Detection of Microplastic contamination in shallow marine habitats using solitary ascidians: Influence of morphology and habitat in a tropical bay, SW Atlantic

Detection of Microplastic contamination in shallow marine habitats using solitary ascidians: Influence of morphology and habitat in a tropical bay, SW Atlantic | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Detection of Microplastic contamination in shallow marine habitats using solitary ascidians: Influence of morphology and habitat in a tropical bay, SW Atlantic Rayane Sorrentino, Paulo Cezar Azevedo da Silva, Brenda dos Santos Ramos, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6889938/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The ubiquity of microplastics (MP) in marine ecosystems and their biological uptake has become a major global concern. Many papers indicate MP ingestion by marine organisms, but few studies address the role of morphological characteristics of species on MP ingestion and retention in filter-feeding species. This study investigates the presence of MP in four ascidians species as models : Styela plicata, Phallusia nigra, Microcosmus exasperatus and Herdmania pallida . They are compared in relation to the shape (simple or branched) and number of oral tentacles, and the presence of branchial folds (from none to 18). Morphology, here, were used as a proxy for mechanisms that may prevent MP ingestion. Furthermore, we compared the concentration of MP among species from sites with and without harbor, and during summer and winter seasons. Specimens were collected in the Ilha Grande Bay, where they are widely distributed in natural and artificial substrates. Microplastics were extracted by density separation, quantified, and categorized by granules, fibers and fragments. The morphological structure present in S. plicata , with simple tentacles and branchial folds retained significantly more MP particles than all other species. This suggests that tentacles morphology and the presence of branchial folds may influence MP capture. Specimens near harbor areas and sampled during winter showed higher abundance of MP, reflecting association of plastic pollution with shipping and unsustainable tourism activities. This is the first assessment of MP contamination in ascidians from tropical bays and from natural environments. Its association with morphology, opens new perspectives for studies dealing with mechanics of filtering activity of marine invertebrates. Oceanography Marine and Freshwater Ecology Ascidian tentacles morphology branchial folds plastic pollution Ilha Grande Bay Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights The studied ascidian species showed different levels of microplastic contamination. Styela plicata had the highest average (0.76 MP.ind .-1 ), while Microcosmus exasperatus had the lowest (0.12 MP.ind .-1 ). The morphology of tentacles (simple) and the presence of branchial folds affect the quantity of ingested microplastic in these solitary ascidians. Areas near harbors displayed higher microplastic abundance in ascidians compared to areas without harbors. Microplastic concentration in ascidians was higher in winter (1.7 – 1.8 MP.ind .-1 ) compared to summer (0.5 – 1.2 MP.ind .-1 ). Introduction Worldwide contamination by microplastics (MP), i.e. synthetic particles lower than 5mm in size, is an issue that concerns researchers in different ecosystems and human health professionals (Jeong et al., 2024 ; Unuofin & Igwaran, 2023 ). On marine environments, MP contamination is now, a ubiquity and a threat, and several marine organisms with commercial use or not, are used as a survey or in monitoring protocols (Alfaro‑Núñez et al., 2021; Simmons et al., 2025). Microplastics exert its influence on aquatic environments through trophic transfer mechanisms and biomagnification. Microplastic enters trophic cascade by diverse ways, mainly through species ingestion and filtration behavior (Unuofin & Igwaran, 2023 ; Marchala et al., 2024 ; Messinetti et al., 2018a ). Microplastic can also be transferred to trophic cascade through animal faeces that can be keep suspended on water column as a organic matter or deposited on seafloor (Li & Meng, 2025; My et al., 2025). In the search for MP bioindicator species, several aspects are used as the use on human feeding, the habitat and biology of species and the way that they ingested MP, the assimilation rate and/or the cleaning mechanisms, morphological characteristics of species, the temporal and spatial availability of species among others (Valente et al., 2025; Pastorino & Barceló, 2024; Avio et al., 2020 ). Among filter feeding marine invertebrates, ascidians are one of the most frequent components of benthic environments. They exist as solitary, colonial, and social animals (Zeng & Swalla, 2005 ), inhabit from intertidal zones to abyssal regions of several marine ecosystems like coral reefs, mangroves, rocky shores, and as biofouling on human constructions, including marine farms (Rocha et al., 2011 ). They filter seawater with suspended organic particles as a food source by the action of the tentacles lying at the base of the inhalant siphon (Petersen, 2007 ; Riisgård & Larsen, 2010a , b ; Wotton, 2020 ). In the sequence, water flows through the branchial basket (also defined as pharyngeal basket), allowing processes such as nutrition, respiration, and clearance management (Petersen, 2007 ). Due to ascidians' distribution, filter-feeding habits, and capacity to accumulate pollutants, they have been used as bioindicators of human impacts on the ocean, from organic pollution (Terlizzi et al., 2002 ), to heavy metals (Tzafriri-Milo et al., 2019), pharmaceutical (Navon et al., 2020 ), Environmental Risk Assessment (Rosner & Rinkevich, 2024 ) and more recently, microplastics, as particles (Silva et al., 2021) or its chemical compounds (Vered et al., 2019 ). Recent research on MP used ascidians as models, but most performed under laboratory conditions, indicating some effects in nutritional states, alterations in metamorphosis and embryonic development, as well as impairments in pigment organs (ocellus) and cellular formation (Messinetti et al., 2018a , b ). Few studies investigate MP in ascidians from the natural environment (Vered et al., 2019 ; Silva et al., 2021). At the same time, there is no study showing a seasonal approach for MP presence on ascidian species, neither exploring differences in its anatomical features related to this issue. To address these questions, we analyzed MP contamination in four ascidian species with distinct morphological characteristics of tentacles and branchial folds, from samples collected on sites with and without harbor facilities from one tropical bay on southwest Atlantic. Those species were Herdmania pallida and Microcosmus exasperatus that have ramified oral tentacles (8 to 23 tentacles and 16 to 20 branchial folds), and Phallusia nigra (none fold), and Styela plicata (8 branchial folds), with simple tentacles. Microplastics were quantified in species to answer three questions: 1) Do accumulation of microplastics by ascidians from natural habitats differs from those on the proximity to artificial ones ? 2) Are there variations in microplastic contamination across these species? 3) Do anatomical features related to shape and number of oral tentacles, and the presence and extent of branchial folds influence this microplastic accumulation? Material and methods Sampled area and fieldwork Ilha Grande Bay (BIG Portuguese abbreviation ) is situated to the southwest of Rio de Janeiro state, and is officially designated as a protected area by the State constitution (Johnsson & Ikemoto, 2015; Silva et al., 2021). The region encompasses both terrestrial and marine ecosystems, characterized by a diverse array of endemic species and an abundance of various organisms. This biodiversity is impacted by potential pollution sources such as fishing and tourism activities, as well as industrial operations including nuclear and oil facilities, shipyards, and marinas (Johnsson & Ikemoto 2015). Nevertheless, in the region, the marine contamination has been impacted by robust demographic expansion and touristic activities, compounded by deficient solid waste disposal practices and inadequate environmental education. Some studies evidenced the presence of plastic and microplastic along the beachfront of BIG (Macedo et al., 2017), as a vector of invasive species (Mantelatto et al., 2020, Póvoa et al., 2022), and within marine organisms such as amphipods and the ascidian Phallusia nigra (Silva et al., 2021). Microplastic pollution exhibits ubiquity even within Environmental Protected Areas, such as the BIG, an area that garnered the designation of World Heritage by UNESCO in 2019 (Silva et al. 2021). This region is distinguished by its pronounced endemism and the presence of well-conserved expanses comprising eleven conservation units (Creed et al. 2007). Sampled areas were defined by species distribution according to the presence and absence of the harbor/marina activities, and the exposure to the ocean side. The designated areas included (1) Continental Urban areas without harbor: Ponta Leste, Piraquara, Praia Vermelha; (2) Continental areas with harbor: Santa Luzia Pier, Portogalo, Marina Brachuy; and (3) oceanside and non-urban areas divides in Small Cove 1 (SC 1) (Dois Rios, Jorge Grego, Lopes Mendes), and Small Cove 2 (SC 2) (Aventureiro, Parnaioca, Ponta da Tacunduba) (Fig. 1). The four species colonize the natural and artificial substrates from Ilha Grande Bay. The main anatomical features of the species are the following: Herdmania pallida (Pyuridae) has 17 to 24 up to third order branched tentacles, and 8 to 9 branchial folds (Nishikawa, 2002; Rocha et al., 2012). Microcosmus exasperatus (Pyuridae) has 8 to 10 pinned first order tentacles, and intermediate ones, with 8 to 10 branchial folds, most commonly 9 (Van Name, 1945). Phallusia nigra (Ascidiidae) is a tropical species, well recognized in fieldwork by your black tunic, which is not fouled by symbionts. It has several simple tentacles, circa 50, but larger animals can have more than 100 tentacles (Van Name, 1945). The branchial sac does not show any internal fold. Styela plicata (Styelidae), is commonly reported from biofouling communities, mainly from artificial substrates, frequently as invasive species. It has also simple tentacles (25-30) but has 4 branchial folds (Van Name 1945; Monniot, 1969). These species were the most conspicuous along the studied site and along the SE Brazilian coast (Rocha et al., 2012; Skinner et al., 2016), representing good models for future large-scale comparisons. From each location and season from the austral winter of 2019, summer of 2020, and winter and summer of 2021, ten individuals of each species were sampled for MP assessment. This number of individuals at each site and season was considered large enough to represent the influence of habitat on ascidian filtration and MP ingestion (Silva et al., 2021) and is similar to those found on other papers (Avio et al., 2020). However, not all these species necessarily coexist at the same site, so, the total number of collected individuals is not equal for each species. Sampling was performed manually by snorkeling, from 0.5 to 5 meters deep and at 1.0 meters deep in pier areas. To prevent the contraction of the organisms and the potential loss of material from the atrial cavity, the individuals were collected using microbiological bags (Kasvi) and subsequently anesthetized with diluted menthol in seawater. Before sampling, plastic bags were cleaned with distilled water to prevent plastic contamination. For preservation and subsequent microplastic extraction purposes, a solution of 10% formaldehyde was introduced. It is important to note that the use of formaldehyde does not impact microplastic particles in question (Courtene-Jones et al. 2017). Microplastic extraction The organisms were dissected, removing the tunic and branchial basket, and the inner body (the body structure containing the stomach, intestine, gonads and other structures) was externally rinsed with distilled water for analysis. The inner bodies of the ten collected individuals of each species at each sampling site were pooled together. Subsequently, the pooled inner bodies were sectioned and combined with hypersaline water (composed of 358.9g of NaCl in 1L of distilled water) for density separation, a process lasting 24 hours. This method is cost-effective, it only separates polymers less dense than 1.2 g cm -3 ( i.e. polyvinyl chloride (PVC)), however, denser polymers have been quantified by this method (Montagner et al. 2021). The supernatant was filtered through a cellulose filter 5µm (Whatman AE98) employing a vacuum pump Primatec 121. The filters were placed in a cleaned petri dish and the microplastics were quantified, measured, and classified into three categories: fragments, fibers, and granules. This was performed using a microscopy Bel Photonics, STEREO-ZOOM SERIE SZ/SZT (5,6x) connected to a camera EUREKAM 5.0 Mpixels. The results were expressed as the “MP items per individual” (MP.ind .-1 ) in each sample and site. Quality assurance and quality control Preventive measures were followed to mitigate external contamination by MP, such as reduction of staff, utilization of cotton lab coats worn by researchers, cleansing of materials using distilled water, and the application of aluminum foil to envelop flasks and containers, thereby forestalling the possibility of cross-contamination via airborne particles. Five recovery samples were applied, using ascidians 5 fibers (nylon, density 1.14 g.cm- 3 ) and 5 fragments (polystyrene, density 1.05 g.cm- 3 ). Four samples represented a recovery of 100% of fragments and fibers, and one sample was 90% of fibers. As a control sample, Petri dishes with distilled water were laid out during the processing of the samples, then filtered and analyzed. The MP found in the control and those that were like the samples were discarded from the results, as in Vered et al., (2019). Data was analyzed and created using Excel (Microsoft). Analysis of MP particles MP particles were analyzed using Fourier transform infrared spectroscopy (FTIR) with attenuated total reflection (ATR) Perkin Elmer (Perkin Elmer Spectrum Version 10.5.2) for polymer identification. The spectroscopic examination encompassed a range from 4000 to 600 cm-1 and the spectral resolution of 4 cm-1. A proportion of 30% of microplastic particles could be analyzed by FT-IR since this equipment detects particles up to 2000 µm in dimension size. The polymers were detected using software spectral references and functional chemical groups. The particles were quantified and compared among species and sites. Statistical Analysis The statistical analyses were conducted using RStudio software (2024.09.1+394). As the data was not normally distributed (Shapiro-Wilk test, p <0.001; and Levene’s Test, p = 0.013). Kruskal-Wallis and Post hoc Dunn Testes were used to compare the variable MP.ind .-1 among species and sites (significant level was considered as < 0.05). Results We investigated the presence of MP on 563 individuals from the four studied species and MP were notably prevalent across all species and sampling sites, totalizing 156 items (83 fragments, 48 fibers, and 23 granules) and an average (and SD) of 0.29 ± 0.3 MP.ind -1 , corresponding to 0.15 ± 0.17 MP.ind -1 of fragments, 0.10 ± 0.25 MP.ind -1 microfibers and 0.03 ± 0.08 MP.ind -1 granules (Table 1). According to the Kruskal-Wallis test ( p < 0.001), the abundance of MP in the species was distinct. Styela plicata, with simple tentacles, exhibited a significant prevalence of MP (0.72 ± 0.44 MP.ind .-1 average ± SD) among all species (Dunn test p < 0.05). The other species did not differ, with P. nigra showing a concentration of 0.23 ± 0.13 MP.ind .-1 , H. pallida 0.20 ± 0.17 and M. exasperates 0.13 ± 0.12 MP.ind .-1 . Fragments were dominant in all species, followed by fibers and granules (Table 1). Blue and black fragments (> 5000 µm) were observed in Styela plicata from Bracuhy, Cais de Santa Luzia, and in Phallusia nigra also from Cais de Santa Luzia, and SC 1, Ponta Leste and Praia Vermelha (Table SM 1). These particles were not countable in statistical analysis, but were observed, categorized and noted. Table 1 The total of individuals sampled in each site, the morphological features of each species separated by the type and number of tentacles and the number of pharyngeal folds at each side of the body of ascidians and, the total items of fragments, fibers, and granules, and the average of MP.ind. -1 reported in each ascidian species. Species Total ind . Tentacles shape (and number) Faringeal folds Fragments Fibers Granules MP.ind .-1 (Average ± SD) S. plicata 98 Simple (25 a 30/ + ) 4 0.35 ± 0.24 0.29 ± 0.14 0.08 ± 0.14 0.72 ± 0.44 P. nigra 208 Simple (50/ 100+) 0 0.13 ± 0.13 0.07 ± 0.05 0.02 ± 0.05 0.23 ± 0.13 H. pallida 102 Branched (8 – 12) 8 - 10 0.08 ± 0.10 0.09 ± 0.05 0.02 ± 0.05 0.20 ± 0.17 M. exasperatus 155 Branched (8 – 10) 8-9 0.08 ± 0.10 0.01 ± 0.07 0.03 ± 0.07 0.13 ± 0.12 The average of MP in ascidians differed among the sites ( p = 0.001). Specimens from harbor areas displayed a greater abundance of MP, Portogalo recorded an average of 0.49 ± 0.60 MP.ind. -1 , Brachuy 0.45 ± 0.19, and Cais de Santa Luzia 0.40 ± 0.31 MP.ind -1 . Ascidians from areas without harbor showed lower concentrations of MP. Ponta Leste exhibiting 0.25 ± 0.14 MP.ind -1 , Piraquara 0.16 ± 0.11 MP.ind -1 , and Praia Vermelha 0.06 ± 0.08 MP.ind .-1 . But, even on the oceanic side, distant from urban centers and facing the ocean, small coves SC 1 (Lopes Mendes/Jorge Grego/Dois Rios); SC 2 (Parnaioca/Aventureiro/Tacunduba) recorded 0.20 ± 0.12 MP.ind -1 and 0.26 ± 0.16 MP.ind .-1 , respectively (Fig. 2 and Fig. 3). After the Post hoc test, Brachuy and Praia Vermelha showed a significant difference among MP averages ( p = 0.01). Fragments were dominant in P. nigra from Cais de Santa Luzia (0.27 ± 0.27 MP.ind -1 ), Bracuhy (0.26 ± 0.21 MP.ind -1 ), SC1 (0.14 ± 0.12 MP.ind -1 ), Piraquara (0.09 ± 0.10 MP.ind -1 ) and Ponta Leste (0.11 ± 0.11 MP.ind .-1 ). On the other hand, fibers were more frequent in Portogalo (0.27 ± 0.61 MP.ind -1 ) and SC2 (0.13 ± 0.17 MP.ind -1 ). Additionally, granules were less reported on all sites, except in Praia Vermelha, which was not reported. Regarding the length of MP, fibers measured 1,716 µm, fragments 1,699 µm, and granules 929 µm (Table 2). The red fibers had the longest average length, measuring 3,065 µm, followed by black fragments with an average of 2,620 µm, red fragments 2,510. The smaller MP were white, black, yellow and purple granules (870, 754, 420, 160 µm, respectively). Styela plicata had the larger MP, reporting black fragments and red fibers measuring an average of 3.190 and 3.065 µm respectively. Phallusia nigra reported an average of 2,800 µm to black fragments (Table SM 2). The most frequent MP in S. plicata , M. exasperatus and P. nigra was blue fragment (32, 23 and 12 items), while in H. pallida was blue fibers (11 items) (Table SM 2). Among sites, the larger average of MP was recorded respectively in Cais de Santa Luzia (red fiber 4,210 µm), Brachuy (black fragment 4,020 µm), SC 1 (black fragments 2,800 µm), and Piraquara (blue fibers 2,632 µm). The higher abundance of blue fragments is consistent in Cais de Santa Luzia (24 itens), Brachuy (11 items), SC1 and Piraquara (9 items). (Table SM 3) Table 2 Minimum, maximum, and average length of microplastics in ascidian species. Types of MP Minimum (µm) Maximum (µm) Average ± SD ( µm) Fibers 310 4450 1716 ± 961 Fragments 250 4680 1699 ± 1232 Granules 160 2920 929 ± 834 The concentration of MP in ascidians among years and seasons did not significantly differ ( p > 0.05). The abundance of MP in ascidians decreased during the years, with 0.3 ± 0.4 MP.ind -1 in 2019, 0.28 ± 0.27 MP.ind -1 in 2020 and 0.27 ± 0.18 MP.ind -1 in 2021 (Fig. 4). The concentration of MP was slightly higher during winter (0.28 ± 0.24 MP.ind -1 ) than in summer (0.28 ± 0.24 MP.ind .-1 ). Fragments were more abundant in all years (Figure 5). Based on the spectroscopic analysis in 30% of the samples, and the discernment of characteristic spectral bands, the microplastics retrieved from the ascidians were categorized as high-density polyethylene (HDPE), homopolymer polypropylene (PPH), and polystyrene (PS), with a prevalence of 75% blue MP, 15% black, and 5% purple MP (2% yellow and 1% transparent, white, and red). Discussion Our results showed the importance of the morphology on the presence of MP on ascidians. The structures of tentacles and branchial basket influenced the higher amount on the widespread and invasive Styela plicata , contrasting with Herdmania pallida , Microcosmus exasperatus and Phallusia nigra . These species differ in the shape of tentacles (simple or branched), and on the presence and number of branchial folds, and were sampled in sites influenced by the absence and presence of harbors. All species recorded at least one microplastic within their bodies, confirming their ability to accumulate MP on nature. Until now, this represents the first field study that provides valuable data on the abundance of MP in these filter-feeding species. The difference in concentration of MP was not discrepant among P. nigra , H. pallida and M. exasperatus , which aligns with observations in other filter-feeding organisms (Pequeno et al., 2021). However, it was observed that species with simple tentacles, especially S. plicata , reported more abundance of MP than those having branched tentacles. Additionally, the presence of folds on the branchial sac and a simple tentacle influences the MP concentration found in S. plicata , which was approximately twice the amount found in P. nigra . Styela plicata , recognized in many regions as an invasive species, was reported to ingest more quantity of MP than P. nigra , M. exasperatus and H. pallida . Although studies have shown micro-nanoplastic ingestion by ascidians (Messinetti et al., 2017, 2019; Eliso et al., 2023 ), investigation in natural contexts is still scarce. Given the observed differences in MP concentration among the four species studied, it is reasonable to suggest that the presence of simple and branched tentacles, and the folds on the branchial sac may influence microplastic accumulation. Gonzalez-Pineda et al. ( 2025 ) tested in lab conditions the ingestion of MP by two Antarctic ascidian species, Cnemidocarpa verrucosa and Molgula pedunculata . The first one with simple oral tentacles and the second, with branched ones, and both species have branchial folds. As the mean density of particles ingested by C. verrucosa was higher, it is plausible to affirm that the tentacle shape influences MP capture and accumulation. Physiological investigations conducted on solitary sea squirts have illuminated the sensitivity of tentacles to both mechanical and chemical stimuli, emphasizing their role as efficient filtering to large particles, such as microplastic, from water before they reach the pharynx (Mackie et al., 2006 ). Members of the Stolidobranchia order, e.g. H. pallida , M. exasperatus , and S. plicata tend to possess more than one sensory organ. S. plicata for example, exhibits two distinct categories of sensory cells: one comprising a singular cilium and short stereocilia of equal length, while the order Phlebobranchia is characterized by two cilia situated within a crescent of stereovilli (Manni et al., 2004 ). In species of the Stolidobranchia order, characterized by branched tentacles, such as H. pallida and M. exasperatus , the pattern of innervation is more complex, due to the branched morphology of the tentacles. Caicci et al. (2007) describe M. socialis as having branched tentacles, two main nerves depart from the central axis of the tentacles (first-order branch), at the base of two rows of coronal cells. This elaborate architecture enhances the surface area of contact with the surrounding water. Given this morphology, we suggest that these species may identify, avoid, and/or reject microplastic particles by a sensitive stimulus. Gonzalez-Pineda et al. ( 2025 ) reported that C. verrucosa can squirt its body, ejecting inorganic material. But M. pedunculata is unable to squirt it and cannot distinguish between organic and inorganic material. The ingestion of MP can be even more complex, as shown by Pennati et al. ( 2022 ), when mixotrophic cryptomonad flagellates exposed to microbeads increased the content of MP on ascidian juveniles. It reinforces the need for more laboratory experiments to attain comprehension of the structural composition of the mechanisms selecting plastic particles by ascidians. However, these results indicate that tentacles, folds, and sensory cells of ascidians influence MP ingestion/capture. The effect of morphology on other filter feeding invertebrates was investigated by several authors in many different marine groups as fishes, bivalves and crustaceans (Jeong et al., 2024 ). Avio et al. (2024) detected differences on MP presence between the Mytilus galloprovincialis and Ostrea edulis , two common itens on human feeding behavior. Xu et al. ( 2024 ) in a review, have indicated that MP accumulate higher on Mytilidae than on Ostreidae species, due their differences on morphology of feeding system. But large differences are reported among species of the same genus ( i.e. Perna , Mytilus , Ostrea , Crassostrea ). The amount of MP detected in our survey (from 0.13 to 0.72 MP.ind -1 ) is within the same range from several other studies, using bivalves as a proxy due to this importance on human feeding (Xu et al., 2024 ). This density was lower than on other Brazilian areas like the closest and very polluted Guanabara bay (Birnstiel et al., 2019) or Santos (Santana et al., 2016 ). Fragments were more abundant than fibers and granules. Similar results were shown by Pequeno et al. (2021), where fragments were common in deposit filter-feeding polychaete Marphysa sanguinea and the bivalve Scrobicularia plana . It is not clear how filter-feeding is related to particle ingestion and frequently this MP abundance is related to the environment. Fragments are formed from the fragmentation of macroplastics, and they are known to persist on sea surfaces due to their floatability properties (Fagiano et al., 2023). These species were sampled in shallow waters (0.5 to 5 meters deep), where they are more exposed to fragments than other types of MP. Cho et al. ( 2021 ), working with bivalves also found the prevalence of fragments. However, Joyce and Falkemberg (2023) found also on bivalves, a predominance of fibers over fragments. These contrasting results shows that ingestion of MP can be very variable on different environments. As indicated from several studies (Zhang et al., 2019 ; Cutroneo et al., 2020 ; Joshi et al., 2024 among others), harbor regions are influenced by port activities, fishing operations, and urban drainage systems, some of the major contributors to MP pollution and fragmentation of macroplastics. So, it was not a surprise to find high concentration of MP on ascidians collected near harbors on studied region. Microplastic quantification revealed a seasonal trend, with higher concentrations observed in the winter of 2019. In the summer of 2020 and 2021, a reduction of microplastic concentration was observed, potentially linked to a decline in tourism and other human activities due to the Sars-Cov-19 pandemic. It is important to highlight that ascidians were not sampled during winter of 2020 due to these restrictions. However, the restrictions were less severe in Brazil during the winter of 2021 and the results suggest that MP concentration is associated with the human use of the natural environment. The winter highs detected here may be related to the summer increase on inadequate plastic discard, and under environmental conditions of high seawater and air temperature, friction with rocky shores, sandbeaches and boats outboard generated impact/turbulence may increase fragmentation, which also explain the prevalence of fragments on our samples. Bom et al. ( 2022 ) with Perna perna in another bay at Brazilian coast detected slightly differences among MP content during the four studied season on the year 2020, with higher amounts on winter, but not differing from autumn and summer. It is difficult to compare data along time and the impact of health restrictions due SARS-COVID 19 from both samples. Analysis revealed that fragments were the most common microplastic type, followed by microfibers and granules, and fragments were also the largest. The identified polymers were polyethylene (PE), polypropylene (PP), and polystyrene (PS). The presence of PE, PP, and PS, the prevalence of blue MP, as well as the average length aligns with findings from other studies (Gago et al., 2018 ; Pequeno et al. 2021). PE, PP, and PS in the marine environment reflect the broad manufacture of these polymers and their distribution worldwide (Pequeno et al., 2021). The distribution of fragments, fibers, and granules in the water collum, especially near harbor areas, contributes significantly to plastic pollution in the marine environment and its subsequent ingestion by numerous organisms. Previous investigations on Ilha Grande bay documented the presence of solid residues (Macedo et al., 2017 ), macroplastics (Macedo et al., 2019 ), and microplastics (Silva et al., 2021), all aligned with the increased tourist activity, urban center development, and the presence of port structures. However, only one paper deals with MP on marine fauna (Silva et al., 2021). Cais de Santa Luzia has a simple wharf building, mainly used for the landing of fishermen (Johnsson & Ikemoto 2015 ), and situated within a cove near a river of the same name, is affected by both a channel accommodating small and large vessels and an unsustainable tourist establishment. Similarly, coastal areas like the marinas area of Portogalo and Brachuy, and crowded sites of Ponta Leste, and Piraquara have been impacted by intense tourism and real estate speculation. The irregular occupation and lack of adequate residue treatment in these regions pose a threat to both marine and coastal ecosystems. The oceanic side has also been impacted by tourism; however, its distance and difficult access limit a larger number of tourists. However, even facing the Atlantic Ocean, microplastics are still presently originated, and distributed by oceanic factors such as ocean and coastal currents, waves and wind (Macedo et al., 2019 ). This spatial variability in human activity and contamination by MP is common in many places worldwide (e.g., Cho et al., 2021 ; Joshi et al., 2024 ) and indicates that humanity has spread MP to even the most remote places, with several unevaluated or underestimated impacts. Conclusion This paper shows that Ascidians can be used as a proxy for MP contamination since its levels of particle densities are close to that one found for several species of bivalves worldwide for example. However, its morphological features can affect the total number of particles and this must be considered when lone species is chosen. In the absence, to the moment, of data concerning MP contamination on other marine animals on BIG, this data can support future studies and monitoring programs, and the discussion by governmental and non-governmental stakeholders about mechanisms to prevent MP contamination, mainly due to the presence of several sea farms on region. And finally, this approach may be extended for other Ascidian species or other species, like the co-occurring mussels Perna perna and the recently introduced Perna viridis (Barbieri et al., 2025; Messano et al., 2025) that is being explored on SW Atlantic without any deeply investigation about differences on MP contamination Declarations Author Contribution R.S.: Conceptualization; Supervision, Investigation; Writing – original draft; Writing – review & editing; P.S.: Methodology; Formal Analysis; Investigation; Writing – original draft; Writing – review & editing; B.R.: Sampling, Methodology, Writing – original draft; L.F.S.: Supervision; Funding acquisition; Writing – original draft , Writing – review & editing; Data availability Data will be made available on request. Competing Interests: Authors declare none competing interest Acknowledgments Authors thank Dr. Rafael B. de Moura for the photos acquired and edition. We also thank Prof. Dr Monica Marques for assistance with the FTIR analysis. This work was financed in part by the Programa de Treinamento e Capacitação Técnica (FAPERJ No 04/2019); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001; Programa de Incentivo à Produção Científica, Técnica e Artística (PROCIÊNCIA); Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), process number E-26/202.768/2019. We are thankful to CEADS UERJ and ESEC Tamoios for the Logistical support during fieldwork. Surveys are performed under licenses from INEA and SISBIO (Auth. #057/2011, 025/2017 and 029/2019) and Estação Ecológica Tamoios (ICMBio - Auth. #36194). References Alfaro-Núñez, A., Astorga, D., Cáceres-Farías, L., Bastidas, L., Soto, L., Valenzuela-Toro, A. M., & Duran, L. R. (2021). Microplastic pollution in seawater and marine organisms across the Tropical Eastern Pacific and Galápagos. 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Biota Neotropica, 11 , 749-759. https://doi.org/10.1590/S1676-06032011000500036 Rocha, R. M., Zanata, T. B., & Moreno, T. R. (2012). Chaves de identificação de famílias e gêneros de ascídias de águas rasas no Atlântico. Biota Neotropica, 12 (1). http://www.biotaneotropica.org.br/v12n1/pt/abstract?identificationkey+bn01712012012 Rosner, A., & Rinkevich, B. (2024). Harnessing ascidians as model organisms for environmental risk assessment. Environments, 11 , 232. https://doi.org/10.3390/environments11110232 Santana, M. F., Ascer, M., Custodio, ` L. G., et al. (2016). Microplastic contamination in natural mussel beds from a Brazilian urbanized coastal region: Rapid evaluation through bioassessment. Marine Pollution Bulletin, 106 (1–2), 183–189. https://doi.org/10.1016/j.marpolbul.2016.02.074 Skinner, L.F., Barboza, D.F., Rocha, R.M. (2016). Rapid Assessment Survey of introduced ascidians in a region with many marinas in the southwest Atlantic Ocean, Brazil. Management of Biological Invasions 7, 13–20. Terlizzi, A., Fraschetti, S., Guidetti, P., & Boero, F. (2002). The effects of sewage discharge on shallow hard substrate sessile assemblages. Marine Pollution Bulletin, 44 (6), 544-550. https://doi.org/10.1016/S0025-326X(01)00282-X Unuofin, J. O., & Igwaran, A. (2023). A review of microplastics contamination in African aquatic environments. Marine Environmental Research , 197 , 102410. https://doi.org/10.1016/j.seares.2023.102410 Van Name, W. G. (1945). The North and South American ascidians. Bull. Amer. Mus. Nat. Hist. 84: 1-476., available online at http://digitallibrary.amnh.org/dspace/handle/2246/1186 Vered, G., Kaplan, A., Avisar, D., Shenkar, N., 2019. Using solitary ascidians to assess microplastic and phthalate plasticizers pollution among marine biota: a case study of the Eastern Mediterranean and Red Sea. Mar. Pollut. Bull. 138, 618–625. https://doi.org/10.1016/j.marpolbul.2018.12.013 Wotton, R.S., 2020. Methods for capturing particles in benthic animals. In: The Biology of Particles in Aquatic Systems . CRC Press, pp. 183–204. Xu, Z., Huang, L., Xu, P., Lim, L., Cheong, K.-L., Wang, Y., & Tan, K. (2024, July 14). Microplastic pollution in commercially important edible marine bivalves: A comprehensive review . Food Chemistry: X , 23 , 101647. https://doi.org/10.1016/j.fochx.2024.101647 Zeng, L., Swalla, B.J., 2005. Molecular phylogeny of the protochordates: chordate evolution. Can. J. Zool. 83(1), 24–33. https://doi.org/10.1139/z05-010 Zhang, C., Zhou, H., Cui, Y., Wang, C., Li, Y., Zhang, D., 2019. Microplastics in offshore sediment in the Yellow Sea and East China Sea, China. Environ. Pollut. 244, 827–833. Additional Declarations The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6889938","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":471030131,"identity":"1a1b8a79-ca54-4f21-9c8a-4aea82b4f662","order_by":0,"name":"Rayane Sorrentino","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIiWNgGAWjYPADAwYGfhCdUECsjgNALZINIC0GRGsBWXQAah0uYN5+xvBzQUWdnPnsw8cefyi4I2d8fnXihwcGDPL8YgewapE5k2MsPePMYWOZc2npBgcMnhmb3Xi7WQLoMMOZsxOwapFgyDGQ5m07kDiDh8dM4oDB4cRtN85uAGlJMLiNQwv/G+PfvP/qgFr4v4G1bJ5xdvMPvFokcsykeRuYQbawgbVs4O/dht8WiWdl1jzHDhtL8LCZSZwxADJu8G6zSDCQwO0X/uTNt3lq6uQkeJifSVT8OSzH3392880fFTby/NLYtTAwcKBHgUQCJFxwA/YHaAL8B/CoHgWjYBSMgpEIALC2XCPaDOw6AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-0937-0214","institution":"Universidade Federal do Etado do Rio de Janeiro","correspondingAuthor":true,"prefix":"","firstName":"Rayane","middleName":"","lastName":"Sorrentino","suffix":""},{"id":471030362,"identity":"00794e5b-e8f6-4109-bd18-85825f23c983","order_by":1,"name":"Paulo Cezar Azevedo da Silva","email":"","orcid":"https://orcid.org/0000-0002-6896-8363","institution":"Universidade do Estado do Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Paulo","middleName":"Cezar Azevedo da","lastName":"Silva","suffix":""},{"id":471030361,"identity":"562a67c6-d4d4-4ba8-9594-c55f6eddefd8","order_by":2,"name":"Brenda dos Santos Ramos","email":"","orcid":"https://orcid.org/0000-0001-5710-1097","institution":"Universifdade do Estado do Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Brenda","middleName":"dos Santos","lastName":"Ramos","suffix":""},{"id":471030363,"identity":"8eb5863d-1e99-43e7-ac4d-10c569c735a1","order_by":3,"name":"Luis Felipe Skinner","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBACPgY2hgMgBj+IkICw8QM2mBbJBlK0gIEBRDExWviPJR4u3GGTb3wj+dgHC4Y7+YS1SKQdODzzTJrlthtpyTMkGJ5ZNhDWwt5wmLftsIHZjRxjoF8OGxDhsOMgLf8NjGfkfyZSCwPQYbxtBwwMJHKYidQikZYA1JJsIHHmGdBhBs8Ia+HnP2b8mbfNzoC/Pfkxs0TFHcJaUACzBIkaGBgYP5CqYxSMglEwCkYEAACN9ja90nUZtQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0971-4870","institution":"Universidade do Estado do Rio de Janeiro","correspondingAuthor":true,"prefix":"","firstName":"Luis","middleName":"Felipe","lastName":"Skinner","suffix":""}],"badges":[],"createdAt":"2025-06-13 16:57:21","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6889938/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6889938/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84762023,"identity":"0dd862af-49c7-4257-937a-78032a8c0793","added_by":"auto","created_at":"2025-06-17 06:14:43","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":211175,"visible":true,"origin":"","legend":"\u003cp\u003eSampling sites in Ilha Grande Bay. The designated areas include: (1) Piraquara de Fora; (2) Marina Brachuy; (3) Santa Luzia Pier; (4) Praia Vermelha; (5) Ponta Leste; (6) Portogalo. Inside the circles (LC1 and LC2) are the two little cove composed by: (7) Lopes Mendes; (8) Jorge Grego Island; (9) Dois Rios; (10) Parnaioca; (11) Ponta da Tacunduba; (12) Aventureiro.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6889938/v1/850bd84aae196fa5f56127a5.jpeg"},{"id":84761268,"identity":"3a58e471-ddb4-4c8b-89e2-97b02bebdd92","added_by":"auto","created_at":"2025-06-17 06:06:43","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57620,"visible":true,"origin":"","legend":"\u003cp\u003eAverage of MP.ind\u003csup\u003e.-1\u003c/sup\u003e recorded in ascidians from Ilha Grande Bay. Small Cave: SC1: Lopes Mendes/Jorge Grego/Dois Rios); SC 2: Parnaioca/Aventureiro/Tacunduba.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6889938/v1/01f20be15962ab5d462238b0.jpeg"},{"id":84762024,"identity":"f06bcc68-f435-4c48-bbf1-ff1312b37c08","added_by":"auto","created_at":"2025-06-17 06:14:43","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":321272,"visible":true,"origin":"","legend":"\u003cp\u003eAverage of MP.ind\u003csup\u003e.-1\u003c/sup\u003e in the sampling sites: Areas on the mainland (PR: Piraquara; PV: Praia Vermelha; PL: Ponta Leste); Oceanic areas SC1 (Little Cove 1): Dois Rios, Lopes Mendes and Jorge Grego and SC2 (Little Cove 2): Aventureiro, Ponta da Tacunduba and Parnaioca). Areas considered as harbor (BR: Marina Brachuy; CS: Cais de Santa Luzia; PG: Portogalo).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6889938/v1/d655a9854fdd7c98528eb91f.jpeg"},{"id":84761285,"identity":"889d6ecd-0908-4504-b308-f22083befb89","added_by":"auto","created_at":"2025-06-17 06:06:44","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":52219,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration of microplastics in ascidian’s species during years and seasons.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6889938/v1/cc84d6f444e2299ea85c1a45.jpeg"},{"id":84761271,"identity":"e7fa0d58-d2df-4710-8268-b0b01097af75","added_by":"auto","created_at":"2025-06-17 06:06:43","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":43683,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration of microplastics particles of microfibers, fragments and granules from summer and winter seasons from 2019 to 2021.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6889938/v1/9803c3b00d8c02bcce90023d.jpeg"},{"id":84763021,"identity":"59c49ac9-1736-458a-9628-a5e61d6670c2","added_by":"auto","created_at":"2025-06-17 06:30:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1448741,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6889938/v1/b43c5170-33ff-409f-a844-cd070f375552.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eDetection of Microplastic contamination in shallow marine habitats using solitary ascidians: Influence of morphology and habitat in a tropical bay, SW Atlantic\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cul class=\"decimal_type\"\u003e\n \u003cli\u003eThe studied ascidian species showed different levels of microplastic contamination. \u003cem\u003eStyela plicata\u003c/em\u003e had the highest average (0.76 MP.ind\u003csup\u003e.-1\u003c/sup\u003e), while \u003cem\u003eMicrocosmus exasperatus\u003c/em\u003e had the lowest (0.12 MP.ind\u003csup\u003e.-1\u003c/sup\u003e).\u003c/li\u003e\n \u003cli\u003eThe morphology of tentacles (simple) and the presence of branchial folds affect the quantity of ingested microplastic in these solitary ascidians.\u003c/li\u003e\n \u003cli\u003eAreas near harbors displayed higher microplastic abundance in ascidians compared to areas without harbors.\u003c/li\u003e\n \u003cli\u003eMicroplastic concentration in ascidians was higher in winter (1.7 \u0026ndash; 1.8 MP.ind\u003csup\u003e.-1\u003c/sup\u003e) compared to summer (0.5 \u0026ndash; 1.2 MP.ind\u003csup\u003e.-1\u003c/sup\u003e).\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eWorldwide contamination by microplastics (MP), i.e. synthetic particles lower than 5mm in size, is an issue that concerns researchers in different ecosystems and human health professionals (Jeong et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Unuofin \u0026amp; Igwaran, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). On marine environments, MP contamination is now, a ubiquity and a threat, and several marine organisms with commercial use or not, are used as a survey or in monitoring protocols (Alfaro‑N\u0026uacute;\u0026ntilde;ez et al., 2021; Simmons et al., 2025).\u003c/p\u003e \u003cp\u003eMicroplastics exert its influence on aquatic environments through trophic transfer mechanisms and biomagnification. Microplastic enters trophic cascade by diverse ways, mainly through species ingestion and filtration behavior (Unuofin \u0026amp; Igwaran, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Marchala et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Messinetti et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e). Microplastic can also be transferred to trophic cascade through animal faeces that can be keep suspended on water column as a organic matter or deposited on seafloor (Li \u0026amp; Meng, 2025; My et al., 2025).\u003c/p\u003e \u003cp\u003eIn the search for MP bioindicator species, several aspects are used as the use on human feeding, the habitat and biology of species and the way that they ingested MP, the assimilation rate and/or the cleaning mechanisms, morphological characteristics of species, the temporal and spatial availability of species among others (Valente et al., 2025; Pastorino \u0026amp; Barcel\u0026oacute;, 2024; Avio et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong filter feeding marine invertebrates, ascidians are one of the most frequent components of benthic environments. They exist as solitary, colonial, and social animals (Zeng \u0026amp; Swalla, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), inhabit from intertidal zones to abyssal regions of several marine ecosystems like coral reefs, mangroves, rocky shores, and as biofouling on human constructions, including marine farms (Rocha et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). They filter seawater with suspended organic particles as a food source by the action of the tentacles lying at the base of the inhalant siphon (Petersen, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Riisg\u0026aring;rd \u0026amp; Larsen, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010a\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Wotton, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the sequence, water flows through the branchial basket (also defined as pharyngeal basket), allowing processes such as nutrition, respiration, and clearance management (Petersen, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDue to ascidians' distribution, filter-feeding habits, and capacity to accumulate pollutants, they have been used as bioindicators of human impacts on the ocean, from organic pollution (Terlizzi et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), to heavy metals (Tzafriri-Milo et al., 2019), pharmaceutical (Navon et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), Environmental Risk Assessment (Rosner \u0026amp; Rinkevich, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and more recently, microplastics, as particles (Silva et al., 2021) or its chemical compounds (Vered et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent research on MP used ascidians as models, but most performed under laboratory conditions, indicating some effects in nutritional states, alterations in metamorphosis and embryonic development, as well as impairments in pigment organs (ocellus) and cellular formation (Messinetti et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003eb\u003c/span\u003e). Few studies investigate MP in ascidians from the natural environment (Vered et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Silva et al., 2021). At the same time, there is no study showing a seasonal approach for MP presence on ascidian species, neither exploring differences in its anatomical features related to this issue. To address these questions, we analyzed MP contamination in four ascidian species with distinct morphological characteristics of tentacles and branchial folds, from samples collected on sites with and without harbor facilities from one tropical bay on southwest Atlantic. Those species were \u003cem\u003eHerdmania pallida\u003c/em\u003e and \u003cem\u003eMicrocosmus exasperatus\u003c/em\u003e that have ramified oral tentacles (8 to 23 tentacles and 16 to 20 branchial folds), and \u003cem\u003ePhallusia nigra\u003c/em\u003e (none fold), and \u003cem\u003eStyela plicata\u003c/em\u003e (8 branchial folds), with simple tentacles. Microplastics were quantified in species to answer three questions: 1) Do accumulation of microplastics by ascidians from natural habitats differs from those on the proximity to artificial ones ? 2) Are there variations in microplastic contamination across these species? 3) Do anatomical features related to shape and number of oral tentacles, and the presence and extent of branchial folds influence this microplastic accumulation?\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSampled area and fieldwork\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIlha Grande Bay (BIG \u003cem\u003ePortuguese abbreviation\u003c/em\u003e) is situated to the southwest of Rio de Janeiro state, and is officially designated as a protected area by the State constitution (Johnsson \u0026amp; Ikemoto, 2015; Silva et al., 2021). The region encompasses both terrestrial and marine ecosystems, characterized by a diverse array of endemic species and an abundance of various organisms. This biodiversity is impacted by potential pollution sources such as fishing and tourism activities, as well as industrial operations including nuclear and oil facilities, shipyards, and marinas (Johnsson \u0026amp; Ikemoto 2015).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNevertheless, in the region, the marine contamination has been impacted by robust demographic expansion and touristic activities, compounded by deficient solid waste disposal practices and inadequate environmental education. Some studies evidenced the presence of plastic and microplastic along the beachfront of BIG (Macedo et al., 2017), as a vector of invasive species (Mantelatto et al., 2020, P\u0026oacute;voa et al., 2022), and within marine organisms such as amphipods and the ascidian \u003cem\u003ePhallusia nigra\u003c/em\u003e (Silva et al., 2021).\u003c/p\u003e\n\u003cp\u003eMicroplastic pollution exhibits ubiquity even within Environmental Protected Areas, such as the BIG, an area that garnered the designation of World Heritage by UNESCO in 2019 (Silva et al. 2021). This region is distinguished by its pronounced endemism and the presence of well-conserved expanses comprising eleven conservation units (Creed et al. 2007).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSampled areas were defined by species distribution according to the presence and absence of the harbor/marina activities, and the exposure to the ocean side. The designated areas included (1) Continental Urban areas without harbor: Ponta Leste, Piraquara, Praia Vermelha; (2) Continental areas with harbor: Santa Luzia Pier, Portogalo, Marina Brachuy; and (3) oceanside and non-urban areas divides in Small Cove 1 (SC 1) (Dois Rios, Jorge Grego, Lopes Mendes), and Small Cove 2 (SC 2) (Aventureiro, Parnaioca, Ponta da Tacunduba) (Fig. 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe four species colonize the natural and artificial substrates from Ilha Grande Bay. The main anatomical features of the species are the following: \u003cem\u003eHerdmania pallida\u003c/em\u003e (Pyuridae) has 17 to 24 up to third order branched tentacles, and 8 to 9 branchial folds (Nishikawa, 2002; Rocha et al., 2012). \u003cem\u003eMicrocosmus exasperatus\u003c/em\u003e (Pyuridae) has 8 to 10 pinned first order tentacles, and intermediate ones, with 8 to 10 branchial folds, most commonly 9 (Van Name, 1945). \u003cem\u003ePhallusia nigra\u003c/em\u003e (Ascidiidae) is a tropical species, well recognized in fieldwork by your black tunic, which is not fouled by symbionts. It has several simple tentacles, circa 50, but larger animals can have more than 100 tentacles (Van Name, 1945). The branchial sac does not show any internal fold. \u003cem\u003eStyela plicata\u003c/em\u003e (Styelidae), is commonly reported from biofouling communities, mainly from artificial substrates, frequently as invasive species. It has also simple tentacles (25-30) but has 4 branchial folds (Van Name 1945; Monniot, 1969). These species were the most conspicuous along the studied site and along the SE Brazilian coast (Rocha et al., 2012; Skinner et al., 2016), representing good models for future large-scale comparisons.\u003c/p\u003e\n\u003cp\u003eFrom each location and season from the austral winter of 2019, summer of 2020, and winter and summer of 2021, ten individuals of each species were sampled for MP assessment. This number of individuals at each site and season was considered large enough to represent the influence of habitat on ascidian filtration and MP ingestion (Silva et al., 2021) and is similar to those found on other papers (Avio et al., 2020). However, not all these species necessarily coexist at the same site, so, the total number of collected individuals is not equal for each species. Sampling was performed manually by snorkeling, from 0.5 to 5 meters deep and at 1.0 meters deep in pier areas. To prevent the contraction of the organisms and the potential loss of material from the atrial cavity, the individuals were collected using microbiological bags (Kasvi) and subsequently anesthetized with diluted menthol in seawater. Before sampling, plastic bags were cleaned with distilled water to prevent plastic contamination. For preservation and subsequent microplastic extraction purposes, a solution of 10% formaldehyde was introduced. It is important to note that the use of formaldehyde does not impact \u0026nbsp;microplastic particles in question (Courtene-Jones et al. 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMicroplastic extraction\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe organisms were dissected, removing the tunic and branchial basket, and the inner body (the body structure containing the stomach, intestine, gonads and other structures) was externally rinsed with distilled water for analysis. The inner bodies of the ten collected individuals of each species at each sampling site were pooled together. Subsequently, the pooled inner bodies were sectioned and combined with hypersaline water (composed of 358.9g of NaCl in 1L of distilled water) for density separation, a process lasting 24 hours. This method is cost-effective, it only separates polymers less dense than 1.2 g cm\u003csup\u003e-3\u003c/sup\u003e (\u003cem\u003ei.e.\u0026nbsp;\u003c/em\u003epolyvinyl chloride (PVC)), however, denser polymers have been quantified by this method (Montagner et al. 2021). The supernatant was filtered through a cellulose filter 5\u0026micro;m (Whatman AE98) employing a vacuum pump Primatec 121. The filters were placed in a cleaned petri dish and the microplastics were quantified, measured, and classified into three categories: fragments, fibers, and granules. This was performed using a microscopy Bel Photonics, STEREO-ZOOM SERIE SZ/SZT (5,6x) connected to a camera EUREKAM 5.0 Mpixels. The results were expressed as the \u0026ldquo;MP \u003cem\u003eitems per individual\u0026rdquo;\u003c/em\u003e (MP.ind\u003csup\u003e.-1\u003c/sup\u003e) in each sample and site.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuality assurance and quality control\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePreventive measures were followed to mitigate external contamination by MP, such as reduction of staff, utilization of cotton lab coats worn by researchers, cleansing of materials using distilled water, and the application of aluminum foil to envelop flasks and containers, thereby forestalling the possibility of cross-contamination via airborne particles.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFive recovery samples were applied, using ascidians 5 fibers (nylon, density 1.14 g.cm-\u003csup\u003e3\u003c/sup\u003e) and 5 fragments (polystyrene, density 1.05 g.cm-\u003csup\u003e3\u003c/sup\u003e). Four samples represented a recovery of 100% of fragments and fibers, and one sample was 90% of fibers. As a control sample, Petri dishes with distilled water were laid out during the processing of the samples, then filtered and analyzed. The MP found in the control and those that were like the samples were discarded from the results, as in Vered et al., (2019). Data was analyzed and created using Excel (Microsoft).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnalysis of MP particles\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMP particles were analyzed using Fourier transform infrared spectroscopy (FTIR) with attenuated total reflection (ATR) Perkin Elmer (Perkin Elmer Spectrum Version 10.5.2) for polymer identification. The spectroscopic examination encompassed a range from 4000 to 600 cm-1 and the spectral resolution of 4 cm-1. A proportion of 30% of microplastic particles could be analyzed by FT-IR since this equipment detects particles up to 2000 \u0026micro;m in dimension size. The polymers were detected using software spectral references and functional chemical groups. The particles were quantified and compared among species and sites.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe statistical analyses were conducted using RStudio software (2024.09.1+394). As the data was not normally distributed (Shapiro-Wilk test, \u003cem\u003ep\u003c/em\u003e \u0026lt;0.001; and Levene\u0026rsquo;s Test, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.013). Kruskal-Wallis and Post hoc Dunn Testes were used to compare the variable MP.ind\u003csup\u003e.-1\u003c/sup\u003e among species and sites (significant level was considered as \u0026lt; 0.05).\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eWe investigated the presence of MP on 563 individuals from the four studied species and MP were notably prevalent across all species and sampling sites, totalizing 156 items (83 fragments, 48 fibers, and 23 granules) and an average (and SD) of 0.29 \u0026plusmn; 0.3 MP.ind\u003csup\u003e-1\u003c/sup\u003e, corresponding to 0.15 \u0026plusmn; 0.17 MP.ind\u003csup\u003e-1\u003c/sup\u003e of fragments, 0.10 \u0026plusmn; 0.25 MP.ind\u003csup\u003e-1\u003c/sup\u003e microfibers and 0.03 \u0026plusmn; 0.08 MP.ind\u003csup\u003e-1\u003c/sup\u003e granules (Table 1). According to the Kruskal-Wallis test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001), the abundance of MP in the species was distinct. \u003cem\u003eStyela plicata,\u0026nbsp;\u003c/em\u003ewith simple tentacles,\u003cem\u003e\u0026nbsp;\u003c/em\u003eexhibited a significant prevalence of MP (0.72 \u0026plusmn; 0.44 MP.ind\u003csup\u003e.-1\u003c/sup\u003e average \u0026plusmn; SD) among all species (Dunn test \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05). The other species did not differ, with \u003cem\u003eP. nigra\u0026nbsp;\u003c/em\u003eshowing a concentration of 0.23 \u0026plusmn; 0.13 MP.ind\u003csup\u003e.-1\u003c/sup\u003e, \u003cem\u003eH. pallida\u003c/em\u003e 0.20 \u0026plusmn; 0.17 and \u003cem\u003eM. exasperates\u003c/em\u003e 0.13 \u0026plusmn; 0.12 MP.ind\u003csup\u003e.-1\u003c/sup\u003e. Fragments were dominant in all species, followed by fibers and granules (Table 1). Blue and black fragments (\u0026gt; 5000 \u0026micro;m) were observed in \u003cem\u003eStyela plicata\u003c/em\u003e from Bracuhy, Cais de Santa Luzia, and in \u003cem\u003ePhallusia nigra\u003c/em\u003e also from Cais de Santa Luzia, and SC 1, Ponta Leste and Praia Vermelha (Table SM 1). These particles were not countable in statistical analysis, but were observed, categorized and noted. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eThe total of individuals sampled in each site, the morphological features of each species separated by the type and number of tentacles and the number of pharyngeal folds at each side of the body of ascidians and, the total items of fragments, fibers, and granules, and the average of MP.ind.\u003csup\u003e-1\u003c/sup\u003e reported in each ascidian species.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"633\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.5906%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpecies\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7559%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal ind\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.2835%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTentacles shape (and number)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.44882%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFaringeal folds\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.8031%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFragments\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.811%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFibers\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.13386%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGranules\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.1732%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMP.ind\u003csup\u003e.-1\u0026nbsp;\u003c/sup\u003e(Average \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.5906%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. plicata\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7559%;\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.2835%;\"\u003e\n \u003cp\u003eSimple (25 a 30/ + )\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.44882%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.8031%;\"\u003e\n \u003cp\u003e0.35\u0026nbsp;\u0026plusmn; 0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.811%;\"\u003e\n \u003cp\u003e0.29\u0026nbsp;\u0026plusmn; 0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.13386%;\"\u003e\n \u003cp\u003e0.08\u0026nbsp;\u0026plusmn; 0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.1732%;\"\u003e\n \u003cp\u003e0.72 \u0026plusmn; 0.44\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.5906%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP. nigra\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7559%;\"\u003e\n \u003cp\u003e208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.2835%;\"\u003e\n \u003cp\u003eSimple (50/ 100+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.44882%;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.8031%;\"\u003e\n \u003cp\u003e0.13\u0026nbsp;\u0026plusmn; 0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.811%;\"\u003e\n \u003cp\u003e0.07 \u0026plusmn; \u0026nbsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.13386%;\"\u003e\n \u003cp\u003e0.02 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.1732%;\"\u003e\n \u003cp\u003e0.23 \u0026plusmn; 0.13\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.5906%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eH. pallida\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7559%;\"\u003e\n \u003cp\u003e102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.2835%;\"\u003e\n \u003cp\u003eBranched (8 \u0026ndash; 12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.44882%;\"\u003e\n \u003cp\u003e8 - 10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.8031%;\"\u003e\n \u003cp\u003e0.08 \u0026plusmn; 0.10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.811%;\"\u003e\n \u003cp\u003e0.09 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.13386%;\"\u003e\n \u003cp\u003e0.02 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.1732%;\"\u003e\n \u003cp\u003e0.20 \u0026plusmn; 0.17\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 15.5906%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eM. exasperatus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.7559%;\"\u003e\n \u003cp\u003e155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12.2835%;\"\u003e\n \u003cp\u003eBranched (8 \u0026ndash; 10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.44882%;\"\u003e\n \u003cp\u003e8-9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.8031%;\"\u003e\n \u003cp\u003e0.08\u0026nbsp;\u0026plusmn; 0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.811%;\"\u003e\n \u003cp\u003e0.01 \u0026plusmn; \u0026nbsp;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.13386%;\"\u003e\n \u003cp\u003e0.03\u0026nbsp;\u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.1732%;\"\u003e\n \u003cp\u003e0.13 \u0026plusmn; 0.12\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe average of MP in ascidians differed among the sites (\u003cem\u003ep\u003c/em\u003e = 0.001). Specimens from harbor areas displayed a greater abundance of MP, Portogalo recorded an average of 0.49 \u0026plusmn; 0.60 MP.ind.\u003csup\u003e-1\u003c/sup\u003e, Brachuy 0.45 \u0026plusmn; 0.19, and Cais de Santa Luzia 0.40 \u0026nbsp;\u0026plusmn; \u0026nbsp;0.31 MP.ind\u003csup\u003e-1\u003c/sup\u003e. Ascidians from areas without harbor showed lower concentrations of MP. Ponta Leste exhibiting 0.25 \u0026plusmn; 0.14 MP.ind\u003csup\u003e-1\u003c/sup\u003e, Piraquara 0.16 \u0026plusmn; 0.11 MP.ind\u003csup\u003e-1\u003c/sup\u003e, and Praia Vermelha 0.06 \u0026plusmn; 0.08 MP.ind\u003csup\u003e.-1\u003c/sup\u003e. But, even on the oceanic side, distant from urban centers and facing the ocean, small coves SC 1 (Lopes Mendes/Jorge Grego/Dois Rios); SC 2 (Parnaioca/Aventureiro/Tacunduba) recorded 0.20 \u0026plusmn; 0.12 MP.ind\u003csup\u003e-1\u003c/sup\u003e and 0.26 \u0026plusmn; 0.16 MP.ind\u003csup\u003e.-1\u003c/sup\u003e, respectively (Fig. 2 and Fig. 3). After the Post hoc test, Brachuy and Praia Vermelha showed a significant difference among MP averages (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.01).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFragments were dominant in \u003cem\u003eP. nigra\u0026nbsp;\u003c/em\u003efrom Cais de Santa Luzia (0.27 \u0026plusmn; 0.27 MP.ind\u003csup\u003e-1\u003c/sup\u003e), Bracuhy (0.26 \u0026plusmn; 0.21 MP.ind\u003csup\u003e-1\u003c/sup\u003e), SC1 (0.14 \u0026plusmn; 0.12 MP.ind\u003csup\u003e-1\u003c/sup\u003e), Piraquara (0.09 \u0026plusmn; 0.10 MP.ind\u003csup\u003e-1\u003c/sup\u003e) and Ponta Leste (0.11 \u0026plusmn; 0.11 MP.ind\u003csup\u003e.-1\u003c/sup\u003e). On the other hand, fibers were more frequent in Portogalo (0.27 \u0026plusmn; 0.61 MP.ind\u003csup\u003e-1\u003c/sup\u003e) and SC2 (0.13 \u0026plusmn; 0.17 MP.ind\u003csup\u003e-1\u003c/sup\u003e). Additionally, granules were less reported on all sites, except in Praia Vermelha, which was not reported.\u003c/p\u003e\n\u003cp\u003eRegarding the length of MP, fibers measured 1,716 \u0026micro;m, fragments 1,699 \u0026micro;m, and granules 929 \u0026micro;m (Table 2). The red fibers had the longest average length, measuring 3,065 \u0026micro;m, followed by black fragments with an average of 2,620 \u0026micro;m, red fragments 2,510. The smaller MP were white, black, yellow and purple granules (870, 754, 420, 160 \u0026micro;m, respectively). \u003cem\u003eStyela plicata\u0026nbsp;\u003c/em\u003ehad the larger MP, reporting black fragments and red fibers measuring an average of 3.190 and 3.065 \u0026micro;m respectively. \u003cem\u003ePhallusia nigra\u0026nbsp;\u003c/em\u003ereported an average of 2,800 \u0026micro;m to black fragments (Table SM 2). The most frequent MP in \u003cem\u003eS. plicata\u003c/em\u003e, \u003cem\u003eM. exasperatus\u003c/em\u003e and \u003cem\u003eP. nigra\u0026nbsp;\u003c/em\u003ewas blue fragment (32, 23 and 12 items), while in \u003cem\u003eH. pallida\u0026nbsp;\u003c/em\u003ewas blue fibers (11 items) (Table SM 2). Among sites, the larger average of MP was recorded respectively in Cais de Santa Luzia (red fiber 4,210 \u0026micro;m), Brachuy (black fragment 4,020 \u0026micro;m), SC 1 (black fragments 2,800 \u0026micro;m), and Piraquara (blue fibers 2,632 \u0026micro;m). The higher abundance of blue fragments is consistent in Cais de Santa Luzia (24 itens), Brachuy (11 items), SC1 and Piraquara (9 items). (Table SM 3)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003eMinimum, maximum, and average length of microplastics in ascidian species.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2766%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTypes of MP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.5319%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMinimum (\u0026micro;m)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.5319%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMaximum (\u0026micro;m)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.6596%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026plusmn; SD (\u003c/strong\u003e\u003cstrong\u003e\u0026micro;m)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2766%;\"\u003e\n \u003cp\u003e\u003cem\u003eFibers\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.5319%;\"\u003e\n \u003cp\u003e310 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.5319%;\"\u003e\n \u003cp\u003e4450 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.6596%;\"\u003e\n \u003cp\u003e1716 \u0026plusmn; 961\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2766%;\"\u003e\n \u003cp\u003e\u003cem\u003eFragments\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.5319%;\"\u003e\n \u003cp\u003e250 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.5319%;\"\u003e\n \u003cp\u003e4680 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.6596%;\"\u003e\n \u003cp\u003e1699 \u0026plusmn; 1232\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 21.2766%;\"\u003e\n \u003cp\u003e\u003cem\u003eGranules\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.5319%;\"\u003e\n \u003cp\u003e160 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 25.5319%;\"\u003e\n \u003cp\u003e2920 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.6596%;\"\u003e\n \u003cp\u003e929 \u0026plusmn; 834\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe concentration of MP in ascidians among years and seasons did not significantly differ (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05). The abundance of MP in ascidians decreased during the years, with 0.3 \u0026plusmn; 0.4 MP.ind\u003csup\u003e-1\u003c/sup\u003e in 2019, 0.28 \u0026plusmn; 0.27 MP.ind\u003csup\u003e-1\u003c/sup\u003e in 2020 and 0.27 \u0026plusmn; 0.18 MP.ind\u003csup\u003e-1\u003c/sup\u003e in 2021 (Fig. 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe concentration of MP was slightly higher during winter (0.28 \u0026plusmn; 0.24 MP.ind\u003csup\u003e-1\u003c/sup\u003e) than in summer (0.28 \u0026plusmn; 0.24 MP.ind\u003csup\u003e.-1\u003c/sup\u003e). Fragments were more abundant in all years (Figure 5).\u003c/p\u003e\n\u003cp\u003eBased on the spectroscopic analysis in 30% of the samples, and the discernment of characteristic spectral bands, the microplastics retrieved from the ascidians were categorized as high-density polyethylene (HDPE), homopolymer polypropylene (PPH), and polystyrene (PS), with a prevalence of 75% blue MP, 15% black, and 5% purple MP (2% yellow and 1% transparent, white, and red).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur results showed the importance of the morphology on the presence of MP on ascidians. The structures of tentacles and branchial basket influenced the higher amount on the widespread and invasive \u003cem\u003eStyela plicata\u003c/em\u003e, contrasting with \u003cem\u003eHerdmania pallida\u003c/em\u003e, \u003cem\u003eMicrocosmus exasperatus\u003c/em\u003e and \u003cem\u003ePhallusia nigra\u003c/em\u003e. These species differ in the shape of tentacles (simple or branched), and on the presence and number of branchial folds, and were sampled in sites influenced by the absence and presence of harbors. All species recorded at least one microplastic within their bodies, confirming their ability to accumulate MP on nature. Until now, this represents the first field study that provides valuable data on the abundance of MP in these filter-feeding species. The difference in concentration of MP was not discrepant among \u003cem\u003eP. nigra\u003c/em\u003e, \u003cem\u003eH. pallida\u003c/em\u003e and \u003cem\u003eM. exasperatus\u003c/em\u003e, which aligns with observations in other filter-feeding organisms (Pequeno et al., 2021). However, it was observed that species with simple tentacles, especially \u003cem\u003eS. plicata\u003c/em\u003e, reported more abundance of MP than those having branched tentacles. Additionally, the presence of folds on the branchial sac and a simple tentacle influences the MP concentration found in \u003cem\u003eS. plicata\u003c/em\u003e, which was approximately twice the amount found in \u003cem\u003eP. nigra\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStyela plicata\u003c/em\u003e, recognized in many regions as an invasive species, was reported to ingest more quantity of MP than \u003cem\u003eP. nigra\u003c/em\u003e, \u003cem\u003eM. exasperatus\u003c/em\u003e and \u003cem\u003eH. pallida\u003c/em\u003e. Although studies have shown micro-nanoplastic ingestion by ascidians (Messinetti et al., 2017, 2019; Eliso et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), investigation in natural contexts is still scarce. Given the observed differences in MP concentration among the four species studied, it is reasonable to suggest that the presence of simple and branched tentacles, and the folds on the branchial sac may influence microplastic accumulation. Gonzalez-Pineda et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) tested in lab conditions the ingestion of MP by two Antarctic ascidian species, \u003cem\u003eCnemidocarpa verrucosa\u003c/em\u003e and \u003cem\u003eMolgula pedunculata\u003c/em\u003e. The first one with simple oral tentacles and the second, with branched ones, and both species have branchial folds. As the mean density of particles ingested by \u003cem\u003eC. verrucosa\u003c/em\u003e was higher, it is plausible to affirm that the tentacle shape influences MP capture and accumulation.\u003c/p\u003e \u003cp\u003ePhysiological investigations conducted on solitary sea squirts have illuminated the sensitivity of tentacles to both mechanical and chemical stimuli, emphasizing their role as efficient filtering to large particles, such as microplastic, from water before they reach the pharynx (Mackie et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Members of the Stolidobranchia order, e.g. \u003cem\u003eH. pallida\u003c/em\u003e, \u003cem\u003eM. exasperatus\u003c/em\u003e, and \u003cem\u003eS. plicata\u003c/em\u003e tend to possess more than one sensory organ. \u003cem\u003eS. plicata\u003c/em\u003e for example, exhibits two distinct categories of sensory cells: one comprising a singular cilium and short stereocilia of equal length, while the order Phlebobranchia is characterized by two cilia situated within a crescent of stereovilli (Manni et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In species of the Stolidobranchia order, characterized by branched tentacles, such as \u003cem\u003eH. pallida\u003c/em\u003e and \u003cem\u003eM. exasperatus\u003c/em\u003e, the pattern of innervation is more complex, due to the branched morphology of the tentacles. Caicci et al. (2007) describe \u003cem\u003eM. socialis\u003c/em\u003e as having branched tentacles, two main nerves depart from the central axis of the tentacles (first-order branch), at the base of two rows of coronal cells. This elaborate architecture enhances the surface area of contact with the surrounding water. Given this morphology, we suggest that these species may identify, avoid, and/or reject microplastic particles by a sensitive stimulus. Gonzalez-Pineda et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) reported that \u003cem\u003eC. verrucosa\u003c/em\u003e can squirt its body, ejecting inorganic material. But \u003cem\u003eM. pedunculata\u003c/em\u003e is unable to squirt it and cannot distinguish between organic and inorganic material. The ingestion of MP can be even more complex, as shown by Pennati et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), when mixotrophic cryptomonad flagellates exposed to microbeads increased the content of MP on ascidian juveniles. It reinforces the need for more laboratory experiments to attain comprehension of the structural composition of the mechanisms selecting plastic particles by ascidians. However, these results indicate that tentacles, folds, and sensory cells of ascidians influence MP ingestion/capture.\u003c/p\u003e \u003cp\u003eThe effect of morphology on other filter feeding invertebrates was investigated by several authors in many different marine groups as fishes, bivalves and crustaceans (Jeong et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Avio et al. (2024) detected differences on MP presence between the \u003cem\u003eMytilus galloprovincialis\u003c/em\u003e and \u003cem\u003eOstrea edulis\u003c/em\u003e, two common itens on human feeding behavior. Xu et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) in a review, have indicated that MP accumulate higher on Mytilidae than on Ostreidae species, due their differences on morphology of feeding system. But large differences are reported among species of the same genus ( i.e. \u003cem\u003ePerna\u003c/em\u003e, \u003cem\u003eMytilus\u003c/em\u003e, \u003cem\u003eOstrea\u003c/em\u003e, \u003cem\u003eCrassostrea\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eThe amount of MP detected in our survey (from 0.13 to 0.72 MP.ind\u003csup\u003e-1\u003c/sup\u003e) is within the same range from several other studies, using bivalves as a proxy due to this importance on human feeding (Xu et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This density was lower than on other Brazilian areas like the closest and very polluted Guanabara bay (Birnstiel et al., 2019) or Santos (Santana et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFragments were more abundant than fibers and granules. Similar results were shown by Pequeno et al. (2021), where fragments were common in deposit filter-feeding polychaete \u003cem\u003eMarphysa sanguinea\u003c/em\u003e and the bivalve \u003cem\u003eScrobicularia plana\u003c/em\u003e. It is not clear how filter-feeding is related to particle ingestion and frequently this MP abundance is related to the environment. Fragments are formed from the fragmentation of macroplastics, and they are known to persist on sea surfaces due to their floatability properties (Fagiano et al., 2023). These species were sampled in shallow waters (0.5 to 5 meters deep), where they are more exposed to fragments than other types of MP. Cho et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), working with bivalves also found the prevalence of fragments. However, Joyce and Falkemberg (2023) found also on bivalves, a predominance of fibers over fragments. These contrasting results shows that ingestion of MP can be very variable on different environments.\u003c/p\u003e \u003cp\u003eAs indicated from several studies (Zhang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Cutroneo et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Joshi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e among others), harbor regions are influenced by port activities, fishing operations, and urban drainage systems, some of the major contributors to MP pollution and fragmentation of macroplastics. So, it was not a surprise to find high concentration of MP on ascidians collected near harbors on studied region.\u003c/p\u003e \u003cp\u003eMicroplastic quantification revealed a seasonal trend, with higher concentrations observed in the winter of 2019. In the summer of 2020 and 2021, a reduction of microplastic concentration was observed, potentially linked to a decline in tourism and other human activities due to the Sars-Cov-19 pandemic. It is important to highlight that ascidians were not sampled during winter of 2020 due to these restrictions. However, the restrictions were less severe in Brazil during the winter of 2021 and the results suggest that MP concentration is associated with the human use of the natural environment. The winter highs detected here may be related to the summer increase on inadequate plastic discard, and under environmental conditions of high seawater and air temperature, friction with rocky shores, sandbeaches and boats outboard generated impact/turbulence may increase fragmentation, which also explain the prevalence of fragments on our samples. Bom et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) with \u003cem\u003ePerna perna\u003c/em\u003e in another bay at Brazilian coast detected slightly differences among MP content during the four studied season on the year 2020, with higher amounts on winter, but not differing from autumn and summer. It is difficult to compare data along time and the impact of health restrictions due SARS-COVID 19 from both samples.\u003c/p\u003e \u003cp\u003eAnalysis revealed that fragments were the most common microplastic type, followed by microfibers and granules, and fragments were also the largest. The identified polymers were polyethylene (PE), polypropylene (PP), and polystyrene (PS). The presence of PE, PP, and PS, the prevalence of blue MP, as well as the average length aligns with findings from other studies (Gago et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Pequeno et al. 2021). PE, PP, and PS in the marine environment reflect the broad manufacture of these polymers and their distribution worldwide (Pequeno et al., 2021). The distribution of fragments, fibers, and granules in the water collum, especially near harbor areas, contributes significantly to plastic pollution in the marine environment and its subsequent ingestion by numerous organisms.\u003c/p\u003e \u003cp\u003ePrevious investigations on Ilha Grande bay documented the presence of solid residues (Macedo et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), macroplastics (Macedo et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and microplastics (Silva et al., 2021), all aligned with the increased tourist activity, urban center development, and the presence of port structures. However, only one paper deals with MP on marine fauna (Silva et al., 2021). Cais de Santa Luzia has a simple wharf building, mainly used for the landing of fishermen (Johnsson \u0026amp; Ikemoto \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and situated within a cove near a river of the same name, is affected by both a channel accommodating small and large vessels and an unsustainable tourist establishment. Similarly, coastal areas like the marinas area of Portogalo and Brachuy, and crowded sites of Ponta Leste, and Piraquara have been impacted by intense tourism and real estate speculation. The irregular occupation and lack of adequate residue treatment in these regions pose a threat to both marine and coastal ecosystems. The oceanic side has also been impacted by tourism; however, its distance and difficult access limit a larger number of tourists. However, even facing the Atlantic Ocean, microplastics are still presently originated, and distributed by oceanic factors such as ocean and coastal currents, waves and wind (Macedo et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This spatial variability in human activity and contamination by MP is common in many places worldwide (e.g., Cho et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Joshi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and indicates that humanity has spread MP to even the most remote places, with several unevaluated or underestimated impacts.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis paper shows that Ascidians can be used as a proxy for MP contamination since its levels of particle densities are close to that one found for several species of bivalves worldwide for example. However, its morphological features can affect the total number of particles and this must be considered when lone species is chosen.\u003c/p\u003e \u003cp\u003eIn the absence, to the moment, of data concerning MP contamination on other marine animals on BIG, this data can support future studies and monitoring programs, and the discussion by governmental and non-governmental stakeholders about mechanisms to prevent MP contamination, mainly due to the presence of several sea farms on region.\u003c/p\u003e \u003cp\u003eAnd finally, this approach may be extended for other Ascidian species or other species, like the co-occurring mussels \u003cem\u003ePerna perna\u003c/em\u003e and the recently introduced \u003cem\u003ePerna viridis\u003c/em\u003e (Barbieri et al., 2025; Messano et al., 2025) that is being explored on SW Atlantic without any deeply investigation about differences on MP contamination\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.S.: Conceptualization; Supervision, Investigation; Writing \u0026ndash; original draft; Writing \u0026ndash; review \u0026amp; editing;\u003c/p\u003e\n\u003cp\u003eP.S.: Methodology;\u0026nbsp;Formal Analysis;\u0026nbsp;Investigation; Writing \u0026ndash; original draft; Writing\u0026nbsp;\u0026ndash; review \u0026amp; editing;\u003c/p\u003e\n\u003cp\u003eB.R.: Sampling, Methodology,\u0026nbsp;Writing \u0026ndash; original draft;\u003c/p\u003e\n\u003cp\u003eL.F.S.: Supervision; \u0026nbsp;Funding acquisition; Writing \u0026ndash; original draft\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; review \u0026amp; editing;\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare none competing interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors thank Dr. Rafael B. de Moura for the photos acquired and edition. We also thank Prof. Dr Monica Marques for assistance with the FTIR analysis. This work was financed in part by the Programa de Treinamento e Capacita\u0026ccedil;\u0026atilde;o T\u0026eacute;cnica (FAPERJ No 04/2019); Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brazil (CAPES) - Finance Code 001; Programa de Incentivo \u0026agrave; Produ\u0026ccedil;\u0026atilde;o Cient\u0026iacute;fica, T\u0026eacute;cnica e Art\u0026iacute;stica (PROCI\u0026Ecirc;NCIA); Funda\u0026ccedil;\u0026atilde;o Carlos Chagas Filho de Amparo \u0026agrave; Pesquisa do Estado do Rio de Janeiro (FAPERJ), process number E-26/202.768/2019. We are thankful to CEADS UERJ and ESEC Tamoios for the Logistical support during fieldwork. Surveys are performed under licenses from INEA and SISBIO (Auth. #057/2011, 025/2017 and 029/2019) and Esta\u0026ccedil;\u0026atilde;o Ecol\u0026oacute;gica Tamoios (ICMBio - Auth. #36194).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlfaro-N\u0026uacute;\u0026ntilde;ez, A., Astorga, D., C\u0026aacute;ceres-Far\u0026iacute;as, L., Bastidas, L., Soto, L., Valenzuela-Toro, A. M., \u0026amp; Duran, L. R. (2021). 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Pollut.\u003c/strong\u003e 244, 827\u0026ndash;833.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Rio de Janeiro State University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ascidian, tentacles morphology, branchial folds, plastic pollution, Ilha Grande Bay","lastPublishedDoi":"10.21203/rs.3.rs-6889938/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6889938/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThe ubiquity of microplastics (MP) in marine ecosystems and their biological uptake has become a major global concern. Many papers indicate MP ingestion by marine organisms, but few studies address the role of morphological characteristics of species on MP ingestion and retention in filter-feeding species. This study investigates the presence of MP in four ascidians species as models\u003c/span\u003e: \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eStyela plicata, Phallusia nigra, Microcosmus exasperatus\u003c/span\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand\u003c/span\u003e \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eHerdmania pallida\u003c/span\u003e. \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eThey are compared in relation to the shape (simple or branched) and number of oral tentacles, and the presence of branchial folds (from none to 18). Morphology, here, were used as a proxy for mechanisms that may prevent MP ingestion. Furthermore, we compared the concentration of MP among species from sites with and without harbor, and during summer and winter seasons. Specimens were collected in the Ilha Grande Bay, where they are widely distributed in natural and artificial substrates. Microplastics were extracted by density separation, quantified, and categorized by granules, fibers and fragments. The morphological structure present in\u003c/span\u003e \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eS. plicata\u003c/span\u003e, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ewith simple tentacles and branchial folds retained significantly more MP particles than all other species. This suggests that tentacles morphology and the presence of branchial folds may influence MP capture. Specimens near harbor areas and sampled during winter showed higher abundance of MP, reflecting association of plastic pollution with shipping and unsustainable tourism activities. This is the first assessment of MP contamination in ascidians from tropical bays and from natural environments. Its association with morphology, opens new perspectives for studies dealing with mechanics of filtering activity of marine invertebrates.\u003c/span\u003e\u003c/p\u003e","manuscriptTitle":"Detection of Microplastic contamination in shallow marine habitats using solitary ascidians: Influence of morphology and habitat in a tropical bay, SW Atlantic","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 06:06:38","doi":"10.21203/rs.3.rs-6889938/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"841abc4e-725c-4529-81e2-0eec4e31d275","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50026104,"name":"Oceanography"},{"id":50026105,"name":"Marine and Freshwater Ecology"}],"tags":[],"updatedAt":"2025-06-17T06:06:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-17 06:06:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6889938","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6889938","identity":"rs-6889938","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}