Environmental Pollution 266 (2020) 115241

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Environmental Pollution

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Contamination of stream fish by plastic waste in the Brazilian Amazon*

* Danielle Regina Gomes Ribeiro-Brasil a, b, , Naiara Raiol Torres a, c, Ana Beatriz Picanço a, David Silva Sousa a, Vanessa Serrao~ Ribeiro a, Leandro Schlemmer Brasil a, d, Luciano Fogaça de Assis Montag a, c a Laboratorio de Ecologia e Conservaçao,~ Instituto de Ci^encias Biologicas, Universidade Federal do Para, Av. Perimetral, 2-224 - Guama, Belem, PA, 66077- 830, Brazil b Programa de Pos-graduaç ao~ em Ecologia, Universidade Federal do Para, Av. Perimetral, 2-224 - Guama, Belem, PA, 66077-830, Brazil c Programa de Pos-graduaç ao~ em Ecologia Aquatica e Pesca, Núcleo de Ecologia Aquatica e Pesca, Universidade Federal do Para, Av. Perimetral, 2651 - Terra Firme, Belem, PA, 66077-530, Brazil d Programa de Pos-graduaç ao~ em Zoologia, Universidade Federal do Para, Av. Perimetral, 2-224 - Guama, Belem, PA, 66077-830, Brazil article info abstract

Article history: Pollution by plastics is a global problem, in particular through the contamination of aquatic environ- Received 22 April 2020 ments and biodiversity. Although plastic contamination is well documented in the aquatic fauna of the Received in revised form oceans and large rivers of the world, there are few data on the organisms of headwater streams, espe- 9 July 2020 cially in tropical regions. In the present study, we evaluated the contamination of small fish by plastics in Accepted 10 July 2020 Amazonian streams. For this, we evaluated the shape and size, and the abundance of plastics in the Available online 19 July 2020 gastrointestinal tracts and gills of 14 fish species from 12 streams in eastern Brazilian Amazon. We used a Generalized Linear Mixed Model (GLMM) to compare the levels of contamination among species and Keywords: fi Fibers between organs. Only one individual of the 68 evaluated (a small cat sh Mastiglanis cf. asopos) contained Freshwater no plastic particles, and no difference was found in the contamination of the gills and digestive tract. Headstreams However, Hemigrammus unilineatus presented less contamination of both the gills and the digestive tract Pollution than the other species, while Polycentrus schomburgkii had less plastic in the gastrointestinal tract, Polymer whereas Crenicichla regani and Pimelodella gerii both had a larger quantity of plastic adhered to their gills in comparison with the other species. Nanoplastics and microplastics adhered most to the gills, while plastic fibers were the most common type of material overall. This is the first study to analyze plastic contamination in fish from Amazonian streams, and in addition to revealing high levels of contamina- tion, some species were shown to possibly be more susceptible than others. This reinforces the need for further, more systematic research into the biological and behavioral factors that may contribute to the greater vulnerability of some fish species to contamination by plastics. Amazonian stream fish show contamination by plastics. The species respond differently. The smaller the particle, the easier it is to adhere to the gills. © 2020 Elsevier Ltd. All rights reserved.

1. Introduction increasing need to understand the characteristics and dimensions of this environmental problem, the concentrations of plastics in the Plastic pollution is a relatively new field of ecological research, environment, the effects of this pollution on the biota, and the although it has gained momentum in recent years due to the measures needed to resolve these questions (McNeish et al., 2018; Su et al., 2019; Yuan et al., 2019). Despite recent advances in the research on the impact of these substances on public health and the fl * This paper has been recommended for acceptance by Eddy Y. Zeng. environment, there is a marked lack of studies on minor uvial * Corresponding author. Laboratorio de Ecologia e Conservaçao,~ Instituto de systems, in particular headwater streams, given that most recent Ciencias^ Biologicas, Universidade Federal do Para, Av. Perimetral, 2-224 - Guama, studies have focused on the marine environment (Wagner et al., Belem, PA, 66077-830, Brazil. 2014; Reid et al., 2019; Jacob et al., 2020) and major river systems E-mail address: [email protected] (D.R.G. Ribeiro-Brasil). https://doi.org/10.1016/j.envpol.2020.115241 0269-7491/© 2020 Elsevier Ltd. All rights reserved. 2 D.R.G. Ribeiro-Brasil et al. / Environmental Pollution 266 (2020) 115241

(Peters and Bratton, 2016; Andrade et al., 2019; Su et al., 2019). stream fish and the understanding of the degree of contamination While these studies have highlighted the problem of plastics in the by plastic waste are fundamental to the maintenance of a region’s aquatic environment resulting from the inadequate disposal of ecosystems. solid waste in riverside urban centers, there is, as yet, no evidence In the present study, we investigated the contamination by on the contamination of the ichthyofauna of headwater systems. plastics in small fish found in headwater streams of the eastern The ingestion of plastics by aquatic organisms is an emerging Amazon region, evaluating the size, shape, and abundance of plastic environmental crisis due to the proliferation of this pollutant in particles in the gastrointestinal tracts and gills of 14 fish species. aquatic ecosystems (Phillips and Bonner, 2015). This proliferation Based on this, we aimed to answer the following questions: (i) results from the contamination of environments by winds, leach- What is the frequency of the occurrence of plastic residues in the ing, and, in particular, the inadequate disposal of solid residues, of gastrointestinal tract and gills the fish that inhabit Amazonian both domestic and industrial origin (Wright et al., 2013; Wagner streams? (ii) Which species are more susceptible to the assimilation et al., 2014; Greven et al., 2016). The inadequate treatment of ef- of plastic particles through their gastrointestinal tract and gills? (iii) fluents is also a major contributor to the input of microparticles into Which types of plastic are assimilated most by the fish that inhabit aquatic environments (Imhof et al., 2013; Yuan et al., 2019). Amazonian streams? and (iv) Which size of plastic particle is most Small streams have an intimate relationship with the terrestrial ingested by Amazonian stream fish? environment, which provides large amounts of allochthonous Based on previous research in ocean environments (Mathalon material, such as fruit, terrestrial insects, seeds, and organic matter and Hill, 2014; Woodall et al., 2014; Yokota et al., 2017; Roch (Rezende and Mazzoni, 2006), but are also relatively vulnerable to et al., 2020) and, more recently, in Amazonian rivers and estu- the leaching of solid waste, including plastics (Silva-Cavalcanti aries (Pegado et al., 2018; Andrade et al., 2019), we predicted that et al., 2017). the stream fish are contaminated by a diversity of types of plastic, The ingestion and/or adherence of plastics exposes organisms to due to the existence of multiple sources of contamination, and that pollutants such as phthalates and bisphenol A e BPA (Barboza et al., contamination patterns are differentiated among species and or- 2020), given that these substances are components of many types gans (Wright et al., 2013; Wagner et al., 2014; Phillips and Bonner, of plastic (Wei et al., 2011; Jung et al., 2020). Chemical additives and 2015; Jabeen et al., 2017; Su et al., 2019). pathogenic microorganisms may also be adsorbed by nano- and microplastics (Wagner and Lambert, 2018). In particular, BPA has 2. Material and methods received increasing attention due to its effects as an endocrine disruptor, which has negative impacts on the endocrine, repro- 2.1. Study area ductive, and nervous systems (Krishnan et al., 1993; Rubin, 2011; Snijder et al., 2013; Valvi et al., 2013; Jung et al., 2020). The fish specimens were collected in the basin of the Guama In fish, plastic particles can cause intestinal damage, such as River, in the municipality of Barcarena, and in the Acara-Capim fissures in the villi (Lei et al., 2018), which permit the passage of basin, in the municipalities of Ipixuna do Para, Concordia do Para, nanoparticles which, upon entering the circulatory system, are and TomeAçu( Fig. 1), in eastern Para, Brazil. This region is subject phagocyted and interfere negatively in the absorption of nutrients to intense anthropogenic pressures, including large-scale defores- and the immune system of the organism (Greven et al., 2016). tation, in addition to its geographical position near the equator in a When ingested, in addition, plastic waste can accumulate in the zone of climatic and ecological transition (Coe et al., 2013; Marengo gastrointestinal tract and lead to the blockage of the intestine. The et al., 2018). The original vegetation, which is still predominant in consequences of intestinal blockage are unclear, but chronic plastic the region, is dense alluvial rainforest, although much of the orig- contamination is known to impact the fitness of species, in inal habitat has been converted to pasture, short and long cycle particular their foraging capacity and reproductive potential (Foley agriculture, urban development, and logging. The region’s climate et al., 2018). is humid tropical, consistent with the “Af” and “Afi” subtypes of the Most of the fish taxa studied up to now are species targeted by Koppen€ classification system, as adapted by Peel et al. (2007), with commercial fisheries, due to the interest in the potential for the annual rainfall of 2000 mm (Cavalcante et al., 2020). transfer of plastics to humans through the diet (Blettler et al., 2019; Su et al., 2019). While small stream fish are not typically consumed 2.2. Selection of the sampling sites by humans, they are of considerable ecological importance due to their occupation of different habitats, the consumption of both We sampled 150-m reaches of 12 streams, classified as first to autochthonous and allochthonous material, their role in nutrient third order in the scheme of Strahler (1957), as adapted by Horton cycling, and even their potential for the control of the larvae of (Scheidegger, 2006), and up to 4 m in width. We selected the study insect disease vectors (Fu et al., 2009). Previous research has also sites in order to best represent the gradient of anthropogenic shown that fish species with distinct ecological niches and patterns disturbance observed within the study region. We sampled the of habitat use have different rates of ingestion of plastics (Mizraji streams during the dry season, in September 2018. et al., 2017). The ingestion of plastic by fish may be accentuated when the 2.3. Sampling of fish fauna is dependent on resources derived from the riparian forest, as in the case of streams (Vannote et al., 1980), principally in areas We collected fish specimens using 55-cm diameter hand nets where the forest has been decimated by urbanization, which fa- (sieve) with a 3 mm mesh between opposite nodes. We adopt this cilitates the transport of solid residues as far as the water. A key active method for greater access to multiple microhabitats in question in the case of stream ecosystems is the exchange of ma- stream (Uieda and Castro, 1999) and for being widely spread in terial between the terrestrial and aquatic environments (Sullivan collect with streams in the Amazon (Leal et al., 2018; Prudente and Manning, 2019), including nutrients coming from the forest et al., 2018; Leitao~ et al., 2018). The fish were euthanized with the (Brejao~ et al., 2013), which is the trophic basis for stream fish (Veit anesthetic Eugenol, fixed in 10% formalin, and after 48 h, they were and Harrison, 2017; Feio et al., 2018), with fish acting as seed dis- transferred to 70% alcohol. We identified the specimens using persers (Correa et al., 2016) and controlling insect populations published taxonomic keys (Le Bail et al., 2000; Albert, 2001; Van (Sarwar, 2015; Why et al., 2016). Given this, the conservation of der Sleen and Albert, 2017). We carried the field expedition out D.R.G. Ribeiro-Brasil et al. / Environmental Pollution 266 (2020) 115241 3

Fig. 1. Location of the streams analyzed in the Guama (A) and Acara-Capim (B) basins, in the northeast and southeast mesoregions of the state of Para, in eastern Brazilian Amazonia. The black dots indicate the study streams. The photos are examples of streams. using license number 4681-1 granted by the Biodiversity Infor- overestimation of contamination, we ran blank samples to obviate mation and System Authorization - SISBIO and approved by the their potential influence on the assessment of the plastic pollution ethics committee of the Federal University of Para (CEUA nº in the organs of the different species, with the observed laboratory 8293020418). background load being subtracted from the value recorded for each sample (Nuelle et al., 2014). The blank samples were Petri dishes 2.4. Biometry and the dissection of the gills and gastrointestinal containing H2O2, which were left exposed to air in the laboratory tracts whilst the laboratory was in use and then conferred to determine the amount of microplastics circulating in the environment, which The standard length of each specimen was measured using a might contaminate the samples and lead to overestimates. digital caliper (0.01 mm precision) and it was weighed on an analytical balance (0.01 g precision). The gills and gastrointestinal 2.6. Microscopy and identification of plastics tract were removed and stored in 5 ml glass vials, washed to remove excess particles and labelled with the species and collec- The filters were observed, measured, and photographed tion site. following the protocol of Avio et al. (2015) and Santos et al. (2016), which consists of the direct observation of the particles in terms of 2.5. Digestion of tissues and filtration their shape, color, and size. We visualized the filters under a ste- reomicroscope at 120 magnification. Approximately 2 ml of hydrogen peroxide (H2O2 30%, previously To classify the shape of the plastic particles, elongated or fila- filtered) was added to each flask containing the gills and the mentous pieces were denominated fibers or microfibers, while gastrointestinal tracts. After the complete digestion of the tissue, those that were spherical in shape (whether rough or smooth) were the material was filtered through a cellulose acetate filter with a classified as microspheres or spheres. All other particles, with porosity of 0.2 mm using a vacuum system following the “Micro- squarish, rectangular or irregular shapes, were classified as frag- plastics: sampling and processing guidebook” from Mississippi ments or microfragments. State University, USA (Sartain et al., 2018). The length (or diameter, for spherical pieces) of each plastic As the fibers found in the samples were optically very similar to particle visualized during the analysis was measured in millime- those found in the procedural blanks, the residual plastic extraction ters. We considered particles between 0.2 and 1 mm were classified methods are susceptible to background contamination from the as nanoplastics, while particles between 1 mm and 5 mm were working environment. To minimize the potential for the classified as microplastics, larger than 5 mm and smaller than 4 D.R.G. Ribeiro-Brasil et al. / Environmental Pollution 266 (2020) 115241

25 mm particles were called mesoplastics, and larger than 25 mm scale for the measurement of the items, and (c) the photographs were called macroplastic following the scheme of Wagner et al. were systematically compared, regarding the morphotype, with (2014). studies that carried out an analysis of the type of plastic (e.g. mFTIR, Hydrogen peroxide is known to digest organic material, and is a FTIR or RAMAN). The images were taken on the same scale to reagent used to digest organs, but this might also lead to a loss of ensure a better comparison between the studies (e.g. Karlsson et al., color in some types of polymer (Nuelle et al., 2014). For this reason, 2017; Su et al., 2019). Part of these procedures were adopted due to color was not considered to be a valid variable for the purposes of the fact that numerous authors (e.g., Wang et al., 2017; Tibbetts the present study. et al., 2018; Seng et al., 2020) have followed the protocol described by Nor and Obbard (2014) and Hidalgo-Ruz et al. (2012). 2.7. Quality assurance and quality control (QA/QC) 2.8. Data analysis Sampling control measures: All the material used during the species sampling was previously washed with filtered water We applied a paired t-test to verify the variation in the quantity (0.22 mm filter). The liquids (alcohol and formaldehyde) were pre- of plastic particles in the different organs, using the number of viously filtered (0.22 mm filter) to fix the specimens. In the sam- plastic particles as the response variable, and the organs (gills or pling, the bottles were opened only when storing the fish. The fish gastrointestinal tract) as the categorical predictors. We applied the were immediately placed in glass jars after being caught. 100% same procedure (paired t-test) for each type of particle (nano- cotton clothing and gloves were used during specimen collection, plastics, microplastics, and mesoplastics), once again with the and the collection equipment (hand sieves) were made of metal. number of plastic particles as the response variable, and the organs Laboratory control measures: random samples of liquid from the (gills or gastrointestinal tract) as the categorical predictors. We also flasks where the fish were stored were filtered and the filters were used the t-test to verify the variation in the shape of the particles, visualized on the same equipment as the samples. During the using the number of particles as the response variable, and the process of visualizing the filters containing samples, an unpro- shape (fiber and fragments) as categorical predictors. We ran the tected filter was placed throughout the process to capture air tests using the t.test function of the stats package of the R program contamination. All the material was protected with aluminum foil (Core R Team, 2019). throughout the process. In addition, the visualization equipment To test the hypothesis that plastic intake rates vary among fish was protected with cotton fabric during the entire working time. species, we developed a Generalized Linear Mixed Model (GLMM) During all phases, all the clothing, coats, and gloves used were 100% using each individual as a sample unit. The number of plastic par- cotton. ticles found in the organs (gills and gastrointestinal content) was Inclusion criteria: to consider an item as plastic, we compared considered as the response variable and a vector with the species the item with images of works that confirmed the type of plastic identity was the predictor variable for the fixed effect. To avoid (e.g. Karlsson et al., 2017; Su et al., 2019). We observe the pattern of biases related to the collecting locality, we used a vector with in- shapes and colors of the items. Thus, we considered as plastics the formation on the sampling sites as a random effect of the model. fibers and filaments that showed the circular shape from one end to We developed the GLMM with the lmer function of the lme4 the other, or any other transversal shape that the item has pre- package (Bates et al., 2015) of the R program (Core R Team, 2019). sented, without thinning or flattening along the structure. In case of filamentary items with twists, these twists were visualized from 3. Results one end of the item to the other. In fragments with different thicknesses, we observed the patterns of colors and designs We analyzed the organs (gastrointestinal tracts and gills) of 68 (stripes, circles, etc.). In the case of spheres, the circular shape was fish specimens, representing 14 species, distributed in 10 families observed and there may be some injury. The same item was and five orders, collected from the 12 study streams (Table 1). The compared with more than one study, when possible. If there was no fish had a mean body mass of 0.89 g ± 1 g and a mean standard similarity between the item and the observations in the studies and length of 19.8 mm ± 5.8 mm. it did not meet the inclusion criteria, the item was not counted. Additionally, if the item falls into the category of what studies do 3.1. Plastic debris in the gastrointestinal tract and gills not consider plastic (e.g., cotton), we also disregard the item. Laboratory observation of the particles: To avoid errors in the We observed plastic particles in 67 (98%) of the 68 individuals interpretation of the plastic particles observed in the samples and analyzed. Particles were only absent from one specimen of the guarantee the accuracy of the results, we adopted a number of rigid small catfish Mastiglanis cf. asopos. We recorded a total of 383 criteria: (i) the samples were immersed completely in hydrogen plastic particles, of which, 201 were located in the gastrointestinal peroxide for 60 days; (ii) during the examination of the samples tracts of the specimens and 182 in their gills. We found plastic under the stereoscopic microscope, the items identified an undi- particles in the gastrointestinal tracts of 88.2% of the specimens, gested animal parts (e.g., claws) were not counted as plastic; (iii) and in the gills of 83.8%. A mean number of 5.6 ± 3.8 particles were each item was pressed with the tip of a needle to verify possible recorded per individual, with a mean of 3.0 ± 2.3 particles in the microstructures, such as that of wood or other material, which gastrointestinal tracts and 2.7 ± 3.0 in the gills (Table 1). There was might otherwise be identified as plastic; (iv) confirmation of the no significant difference between organs in the amount of plastic absence of cellular or organic structures; (v) only fibers of the same particles observed (t ¼0.6; df ¼ 67; p ¼ 0.5). size, with untapered ends, were counted; (vi) only non-shiny par- ticles were counted; (vii) fibers with segments were not counted, 3.2. Variation in the ingestion of plastic debris among species and (viii) fibers that appeared to contain twisted filaments were counted. To guarantee the quality of the systematic analysis, the The GLMM indicated that four species of the 14 species exam- following criteria were adopted: (a) all the samples were examined ined are significantly more or less susceptible to contamination by by a pair of observers, following strictly the criteria described plastic. Hemigrammus unilineatus (t ¼ 2.1; df ¼ 54; p ¼ 0.04) and above; (b) the two observers invariably used exactly the same Polycentrus schomburgkii (t ¼ 2.6; df ¼ 54; p ¼ 0.01) had signifi- apparatus to observe and photograph the samples, and the same cantly fewer plastic particles in their gastrointestinal tracts than the D.R.G. Ribeiro-Brasil et al. / Environmental Pollution 266 (2020) 115241 5

Table 1 List of the species collected during the present study of 12 small streams in eastern Brazilian Amazonia, showing the mean standard length (SL), body mass (M), and number of plastic particles recorded in the gastrointestinal tracts and gills of each species. N ¼ number of individuals analyzed; SL ¼ Standard Length; Gastroint. ¼ plastic particles in the gastrointestinal tract, Gills ¼ plastic particles in the gills, SD ¼ standard deviation.

Taxon/Authority N SL (mm) Mass (g) Gastroint. Gill Mean ± SD Mean ± SD

Characiformes Characidae Hemigrammus unilineatus (Gill, 1858) 5 23.1 ± 4.6 1.3 ± 0.2 4.4 ± 2.1 1.7 ± 2.1 Iguanodectidae Bryconops melanurus (Bloch, 1794) 5 54.6 ± 22.4 2.0 ± 1.5 1.6 ± 1.3 2.4 ± 1.9 Iguanodectes rachovii Regan, 1912 5 22.0 ± 5.1 0.2 ± 0.1 3.8 ± 2.2 1.4 ± 0.1 Erythrinidae Hoplias malabaricus (Bloch, 1794) 5 33.6 ± 3.8 0.6 ± 0.2 3.0 ± 2.6 0.7 ± 0.6 Gasteropelecidae Carnegiella strigata (Günther, 1864) 5 21.9 ± 0.9 0.4 ± 0.1 3.4 ± 3.5 2.6 ± 3.6 Lebiasinidae Copella arnoldi (Regan, 1912) 5 26.1 ± 3.7 0.3 ± 0.1 2.4 ± 0.9 1.2 ± 1.1 Cichliformes Cichlidae Aequidens tetramerus (Heckel, 1840) 5 32.96 ± 3.7 1.4 ± 0.5 1.4 ± 1.5 1.2 ± 1.1 Crenicichla gr regani Ploeg, 1989 5 36.6 ± 7.7 1.0 ± 0.4 3.8 ± 1.6 5.0 ± 2.8 Nannacara taenia Regan, 1912 5 27.4 ± 2.6 0.8 ± 0.3 2.0 ± 1.4 1.6 ± 0.1 Cyprinodontiformes Rivulidae Laimosemion strigatus (Regan, 1912) 6 17.0 ± 3.9 0.2 ± 0.1 2.3 ± 1.6 2.8 ± 2.9 Perciformes Polycentridae Polycentrus schomburgkii Müller & Troschel, 1849 6 21.4 ± 4.6 0.4 ± 0.3 4.8 ± 4.3 1.7 ± 2.1 Siluriformes Callichthyidae Megalechis thoracata (Valenciennes, 1840) 5 46.9 ± 19.4 3.9 ± 5.0 2.0 ± 1.0 1.4 ± 0.5 Heptapteridae Mastiglanis cf asopos Bockmann, 1994 5 24.9 ± 3.2 0.2 ± 0.1 2.5 ± 2.6 4.0 ± 4.2 Pimelodella geryi Hoedeman, 1961 5 34.1 ± 3.6 0.5 ± 0.2 3.8 ± 1.3 7.5 ± 5.2

other species (Table 2, Fig. 3b). The contamination of the gills of the study. Hemigrammus unilineatus was also significantly lower than in the other species analyzed (t ¼ 1.2; df ¼ 54; p ¼ 0.04). By contrast, Crenicichla regani (t ¼ 2.2; df ¼ 54; p ¼ 0.03) and Pimelodella geryi (t ¼ 3.5; df ¼ 54; p < 0.01) had significantly larger amounts of 3.4. Size plastics debris plastics particles in their gills than the other species (Table 2, Fig. 3a). The particles ranged in size from 0.2 mm to 13.8 mm, with 22% being classified as nanoplastics (<1 mm), 46.7% as microplastics (1.1e5.0 mm), and 33.3% as mesoplastics (>5mme25 mm). We 3.3. Shape of the plastic particles found no significant difference in the amount of mesoplastics found in the gastrointestinal tracts and gills (t ¼ 1.9, df ¼ 26, p ¼ 0.06). We recorded 358 fibers and 25 fragments (Fig. 2), with fibers However, both microplastics (t ¼ 2.7, df ¼ 16.1, p ¼ 0.01) and being significantly more common than fragments overall (t ¼ 10.6, nanoplastics (t ¼ 2.2, df ¼ 26, p ¼ 0.04) were adsorbed significantly df ¼ 145.6, p < 0.01), representing 93.5% of the particles recorded in more in the gills (Fig. 4a) than the gastrointestinal tract (Fig. 4b).

Table 2 Summary of the results of the GLMM using species as the predictor variables and the number of plastic particles in the organs (gills and gastrointestinal tract) as the response variable. The collecting localities were used as a random factor. SE: Standard Error, DF: Degrees of Freedom. In bold, significant values (p-values < 0.05).

Variable Plastics in gastrointestinal tract Plastics in gills

Estimate SE DF t P Estimate SE DF t p

(intercept) 1.4 1.06 54 1.32 0.19 1.2 1.21 54 0.99 0.33 Bryconops melanurus 0.2 1.42 54 0.14 0.89 1.2 1.64 54 0.73 0.47 Carnegiella strigata 2.0 1.49 54 1.34 0.19 1.4 1.72 54 0.82 0.42 Copella arnoldi 1.0 1.42 54 0.71 0.48 0.0 1.64 54 0 1 Crenicichla regani 2.4 1.49 54 1.61 0.11 3.8 1.72 54 2.21 0.03 Hemigrammus unilineatus 3.0 1.49 54 2.01 0.04 3.4 1.72 54 1.98 0.04 Hoplias malabaricus 1.6 1.64 54 0.98 0.33 0.5 1.90 54 0.28 0.78 Iguanodectes rachovii 2.4 1.42 54 1.69 0.10 0.2 1.64 54 0.12 0.90 Laimosemion strigatus 0.9 1.44 54 0.65 0.52 1.63 1.65 54 0.99 0.33 Mastiglanis asopos 1.1 1.58 54 0.70 0.49 2.8 1.81 54 1.54 0.128 Megalechis thoracata 0.6 1.42 54 0.42 0.67 0.2 1.64 54 0.12 0.90 Nannacara taenia 0.6 1.42 54 0.42 0.67 0.4 1.64 54 0.24 0.81 Pimelodella geryi 2.3 1.58 54 1.49 0.14 6.3 1.81 54 3.48 0.01 Polycentrus schomburgkii 3.4 1.36 54 2.53 0.01 0.4 1.57 54 0.30 0.77 6 D.R.G. Ribeiro-Brasil et al. / Environmental Pollution 266 (2020) 115241

Fig. 2. Examples of plastic particles in the form of fibers and fragments recorded in the gastrointestinal tracts and gills of fish collected from streams in the Guama and Acara-Capim basins, in eastern Brazilian Amazon.

4. Discussion the present study having at least one plastic item in the gastroin- testinal tract. This value is also very much higher than those The present study recorded high contamination by nanoplastics, recorded by Andrade et al. (2019) in the serrasalmids of the Xingu microplastics, and mesoplastics in fish from small Amazonian River (25%) and by Pegado et al. (2018) in fish from the Amazon streams. However, significant variation was found in the levels of estuary (30%). These differences between streams and more contamination between organs (gastrointestinal tract and gills) and expansive environments, such as rivers and estuaries, may be among species, corroborating our hypothesis. Overall, the study related, in part, to the reduced volume of water found in the confirmed that, as in oceans (Wright et al., 2013; Woodall et al., streams, and their greater proximity to potential sources of 2014; Galloway et al., 2017), estuaries (Phillips and Bonner, 2015; contamination, such as urban centers (McNeish et al., 2018), and Blettler et al., 2017; Pegado et al., 2018), and rivers (Zbyszewski and possibly also the reduced availability of feeding resources in the Corcoran, 2011; Andrade et al., 2019; Yuan et al., 2019), small environment, in comparison with the input of plastic waste, as headwater streams in the Amazon are being contaminated by this observed in the experimental study of Scherer et al. (2017). In this global pollutant. Although the Amazon basin is, in general, rela- study, aquatic invertebrates with different feeding strategies were tively sparsely populated, the level of contamination recorded in exposed to fluorescent polystyrene spheres of 1, 10, and 90 mm the present study was surprisingly similar to those recorded in (3e3000 particles/mL) together with natural particles, including other regions with much higher population density in a number of sediments and food items, and the results showed that the pres- different countries (Table 3). Clearly, contamination of headwater ence of the natural material reduced the assimilation of plastic systems has profound implications for the whole watershed and its particles significantly (Scherer et al., 2017). biodiversity. Urbanization is the type of land use that most contributes to the To date, relatively few studies have evaluated the occurrence of contamination of freshwater ecosystems by plastic waste plastic waste in Amazonian fish (e.g. Pegado et al., 2018; Andrade (McCormick et al., 2016; Peters and Bratton, 2016; Silva-Cavalcanti et al., 2019), and none of this research has focused on stream- et al., 2017). Urban streams also tend to have the least cover of ri- dwelling species, nor the assimilation of residues in the gills. The parian vegetation. This results in a reduction of the input of organic results of the present study have shown clearly that stream fish are material from this vegetation (including leaves, fruit, and in- ingesting plastic waste in larger quantities than the fish that inhabit vertebrates, which provide the fish fauna with feeding resources), Amazonian rivers and estuaries. On average, the stream fish as well as the loss of its capacity to impede the infiltration of sed- examined in the present study had ingested three plastic items iments and pollutants from the surrounding landscape (Roy et al., (range: 0e12). This value is higher than that recorded in much 2005; Scherer et al., 2017, 2020). Urban streams are also widely more expansive aquatic environments, such as the Xingu River, a used for domestic activities, such as the washing of laundry and major Amazon tributary, where Andrade et al. (2019) recorded a dishes, as well as for leisure, which intensify in areas with more mean of 2.08 items per fish in piranhas and pacus (Serrasalmidae), intense urban development, which contributes to the contamina- and the Amazon estuary, where Pegado et al. (2018) recorded a tion of the aquatic environment by microfibers derived from the mean of 1.2 items. fabrics that come into contact with the water (Jemec et al., 2016; These findings are reinforced by the high frequency of Mason et al., 2016). contaminated individuals, with 87% of the stream fish examined in The plastic particles ingested by the fish not only have no D.R.G. Ribeiro-Brasil et al. / Environmental Pollution 266 (2020) 115241 7

the present study, it would appear to be necessary to establish research programs to monitor the contamination of Amazonian streams by plastic waste. One important limitation for the under- standing of the impacts of this contamination is the paucity of data on the ecology of the fish that inhabit these streams, in particular their foraging strategies and diets (Brejao~ et al., 2013). In the present study, two species, Hemigrammus unilineatus (Characidae) and Polycentrus schomburgkii (Polycentrydae), inges- ted significant fewer plastic particles than the other species (Table 2, Fig. 3). Hemigrammus unilineatus is an active, voracious predator, which is typically found in calm backwaters, where it feeds primarily on small invertebrates (Catarino and Zuanon, 2010). By contrast, P. schomburgkii has morphological specializations that permit the fish to camouflage itself in the foliage, where it waits to ambush its prey (Catarino and Zuanon, 2010). Both species are predators that use different feeding tactics, but can be found in similar environments, among the leaves in streams backwater areas (Catarino and Zuanon, 2010; Brejao~ et al., 2013). Besides that, the lentic environments and habitat use may also be an important factor, given that P. schomburgkii may be found camouflaged in the foliage of backwater habitats, where plastic particles may be less mobile and tend to sediment to the bottom (Yuan et al., 2019). This would mean that there may be fewer moving particles in the water column that are likely to be ingested or, in the specific case of H. unilineatus, adsorbed in the gills. In addition, these families have the presence of taste buds (Dabrowski, 1984) that allow them greater selectivity in food. The unpleasant taste would thus be able to avoid the ingestion of certain substances, based on their palat- ability. Even though plastic particles may resembles potential prey (Kim et al., 2019), then, some fish may be able to identify and expel them prior to ingestion. The expulsion of unpalatable particles has been observed in the zebrafish, Danio rerio, for example, in a lab- oratory setting (Kim et al., 2019). While the accidental ingestion of plastic particles has been widely reported in freshwater fish (Phillips and Bonner, 2015; Fig. 3. Summary of the contamination by plastic particles in the organs of fish Peters and Bratton, 2016; Jabeen et al., 2017; Silva-Cavalcanti et al., collected from Amazonian streams; a) number of plastic particles in the gills, and b) 2017; Pegado et al., 2018; Andrade et al., 2019), the assimilation of number of plastic particles in the gastrointestinal tract. In both figures, the red line represents the mean number of particles per species, the red bars indicate the species these contaminants through other routes, such as the gills, has with the largest amount of plastic particles and the green bars, those with the smallest received much less attention (Table 3). By contrast, two species, amount of microplastics.(For interpretation of the references to color in this figure C. regani and P. geryi, had significantly more plastic particles legend, the reader is referred to the Web version of this article.) adhered to their gills than the other species (Table 2, Fig. 3). This is probably because both species have a serrated operculum with irregular projections (Varella et al., 2012) and, in the specific case of nutritional value, but also hamper the absorption of nutrients by C. regani micro-spines in the branchial arches (Kullander, 2003), the fish (Chen et al., 2017). This impact provokes alterations in the which may favor the accumulation of particles in the gills. In the lipid and energetic metabolism of the fish, reducing the energy case of P. geryi, on the other hand, the behavior of the species, available to the fish to devote to foraging and reproductive activ- which spends most of its time hidden under rocks (Pinna and Keith, ities (Sa et al., 2015; Lu et al., 2016). The ingestion of plastic may also 2019), may increase the probability of the gills coming into contact cause the death of the fish through entanglement or asphyxia with plastic particles present in the sediment, and thus of these (Vendel et al., 2017). Given all these considerations, and the evi- particles adhering to the gill tissue. Nanoparticles may pass dence of the contamination of stream fish by plastics obtained by through the gills and accumulate in the organs through the

Fig. 4. Plastic particles of different sizes found in the organs of fish specimens collected from 12 Amazonian streams: (a) nanoplastics and (b) microplastics. 8 D.R.G. Ribeiro-Brasil et al. / Environmental Pollution 266 (2020) 115241

Table 3 The findings of some previous studies of the occurrence of plastic pollutants in fish in different environments, regions, and countries. SD ¼ standard deviation.

Reference Organs Average ± SD Environments Demography density Local/countries

Andrade et al. (2019) Stomach 6.0a Xingu river 4.4 hab/Km2 (IBGE, 2019) Amazon/Brazil Pegado et al. (2018) Stomach 1.2 ± 5.0 Amazon estuary 4.4 hab/Km2 (IBGE, 2019) Amazon/Brasil Peters and Bratton (2016) Stomach 1.6 b Brazos river 109.9 hab/Km2 (US Census, 2018) Texas/USA Phillips and Bonner (2015) Gastrointestinal 4.6 ± 3.9 Neches river 109.9 hab/Km2 (2017, estimated) Texas/USA Silva-Cavalcanti et al. (2017) digestive tract 3.6 ± 3.4 Pajaú river 7.0 hab/Km2 (IBGE, 2019) Pernambuco/Brazil Su et al. (2019) Head 0.1 ± 0.4 urban lakes 3.3 hab/Km2 Australia Su et al. (2019) Body 0.6 ± 1.3 urban lakes 3.3 hab/Km2 Australia This study Gills 2.7 ± 3.0 Amazon stream 4.4 hab/Km2 (IBGE, 2019) Amazon/Brazil This study Gastrointestinal 3.0 ± 2.3 Amazon stream 4.4 hab/Km2 (IBGE, 2019) Amazon/Brazil

Mean ± standard deviation per individual. a Mean standard deviation per species. b The study shows only mean, without standard deviation. circulatory system (Watts et al., 2014; Lu et al., 2016). resident in urban centers and in areas with no public sanitation The adsorption of plastic particles by the gills was related sys- system or effective sewage treatment (Barthem et al., 2004; Melo tematically with particle size (Fig. 4), that is, the smaller the par- et al., 2019), which likely facilitates the contamination of Amazo- ticle, the more easily it will be adsorbed to the gills (Lu et al., 2016; nian streams by plastic waste. As the streams surveyed in the Su et al., 2019). Smaller particles, such as mesoplastics and nano- present study discharge into rivers which, in turn, flow into estu- plastics, are more mobile in the water (Jemec et al., 2016; Dawson aries and eventually reach the oceans (Scheidegger, 2006; Eriksen et al., 2018; Yuan et al., 2019; Jacob et al., 2020) and may thus be et al., 2014; Lebreton et al., 2017), it seems likely that much of the adhered more easily by the gills during the movement of the gill plastic waste ingested by the fish in these environments is derived and operculum during the exchange of ions, gases and water (Watts from streams. et al., 2014; Cole and Galloway, 2015; Chen et al., 2017; Jacob et al., fi 2020). Similar ndings to those of the present study have been 5. Conclusions recorded in the freshwater zebrafish, D. rerio (Huang et al., 2016; Lu et al., 2016; Chen et al., 2017) and the marine crab, Carcinus maenas In the present study, we showed that fish from Amazonian (Watts et al., 2014). Although the particles were both ingested and headwater streams are contaminated with nanoplastic, micro- accumulated in the gills, the potential toxic effects of these sub- plastic, and mesoplastic particles in much the same way as fish stances were not evaluated in the present study, and represent an from other aquatic environments, although we found no evidence important focus for further research. of differences in the levels of contamination by plastic waste by The shape of the plastic particle is potentially also an important ingestion or adhesion to the gills. We can confirm, however, that parameter, given that an ample variety of shapes has been detected certain species are more, or less, susceptible to the assimilation of in the aquatic environment (Wagner and Lambert, 2018). Fibers microplastics than others. These differences may be determined by were the type of particle most ingested and adsorbed in the present interspecific variations in behavioral patterns and foraging strate- study (Fig. 2 and 93.5% of the particles), which is consistent with gies. Fibers were the most common plastic particles, overall, and the results of a number of previous studies in freshwater environ- the smaller particles (nano- and microplastics) were more likely to ments (Jemec et al., 2016; Mason et al., 2016; Grigorakis et al., 2017; adhere to the gills. Our results indicate that stream fishes are fi Foley et al., 2018). The accumulation of plastic bers in these stream extremely vulnerable to microplastic pollution and that urbaniza- fi sh is particularly preoccupying, given that this type of particle is tion is a major factor contributing to the pollution of freshwater seen by many authors as the most dangerous, given its ease of environments with plastic waste. Given this, more comprehensive accumulation (Qiao et al., 2019). This appears to be an intrinsic data on the biology and behavior of the fish will be necessary to quality of this type of particle, given that a similar pattern has been identify the factors that determine differences in their vulnerability recorded in both invertebrates (Jemec et al., 2016; Dawson et al., to contamination by plastics. These findings reinforce the need to 2018) and vertebrates (Qiao et al., 2019). In fact, Qiao et al. (2019) monitor the streams to ensure that there is no increase in the fi show that microplastic bers not only accumulated more easily, contamination rates which might impact the health, behavior, and but also caused more serious intestinal toxic effects than fragments reproduction of the species. It will also be necessary to involve local fi and spheres. In freshwater environments, plastic bers are derived human populations in the management of solid waste through primarily from the washing of clothes (Jemec et al., 2016; Kelly environmental education initiatives that can engage the whole et al., 2019; Falco et al., 2020; Fontana et al., 2020), as well as do- community, including both public and private institutions, and fl mestic and industrial ef uents (Mason et al., 2016; Fontana et al., encourage a reduction in the consumption of disposable products, fi 2020), given that neither washing machine lters nor wastewater such as single-use plastics, as well as the reuse and recycling of treatment plants are designed to retain particles of this size (Kelly products that would otherwise be discarded as waste. Given the et al., 2019; Fontana et al., 2020). current scenario, it will be necessary to consider legal measures for The two microbasins evaluated in the present study, that is, the the regulation and control of the presence of these substances in Acara-Capim and the Guama, receive approximately 274.5 million aquatic environments in order to guarantee the quality of these fl liters of urban ef uents per day, of which, only 8% pass through a environments and the continued functioning of their ecosystems. sanitation system, and only 3% are treated before being discharged into the streams (Aviz et al., 2011). A number of studies have shown CRediT authorship contribution statement that pollution by urban effluents is causing the loss of biodiversity, and the quality of the water and soil in the Amazon region Danielle Regina Gomes Ribeiro-Brasil: Conceptualization, (Barthem et al., 2004; Couceiro et al., 2007; Aviz et al., 2011; Melo Methodology, Formal analysis, Investigation, Resources, Writing - et al., 2019). A large proportion of the region’s population is original draft, Writing - review & editing, Project administration. D.R.G. Ribeiro-Brasil et al. / Environmental Pollution 266 (2020) 115241 9

Naiara Raiol Torres: Methodology, Investigation, Writing - review lake. Environ. Monit. Assess. 189, 581. https://doi.org/10.1007/s10661-017- & 6305-8. editing. Ana Beatriz Picanço: Methodology, Investigation. David ~ ~ Brejao, G.L., Gerhard, P., Zuanon, J., 2013. Functional trophic composition of the Silva Sousa: Methodology, Investigation. Vanessa Serrao Ribeiro: ichthyofauna of forest streams in eastern Brazilian Amazon. Neotrop.Ichthyol. Methodology, Investigation. Leandro Schlemmer Brasil: Concep- 11, 361e373. Catarino, M.F., Zuanon, J., 2010. Feeding ecology of the leaf fish Monocirrhus pol- tualization, Methodology, Formal analysis, Investigation, Writing - fi & yacanthus (Perciformes: Polycentridae) in a terra rme stream in the Brazilian review editing. Luciano Fogaça de Assis Montag: Conceptuali- Amazon. Neotrop. Ichthyol. 8 (1) https://doi.org/10.1590/S1679- zation, Methodology, Formal analysis, Investigation, Resources, 62252010000100022. Writing - original draft, Writing - review & editing, Supervision, Cavalcante, R.B.L., Ferreira, D.B. da S., Pontes, P.R.M., Tedeschi, R.G., da Costa, C.P.W., de Souza, E.B., 2020. Evaluation of extreme rainfall indices from CHIRPS pre- Funding acquisition. cipitation estimates over the Brazilian Amazonia. Atmos. Res. 238, 104879. https://doi.org/10.1016/j.atmosres.2020.104879. Declaration of competing interest Chen, Q., Gundlach, M., Yang, S., Jiang, J., Velki, M., Yin, D., Hollert, H., 2017. Quantitative investigation of the mechanisms of microplastics and nanoplastics toward zebrafish larvae locomotor activity. Sci. Total Environ. 584 (585), The authors declare that they have no known competing 1022e1031. https://doi.org/10.1016/j.scitotenv.2017.01.156. financial interests or personal relationships that could have Coe, M.T., Marthews, T.R., Costa, M.H., Galbraith, D.R., Greenglass, N.L., fl Imbuzeiro, H.M.A., Levine, N.M., Malhi, Y., Moorcroft, P.R., Muza, M.N., appeared to in uence the work reported in this paper. Powell, T.L., Saleska, S.R., Solorzano, L.A., Wang, J., 2013. Deforestation and climate feedbacks threaten the ecological integrity of south-southeastern Acknowledgments Amazonia. Philos. Trans. R. Soc. B Biol. Sci. 368 https://doi.org/10.1098/ rstb.2012.0155. Cole, M., Galloway, T.S., 2015. Ingestion of nanoplastics and microplastics by Pacific We thank Hydro Alunorte for funding and for offering us the oyster larvae. Environ. Sci. Technol. 14625e14632. https://doi.org/10.1021/ opportunity to develop this project in the Amazon through various acs.est.5b04099. research projects. 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