coastal waters[END_REF]. The increase in urbanisation in the Santa Cruz Channel [START_REF] Pelage | Coastal land use in Northeast Brazil: mangrove coverage evolution over three decades[END_REF] surely amplified the sewage discharge in this area. Pellets can be found floating in the water column, and they can even have fish eggs attached to them [START_REF] Ivar Do Sul | The present and future of microplastic pollution in the marine environment[END_REF]. Predatory fishes such as C. undecimalis can ingest the pellets directly by confusing them with their natural prey. Moreover, when feeding, opportunistic predators ingest a large amount of prey, which might increase the momentary build-up of MPs particles prior to egestion.

Predator species are more vulnerable to microplastic contamination due to the trophic transference, which occurs when they ingest contaminated prey [START_REF] Eriksson | Origins and biological accumulation of small plastic particles in fur seals from Macquarie Island[END_REF][START_REF] Ferreira | Plastic debris contamination in the life cycle of Acoupa weakfish (Cynoscion acoupa) in a tropical estuary[END_REF][START_REF] Chagnon | Plastic ingestion and trophic transfer between Easter Island flying fish (Cheilopogon rapanouiensis) and yellowfin tuna (Thunnus albacares) from Rapa Nui (Easter Island)[END_REF][START_REF] Nelms | Investigating microplastic trophic transfer in marine top predators[END_REF]Ferreira et al., 2019).

Consequently, as we observed among the three analysed demersal fish species, the predator, C. undecimalis, had the highest contamination rate (3.3 ± 2.9 MPs fish -1 ) despite being in its early life stage (juvenile), followed by G. stomatus (1.7 ± 1.5 MPs fish -1 ) and B. ronchus (1.2 ± 1.3 MPs fish -1 ). Differences in plastic ingestion might be associated with the feeding strategy of species [START_REF] Wright | Microplastic ingestion decreases energy reserves in marine worms[END_REF][START_REF] Miller | Bioaccumulation and biomagnification of microplastics in marine organisms: A review and meta-analysis of current data[END_REF]. Our study tested and confirmed the hypothesis that microplastic ingestion varies with the different feeding strategies. However, regardless of the diet preferences of the species, in our study area, it is reported many sources of MPs contaminants, which probably accumulate in the sediment and water column, negatively affecting the life-strategies of fish species and mostly the juvenile stages which utilise estuaries as a nursery ground. Thus, the predators are more prone to be contaminated by microplastics through two main exposure routes: (1) the highly contaminated estuarine habitats and (2) the ingestion of contaminated prey. Anthropogenic activities in the ECSC (urban areas, manufacturers, aquaculture plants, and sugarcane fields) are found surrounding the whole floodplain, and these activities might be an important source of microplastics and other contaminants (e.g., pesticides and heavy metals). Indeed, the ECSC has registered many impacts such as habitat loss and mercury releases [START_REF] Pelage | Coastal land use in Northeast Brazil: mangrove coverage evolution over three decades[END_REF][START_REF] Araújo | Bioavailability and sequential extraction of mercury in soils and organisms of a mangrove contaminated by a chlor-alkali plant[END_REF][START_REF] Albuquerque | Tracking Hg historical inputs by Pb-210 geochronology for the Itapessoca Estuarine Complex, Pernambuco, Brazil[END_REF], which likely affect the estuarine community. Besides, our study has identified high contamination by microplastics in fish species that are a relevant source of protein locally and regionally. Despite being a Marine Protected Area, which provides essential ecosystems services, there is a lack of awareness and public policies, highlighting the importance of monitoring and management policies to control and mitigate social and health problems.

Further studies are needed regarding the microplastic impacts on marine fauna and whether they could transfer adsorbed pollutants such as persistent organic pollutants (POPs), heavy metals, and plastic additives to the food web are necessary, as microplastics particles can be transferred along the trophic chain, the chances to accumulate other pollutants in the food web increases. In fishery resources, this question is a public health matter because it is linked to human uptake of these pollutants. Our study also emphasises the importance of implementing protocols to extract microplastics in biological samples, which guarantee the quality of samples, avoid under or overestimation and airborne contamination, and which can be easily replicable.

Acknowledgement

Figure 1 .

 1 Figure 1. Map of the Estuarine Complex of the Santa Cruz Channel (ECSC) adapted from Pelage et al., 2019.

Figure 2 .

 2 Figure 2. Flowchart with the stepwise of the extraction protocol.

Figure 3 .Figure 4 .Figure 5 .

 345 Figure 3. Mean number of microplastics ingested by fishes collected in the Estuarine Complex of the Santa Cruz Channel. The asterisks represent the statistical differences with a significance of 0.05.

  This study was supported by the LMI TAPIOCA program CAPES/COFECUB (88881.142689/2017-01); FUNBIO and Humanize under the grant "Programa Bolsas Funbio -Conservando o Futuro 2019 (02/2019)"; FACEPE (Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco); and MicroplastiX Project JPI-Oceans/CONFAP/FACEPE (n°APQ-0035-1.08/19). We thank the CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), which provided research grants to Flávia Lucena Frédou (CNPq n° 308554/2019-1) and Thierry Frédou (CNPq 307422/2020-8); FACEPE for granting a scholarship (BFP-0049-5.06/20) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for granting a scholarship at doctoral level for the first author. All authors thanks to the members of the laboratory BIOIMPACT for the support in the sampling procedures and the fishermen of Itapissuma for the sampling collect.

Table

  ).

	Family/Species		Ecological parameters		Biometry			Microplastics occurrence
		N	Habitat use	Feeding habit	TL (cm) Min-Max	SL (cm) Min-Max	TW (g) Min-Max	MPs	FO%	TL (mm) Min-Max
	Gobiidae
	Gobionellus stomatus (Starks, 1913)	31	Demersal	DV	9.3 -11.9	7.0 -9.0	3.78 -7.89	52	67	0.02 -5.00
	Sciaenidae
	Bairdiella ronchus (Cuvier, 1830)	21	Demersal	ZB	12.5 -16.5	10.0 -14.0	22.2 -62.4	26	74	0.11 -3.35
	Centropomidae
	Centropomus undecimalis (Bloch, 1792)	30	Demersal	PV	8.9 -14.0	7.3 -11.0	5.8 -16.78	98	77	0.06 -3.79

web experiment: CYP1A induction and visual tracking of persistent organic pollutants. Environmental Toxicology and Chemistry, 35(7), 1656-1666. doi.org/10.1002/etc.3361 Bellas, J., Martínez-Armental, J., Martínez-Cámara, A., Besada, V., & Martínez-Gómez, C., 2016. Ingestion of microplastics by demersal fish from the Spanish Atlantic and Mediterranean coasts. Marine pollution bulletin, 109(1), 55-60.