Presence and quantification of the microplastics and bisphenol-A in sediments along the Guanabara Bay, Brazil Presença e quantificação de microplásticos e bisfenol-A em sedimentos ao longo da Baía de Guanabara, Brasil

Microplastics (MP) and endocrine disruptors, such as bisphenol-A (BPA) have both become significant environmental concerns worldwide, although assessments in estuarine environments are still scarce. In this regard, Guanabara Bay is one of the most important Brazilian estuarine systems, displaying significant economic and environmental relevance, although it has suffered increasing anthropogenic effects for decades. Thus, this study aimed to assess the occurrence of both MP and BPA in the sediments of this estuarine system though stereomicroscope identification and high-performance liquid chromatography. Both pollutants were detected in all sediment samples. A total of 3 to 11 MP particles (<5 mm) per 50 cm 2 were detected, higher than in other studies worldwide. MPs were categorized as fibers and fragments, mostly green, possibly from fisher rope and net degradation. BPA ranged from 0.36 to 19.75 ng g -1 , higher near Governador Island and the Rio de Janeiro harbor. Potential correlations between MPs and BPA, however, could not be determined. Both MP and BPA represent significant environmental concerns in the already highly impacted Guanabara Bay estuarine system, and further assessments are paramount to understand potential ecotoxicological hazards associated to these pollutants.

Concerning solid waste, plastic pollution has become a worldwide issue (USEPA, 2016), with about 6.4 million tons are dumped into seas and oceans each year, 80% of which is marine waste (MARTÍN-LARA et al., 2021). Due to their buoyant and persistent properties, plastics can become widely dispersed via hydrodynamic processes, reaching distant sites from their initial input area (HOLMES et al., 2012). A clear example of this comprises, the floating garbage patches that form ocean plastic patches (UNEP 2005;HOLMES et al., 2012;KINSLEY, 2017;LEAL-FILHO et al., 2021).
Microplastics (MPs) comprise small plastics ranging from 0.1 to 5.000 μm, categorized as primary when originally manufactured in that size, while secondary microplastics originate from the fragmentation of larger plastics (COLE et al., 2011;GESAMP, 2015). Fragmentation processes can consist in both physical and chemical degradation, such as abrasive forces, oxidation, hydrolysi and the effect of UV light, or biodegradation, caused by fungi, bacteria or algae (KLEIN et al., 2018). Once in the ocean, MPs can undergo further changes in density, resulting in increased mass that is then deposited on beaches and in bottom sediments (VAN CAUWENBERGHE et al., 2015). Marine MP contamination drastically affects the many organisms (e.g., bivalves, fishes, turtles, cetaceans) that ingest these particles daily (DERRAIK, 2002;CARSON et al., 2011;THOMPSON, 2015). Several studies concerning MP exposure in different aquatic species under laboratory conditions have indicated significant physiological effects, as depicted in Table 1.
Evaluate the uptake, distribution, and toxicity of polystyrene in developing zebrafish Decreased heart rate, altered swimming behavior PITT et al. (2018) Evaluate the effects of polyethylene ingestion in the mussels and lugworms Inflammatory response and granulocytomas were observed after the intake of particles up to 80 μm VAN CAUWENBERGHE et al. (2015) Evaluate the effects of 30 nm polystyrene particle ingestion (0, 0.1, 0.2, and 0.3 g L -1 ) in mussels Filtering activity was reduced in presence of polystyrene. When exposed to 0.1 gL -1 , polystyrene was recognized as a low nutritional food by mussels MOOS et al. (2012) Evaluate the ingestion, translocation, and accumulation of MPs debris (3.0 or 9.6 μm) in the mussels MP accumulation was observed in the gut and MP translocation from the gut to the circulatory system WEGNER et al., 2012 Evaluate the presence of MPs in soft mussel tissues 0.36 ± 0.07 particles per gram in mussel soft tissues BROWNE et al., 2008 Evaluation of the effects of microscopic unplasticised polyvinylchloride (UPVC) Energy reserve depletion corresponding to 5% of sediment weight was observed after chronic exposure to UPVC. Accumulation of UPVC in the long gut and inflammation with enhanced phagocytic response was detected after chronic exposure BESSELING et al., 2013 In addition, MPs contain chemicals additives and can adsorb several other contaminants, both organic and inorganic, also representing ecotoxicological risks, as both the MPs and their leached chemicals can bioaccumulate and biomagnify throughout the trophic chain (HOLMES et al., 2012;AUTA, 2017;ANBUMANI et al., 2018;WANG, et al., 2020). The main plastic additives found in the environment are phthalates, bisphenol-A (BPA), polybrominateddiphenyl ethers (PBDEs), nonylphenols (NP), andalkylphenolethoxylates (APEs) (USEPA, 2016;HERMABESSIERE et al., 2017). These compounds are considered endocrine disruptors, altering endocrine system functions in both animals and humans, affecting their growth and reproduction while also leading to several diseases, such as cancer, fertility disorders, and abnormal sexual development (BILA; DEZOTTI, 2006;CUNHA, et al., 2016). BPA exposure, for example, leads to both development and reproduction effects, even at low concentrations, i.e., decreasing levels of male hormones, reduced sperm density and motility, testicular cell death, and inhibition of egg production and spermatogenesis (KANG et al., 2007;CANESI;FABBRI, 2015;ENCARNAÇÃO et al., 2019;WU;SEEBACHER, 2020). BPA may also respond differently depending on organismal life stage and physiological state, making it difficult to trace its possible mechanisms of action (CANESI;FABBRI, 2015).
Studies on microplastics associated to endocrine disruptor concentrations in estuarine environments are, however, still scarce worldwide. Therefore, this study aimed to investigate MP distribution and the presence of BPA in the coastal sediments of one of the most important water systems on the Brazilian coast, Guanabara Bay, with significant ecological and socio-economic relevance (ZHANGA, et al., 2016;CAJARAVILLE et al., 2016), even though it is subject to intense contamination from several polluting sources, leading to extremely high aquatic biota risks due to exposure to numerous contaminants with toxic potential (SINGARE, 2016).

STUDY AREA
Guanabara Bay, located in the state of Rio de Janeiro, Southeastern Brazil (22º40'S and 23º00'S latitude and 043º00'-043º18'W longitude), is one of the largest Brazilian coastline bays, comprising about 384 km 2 ( Figure 1). It is surrounded by the highly urbanized cities of Rio de Janeiro, Duque de Caxias, Niterói and São Gonçalo, as well as three other municipalities (AMADOR, 2012;SOARES-GOMES et al., 2016). Its connection with the open sea is through a narrow entrance only 1.6 km wide, which directly influences the bay's hydrodynamics (CARVALHO; BAPTISTA NETO, 2016). Its maximum depth is of 58 m in the central region, becoming shallower to the north, near Ponte Rio-Niterói, with an average depth of 5.7 m (KJERFVE et al., 1997;MELO et al., 2015). This depth change is due to high sedimentation rates originating from human activities in surrounding areas (CARVALHO; BAPTISTA NETO, 2016).
The Guanabara Bay drainage basin extends 4080 km 2 consisting of 91 rivers and channels, encompassing the Rio de Janeiro, Nova Iguaçu, Belford Roxo, Duque de Caxias, Magé, Petrópolis, Itaboraí, São Gonçalo, Niterói, Rio Bonito, Teresópolis and Cachoeiras de Macacu municipalities, with a population concentration equivalent to 80% of the population of the entire state of Rio de Janeiro. Many of these rivers, mainly in the metropolitan area, are highly polluted. In addition, this estuarine system receives drainage from two harbors, refineries, and over 12,000 industries which account for 25% of all organic pollution released into the bay (KJERFVE et al., 1997;BAPTISTA NETO et al., 2006), as well as industrial and domestic sewage effluents, urban and agricultural runoff, as well as atmospheric fallout (COSTA, 2018). However, although this is a well-studied area, there is still much to investigate about its contamination levels (BAPTISTA NETO et al. (2006).

SAMPLING
Sediment samplings were carried out in December 2014, at six sampling points using a Van Veen grab sampler (KC Denmark AS) and a spatula (both stainless steel). For the MPs analysis, 50 cm² of the surface sediment layer about 1 cm deep were obtained according to CAUWENBERGHE et al. (2013), and surface sediment aliquots were separated for the BPA analyses. The depth of the sampling stations ranged from 2.8 to 5.9 m. The sediment samples were then transferred to previously decontaminated amber glass bottles and stored at 4 °C until further analyses. Figure 1 and Table 2 depict the location and basic information of each sampling station.

LABORATORY ANALYSES
All materials and glassware used for sample collection and preparation were thoroughly decontaminated with a 10 % nitric acid solution to avoid interference from potential contaminants.
Concerning the MP analysis, sediment samples were prepared according to THOMPSON et al. (2004) andMASURA et al. (2015). Flotation was used to separate less dense MP particles from the sediments following 30% hydrogen peroxide (H2O2) addition to remove organic matter, as the samples were muddy, containing high organic matter concentrations and impairing MPs separation. Briefly, sediment aliquots were mixed with a hypersaline solution (140 g L -1 NaCl) at 1.5-to 2-fold the sediment volume in a beaker, stirred for four minutes and left to rest it was until sediment decantation and MP floating. The supernatants were filtered employng a vacuum filtration system using filter paper (47 mm) and oven-dried at 60 °C. The paper filters were then stored in Petri dishes until the analysis. The obtained MPs were separated and manually identified under a Zeiss STEMI 2000 C stereomicroscope for size class determinations and categorization according to their appearance, characteristics, and origin (such as from fishing activities, fibers, fragments, Styrofoam, or pre-production pellets).
For the BPA determinations, 10 g aliquots of the dried sediment were extracted using 10 mL of methanol in an ultrasonic bath for 5 min. The samples were subsequently centrifuged at 2,500 g for 5 min and the supernatants were transferred to a 200 mL volumetric flask. This procedure was repeated three times. The sample volume was then made up to 200 mL with ultrapure water and acidified to pH 2.0. Subsequently, BPA was extracted by solidphase extraction using StrataX cartridges (500 mg per 6 mL, Phenomenex ® ) and a manifold (Agilent Technologies ® ). The cartridges were preconditioned with 3 x 2 mL hexane, 1 mL acetone, 3 x 2 mL methanol and 5 x 2 mL ultrapure water at pH 3.0. The samples were then percolated under vacuum at about a 10 mL min -1 flow and maintained under vacuum for 30 minutes after extraction. Analyte elution was then performed using 4 mL of acetone. The extracts were finally evaporated under a nitrogen flow to dryness and resuspended with 500 µL of acetonitrile.
The BPA sediment determination methodology was previously validated according to guidelines reported by Silva et al. (2016), based on Brazil's National Institute of Metrology, Standardization and Industrial Quality (INMETRO, 2010) method. This method employs a high-performance liquid chromatography and a fluorescence detector (HPLC/FLU) (Waters Corporation ® ) using a 60% acetonitrile and 40% ultrapure water mobile phase, a 20 µL injection volume and three replicates for each sample. The eluent flow rate was set at 1 mL min -1 , with emission wavelength set at 300 nm and excitation wavelength set at 223 nm. The stationary phase comprised a Novapak PAH chromatographic column (4.6 x 250 mm, 5 microns) maintained at 40 °C ± 1 throughout 8 min runs in isocratic mode. The BPA concentrations were identified by comparing peak retention times with corresponding standard solution peaks. Analytical curve correlation coefficient (R²) values were always > 0.99. The limit of detection was 1.899 µg L -1 and the limit of quantification was 62.5 µg L -1 , with recoveries ranging from 97 to 106%.

RESULTS AND DISCUSSION
The presence of MP particles was observed in all sediments sampled from Guanabara Bay (Figure 2), categorized mainly as microplastics and fibers.
The detected MPs ranged from 3 to 11 particles per 50 cm 2 (Figure 3 and Table 3). Most of the identified MPs originated from local sources such as effluents, fishing, and port activities. MP migration from the water column to the sediment is probable, as indicated in a study conducted in the northeastern Atlantic Ocean, that detected MPs in 23 of 30 samples collected from bottom sediment (THOMPSON et al., 2004).   Guanabara Bay MP fragment concentrations are higher than in other studies conducted worldwide, which report an average abundance of 1 MP per 50 cm 2 (CAUWENBERGHEET et al., 2013), demonstrating that this coastal marine area suffers from higher MP concentrations due to intense anthropogenic activities. Sampling station 6 near the Rio de Janeiro harbor, a significantly impacted area, presented the highest MP concentration, of 11 particles per 50 cm 2 . In the present study, most of the detected MPs were green, indicating they may originate from the degradation of fisher ropes and nets, which are mostly this color.
High MP abundances have been previously reported at Guanabara Bay, for example, by ALVES and FIGUEIREDO (2019), who reported from 160 to 1,000 items kg -1 or 4,367 to 25,794 items m 2 in all sediment samples analyzed in their study, with translucent polyester microfibers being the most frequent MPs. In another assessment, CASTRO et al. (2020) evaluated the distribution of plastic items in different matrices (surface waters and bottom and beach sediments) and presenting different hydrodynamic properties, from Jurujuba and Itaipu Coves, both located within Guanabara Bay. The samples were composed of MPs (83%), mesoplastics (13%), and macroplastics (4%), with sizes ranging from 100 μm to 170,000 μm (17 cm), with bottom sediments containing mostly fibers/lines and MP fragments. Another investigation conducted by CARVALHO and BAPTISTA NETO (2016) analyzed microparticles on 35 surrounding Guanabara Bay beaches, reporting concentrations ranging. from 12 to 1300 per m 2 , comprising mostly MP fragments (56%). followed by Styrofoam fragments (26.7%), pellets (9.9%), and fibers (7.2%). OLIVATTO et al. (2019) determined the presence of MPs and identified plastic residues in all surface water Guanabara Bay samples analyzed in their study, with average concentrations of 1.40 to 21.3 particles per m 3 . In another assessment, BAPTISTA-NETO et al. (2019) detected the presence of MPs in 100 % of analyzed sediment samples from the inner continental shelf of Rio de Janeiro, close to the Guanabara Bay estuary system, composed mainly of secondary MPs, with almost 50% categorized as fibers, followed by plastic films, plastic fragments, and pellets. Studies have also been carried out in Guanabara Bay aiming to assess the impact of MPs on aquatic organisms and potential trophic chain effect, mainly through ingestion as the ingestion of MPs, which is an important pathway for these particles to enter the trophic chain. In this regard, CASTRO et al, (2016) and more recently BIRNSTIEL et al., (2019), analyzed MPs in a mussel farming area in Guanabara Bay (Jurujuba Cove), both reporting high MP concentrations in these bivalves.
Concerning BPA, this compound was detected in all sediment samples ranging from 0.40 to 20 ng g -1 (Figure 4). The highest concentrations were observed in sediments from sampling stations 2 (20 ng g -1 ) and 6 (19 ng g -1 ), located near Governador Island and the Rio de Janeiro harbor, respectively. BPA concentrations have been reported worldwide for different matrices such as surface water (rivers, lakes, and lagoons), suspended particulate matter, and sediments (CUNHA, et al., 2021;WEN et al., 2018). For example, BPA concentrations in sediment samples from the Daliao River ranged from 3.7 to 25.3 ng g -1 (CALDWELL et al., 2012) and in the Yangtze River, from 1.2 to 6.5 ng g -1 (SHI; ZHANG, 2012), both in China, while samples from the Bay of Biscay, in Spain, ranged from 0.01 to 0.04 ng g -1 (PUY-AZURMENDI et al., 2013). In Brazil, NASCIMENTO (2016) analyzed two important tributaries of Guanabara Bay and nine points within Guanabara Bay, comprising both surface and deep waters, detecting BPA concentrations from 22.33 to 1325.16 ng L -1 at Mangue Channel and Maracanã River, both Guanabara Bay tributaries, and at Guanabara Bay itself, from 14.06 to 298.55 ng L -1 to surface water and from 51.24 to 465.55 ngL -1 in deep water. In the state of São Paulo, also in Southeastern Brazil, BPA concentrations in surface waters in the city of Campinas BPA ranged from 0.005 to 1.760 μg L -1 (SODRÉ et al.;, 2010, while samples from the state of Minas Gerais ranged from 8.6 to 168.3 ng L -1 , with an applied risk analysis indicating toxic BPA (MOREIRA et al., 2011). Associations between MP and BPA, as well as with other pollutants, have been reported previously as leading to combined toxic effects (Pittura et al., 2018;Sun et al., 2021;Tang et al., 2020), directly affecting physiological processes in aquatic species (Aarab et al., 2006;Juhel et al., 2017). Furthermore, BPA has been proven to significantly adsorb to MPs and, consequently by aquatic species, suggesting that both pollutants interact in organisms (Chen et al., 2017). These associations, however, could not be determined herein, due to the need for expensive and time-consuming chemical MP analyses. Thus, further research is required in this regard at Guanabara Bay, to determine the toxic potential of BPA associated to MP.

CONCLUSION
Both MPs and BPA were detected in all sediment samples obtained along the Guanabara Bay. Guanabara Bay MP fragment concentrations were higher than in other studies conducted worldwide, especially near the Rio de Janeiro harbor, which presented the highest MP concentrations. Most of the detected MPs were green, possibly from fisher rope and net degradation. BPA concentrations were higher near Governador Island and the Rio de Janeiro harbor, both highly impacted areas. Although potential correlations between MPs and BPA could not be determined herein, both represent significant environmental concerns in this already highly impacted estuarine system, and further assessments in this regard are paramount.

ACKNOWLEDGEMENTS
This study was supported in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) -Finance Code 001, the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).