Quantitative analysis of pellets on beaches of the São Paulo coast and associated non-ingested ecotoxicological effects on marine organisms

Regional Studies in Marine Science 29 (2019) 100705

Contents lists available at ScienceDirect

Regional Studies in Marine Science journal homepage: www.elsevier.com/locate/rsma

Quantitative analysis of pellets on beaches of the São Paulo coast and associated non-ingested ecotoxicological effects on marine organisms ∗

G.M. Izar a , , L.G. Morais b , C.D.S. Pereira a , A. Cesar a , D.M.S. Abessa b , R.A. Christofoletti a a

Department of Ocean Science, Federal University of São Paulo (UNIFESP), Silva Jardim Street, 136, 11015-020, Santos, São Paulo, Brazil Department of Marine Science and Coastal Management, São Paulo State University (UNESP), Praça Infante Dom Henrique, s/n, 11330-900, São Vicente, São Paulo, Brazil b

article

info

Article history: Received 5 July 2018 Received in revised form 27 May 2019 Accepted 30 May 2019 Available online 3 June 2019 Keywords: Plastic pellets Microplastics Coastal impact Toxicity Pollutants Sources

a b s t r a c t We evaluated the densities of plastic pellets on beaches along the coast of São Paulo State in Brazil in order to establish a relationship between their spatial distribution and their distance from the main source of pellets. We hypothesized that pellets would be concentrated close to their main source (in this case, the Port of Santos). We also tested whether beached pellets could induce toxicity in water and sediment samples, as well as whether pellet color, sample site (beach), and pellet density were correlated with toxicity. We observed a decreasing gradient of pellet density along the São Paulo coast, with higher densities detected within Santos Bay, which is close to the Port of Santos. Beached pellets induced water-based toxicity in the sea urchin Lytechinus variegatus and the copepod Nitocra sp. at high densities but were not capable of producing toxicity at environmentally realistic densities. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Plastic residue represents more than 90% of the world’s marine litter (Milijö, 2001), and it is estimated that the amount of plastic in the oceans has grown at a rate of 8% per year (Ogata et al., 2009). Plastic pellets consist of industrial raw material and are a few millimeters in diameter. They are generally round or cylinder shaped and are transported to manufacturing sites to be molded into their final plastic products (Mato et al., 2001). Plastic pellets can accidentally be released into the sea, and their worldwide dispersal is associated with the transportation of massive amounts of these plastics, largely along maritime routes. Each pellet-carrying cargo ship is estimated to carry approximately one billion pellets per trip (Hammer et al., 2012). Sources of pellets in natural environments can also include industrial production (Karlsson et al., 2018), input through rivers (Lechner and Ramler, 2015), and leakage during road and railway transport (US EPA, 1992). This variety of sources raises concerns about plastic pellets around the world, as they have already been found in all of the world’s oceans (Mato et al., 2001), as well as in most coastal regions and on the corresponding beaches. The distribution of pellets in coastal regions is understood to be associated with their sources, which are usually located on land (Gregory, 1999), in regions near port facilities, in cities, and in industrial areas. Thus, the distance between coastal sites ∗ Corresponding author. E-mail address: [email protected] (G.M. Izar). https://doi.org/10.1016/j.rsma.2019.100705 2352-4855/© 2019 Elsevier B.V. All rights reserved.

and sources of plastic pellets may be an important factor to consider when evaluating and monitoring the impacts of this type of marine litter on the environment. The possibility of such a relationship has been documented previously in the literature (Khordagui and Abu-Hilal, 1994; Gregory, 1978; Colton, 1974; Ross et al., 1991; Shiber, 1979, 1982; Moore et al., 2001; Claessens et al., 2001) but had never been effectively tested. The findings in the literature on the distribution of pellets along beaches and in coastal environments are conflicting. There is no consensus regarding spots with higher accumulation, and pellet density varies considerably (Hidalgo-Ruz et al., 2012). Thus, comparing results from different studies is difficult (Derraik, 2002). Turra et al. (2014) proposed that pellet density could be presented as the number of pellets per m3 . Definitions of pellet accumulation spots remain inconsistent in the literature, since these definitions depend on the type of beach where the pellets have accumulated. While some studies evaluate the entire beach (transects perpendicular to the coastline), others have focused on different portions of the beach—the supralittoral zone, the high tide line or the sublittoral zone (Hidalgo-Ruz et al., 2012; Van Cauwenberghe et al., 2015). Most of the studies on pellets and microplastics conducted worldwide have sampled plastics along the high tide line (Hidalgo-Ruz et al., 2012). Recently, Moreira et al. (2016), Turra et al. (2014), and Heo et al. (2013) have suggested that the sampling of pellets and microplastics should be concentrated in the supralittoral zone and close to the upper limit of the backshore, because this area best represents the accumulation of these materials on the standing stock.

2

G.M. Izar, L.G. Morais, C.D.S. Pereira et al. / Regional Studies in Marine Science 29 (2019) 100705

Moreira et al. (2016) also argued that further research on pellets should be focused on monitoring the behavior and dynamics of pellets on the standing stocks of sandy beaches in order to better understand their spatial distribution patterns. The depths at which sampling has been performed have been quite varied, though many studies have sampled pellets in the surface layers of sand (first 5 cm of the surface) (Hidalgo-Ruz et al., 2012). Thus, identifying the most appropriate beach zone to sample plastic pellets remains a challenge for understanding and evaluating this type of pollutant and its environmental impacts. In addition to economic losses and esthetic damages to beaches, the presence of pellets within coastal environments represents a huge concern for marine wildlife (Pruter, 1987; Shiber and Barrales-Rienda, 1991; Milijö, 2001; Laist, 1987; Hammer et al., 2012). Pellets can easily be mistaken for food and eaten by marine animals, such as birds (Moser and Lee, 1992), turtles (Bjorndal et al., 1994; Tomás et al., 2002; Tourinho et al., 2010), fish (Davison and Asch, 2011), and cetaceans (Baird and Hooker, 2000). Pellet ingestion can cause serious damage to marine organisms by obstructing the intestine, reducing appetite, and exposing the animal to dangerous chemical compounds (Endo et al., 2005; Milijö, 2001). Thus, studies on pellet distribution are important to better identify areas where the marine biota will be most likely to ingest them. Another aspect that is still not clearly understood is the pellet’s ability to adsorb potentially toxic contaminants (Pereira et al., 2011) and release them back to the environment, causing toxicity to marine organisms. Until recently, there was little information on how contaminants released by pellets could affect marine biota. In recent studies, toxicity has been reported in embryos of the sea urchin Lytechinus variegatus exposed to both virgin and beach-stranded pellets (Nobre et al., 2015), while acute toxicity was found to be induced in Nitocra spinipes when exposed to different plastic compounds (Bejgarn et al., 2015). However, lists of chemical compounds associated with plastic pellets are still scarce, making it difficult to properly assess the environmental risks associated with this debris (Li et al., 2015). Some compounds that have been identified in plastic pellets include bisphenol A (BPA), phthalates, and flame retardants such as polybrominated diphenyl ethers (PBDE) and tetrabromobisphenol A (TBBPA), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and dichlorodiphenyltrichloroethane (DDT) and its breakdown products, which include dichlorodiphenyldichloroethylene (DDE), dichlorodiphenyldi chloroethane (DDD), and metals (Ashton et al., 2010; Endo et al., 2005; Gorman et al., 2019; Hammer et al., 2012; Holmes et al., 2012; Jayasiri et al., 2015; Koch and Calafat, 2009; Ogata et al., 2009; Taniguchi et al., 2016; Teuten et al., 2009). Pellet color may have an additional influence on toxicity because of the direct relationship between color and the concentration of contaminants adsorbed. Yellow pellets have higher concentrations of PCBs and generally exhibit this color when the pellets have been present in the coastal environment for longer periods (Endo et al., 2005). Endo et al. (2005) report that contaminants adsorbed onto pellets may represent local contamination levels, and that sampling sites could also be considered a factor that influences toxicity (Endo et al., 2005; Ogata et al., 2009). On the other hand, white and virgin pellets are manufactured with certain chemical additives (the case of polyolefin, for example). Virgin pellets are processed by compounding, and during this step, numerous additives and color pigments are added. Therefore, these pellets may have a considerable concentration of chemical additives (Hammer et al., 2012; Koch and Calafat, 2009), which may cause them to produce high levels of toxicity in aquatic organisms (Nobre et al., 2015). Persistent organic pollutants (POPs) can be found in pellets at concentrations 106

times higher than those detected in the surrounding seawater (Mato et al., 2001). Thus, pellets may be considered geochemical carriers of hydrophobic pollutants and are able to transfer these contaminants to marine organisms (Mato et al., 2001). Brazil is home to the Port of Santos, one of the largest and most important ports in Latin America. It is responsible for 28% of the annual trade in exported products in Brazil, and regarding plastic pellets it handles 50,000 tons/month (Fisner et al., 2013), and is a major source of pellets in the adjacent coastal regions (Taniguchi et al., 2016). The vicinity of the Port of Santos also shelters a large industrial complex within in the city of Cubatão, which also contributes to the discharge of contaminants and marine litter in the region, including plastic pellets that can be carried along the Cubatão River to the Santos Bay. Plastic pellets have been documented along the entire length of the São Paulo coast (Taniguchi et al., 2016), with high densities reported on the beaches closest to the Port of Santos (Fisner et al., 2017; Moreira et al., 2016; Turra et al., 2014). This finding suggests that there is a possible relationship between pellet density and distance from the source. The coast of São Paulo State has another port (the Port of São Sebastião, located approximately 130 km north of the Port of Santos), though it does not transport or store plastic pellets; thus, it does not represent a source of this material. Another potential source of plastic pellets could be the Port of Paranaguá, which is located approximately 310 km south of the Port of Santos and which is involved in pellet transportation. This study sought to evaluate pellet density on beaches along the coast of São Paulo in an attempt to establish a relationship between pellet distribution and the distance from the main source of pellets. We tested the hypothesis that pellet density is higher on beaches closest to the Port of Santos and lower on beaches located farther away from the port. We also tried to determine the most appropriate beach zone for sampling plastic pellets (i.e. zones where plastic pellets are most likely to accumulate). We also tested whether water or sediment samples could become toxic to marine organisms after contact with beached pellets. We evaluated whether certain factors could contribute to this toxicity, such as pellet color, sampling site (beach), and pellets density. Our ecotoxicological hypotheses were that high amounts of pellets would be capable of transferring contaminants to water and sediment, thus producing toxicity for the marine organisms tested, that pellets of different colors (white and yellow) would have different toxic effects on the exposed organisms, that pellet toxicity would reflect the degree of pollution degree at the respective sampling site; and that the pellets found on the beaches of São Paulo State would reach densities high enough to be toxic to marine organisms. 2. Materials and methods 2.1. Pellet Analysis on the Coast of São Paulo State 2.1.1. Determining the most appropriate beach zone for sampling pellets to determine pellet density Critical issues when studying plastic pellet pollution include identifying their vertical and horizontal distribution along the beach, understanding whether pellets tend to accumulate on certain portions of the beach, and determining where they can become pollutants of concern. To evaluate pellet distribution in this study, we selected three beaches with similar conditions in terms of exposure (sheltered beaches), length (less than 300 m), and human impact (difficult access; lack of public cleaning services and/or sand removal). The beaches chosen in this step are located in the Baixada Santista Metropolitan Region on the coast of São Paulo State and close to Port of Santos. The beaches known as Itaquitanduva and Góes are located within the Santos

G.M. Izar, L.G. Morais, C.D.S. Pereira et al. / Regional Studies in Marine Science 29 (2019) 100705

3

Fig. 1. Map of the Santos Bay showing the Port of Santos and the three beaches selected for pellet sampling.

Bay (less than 10 km from the port), while Preta Beach is farther from the port (≈20 km) in the city of Guarujá (Fig. 1). As with other studies in the literature, pellets were sampled from two different and commonly studied beach zones at each beach (Hidalgo-Ruz et al., 2012). The first zone was the high tide line, which was defined as the portion below highest line of natural or anthropogenic material accumulation on the backshore, and which represent pellets that had recently accumulated on the beach and had been more strongly influenced by waves and tides. The second zone was the upper limit of the backshore, which was defined as the uppermost portion of the sandy beach close to vegetation or anthropogenic constructions (boardwalks, walls, or houses), which represented the best beach zone for pellet accumulation (standing stocks); this zone tends to be less influenced by waves and tides and is affected by such factors only during extreme events of wave run-up and storm surges. In each zone, pellets were collected to a depth of up to 25 cm of the sediment column. This depth includes the layer that has the most contact with seawater, meaning it most accurately represents the potential ecotoxicity to which the marine organisms tested are exposed. Samples were collected at three different depths (Surface Column [0 cm–5 cm], Middle Column [6 cm–19 cm], and Deep Column [20 cm–25 cm]) and from the center (in length) of each beach using a cylindrical corer with a 7.5 cm radius; 5 replicates were obtained from each beach zone and at each depth. The removed sediment was separated by depth and placed in a bucket with seawater, in which the pellets floated to the top due to the density difference and the material was then sieved through a 1 mm mesh. The total number of pellets was counted for each beach zone and depth (in pellets per corer), and the data was analyzed with a 3-factor orthogonal ANOVA: Beach (Factor 1, random, orthogonal, 3 levels), Sample Area (Factor 2, fixed, orthogonal, 2 levels) and Depth (Factor 3, fixed, orthogonal, 3 levels). 2.1.2. Determining associations between pellet density and distance from port We treated the Port of Santos as the main source of plastic pellets in the study area based on the literature regarding coastal circulation patterns (Castro Filho et al., 1987; de Souza and Robinson, 2004; Harari et al., 2006; Harari and Camargo, 1998), distance, and presence of primary sources of these materials. As mentioned previously, the Port of Paranaguá could be

a potential source of pellets on the beaches in the Baixada Santista Metropolitan Region; however, the Brazilian Coastal Current (BCC) mainly flows southward along the coast of São Paulo State (de Souza and Robinson, 2004) and is driven by the predominant winds (Harari et al., 2006; Harari and Camargo, 1998). The coastal current can invert its direction and flow north, but only for short periods of time when cold fronts influence the circulation (Castro Filho et al. 1996). Since the marine transport of pellets is less likely to originate from the Port of Paranaguá, and because of its greater distance from the sampling area, we did not treat the Port of Paranaguá as a main source of pellets to our study area. To determine associations between pellet density and distance from the port, we adopted a sample area extending along the coast of São Paulo State from Ilha Comprida to Ubatuba, an area which encompassed eight cities (Ilha Comprida [IC], Peruíbe, Itanhaém, Praia Grande, Santos, Guarujá, São Sebastião and Ubatuba) distributed along almost 350 km of coastline. To test our hypothesis regarding the pellets’ density gradient, 12 beaches were selected (IC Pedrinhas, IC Centro, Prainha, Praia dos Pescadores, Itaquitanduva, Góes, Guaiúba, Mar Casado, Engenho, Cigarras, Tenório, and Cambury) (Fig. 2); these beaches are located at different distances to the north and south of the Port of Santos (<5 km, 10 km, 55 km, 90 km, 140 km and 170 km). The beaches were selected based on their morphological characteristics and following the United Nations Environment Program (UNEP) protocol (Cheshire et al., 2009): low mean slope (between 15◦ and 45◦ ), beach width ranging from 100 m to 1 km, free access to the sea, and without any anthropogenic structures blocking wave and current flow. The exceptions were the two southernmost beaches, which were located in an area with very extensive beaches without geographical breaks, but which were the desired distance from the port. At each beach, a sample of pellets was collected from the surface sand layer (5 cm) from the upper limit of the backshore using a cylindrical 7.5 cm radius corer positioned in the median (central) portion of each beach; 20 replicates were collected in each site. The pellets were separated from the sediment by density using the flotation method (Manzano, 2009) and counted at the sampling site. Pellet colors were defined visually between the shades found (white and transparent, yellow, orange, brown, or black). The total number of pellets from each beach was converted into pellets per m3 , as proposed by Turra et al. (2014), and the number of pellets in each replicate was recorded. The

4

G.M. Izar, L.G. Morais, C.D.S. Pereira et al. / Regional Studies in Marine Science 29 (2019) 100705

Fig. 2. Map of the São Paulo coast showing the Port of Santos and the 12 beaches selected for pellet sampling to determine the relationship between pellet density and port distance.

mean numbers of pellets were calculated for each beach and were then associated with the distance of the beach from the Port of Santos (in km) using linear regression. The data in both axes were adjusted in a Log10 (X +1) conversion in order to obtain a better linear adjustment. 2.2. Ecotoxicological effects of Non-Ingested pellets on marine organisms To test the toxicity of contaminants released by the pellets, we exposed three different species of marine organisms (embryos of the sea urchin Lytechinus variegatus, ovigerous females of the infaunal copepod Nitocra sp., and adults of the burrowing amphipod Tiburonella viscana) to the contaminants. The organisms were chosen due to their sensitivity to contaminants, the ease in raising and handling them in a laboratory setting or in obtaining them from a natural environment, and because they are representative of different habitats of the coastal environment. Moreover, these organisms are widely used in ecotoxicological studies and are well-established biological models with defined protocols for toxicity testing. The organisms were exposed to the pellets in two tests. In the first test, embryos of the sea urchin L. variegatus and the copepod Nitocra sp. were used to evaluate exposure in aqueous solutions and test the acute and/or chronic ecotoxicological effects of extremely high pellet density exposure. This step also considered some possible influences on pellet toxicity, such as color and sample site. In the second test, we evaluated exposure through whole-sediment testing; the organism used as a model was the amphipod T. viscana, and the extent of exposure tested was based on the pellet densities previously found in this study along the coast of São Paulo and the maximum densities reported in the literature. The function of the second test was to determine whether pellets would become toxic to marine organisms under real exposure conditions. Pellets were collected from two areas with different levels of contamination and toxicity to marine organisms; in both places, high quantities of pellets were quickly obtained. The first area was the Santos Bay, a region historically contaminated by different types of pollutants such as metals, PAHs, detergents, pharmaceuticals, and personal care products (Abessa et al., 2008; Pereira et al., 2016; dos Santos et al., 2018), with high toxicity

reported in the water column (Moreira and Abessa, 2014; Sousa et al., 2014) and in the sediments (Sousa et al., 2014; Araujo et al., 2013; Maranho et al., 2008). The second area was Itaguaré Beach, a reference site in the city of Bertioga (CETESB, 2016), located near a state park known as the Parque Estadual Restinga de Bertioga and within a Marine Protected Area known as the Área de Proteção Ambiental Marinha Litoral Centro (APAMLC), which is relatively far from contamination sources and which is often used as a control site in ecotoxicological studies (Ferraz, 2013). A second site from the city of Bertioga was sampled: Riviera de São Lourenço Beach, where 100% of the sewage is collected and treated through tertiary treatment and disposed far from the beach at the Itapanhaú River. In each area, samplings were conducted twice (June and September 2015) through an active search on the high tide line, and the pellets were kept refrigerated at 5 to 10 ◦ C. 2.2.1. Testing pellet toxicity for marine organisms in cases of NonIngested exposure To determine the extent to which the pellets were toxic to the marine organisms exposed to them, L. variegatus embryos and adult ovigerous females of Nitocra sp. were exposed to pellets in an aqueous medium. For each of these tests, the pellets were separated by origin and by color (white or yellow). In both assays, 10 mL of test solution was used. The solution consisted of 8 mL of filtered seawater and 2 mL of pellets; this quantity is equivalent to approximately 40 pellets. Details of the exposure conditions are provided below. In the test with L. variegatus, a net was used to keep the pellets separate from the organisms, since sea urchin embryos are sensitive to direct contact with small particles during the initial planktonic stages. Four replicates were prepared for each treatment, as was a clean seawater control treatment and a net control treatment. Pellets were placed in the lower section of the test chamber. A sterilized synthetic net (0.5 mm pores) was placed over the pellets in order to reserve the upper portion of the chamber for only the aqueous solution in which the sea urchin embryos were kept and could swim freely. Newly fertilized L. variegatus eggs were exposed to pellets for 24 h, and at the end of each assay, buffered formalin was added to each test chamber. The first 100 larvae were then counted (US EPA, 1991; Brazilian National Standards Organization [ABNT]/Standard No. NBR 15350

G.M. Izar, L.G. Morais, C.D.S. Pereira et al. / Regional Studies in Marine Science 29 (2019) 100705

, 2012) and classified according to their morphologies: (a) normal: larvae that reached the Pluteus stage; or (b) affected: embryos with retarded development or which were abnormal in shape. Perina et al. (2011) have provided a helpful description of the possible abnormalities of L. variegatus embryos. In the experiments with Nitocra sp., pellets were arranged in the water column without net separation. In this case, the pellets remained floating within the test chambers to allow for direct contact with the organisms. Five ovigerous females were exposed for 10 days, and the offspring (nauplii and copepodites) were counted and divided by the number of adult females (Lotufo and Abessa, 2002). For both tests, the data were analyzed in steps. In the first step, we used a one-way ANOVA to confirm the toxicity by verifying the differences between the treatments and the controls (only water, and water and net for L. variegatus assay; and only water for Nitocra sp.). In the next step, we analyzed the data using a two-way multi-factor ANOVA: Color (Factor 1, fixed, 2 levels) and Beach (Factor 2, fixed, 4 levels). The second analysis was performed without considering the controls in order to identify the influence of the factors on pellet toxicity. 2.2.2. Densities at which pellets become toxic to marine organisms We conducted two bioassays with Tiburonella viscana (Melo and Abessa, 2002; ABNT/NBR 15638 , 2008) to test the acute toxicity of sediments contaminated by pellets at different densities, as explained previously. Reference inert sediment from Engenho D’Água Beach in the city of Ilhabela was used for the bioassays, and this sediment was contaminated with pellets using the spiking method described by Simpson et al. (2004). The first bioassay was based on the densities of the pellets found along the São Paulo coast (1, 2, 5, and 10 pellets per 100 g). The exception was those found at Itaquitanduva Beach, where the pellet density was considered an outlier in the area. In the second experiment, which was based on the results of the first bioassay, we extended the range of pellet densities in order to include other realistic values, including those found on Itaquitanduva Beach, as well as the highest densities found in the literature (Mcdermid and Mcmullen, 2004), the density associated with acute toxicity in the first bioassay, and finally, the most common density on São Paulo State beaches. Thus, in this experiment, the sediments tested had pellet densities of 2, 10, 60, and 120 pellets per 100 g of sediment. To calculate the pellet density used in each bioassay with T. viscana, the densities were converted to pellets per m3 (Turra et al., 2014) and then transformed into a value corresponding to a proportion of 100 ml of sediment. Pellets were selected randomly for each density tested, regardless of color or beach of origin. All acute toxicity tests lasted 10 days and consisted of the exposure of 40 healthy T. viscana adults per treatment, which were divided into 4 replicates for each pellet density (each replicate contained 10 individuals). Mortality rates were determined by the number of dead or missing organisms after 10 days. The results of both bioassays were analyzed by a one-way ANOVA: Treatments (Factor 1, fixed, 5 levels). 3. Results We collected a total of 13,138 pellets from the four different sampling surveys conducted in 2015. The most common pellets’ color was white (50%), followed by yellow (30%) and orange (10%). Pellets of other colors (brown, black, and pigmented) were less common (10%).

5

Table 1 Results of the three-factor ANOVA: comparison of pellet densities between beaches influenced by the following factors: Beach (Factor 1, random, 3 levels), Sample area (Factor 2, fixed, 2 levels), and depth (Factor 3, fixed, 3 levels). Bold numbers represent significant p values. Source

Df

MS

F

p

Beach Sample area Depth Beach x Depth Beach x Sample area Sample Area x Depth Beach x Sample area x Depth Residual

2 1 2 4 2 2 4 72

25.14 4.56 11.56 0.71 0.38 4.17 0.63 0.34

52.63 11.74 16.07 1.14 0.61 6.61 1.81

0.156 0.075 0.012 0.450 0.584 0.053 0.135

Fig. 3. Pellet distribution at sections of the beach and sediment depths. Comparison of pellet densities between the higher high tide line and the upper limit of the backshore at three different depths. The data from the three beaches sampled were integrated into this graph due to non-interference of the Beach factor for pellet dispersion. Sample corer had a 7.5 cm radius.

3.1. Quantitative Analysis of Pellets on the São Paulo Coast A significant difference in pellet distribution was observed at the different sediment depths regardless of other factors such beach and sample site (Table 1). The surface layer of the sediments had the highest pellet density. However, even close to the threshold of significance (p = 0.053), the interaction between depth and sample site suggests a trend in the distribution of pellets, with higher pellet densities on the high tide line than on the backshore, where the densities are more homogeneous between depths (Fig. 3). The tests revealed a clear trend of decreasing pellet densities on São Paulo beaches the farther the beach was located from the Port of Santos (R2 = 0.4369, p < 0.05, y = 4.3702–1.0804x), regardless of the beach’s northernly or southerly location (Fig. 4). Beaches located inside the Santos Bay, which are strongly influenced by the port, were found to have higher pellets densities, followed by the beaches located close to the port (10 km) but outside of the bay. Itaquitanduva Beach (10 km) was found to have a much higher mean number of beached pellets when compared to the averages found on the other beaches (Fig. 5). Cambury (170 km the northernmost beach included in this study) was the only beach on which no pellets were found. 3.2. Ecotoxicological effects of pellets on marine organisms in an aqueous solution The results of the toxicity test with L. variegatus specimens differed between some of the treatments (one-way ANOVA, F = 12.28, df = 9, p < 0.001, Tukey post-hoc), indicating that toxic effects were experienced by organisms exposed to pellets of different colors from Góes Beach and Riviera de São Lourenço Beach, and to white pellets from Itaguaré Beach (Fig. 6A).

6

G.M. Izar, L.G. Morais, C.D.S. Pereira et al. / Regional Studies in Marine Science 29 (2019) 100705 Table 3 Two-way ANOVA comparing pellet toxicity between the treatments (Nitocra sp. female fertility) under the influence of the factors color (Factor 1, fixed, 2 levels) and beach (Factor 2, random, 4 levels). Bold numbers represent significant p values.

Fig. 4. Relationship between the densities of pellets (pellets/m3 ) on different beaches along the coast of São Paulo State and their respective distances from the Port of Santos (Km). A logarithmic relationship (adjusted Log for linear regression: R2 = 0.4369, p < 0.05, y = 4.3702–1.0804x) between densities of beach-stranded pellets and the distance of the beach from the Port of Santos was determined. Data were log-transformed to base 10. Table 2 Two-way ANOVA comparing pellet toxicity in the treatments (L. variegatus larval abnormalities) under the influence of the factors color (Factor 1, fixed, 2 levels) and beach (Factor 2, random, 4 levels). Bold numbers represent significant p values. Source

df

MS

F

p

Color Beach Color x Beach Residual

1 3 3 24

371.28 4287.36 604.95 313.24

1.18 13.68 1.93

<0.001

0.287 0.151

When the treatments were compared, only the Beach factor was found to be relevant to the toxicity experienced by L. variegatus (Table 2). This factor was clearly divided into three levels of toxicity (Fig. 6B), in that the pellets sampled from Góes Beach were found to be highly toxic, while the pellets from Riviera de São Lourenço Beach and Itaguaré Beach exhibited intermediate toxicity, and the pellets from Itaquitanduva Beach exhibited no toxicity. In the tests with Nitocra sp., no treatment was found to be toxic to adult organisms (one-way ANOVA, F = 1.48, df = 8, p = 0.206). However, there was a difference in female fecundity in all treatments relative to controls (one-way ANOVA, F = 15.89, df = 8, p < 0.001, Tukey post-hoc), a result that shows that exposure to pellets induced chronic toxicity in this species in all

Source

df

MS

F

p

Color Beach Color x Beach Residual

1 3 3 24

1.62 709.59 163.86 82.05

0.01 8.64 1.99

0.889 <0.001 0.141

treatments (Fig. 6C). The Beach factor was found to be highly significant (p < 0.001) for female fertility (Table 3), as well as for embryos of L. variegatus, results which reinforce the presence of a local variability pattern for pellet toxicity and which exclude the Color factor. The yellow pellets from Itaguaré Beach were less toxic than those from Itaquitanduva Beach (which were both yellow and white) and were also less toxic than yellow pellets from Góes Beach (Fig. 6D). In the T. viscana assay, only 10 pellets per 100 g of sediment induced acute toxicity in the first test (one-way ANOVA, F = 3.61, df = 15, p < 0.05, Tukey post-hoc) (Fig. 7A). In the second test, in which densities were higher, no toxicities were observed (one-way ANOVA, F = 5.25, df = 4, p = 0.068) (Fig. 7B). 4. Discussion The pellet distribution pattern found in this study is similar to those reported in studies at other regions, with a higher quantity of pellets in the upper limit of the backshore (Heo et al., 2013; Thornton and Jackson, 1998; Zurcher, 2009; Turner and Holmes, 2011; Turra et al., 2014). For the pellets on the standing stock, coastal dunes were considered better sampling areas than other parts of the beach (Moreira et al., 2016). However, the same pattern was not found in the surface beach layer (0 cm to 5 cm), where the highest quantities of pellets were found at the hide tide line, but with substantial variability between replicates. This variability could be attributed to the greater influence of waves and tide dynamics at the hide tide line, where the tide pulls out and moves the pellets around. Therefore, we argue that the upper limit of the backshore is the best area for sampling pellets on sandy beaches when a coastal dune does not exist or is occupied by human structures and/or if the region has prevailing onshore

Fig. 5. Map of the São Paulo State coast, showing the respective densities of plastic pellets along the state’s beaches.

G.M. Izar, L.G. Morais, C.D.S. Pereira et al. / Regional Studies in Marine Science 29 (2019) 100705

7

Fig. 6. (A) Percentage of normally developed L. variegatus embryos in the toxicity tests. (B) Data analysis without controls and with different treatments for two factors (sampling site and color of pellets). (C) Fecundity of Nitocra sp. exposed to different pellet treatments (sampling site and pellet colors). (D) Data analysis without controls and with different treatments for two factors (sampling site and pellet colors). *Asterisks indicate significant toxicity (p < 0.05) relative to the respective controls (one-way ANOVA). Different letters indicate significant differences between beaches in terms of pellet toxicity.

Fig. 7. Survival of Tiburonella viscana exposed to sediments with plastic pellets; (A) experiment with different beach-stranded pellet densities found on São Paulo State beaches (pellets/100 g of sediment). (B) experiment with higher pellet densities. *Asterisks indicate treatments that induced acute toxicity in organisms (p < 0.05) relative to their respective exposure controls (one-way ANOVA)

winds. Our results corroborate the literature in that they indicate where the standing stocks with the most pellets are located, even considering the fact that we sampled pellets from surface layers of the beach sediments (only a few centimeters deep). Turra et al. (2014) and Fisner et al. (2017) proposed that pellets are best sampled up to 1 meter in depth. However, even when our samples did not reach this depth, we observed a stable pattern in pellet density at all depths across all of the beaches tested. This difference can be attributed to the daily beach cleaning that occurs at Santos Beach, which may remove the pellets from the surface layers or bury them at deeper layers, as has been explained by Moreira et al. (2016). Pellet concentrations across the coastal area of São Paulo State were higher surrounding the Port of Santos, where active pellet transport occurs. However, pellets were found at more distant locations as well, as these plastics can be spread rapidly. Beaches

inside the Santos Bay presented the highest pellet densities, reflecting their proximity to the sources of pellets. Fewer pellets were found the farther the sampling area was from the port. Despite the lack of temporal replicates, it was possible to identify this gradient, because higher pellet densities occurred in beaches located closest to the Port of Santos and inside the Santos Bay, and decreasing densities were observed on the most distant beaches in both northward and southward directions (for example, on Cambury Beach, the northernmost sampling site, no pellets were collected). Both the transport and deposition of pellets along the coastline are likely influenced by a set of factors, such as surface wind regime, surface currents, waves, and the geographical position of the beach. Thus, the results confirmed our initial hypothesis regarding the occurrence and distribution of pellets along the coast. The Port of Santos, considered the largest port in Latin America, is likely the main source of pellets on the São Paulo

8

G.M. Izar, L.G. Morais, C.D.S. Pereira et al. / Regional Studies in Marine Science 29 (2019) 100705

coast; the densities of pellets found on the beaches located within the Santos Bay are among the highest densities in the world. This result is in accordance with previous findings obtained by Colton (1974), Ross et al. (1991), and Claessens et al. (2001), who reported that areas surrounding ports are common spots for microplastic abundance in coastal environments. However, some authors have found different results and have reported large quantities of microplastics in remote areas, far from ports and industrial regions (Gregory, 1983, 1999; Mcdermid and Mcmullen, 2004). These inconsistencies in the literature suggest that other factors may interfere with the dispersion of microplastics. For example, in findings that differed from those reported by Moreira et al. (2016), the densities of pellets on the beaches from the southern and northern regions of the São Paulo coast were similar in our study. The different strategies used to sample pellets may explain this difference: in our study, the backshore was sampled, while Moreira et al. (2016) sampled the coastal dunes. These differences also provide secondary hypotheses for new studies: Do beach morphodynamics constitute an influencing factor on pellet distribution and deposition? Can the presence of sites that facilitate deposition, such as coastal dunes, affect pellet distribution? As shown previously, the distance between the study site and the source of pellet pollution appears to be an important factor for pellet dispersion, but this dispersion may also be influenced by other factors, such as wind and surface marine currents, which were not tested in this study. Winds may influence pellet transport and deposition, although this influence was not identified in a study of microplastics dispersion toward beaches downwind by Browne et al. (2010). Winds acting directly on the surface of the water (Colling, 2001) have an almost direct effect on the pellets and microplastics floating on the water, influencing their dispersion along different beaches (Kukulka et al., 2012; Thornton and Jackson, 1998; Kim et al., 2015; Ross et al., 1991; Heo et al., 2013; Ivar do Sul et al., 2009). In the Santos Bay, the prevailing winds flow westward, and the surface ocean currents tend to flow counterclockwise (Harari and Camargo, 1998; Harari and Gordon, 2001). The short distance from the source of pellet pollution combined with the wind and surface currents in the region results in the large number of pellets found on Itaquitanduva Beach. In a global context and using extrapolation to compare pellet densities (in pellets per m3 ) as proposed by Turra et al. (2014), two of the beaches studied herein (Góes and Itaquitanduva, both of which are located inside the Santos Bay) were found to have high pellet densities. Itaquitanduva Beach had the third highest pellet density out of all of the beaches reported in the literature, behind only Cargo Beach, located in the Midway Atoll northwest of the Main Hawaiian Islands (Mcdermid and Mcmullen, 2004) and the northeastern beach on South Soko Island, located on the west coast of Hong Kong (Zurcher, 2009). Because Itaquitanduva Beach is located within a State Park (a Protected Area), the extent of its plastic pellet pollution has further implications for the conservation of the area. Santos Beach (Turra et al., 2014), which is also located inside the Santos Bay, was found to have pellets at one third of the density of the pellet density found on Góes Beach and at one nineteenth of the density found on Itaquitanduva Beach. This comparison can be explained by the fact that the method used in the prior study on Santos Beach sampled all of the beach zones, not only the backshore, a difference which produced a lower average pellet density for Santos Beach. Moreover, Santos Beach has a public cleaning service, which may remove a certain amount of pellets. Unfortunately, the public screening and sweeping services do not remove all microplastics. The removal of marine debris by mechanical shovels can remove the larger granules but can simultaneously bury pellets deeper into the sand layers. This result exemplifies the difficulty of comparing

pellet density data obtained using different sampling methods, as well as the importance of uniformity in units and sampling. This comparison could also confirm the influence of wind and surface currents on pellet dispersion, since these three beaches are located inside in the same bay, but only Itaquitanduva faces the predominant direction of the winds and surface currents of the region (Harari and Gordon, 2001). Sediment toxicity spiked only when the highest concentration of pellets was tested in the first experiment (10 pellets per 100 g of sediment). However, in the second bioassay, even when much higher concentrations of pellets were considered, no toxicity was found. These results suggest that the densities of pellets found on beaches in São Paulo State were not high enough to induce acute toxicity in T. viscana, nor was the highest pellet density found in the literature. It is important to note, however, that pellets induced toxicity when water exposure was included in the test. This finding has important ecological concerns because of the potential effect of this toxicity on the structure and quality of aquatic communities. The toxicity experienced by L. variegatus embryos and Nitocra sp. was observed only when extremely high pellet densities were tested, densities which have not yet been found in any coastal environments. However, considering the well-known 8% annual growth trend of plastics in the world’s oceans (Ogata et al., 2009), it is not unreasonable to predict that such densities could be achieved in the very near future. Our results also differed from those obtained by Nobre et al. (2015), who observed a higher toxicity produced by virgin pellets when compared to beach-stranded pellets. In our study, this difference was not found. This result suggests that, once released into the aquatic environment, virgin pellets are likely to release chemicals into the water, consequently reducing their toxic potential as produced by chemical additives. It is known that some industrial additives undergo fast biodegradation when dissociated and released into the marine environment (Teuten et al., 2009). The white pellets found in this study have been in the environment for some time, thus becoming weathered and exchanging chemicals with the medium. On the other hand, the long presence of the pellets in the sea can also result in increased structural degradation, increasing pellet surface area and, consequently, their hydrophobic contaminant adsorption capacity (Endo et al., 2005; Ogata et al., 2009). In this sense, pellets crossing clean waters will tend to reduce the amounts of adsorbed chemicals, while those crossing polluted waters will tend do adsorb more chemicals. In our study, few differences were observed in the comparison of toxicity levels between white and colored pellets. Pellet toxicity was found to depend not only on color, but also on the beach and species tested. Therefore, when considering beach-stranded pellets, both the loss of additives and the adsorption of new compounds may have influenced pellet toxicity, despite the tendency of yellow pellets to have a higher concentration of adsorbed POPs (Endo et al., 2005). However, due to their high Kow (affinity for lipophilic compounds and strong hydrophobicity), many POPs can remain heavily adsorbed on the pellets and will not be released into water, sediment, or biota. Despite the fact that pellets from all of the beaches tested induced toxicity in the harpacticoid copepod Nitocra sp., in the two toxicity tests with the aqueous solutions (i.e. the tests on both L. variegatus and Nitocra sp.), the differences in the toxicity observed were dependent on the pellets’ collection site. According to Mato et al. (2002) and Endo et al. (2005), pellets can be used as indicators of local marine pollution; thus, we expected to find higher toxicity among pellets collected on the beaches inside and close to the Santos Bay. This pattern was obtained in the tests with Nitocra sp., but not in the assay with L. variegatus embryos. We therefore cannot associate pellet toxicity with local

G.M. Izar, L.G. Morais, C.D.S. Pereira et al. / Regional Studies in Marine Science 29 (2019) 100705

contamination. Pellets from Itaguaré Beach and other neighboring beaches (such as Guarujá) were found to have high levels of PCBs and DDTs, which were even higher than those reported for pellets from Itaquitanduva Beach (Taniguchi et al., 2016). This can at least partially explain the differences in the patterns between the two tests. Pellets from Itaguaré and Riviera de São Lourenço, the least contaminated sites (CETESB, 2016), were found to have higher levels of POPs than the pellets from Itaquitanduva Beach and Góes Beach, two highly contaminated areas, because they could have adsorbed high levels of POPs inside the Santos Bay just after their release to the environment, and on their way to the beach where they were eventually deposited. This possibility raises concerns that pellets could carry chemicals to remote areas, a phenomenon also observed by Heskett et al. (2012). Further studies are necessary to establish a relationship between pellet contamination, their sources, and the levels of environmental contamination both in the pellet origins and at the sites from where they are eventually sampled. In any case, the levels of POPs on pellets from Itaguaré Beach show that contaminated pellets had reached that site, and this idea of transport could explain the toxicity observed. The difference between the responses exhibited by the two organisms tested may reflect the way in which they were exposed to the pellets: Nitocra sp. was directly exposed and remained in contact with the pellets, whereas the L. variegatus embryos were tested in the assays without direct contact with the pellets; they were exposed only to chemicals released from the pellets into the overlying water. No toxicity was observed only in the case of the assay involving L. variegatus and the pellets from Itaquitanduva Beach, despite the fact that toxic effects were expected due to the beach’s location and pellet density. The contaminants may have been adsorbed onto the pellets and not released into the water. The toxicity associated with pellets from Góes Beach is concerning, as both organisms tested experienced negative effects (L. variegatus and Nitocra sp.). This site is directly influenced by the port and local shipyards, and it is also exposed to pollution resulting from the release of untreated sewage (Lamparelli et al., 2001). Thus, the pellets may have adsorbed additional chemicals, such as pharmaceuticals and personal care products (PPCPs) and antifouling biocides. More studies on this beach and its surrounding region are important to understand the severity of the impacts generated. The toxicity of the pellets collected from Riviera de São Lourenço Beach and Itaguaré Beach, the most well conserved beaches in this study, provide further support to the theory that pellets are able to carry local contamination to remote locations (Taniguchi et al., 2016). Overall, the results of this study support previous findings that pellets tend to accumulate on beaches located close to ports that transport them, and that their quantities tend to decrease the farther the beach is from said port. The presence of pellets at high concentrations may be sublethally toxic to marine organisms, an effect which has negative implications for the health of the ecosystem, particularly when the increasing amounts of plastics predicted to be introduced to the seas are considered. White pellets were not found to be more toxic than yellow pellets, a result which was contrary to expectations. The site where pellets are collected may be an important factor in determining pellet toxicity, since the results obtained herein reflect a tendency of pellet toxicity to reflect local contamination in historically contaminated areas. However, pellets collected in regions with low levels of contamination were also found to be toxic; this finding suggests that pellets are able to transport contamination to remote areas and contamination-free regions; it is in opposition to our original hypothesis. Pellets can also be toxic in contamination-free regions.

9

This study provides relevant information on pellet dispersion and the impacts of pellets on coastal environments. The findings confirm the existence of a relationship between pellet densities on beaches and the beach’s distance from the source of pellet pollution. Moreover, plastic pellets seem to be toxic to benthic organisms in that they affect their reproduction and development and even produce lethal effects. These results are alarming because they suggest that plastic pellets are chemical carriers for pollutants, and that their presence in the marine environment may have negative effects on the function and structure of benthic marine communities. Acknowledgments We would like to thank the Brazilian National Council for Scientific and Technological Development (CNPq), Brazil for the financial support. We would also like to thank the ecotoxicological team in Santos, especially our colleagues from the Center of Investigation in Aquatic Pollution and Ecotoxicology (NEPEA). A. Cesar; C.D.S. Pereira, D.M.S. Abessa, and R.A. Christofoletti are grateful to CNPq for the fellowships (PQ 310354/2016-1; PQ 306705/2016-8; PQ 311609/2014-7, and PQ 311560/2016-4). We would also like to thank Aleicia Holland, PhD (La Trobe University, Australia) and Tan Tjui-Yeuw, who kindly revised the final version of this manuscript. References Abessa, D.M.S., Carr, R.S., Souza, E.C.P.M., Rachid, B.R., Zaroni, L.P., Gasparro, M., Pinto, Y.A., Bicego, M.C., Hortellani, M.A., Sarkis, J.E.S., Muniz, P., 2008. Integrative ecotoxicological assessment of contaminated sediments in a complex tropical estuarine system. In: Mar. Pollut. Bull. New Research. Nova Science Publishers Inc., New York, pp. 125–159. Araujo, G.S., Moreira, L.B., Morais, R.D., Davanso, M.B., Garcia, T.F., Cruz, A.C.F., Abessa, D.M.S., 2013. Ecotoxicological assessment of sediments from an urban marine protected area (Xixová-Japuí State Park, SP, Brazil). Mar. Pollut. Bull. http://dx.doi.org/10.1016/j.marpolbul.201308005, online. Ashton, K., Holmes, L., Turner, A., 2010. Association of metals with plastic production pellets in the marine environment. Mar. Pollut. Bull. 60 (11), 2050–2055. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS ABNT, ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS ABNT. NBR 15638, 2008. Qualidade da Água – Determinação da toxicidade aguda de sedimentos marinhos ou estuarinos com anfípodos, 17. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS ABNT, ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS ABNT. NBR 15350, 2012. Ecotoxicologia Aquática. – Toxicidade crônica de curta duração – Método de ensaio com Ouriço-do-mar (Echinodermata: Echinoidea), 21. Baird, R.W., Hooker, S.K., 2000. Ingestion of plastic and unusual prey by a juvenile harbour porpoise. Mar. Pollut. Bull. 40 (8), 719–720. Bejgarn, S., Macleod, M., Bogdal, C., Breitholtz, M., 2015. Toxicity of leachate from weathering plastics: An exploratory screening study with Nitocra spinipes. Chemosphere 132, 114–119. Bjorndal, K.A., Bolten, A.B., Lagueux, C.J., 1994. Ingestion of marine debris by juvenile sea turtles in coastal florida habitats. Mar. Pollut. Bull. 28 (3), 154–158. Browne, M.A., Galloway, T.S., Thompson, R.C., 2010. Spatial patterns of plastic debris along estuarine shorelines. Environ. Sci. Technol. 44 (9), 3404–3409. Castro Filho, B.M., Miranda, L.B., Miyao, S.Y., 1987. Condições hidrográficas na plataforma continental ao largo de Ubatuba: Variações sazonais e em média escala. Bol. lnstituto Ocean 35, 135–151. COMPANHIA DE TECNOLOGIA DE SANEAMENTO AMBIENTAL CETESB, 2016. Qualidade das águas superficiais do Estado de São Paulo. Parte 2 - Águas salinas e salobras 2015. p. 164. Cheshire, A.C., Adler, E., Barbière, J., Cohen, Y., Evans, S., Jarayabhand, S., Jeftic, L., Jung, R.T., Kinsey, S., Kusui, E.T., Lavine, I., Manyara, P., Oosterbaan, L., Pereira, M.A., Sheavly, S., Tkalin, A., Aradarajan, S., Wenneker, B., Westphalen, G., 2009. UNEP/IOC Guidelines on Survey and Monitoring of Marine Litter. In: UNEP regional seas reports and studies, p. 131. Claessens, M., De Meester, S., Van Landuyt, L., De Clerck, K., Janssen, C.R., 2001. Occurrence and distribution of microplastics in marine sediments along the belgian coast. Mar. Pollut. Bull. 62 (10), 2199–2204. Colling, A.A., 2001. Ocean Circulation, second ed. Open University Oceanography Course Team, ButterWorth-Heinemarin, p. 286. Colton, Jr., J.B., 1974. Plastics in the ocean. Oceanus 18 (1), 61–64.

10

G.M. Izar, L.G. Morais, C.D.S. Pereira et al. / Regional Studies in Marine Science 29 (2019) 100705

Davison, P., Asch, R.G., 2011. Plastic ingestion by mesopelagic fishes in the North Pacific Subtropical Gyre. Mar. Ecol. Prog. Ser. 432, 173–180. Derraik, J.G.B., 2002. The pollution of the marine environment by plastic debris: a review. Mar. Pollut. Bull. 44, 842–852. Endo, S., Takizawa, R., Okuda, K., Takada, H., Chiba, K., Kanehiro, H., Ogi, H., Yamashita, R., Date, T., 2005. Concentration of polychlorinated biphenyls (PCBs) in beached resin pellets: Variability among individual particles and regional differences. Mar. Pollut. Bull. 50, 1103–1114. Ferraz, M.A., 2013. Desenvolvimento E Aplicação De Um Método Para a Avaliação E Identificação Da Toxicidade Usando O Organismo Bentônico Nitocra Sp (Crustacea: Copepoda: Harpacticoida) (M.sc. Dissertation). 2013, (84), Universidade Santa Cecília University, Santos, Brazil. Fisner, M., Majer, A.P., Balthazar-Silva, D., Gorman, D., Turra, A., 2017. Quantifying microplastic pollution on sandy beaches: the conundrum of large sample variability and spatial heterogeneity. Environ. Sci. Pollut. Res. 24 (15), 13732–13740. Fisner, M., Taniguchi, S., Majer, A.P., Bícego, M.C., Turra, A., 2013. Concentration and composition of polycyclic aromatic hydrocarbons (pahs) in plastic pellets: implications for small-scale diagnostic and environmental monitoring. Mar. Pollut. Bull. 76 (1–2), 349–354. Gorman, D., Moreira, F.T., Turra, A., Fontenelle, F.R., Combi, T., Bícego, M.C., Martins, C.C., 2019. Organic contamination of beached plastic pellets in the south atlantic: Risk assessments can benefit by considering spatial gradients. Chemosphere 223, 608–615. Gregory, M.R., 1978. Accumulation and distribution of virgin plastic granules on New Zealand beaches. N.Z. J. Mar. Freshw. Res. 12 (4), 399–414. Gregory, M.R., 1983. Virgin plastic granules on some beaches of eastern Canada and Bermuda. Mar. Environ. Res. 10, 73–92. Gregory, M.R., 1999. Plastics and south pacific island shores: environmental implications. Ocean Coast. Manag. 42, 603–615. Hammer, J., Kraak, M.H.S., Parsons, J.R., 2012. Plastics in the marine environment: The dark side of a modern gift. Rev. Environ. Contam. Toxic. 220, 1–44. Harari, J., Camargo, R. de, 1998. Modelagem numérica da região costeira de Santos (SP): Circulação de maré. Rev. Bras. Oceanogr. 46 (2), 135–156. Harari, J., Camargo, R., Franca, C.A.S., Mesquita, A.R., Picarelli, S.S., 2006. Numerical modeling of the hydrodynamics in the coastal area of sao paulo state Brazil. J. Coast. Res. 39, 1560–1563. Harari, J., Gordon, M., 2001. Simulações Numéricas da Dispersão de Substâncias no Porto e Baía de Santos, sob a Ação de Marés e Ventos. Rev. Bras. Recursos Hídricos 6 (4), 115–131. Heo, N.W., Hong, S.H., Han, G.M., Hong, S., Lee, J., Song, Y.K., Jang, M., Shim, W.J., 2013. Distribution of small plastic debris in cross-section and high strandline on heungnam beach. South Korea Ocean Sci. J. 48 (2), 225–233. Heskett, M., Takada, H., Yamashita, R., Yuyama, M., Ito, M., Geok, Y.B., Ogata, Y., Kwan, C., Heckhausen, A., Taylor, H., Powell, T., Morishige, C., Young, D., Patterson, H., Robertson, B., Bailey, E., Mermoz, J., 2012. Measurement of persistent organic pollutants (POPs) in plastic resin pellets from remote islands: Toward establishment of background concentrations for international pellet watch. Mar. Pollut. Bull. 64 (2), 445–448. Hidalgo-Ruz, V., Gutow, L., Thompson, R.C., Thiel, M., 2012. Microplastics in the marine environment: A review of the methods used for identification and quantification. Environ. Sci. Technol. 46 (3060–3075). Holmes, L.A., Turner, A., Thompson, R.C., 2012. Adsorption of trace metals to plastic resin pellets in the marine environment. Environ. Pollut. 160, 42–48. Ivar do Sul, J.A., Spengler, A., Costa, M.F., 2009. Here, there and everywhere. small plastic fragments and pellets on beaches of fernando de noronha (Equatorial Western Atlantic). Mar. Pollut. Bull. 58 (8), 1236–1238. Jayasiri, H.B., Purushothaman, C.S., Vennila, A., 2015. Bimonthly variability of persistent organochlorines in plastic pellets from four beaches in mumbai coast, India. Environ. Monit, Assess. 187 (7), 469. Karlsson, T.M., Arneborg, L., Broström, G., Almroth, B.C., Gipperth, L., Hassellöv, M., 2018. The unaccountability case of plastic pellet pollution. Mar. Pollut. Bull. 129 (1), 52–60. Khordagui, H.K., Abu-Hilal, A.H., 1994. Industrial plastic on the southern beaches of the Arabian Gulf and the western beaches of the Gulf of Oman. Environ. Pollut. 84 (3), 325–327. Kim, I.S., Chae, D.H., Kim, S.K., Choi, S., Woo, S.B., 2015. Factors influencing the spatial variation of microplastics on high-tidal coastal beaches in Korea. Arch. Environ. Contam. Toxic. 69 (3), 299–309. Koch, H.M., Calafat, A.M., 2009. Human body burdens of chemicals used in plastic manufacture. Philos. Trans. R. Soc. B 364 (1526), 2063–2078. Kukulka, T., Proskurowski, G., Morét-Ferguson, S., Meyer, D.W., Law, K.L., 2012. The effect of wind mixing on the vertical distribution of buoyant plastic debris. Geophys. Res. Lett. 39 (7). Laist, D.W., 1987. Overview of the biological effects of lost and discarded plastic debris in the marine environment. Mar. Pollut. Bull. 18 (6B), 319–326. Lamparelli, M.L., Costa, M.P., Prósperi, V.A., Bevilácqua, J.E., Araújo, R.P.A., Eysink, G.G.L., Pompéia, S., 2001. Sistema Estuarino de Santos e São Vicente. Relatório Técnico. CETESB, São Paulo, SP, p. 163.

Lechner, A., Ramler, D., 2015. The discharge of certain amounts of industrial microplastic from a production plant into the river danube is permitted by the Austrian legislation. Environ. Pollut. 200, 159–160. Li, H.X., Getzinger, G.J., Ferguson, P.L., Orihuela, B., Zhu, M., Rittschof, D., 2015. Effects of toxic leachate from commercial plastics on larval survival and settlement of the Barnacle Amphibalanus amphitrite. Environ. Sci. Technol. 50 (2), 924–931. Lotufo, G.R., Abessa, D.M.S., 2002. Testes de toxicidade com sedimento total e água intersticial estuarinos utilizando copépodos bentônicos. In: Nascimento, I.A., Sousa, E.C.P.M., Nipper, M. (Eds.), Métodos Em Ecotoxicologia Marinha. AplicaÇões No Brasil. Editora Artes Gráficas e Indústria Ltda, São Paulo, pp. 151–162. Manzano, A.B., 2009. DistribuiÇãO, Taxa de Entrada, ComposiÇãO Química E IdentificaÇãO de Fontes de GrâNulos PláSticos Na Enseada de Santos, SP, Brasil (M.sc. Dissertation). São Paulo University, São Paulo, Brazil, p. 122. Maranho, L.A., Davanso, M.B., Abessa, D.M.S., 2008. Avaliação da qualidade de sedimentos do porto de Santos: Determinação da toxicidade crônica e aguda. Holos Environ. 8 (2). Mato, Y., Isobe, T., Takada, H., Kahnehiro, H., Ohtake, C., Kaminuma, T., 2001. Plastic resin pellets as a transport mediumfor toxic chemicals in the marine environment. Environ. Sci. Technol. 35, 318–324. Mato, Y., Takada, H., Zakaria, M.P., Kuriyama, Y., Kanehiro, H., 2002. Toxic chemicals contained in plastic resin pellets in the marine environment— spatial difference in pollutant concentrations and the effects of resin type. Environ. Sci. 15, 415–423. Mcdermid, K.J., Mcmullen, T.L., 2004. Quantitative analysis of small-plastic debris on beaches in the hawaiian archipelago. Mar. Pollut. Bull. 48, 790–794. Melo, S.L.R., Abessa, D.M.S., 2002. Ensaios de ecotoxicidade com sedimentos marinhos utilizando anfípodos. In: Nascimento, I.A., Souza, E.C.P.M., Nipper, M.G. (Eds.), Métodos Em Ecotoxicologia Marinha: AplicaÇões No Brasil. Artes Gráficas e Indústria Ltda, São Paulo, pp. 163–178. Milijö, A., 2001. Marine Litter - Trash that Kills. Risbergs Uddevalla, Sweden, p. 20. Moore, C.J., Moore, S.L., Leecaster, M.K., Weisberg, S.B., 2001. A comparison of plastic and plankton in the North Pacific Central Gyre. Mar. Pollut. Bull. 42, 1297–1300. Moreira, L.B., Abessa, D.M.S., 2014. Water toxicity assessment of santos bay under different climate conditions. Ecotox. Environ. Contam 9 (1), 51–57. Moreira, F.T., Balthazar-Silva, D., Barbosa, L., Turra, A., 2016. Revealing accumulation zones of plastic pellets in sandy beaches. Environ. Pollut. 1–9. http://dx.doi.org/10.1016/j.envpol.2016.07.006, online. Moser, M.L., Lee, D.S., 1992. Fourteen-year survey of plastic ingestion by Western North Atlantic Seabirds. Colonial Waterbirds 15 (1), 83–94. Nobre, C.R., Santana, M.F.M., Maluf, A., Cortez, F.S., Cesar, A., Pereira, C.D.S., Turra, A., 2015. Assessment of microplastic toxicity to embryonic development of the sea urchin Lytechinus variegatus (Echinodermata: Echinoidea). Mar. Pollut. Bull. 92, 99–104. Ogata, Y., Takada, H., Mizukawa, K., Hirai, H., Iwasa, S., Endo, S., Mato, Y., Saha, M., Okuda, K., Nakashima, A., Murakami, M., Zurcher, N., Booyatumanondo, R., Zakaria, M.P., Dung, L.Q., Gordon, M., Miguez, C., Suzuki, S., Moore, C., Karapanagioti, H.K., Weerts, S., Mcclurg, T., Burres, E., Smith, W., Velkenburg, M.V., Lang, J.S., Lang, R.C., Laursen, D., Danner, B., Stewardson, N., Thompson, R.C., 2009. International pellet watch: Global monitoring of persistent organic pollutants (POPs) in coastal waters. 1. Initial phase data on PCBs, DDTs, and HCHs. Mar. Pollut. Bull. 58, 1437–1446. Pereira, C.D.S., Maranho, L., Cortez, F.S., Pusceddu, F.H., Santos, A.R., Ribeiro, D.A., Cesar, A., Guimarães, L.L., 2016. Occurrence of pharmaceuticals and cocaine in a Brazilian coastal zone. Sci. Total Environ. 548, 148–154. Pereira, F.C., Oliveira, A.L., Turra, A., 2011. Gestão de resíduos sólidos no ambiente marinho: pellets plásticos. In: V Simpósio Brasileiro de Oceanografia. Santos, SP, Brasil. Perina, F.C., Abessa, D.M.S., Pinho, G.L.L., Fillmann, G., 2011. Comparative toxicity of antifouling compounds on the development of sea urchin. Ecotoxicology 20 (8), 1870–1880. Pruter, A.T., 1987. Sources, quantities and distribution of persistent plastics in the marine environment. Mar. Pollut. Bull. 18 (6B), 305–310. Ross, J.B., Parker, R., Strickland, M., 1991. A survey of shoreline litter in Halifax Harbour 1989. Mar. Pollut. Bull. 22, 245–248. dos Santos, D.M., Buruaem, L., Goncalves, R.M., Williams, M., Abessa, D.M.S., Kookana, R., de Marchi, M.R.R., 2018. Multiresidue determination and predicted risk assessment of contaminants of emerging concern in marine sediments from the vicinities of submarine sewage outfalls. Mar. Pollut. Bull. 129, 299–307. Shiber, J.G., 1979. Plastic pellets on the coast of Lebanon. Mar. Pollut. Bull. 10 (1), 28–30. Shiber, J.G., 1982. Plastic pellets on Spain’s ’costa del sol’ beaches. Mar. Pollut. Bull. 13 (12), 409–412. Shiber, J.G., Barrales-Rienda, J.M., 1991. Plastic pellets, tar, and megalitter on beirut beaches, 1977-1988. Environ. Pollut. 71, 17–30.

G.M. Izar, L.G. Morais, C.D.S. Pereira et al. / Regional Studies in Marine Science 29 (2019) 100705 Simpson, S.L., Angel, B.M., Jolley, D.F., 2004. Metal equilibration and bioavailability in laboratory-contaminated (spiked) sediments used for the development of whole-sediment toxicity tests. Chemosphere 54 (5), 597–609. Sousa, E.C.P.M., Zaroni, L.P., Gasparro, M.R., Pereira, C.D.S., 2014. Review of ecotoxicological studies of the marine and estuarine environments of the Baixada Santista (São Paulo, Brazil). Braz. J. Oceanogr. 62 (2), 133–147. de Souza, R.B., Robinson, I.S., 2004. Lagrangian and satellite observations of the Brazilian coastal current. Cont. Shelf Res. 24, 241–262. Taniguchi, S., Colabuono, F.I., Dias, P.S., Oliveira, R., Fisner, M., Turra, A., Izar, G.M., Abessa, D.M.S., Saha, M., Hosoda, J., Yamashita, R., Takada, H., Lourenço, R.A., Magalhães, C.A., Bícego, M.C., Montone, R.C., 2016. Spatial variability in persistent organic pollutants and polycyclic aromatic hydrocarbons found in beach-stranded pellets along the coast of the state of São Paulo, southeastern Brazil. Mar. Pollut. Bull. 106 (1), 87–94. Teuten, E.L., Saquing, J.M., Knappe, D.R.U., Barlaz, M.A., Jonsson, S., Björn, A., Rowland, S.J., Thompson, R.C., Galloway, T.S., Yamashita, R., Ochi, D., Watanuki, Y., Moore, C., Viet, P.H., Tana, T.S., Prudente, M., Boonyatumanond, R., Zakaria, M.P., Akkhavong, K., Ogata, Y., Hirai, H., Iwasa, S., Mizukawa, K., Hagino, Y., Imamura, A., Saha, M., Takada, H., 2009. Transport and release of chemicals from plastics to the environment and to wildlife. Philos. Trans. R. Soc. B 364, 2027–2045. Thornton, L., Jackson, N.L., 1998. Spatial and temporal variations in debris accumulation and composition on an estuarine shoreline, Cliffwood Beach, New Jersey, USA. Mar. Pollut. Bull. 36 (9), 705–711.

11

Tomás, J., Guitart, R., Mateo, R., Raga, J.A., 2002. Marine debris ingestion in loggerhead sea turtles, Caretta caretta, from the Western Mediterranean. Mar. Pollut. Bull. 44, 211–216. Tourinho, P.S., Ivar do Sul, J.A., Fillmann, G., 2010. Is marine debris ingestion still a problem for the coastal marine biota of southern Brazil?. Mar. Pollut. Bull. 60, 396–401. Turner, A., Holmes, L., 2011. Occurrence, distribution and characteristics of beached plastic production pellets on the island of Malta (central Mediterranean). Mar. Pollut. Bull. 62 (2), 377–381. Turra, A., Manzano, A.B., Dias, R.J.S., Mahiques, M.M., Barbosa, L., BalthazarSilva, D., Moreira, F.T., 2014. Three-dimensional distribution of plastic pellets in sandy beaches: shifting paradigms. Nature 4 (4435). U.S. United State Environmental Protection Agency (US EPA), 1991. Technical Support Document for Water Quality-based Toxics Control. Office of Water, Washington, DC. EPA/505/2-90-001. U.S. United State Environmental Protection Agency (US EPA), 1992. Plastic Pellets in the Aquatic Environment: Sources and Recommendations. Washington, DC. EPA 842-B-92-010. Van Cauwenberghe, L., Devriese, L., Galgani, F., Robbens, J., Janssen, C.R., 2015. Microplastics in sediments: A review of techniques, occurrence and effects. Mar. Environ. Res 1–13. http://dx.doi.org/10.1016/j.marenvres.2015.06.007, online. Zurcher, N.A., 2009. Small Plastic Debris on Beaches in Hong Kong: An Initial Investigation (M.sc. Dissertation). The University of Hong Kong, Hong Kong, China, p. 81.