# Plastisphere composition in a subtropical estuary: Influence of season, incubation time and polymer type on plastic biofouling

Tobias Sérvulo, Joe D Taylor, Maíra C Proietti, Lucas D S Rodrigues, Igor P Puertas, Roberta A Barutot, Ana L D F Lacerda

► To cite this version:

Tobias Sérvulo, Joe D Taylor, Maíra C Proietti, Lucas D S Rodrigues, Igor P Puertas, et al. Plastisphere composition in a subtropical estuary: Influence of season, incubation time and polymer type on plastic biofouling. Environmental Pollution, 2023, 332 (121873), pp.121873. 10.1016/j.envpol.2023.121873. hal-05493764

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# Plastisphere composition in a subtropical estuary: Influence of season, incubation time and polymer type on plastic biofouling

Tobias Sérvulo<sup>a,*,1</sup>, Joe D. Taylor<sup>b</sup>, Maíra C. Proietti<sup>a,2</sup>, Lucas d.S. Rodrigues<sup>a,3</sup>,
Igor P. Puertas<sup>a,4</sup>, Roberta A. Barutot<sup>a</sup>, Ana L.d.F. Lacerda<sup>a,c,5</sup>

E-mail address: tobias.tsr@usp.br (T. Sérvulo).

1 Present address: Laboratório de Oceanografia Microbiana - MicrOcean Lab, Instituto Oceanográfico – IO, Universidade de São Paulo – USP, São Paulo, Brazil.

2 Present address: The Ocean Cleanup, Rotterdam, the Netherlands.

3 Present address: Departament d'Estadística i Investigació Operativa, Universitat de València, Valencia, Spain.

4 Present address: Laboratório de Anfíbios e Répteis - LAR, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, Brazil.

5 Present address: Laboratoire d'Océanographie de Villefranche, Sorbonne Université, Villefranche-sur-Mer, France.

**Keywords:**

Plastics

Plastisphere

DNA

metabarcoding

Patos Lagoon

estuary

**A B S T R A C T**

Plastics are abundant artificial substrates in aquatic systems that host a wide variety of organisms (the plasti-sphere), including potential pathogens and invasive species. Plastisphere communities have many complex, but not well-understood ecological interactions. It is pivotal to investigate how these communities are influenced by the natural fluctuations in aquatic ecosystems, especially in transitional environments such as estuaries. Further study is needed in subtropical regions in the Southern Hemisphere, where plastic pollution is ever increasing. Here we applied DNA-metabarcoding (16S, 18S and ITS-2) as well Scanning Electron Microscopy (SEM) to assess the diversity of the plastisphere in the Patos Lagoon estuary (PLE), South Brazil. Through a one-year *in situ* colonization experiment, polyethylene (PE) and polypropylene (PP) plates were placed in shallow waters, and sampled after 30 and 90 days within each season. Over 50 taxa including bacteria, fungi and other eukaryotes were found through DNA analysis. Overall, the polymer type did not influence the plastisphere community composition. However, seasonality significantly affected community composition for bacteria, fungi and general eukaryotes. Among the microbiota, we found *Acinetobacter* sp., *Bacillus* sp., and *Walloemia mellicola* that are putative pathogens of aquatic organisms, such as algae, shrimp and fish, including commercial species. In addition, we identified organisms within genera that can potentially degrade hydrocarbons (e.g. *Pseudomonas* and *Cla-dosporium* spp). This study is the first to assess the full diversity and variation of the plastisphere on different polymers within a subtropical Southern Hemisphere estuary, significantly expanding knowledge on plastic pollution and the plastisphere in estuarine regions.

**Introduction**

Plastic pollution is currently one of the major issues affecting aquatic systems, causing serious impacts in marine ecosystems (Everaert et al., 2020). Transitionary environments such as estuaries have a great importance for the transfer and retention of plastics between continents and oceans (UNEP - United Nations Environment Programme, 2016), and it is predicted that between 0.8 and 2.7 million metric tons of

mismanaged plastics annually flush from rivers toward the oceans (Meijer et al., 2021). The packaging industry is one of the main sources of plastics found in marine ecosystems (36%) (GESAMP, 2019), with polyethylene and polypropylene being the most common polymers used in packaging and single-use plastics (Fruergaard et al., 2023; Van Emmerik, et al., 2018). Due to society's high demand for these products (Plastics Europe, 2021) and their mismanagement on land, they are frequently found in estuarine and coastal environments (Van Emmerik,

<footnote><sup>a</sup> *Projeto Lixo Marinho - Instituto de Oceanografia, Universidade Federal do Rio Grande - FURG, Rio Grande, Brazil*</footnote>

<footnote><sup>b</sup> *UK Centre for Ecology and Hydrology, Wallingford, UK*</footnote>

<footnote><sup>c</sup> *University of Salford, Salford, Greater Manchester, United Kingdom*</footnote>

<footnote>* Corresponding author.</footnote>
<figure><img src="image_2.png" /></figure>
et al., 2018; Fruergaard et al., 2023). Once in aquatic systems, plastics act as artificial substrates for a variety of organisms, allowing the establishment of diverse communities in an often self-sufficient ecosystem (Delacuvellerie et al., 2022). This new ecosystem hosts pri-mary producers, predators, symbionts, parasites and decomposers, responsible for many ecological interactions (Amaral-Zettler et al., 2020). The establishment of these plastic-adhered organisms from across the tree of life create epilastic communities (Reisser et al., 2014), often referred to as the “plastisphere” (Zettler et al., 2013; Rogers et al., 2020).

There is still no consensus about the influence of plastic character-istics (e.g. polymeric composition, texture, color, additives, opacity) (Debroas et al., 2017; Bhagwat et al., 2021) or the incubation time of plastics in aquatic systems (Rogers et al., 2020) on the plastisphere colonization process. It has been suggested that environmental condi-tions are the determinant factors in plastic colonization (Oberbeckmann et al., 2014; De Tender et al., 2015; Mincer et al., 2016). Abiotic factors, such as temperature and salinity, are known to influence biofilm growth and diversity on plastics (Chen et al., 2019; Li et al., 2021; Philippe et al., 2023). Oberbeckmann et al. (2018) have demonstrated that warmer waters and higher salinity can influence bacterial assemblages on microplastics. Moreover, other studies analyzing seasonal effects of plastics’ microbial composition have suggested that the differences in temperature and salinity between winter and summer are main drivers

for changes in the biofilm's microbial communities (Pinnell and Turner, 2020; Zhang et al., 2022; Marsay et al., 2023). There are apparently no plastic-specific organisms, suggesting that epilastic organisms are opportunistic rather than specific colonizers of any plastic type (Laverty et al., 2020; Rogers et al., 2020; Oberbeckmann et al., 2021; Latva et al., 2022). The diversity of organisms living on this new and synthetic substrate, including potential pathogens, invasive species and bio-degraders, may have ecological and economic impacts (Kirstein et al., 2016; Amaral-Zettler et al., 2018, 2020; Delacuvellerie et al., 2019, Zhang and Zhong, 2021). Additionally, due to the fact that epilastic communities are self-sufficient systems, the plastisphere may modify natural environments and surpass biogeographical barriers. This ecosystem can facilitate metal accumulations on plastics (Richard et al., 2019), as well as influence carbon, nitrogen and phosphorus cycles (Yang et al., 2023; Galgani and Loiselle, 2021). The estuarine plastisphere can also display high production of nitrous oxide (N₂O) - one of the main greenhouse gasses - highlighting its potential role in climate change (Su et al., 2022).

There is a knowledge gap regarding the plastisphere in the western South Atlantic Ocean (Agostini et al., 2021; Lacerda et al., 2022), and even less is known about the diversity of biofouling organisms on plastics from estuarine environments at the region (Pinheiro et al.,

2021a). Only a few studies have looked at the plastisphere in estuarine regions using molecular tools (Jiang et al., 2018; Sosa & Chen, 2022; Delacuvellerie et al., 2022), and to our knowledge none have been conducted in Brazilian estuaries. In 2019 the “National Plan to Combat

Litter at Sea” (Ministério do Meio Ambiente, 2019) was created in Brazil

and despite this initiative, the abundance and impact of plastics and their associated communities, especially in estuarine areas, are still underestimated (Pinheiro et al., 2021a). Therefore, this study aimed to describe the plastisphere diversity in the Patos Lagoon Estuary (PLE), which is part of the world's largest choked lagoon (Kjerfve, 1986). This

study also aimed to evaluate if season, incubation time and polymer type

affect the diversity of life that inhabits the plastisphere. We assessed the community composition by using multi-marker DNA metabarcoding to target both prokaryotes and eukaryotes. We combined molecular data with Scanning Electron Microscopy (SEM) to morphologically confirm the biofilm found with the DNA metabarcodes.
## Material and methods

### *Patos Lagoon Estuary (PLE) site*

The PLE is located in the extreme south of Brazil, and is connected to the Atlantic Ocean through a narrow channel (Seeliger and Odebrecht, 2010). Our experiment was placed in shallow waters of an island (Ilha dos Marinheiros) located near the PLE mouth. The Patos Lagoon estuary has a well-marked seasonality, and each season has unique hydrody-namics and physical-chemical characteristics (Monteiro et al., 2011). The PLE hydrodynamics is influenced mainly by wind and continental runoff. Summer and autumn are commonly drought periods, with lower continental runoff and a greater input of marine water into the estuary, while winter and spring have peaks of river discharge (Mołller et al., 2001; Mołller and Fernandes, 2010). Due to intense port and fishing activities, as well as continuous urban development, the PLE receives many domestic, agricultural, and industrial effluents that have increased pollution levels and, consequently, have the potential to change the composition of local fauna and flora (Odebrecht et al., 2010a and 2010b; Muelbert et al., 2010).

### *In situ experiment*

We placed 5 cm × 5 cm plates of two polymer types (polyethylene – PE, and polypropylene – PP) in a floating structure attached to a pier at Ilha dos Marinheiros (32°1′37″ S, 052′11′47″ W) (Fig. 1). We choose these polymers as they are the most commonly found in estuarine and marine waters (Fruergaard et al., 2023). Plates were 1.5 mm thick, manufactured from pure pellets of each polymer. A new set of plates was placed at the beginning of each season, meaning that we assessed colonization after each time period. Within each season, plates were recovered after 30 and 90 days. After sampling, each plate was indi-vidually placed in an aluminium bag and transported in a cool box to the Marine Molecular Ecology Laboratory of the Rio Grande Federal Uni-versity - FURG. A 1 cm × 1 cm piece was cut from each plate with sterile scissors, placed in a tube containing DNA extraction buffer, and stored at

-20 °C until DNA extraction. The remaining plates were fixed in Glutaraldehyde 4%, and a 0.5 cm × 0.5 cm piece of each plate was destined to scanning electron microscopy (18 plates evaluated).

DNA extraction, Polymerase Chain Reaction (PCR) and High-Throughput Sequencing.

The DNA of epiplastic communities from 32 samples was extracted with the DNeasy PowerSoil kit (Qiagen). DNA extractions were carried out according to the manufacturer's instructions, with two modifica-tions: the digestion step, in which we added 10 µl (1000 U/µl) of lysozyme to improve extraction efficiency (Debeljak et al., 2017), and the DNA elution step, in which we used a lower buffer volume (30 µl). The quality and

concentration of the extracted DNA were checked by spec-trophotometry using a Biodrop DUO (Harvard Bioscience). The rRNA genes 16S–V4 and 18S V4 and V9 regions were amplified through Po-lymerase Chain Reactions (PCRs) in order to identify prokaryotes (16S) and eukaryotes (18S); two molecular markers targeting the 18S rRNA gene were used for eukaryotes due to the fact they show different taxa resolutions. Choi & Park (2020) demonstrated that the 18S–V9 marker is 20% more likely to identify a greater number of OTUs than the V4 re-gion, indicating that the 18S–V9 marker has a more detailed resolution, especially for rare taxa. The ITS-2 region was amplified by a semi-nested approach to identify Fungi. We used primers 515f (5'-GTGYCAGCMGCCGCGGTAA-3') and 806r (GGAC TACNVGGGTWTCTAAT) to amplify the 16S–V4 region (Walters et al., 2016); primers TAReuk454 (5'-CAGCASCYGCGGTAATTCC-3') and TAReukRev3 (5'-ACTTTCGTTCTTGATYRA-3') to amplify 18S–V4
<figure><img src="image_3.png" /></figure>

**Fig. 1.** Sampling area where the *in situ* experiment on plastics biofouling was placed (2016 – 2017) in the Patos Lagoon estuary (left panel, purple rectangle) at Ilha dos Marinheiros (right panel, purple dot). The map was constructed in R studio v1.1.456 software by using the package ggmap v3.0.0 (Kahle and Wickham, 2013). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

region (Stoeck et al., 2010); and primers 1391f (5'-GTACA CACCGCCCGTC-3') and EukB (5'-TGATCCTTCTGCA

GGTTCACCTAC-3') to amplify 18S–V9 region (Amaral-Zettler et al.,

2009). To amplify ITS-2, the first PCR was carried out using the fungal specific forward primer ITS1f (5'-CTTGGTCATTTAGAGGAAGTAA-3') and the general primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3'); PCR products were then diluted 1:50 and 1 µl was used in a second PCR with primers gITS7 (5'-GTGARTCATCGARTCTTTG-3') and ITS4. Specific PCR conditions are detailed in Lacerda et al. (2020, 2022). High throughput sequencing was run on Illumina Miseq using the V3 2 × 300 bp Illumina sequencing kit (Illumina) at the University of York's Ge-nomics and Bioinformatics Laboratory.

*Scanning electron microscopy (SEM)*

Plastic fragments fixed in Glutaraldehyde 4% were dehydrated in absolute ethanol (Reagent-grade, MERK), then taped to an aluminium sheet with carbon tape and coated with a 20–30 nm gold layer. The morphology of

plastisphere organisms was observed using a JEOL mi-croscope (JSM 6610LV, JEOL, Tokyo), operated at 10–20 kV at a

working distance of 10–26 mm. Each item was imaged at a magnifica-tion of 25×, followed by imaging at different magnifications (20x to 40,000x) to better record different taxonomic groups.

*Analysis of DNA metabarcodes*

DNA sequences were analysed using a combination of USEARCH v7.0.1090 (32Bit) (Edgar, 2010) and QIIME v1.8.0 (Caporaso et al.,
2010). Forward and reverse reads were merged using USEARCH. Cuta-dapt (Martin, 2011) was used to separate each individual primer set from the data set, removing primers and adapters. For each primer set, fastq files were quality filtered (low quality, expected error > 0.5 and short sequences < 200 bp), with a 200 bp minimum length for 16S, ITS2 and length truncated for 18S (370 bp for V4 and 150 bp for V9) and then converted to FASTA files. The FASTA files were dereplicated, abundance sorted and had their singleton sequences removed. Operational taxo-nomic units (OTUs) were clustered using the UPARSE clustering algo-rithm at 97% (Edgar, 2013). Chimeras were filtered using UCHIME (Edgar et al., 2011) and OTUs were then mapped back to the original reads and an OTU table produced. OTUs were classified using UCLUST against SILVA 132 database (Quast et al., 2013) for 16S, PR2 for 18S (Guillou et al., 2013), and UNITE ITS database for ITS-2 (7.1 2016/11/20 release; Ko'ljalg et al., 2005). Before further analysis, OTUs with less than 3 reads were removed. After that, samples that remained with less than 500 reads in the 16S dataset were excluded from the downstream analysis, as well as samples with less than 100 read-s/sample for the other datasets (ITS2, 18S-V4 and 18S-V9).

## Statistical analysis

Data normalization was performed after quality control and removal of low-quality reads, and normalization was made by using the number of reads per OTU in comparison to the total number of reads per sample. Statistical analysis were conducted based on the relative abundance of sequences in each sample. OTU richness and community composition were evaluated according to the following categories: polymer type (PE
and PP), incubation time (30 and 90 days) and season (spring, summer, autumn, and winter). Multiple Generalized Linear Models (GLMs) under Negative Binomial distribution and log-link function were developed for each DNA marker to verify differences in OTU richness by categories (without interaction terms in linear predictors). Then, the models were compared from null to full model and the one with the best fit was chosen using the Akaike information criteria (AIC) (Zuur et al., 2013). Permutational Multivariate Analyses of Variance (PERMANOVA) were used to compare the community composition among the different cat-egories (fixed factors), with 9999 permutations (Anderson et al., 2006) using the Bray-Curtis binary index for 16S and the Jaccard distance for the eukaryotic markers. Principal Coordinate Analysis (PCoA) was per-formed to verify seasonal differences in the community composition, based on either Bray-Curtis dissimilarity or Jaccard distance matrices. Differences with p ≤ 0.05 were deemed statistically significant. The *ggplot2* package (Wickham, 2009) was used in *R* 1.1.456 to build dot-plots with OTU richness, PCoA plots with community composition, balloon plots with the frequency of occurrence of eukaryotes, as well as barplots with the relative abundance of bacteria and fungi. For SEM analysis, between 3 and 15 images were obtained per sample, and 100 images were analysed to group the organisms into taxonomic groups (i. e., diatoms, bacteria) based on their morphology.

# Results

## Generation of sequence reads for 16S, 18S V4 and V9, and ITS-2 region

After sequencing and quality filtration, the 16S rRNA dataset had 485,966 DNA reads comprising 9316 OTUs from 14 bacterial phyla and some unassigned bacteria. The number of bacterial OTUs per sample ranged from 71 to 5702 (average 3504.05 ± 1912.1 SE). For Fungi identification, the ITS-2 dataset presented 831,077 DNA reads, composed of 149 fungal OTUs, where the number of OTUs per samples ranged from 5 to 39 (average OTUs/sample 16.4 ± 9.6 SE) from six phyla, as well as undefined OTUs.

For the general eukaryotes, after sequencing and quality filtration, the 18S-V4 dataset had 265,383 DNA reads comprising 1338 OTUs from 20 eukaryotic groups and some unassigned eukaryotes, whilst the

18S-V9 dataset had 1,045,062 reads classified into 881 OTUs from 39 eukaryotic groups and some unassigned eukaryotes. For the 18S-V4, the number OTUs per ample ranged from 41 to 782 (average OTUs/sample 283 ± 226.1 SE). Finally, for the 18S-V9 dataset, a range of 24 to 215 OTUs per sample (average 99 ± 65.3 SE) was observed.

## Influence of incubation time in the plastisphere

A new set of plates was incubated at the beginning of each season, so the evaluated plastisphere was not from the same plastics over seasons.
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** OTU richness of the plastisphere from Patos Lagoon estuary, South Brazil, described through multi-DNA metabarcoding (16S, ITS-2, 18S V4 and V9 regions), according to season (spring, summer, autumn, winter), polymer type (magenta: PE – polyethylene; green: PP – polypropylene), and incubation time (30 and 90 days). Significant variations of OTU richness for all markers between seasons. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
Within each season, samples were collected in different incubation times; we found no evidences of differing OTU richness between these times. However, we did find significant variation in plastisphere composition (Fig. 2) (GLM and PERMANOVA results, Supplementary Material 2). It is important to note that the same number of samples was collected in all periods (30 and 90 days) within each season; however, there were differences in the number of samples between datasets as some samples did not sequence successfully.

## Richness and composition of the plastisphere according to polymer (PE x PP)

### Bacterial (16S) and fungal (ITS-2) communities

OTU richness differed for bacteria according to polymer types (Fig. 2; Supplementary Material 2). However, there was no difference overall in the bacterial and fungal community composition between PE and PP (16S - PERMANOVA: F = 0.563, p > 0.05; ITS-2 PERMANOVA: F = 0.7502, p > 0.05) (Fig. 4; Supplementary Material 2). For the 16S dataset, Proteobacteria (mostly α-Proteobacteria and γ-Proteobacteria)

had the highest relative abundances in samples for both polymers (Fig. 3B). For the ITS-2 marker, Lecanoromycetes (Ascomycota) and Tremellomycetes (Basidiomycota) were the dominant groups in both polymer types, representing together over 40% of the fungal OTUs (Fig. 3D).

### Eukaryotic (18S - V4 and V9 regions) communities

The two 18S markers showed no evidences of community composition varying according to polymer types (Fig. 4) (Supplementary material 2). The general eukaryotic OTUs richness (Fig. 2), however, varied between polymer types in both eukaryotic datasets (Supplementary Material 2).

## Richness and composition of the plastisphere according to season

### Bacterial (16S rRNA gene) and fungal (ITS-2) communities

For the 16S dataset, the GLM showed season and polymer as the best predictor of OTU richness (Fig. 2; Supplementary Material 2) with a higher richness in autumn and winter compared to spring and summer. There was a difference in bacterial community composition between seasons (Fig. 4; PERMANOVA: F = 43.446, p < 0.05). Within the 16S libraries, Proteobacteria (mostly α-Proteobacteria and γ-Proteobacteria) was the most abundant phylum in all seasons (63% in spring, 38% summer, 46% autumn, and 52% winter) (Fig. 3). The other two most abundant bacterial phyla in most seasons were Cyanobacteria (5% spring, 9% summer, 10% autumn, 12% winter) and Planctomycetes (6% spring, 19% summer, 24% autumn, 12% winter). Other phyla such as Firmicutes and Bacteroidetes had lower contributions. All 14 bacterial phyla identified along the experiment were observed in winter and autumn; in spring, 11 out of 14 and in summer, 12 out of 14 phyla were found. In spring, Proteobacteria and Verrucomicrobia were the most
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Relative abundances of bacterial (rRNA 16S) and fungal (ITS-2) taxa detected in the estuarine plastisphere of Patos Lagoon, according to season (left panels) and polymer type (right panels: PE – polyethylene; PP – polypropylene).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Principal Coordinate Analysis (PCoA) based on the Bray-Curtis dissimilarity for the 16S rRNA gene, and on the Jaccard distance matrix for the ITS-2, 18S-V4 and 18S-V9, showing the community composition of the estuarine plastisphere at Patos Lagoon, according to season (color) and polymer type (shape: Polyethylene - PE and Polypropylene - PP). Smaller colorful dots represent centroids. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

frequent groups (Frequency of occurrence – FO 100%), while the other phyla showed lower FOs. In winter, all groups showed FO ≥ 33%. Only 5

phyla out of the 14 had FO 100%, whereas in autumn the most abundant groups (Proteobacteria, Planctomycetes, Cyanobacteria and Verruco-microbia) presented FOs of at least 83%. Finally, during the summer, 11 bacterial phyla presented FO of 100% (Chlamydiae, Epsilonbacteraeota and Patescibacteria were the exception) (Fig. 2, Supplementary Material 1).

For Fungi, there was difference in OTU richness per season (Fig. 2; Supplementary Material 2), and also a seasonal variance of the fungal community composition (Fig. 4; PERMANOVA: F = 1.5284, p < 0.05).

For spring, summer and winter, the most abundant fungal phyla was

Ascomycota (relative abundances of 76% in spring; 62% summer; 41% winter; 23% autumn). In autumn, the most abundant phyla was Basi-diomycota (45%). The highest

diversity was found during the autumn, when OTUs from six phyla were found. We observed the dominance of Ascomycota and Basidiomycota during spring and summer. Autumn and winter had other significant representatives, such as Chytridiomycota which was the second most abundant during these seasons (Fig. 3). Zoopagomycota and Neocallimastigomycota were present only in autumn samples, with one OTU each: *Coemansia braziliensis* and *Neo-callimastix* sp., respectively. Many unassigned fungi were found in all
seasons. The most abundant OTU within the ITS-2 dataset was *Xanthoria parietinais*, a lichened fungus. Some groups of fungi that we found are putative pathogens, such as *Meyerozyma guilliermondii* and *Leptosphaer-ulina australis* (Yan et al., 2021; Zhang & Zhong, 2021). Moreover, we also found fungi that can act in the biodegradation of plastics, including *Aspergillus*, *Pleosporales* and *Cladosporium* species (Munir et al., 2018; Ekanayaka et al., 2022; Kale et al., 2015). Frequency of occurrence for fungi was quite varied amongst seasons. Ascomycota and Basidiomycota were the only fungal phyla present in every season (Fig. 3, Supplementary Material 1).

*General eukaryotic (18S-V4 and 18S-V9 rRNA genes) communities* Significant differences in OTU richness were observed for both 18S eukaryotic datasets between seasons (Fig. 2; Supplementary Material 2).

The eukaryotic community composition also varied for both eukaryotic markers between seasons (Fig. 4; 18S-V4 PERMANOVA: F = 4.8822, p < 0.05; 18SV9 PERMANOVA: F = 2.1626, p < 0.05). For the 18S-V4, all 20 eukaryotic groups detected were observed in spring, but only *Crus-tacea* and *Ciliophora* had a FO of 100% (Fig. 5). During the winter, 16 eukaryotic groups were present, all with FO > 15%. However, only *Chlorophyta* and *Diatoms* were frequent in all samples (FO 100%). In the autumn, 19 eukaryotic groups were identified, the most frequent being
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Frequency of occurrence (FO %) of eukaryotic taxa (Groups) found in the estuarine plastisphere of the Patos Lagoon, South Brazil, according to seasons (autumn, spring, summer and winter) per molecular marker (18S–V4 and 18S–V9 rRNA genes).</figcaption></figure>

Chlorophyta and Ciliophora (FO 100%). In summer, 7 eukaryotic groups were found, of which only Crustacea was present in all samples (Fig. 5). The 18S–V9 marker displayed a higher diversity of taxa compared to 18S–V4 (Fig. 5), with 19 exclusive groups: Apusozoa, Bryozoa, Cen- troheliozoa, Chaetognatha, Charophyta, Chrysophyta, Cryptophyta, Ctenophora, Excavata, Dinoflagellata, Foraminifera, Haptophyta, Insecta, Mollusca, Opalozoa, Radiolaria, Rhodophyta, Teleostei and Telonemia. In spring, 25 out of the 39 eukaryotic groups identified by 18S-V9 were present. Crustacea, Ascidiacea, Chlorophyta and Diatoms showed FO 100%. Winter presented the highest diversity of taxa, with 37 eukaryotic groups (all but Bryozoa and Insecta), but only Crustacea, Ascidiacea and Fungi presented FO 100%. During autumn, 32 groups were identified. Of these, Crustacea, Ascidiacea, Fungi and Ciliophora had FO 100%. Finally, for summer, 28 eukaryotic groups were found, with Crustacea, Ascidiacea, Fungi, Ciliophora, Chlorophyta and Diatoms present in all samples (FO 100%) (Fig. 5).

Morphological evaluation of the microbial communities

Bacteria and diatoms were the dominant groups observed in SEM images. We identified colony-forming organisms such as cyanobacteria-like colonies (Fig. 6 A), as well as many pennate (Fig. 6 F, G, I, K, L) and centric diatoms (Fig. 6 H, J), confirming the high abundance of these groups that were identified by DNA analysis.

Discussion

The colonization of plastic debris in aquatic environments has been recognized since the 1970s (Amaral-Zettler et al., 2020), but few studies have detailed the biodiversity of this novel ecosystem by combining molecular markers for both prokaryotes (bacteria) and eukaryotes (Bryant et al., 2016; Debroas et al., 2017; Dussud et al., 2018; Lacerda et al., 2020, 2022; Audre´zet et al., 2022), especially in estuaries. The present work described the plastisphere using a colonization experiment in the Patos Lagoon estuary (PLE), where few studies have evaluated
biofouling on plastics (Pinheiro et al., 2021a) and, to date, none included a detailed description through DNA metabarcoding analysis. In fact, to our knowledge, this is the first time such characterization has been done in South America.

Assessment of plastisphere diversity in the Patos Lagoon estuary

The most abundant bacterial phyla we found (Proteobacteria, Cya-nobacteria, Bacteroidetes and Planctomycetes) have also been described as the most abundant in different regions across the globe, such as in the North Atlantic and North Pacific, Baltic Sea, Mediterranean Sea and South Atlantic (Zettler et al., 2013; Oberbeckmann et al., 2014, 2016; Amaral-Zettler et al., 2015; Bryant et al., 2016; Davidov et al., 2020; Lacerda et al., 2022; Wallbank et al., 2022). Indeed, a global analysis of bacteria in the plastisphere described Proteobacteria, Cyanobacteria and Bacteroidetes as the most representative groups (Wright et al., 2020a). Regarding the most frequent eukaryotic groups we observed (Crusta-ceans, Ascidiacea, Fungi, Chlorophyta, Ciliates and Diatoms), similar results were found in other regions, such as the Atlantic Ocean (North and South) (Zettler et al., 2013; Debroas et al., 2017; Lacerda et al., 2022), from brackish waters in Germany (Kettner et al., 2019) and on the New Zealand coast (Audr´ezet et al., 2022). The knowledge on fungal diversity of the plastisphere is still limited; nevertheless, another study also found that groups such as Ascomycota and Chytridiomycota dominated the fungal assemblages of the plastisphere in fresh and brackish waters in Germany (Kettner et al., 2017). In our previous work developed in the open ocean of South Brazil (Lacerda et al., 2022), Ascomycota and Basidiomycota were the most frequent fungal phyla on plastics. This is also in agreement with what we found in the estuarine plastisphere at PLE.

Pinheiro et al. (2021a) assessed biofouling on litter (including plastics) at a saltmarsh near the PLE mouth. These authors visually identified macroscopic epilastic organisms and classified them based on their morphology, finding crustaceans, algae, mollusks and insects (eggs). In our work, we also found these groups, but combining molecular markers
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Scanning Electron Microscopy (SEM) images of the microbial community found in the plastisphere of Patos Lagoon estuary: (A, B) bacteria colonies; (C, D, E) clusters of centric and pennate diatoms; (F, G, I, K, L) individual pennate diatoms; (H, J) individual centric diatoms.</figcaption></figure>

led to the identification of a much wider diversity of organisms associated with plastics at the region, and indeed we identified over 50 prokaryotic and eukaryotic groups.

SEM images allowed us to evaluate the morphology of the estuarine plastisphere from Patos Lagoon, to compare with the DNA-metabarcoding datasets. Among them, pennate diatoms stood out, which is mostly a benthic group associated with natural substrates such as sediments. It is important to note that, although it is a valuable tool for the morphological analysis of the

plastisphere, SEM requires a very large magnification of the evaluated surfaces, which considerably reduces the area of observation and hinders a quantitative description of organisms.
*Influence of season, polymer type and incubation time on
plastisphere development*

We found seasonal variation in the community
composition for all markers datasets, and this can be
related to environmental factors. In fact, during winter
where there is a greater river discharge, we observed
more taxa commonly found in freshwater or terrestrial
environments, such as Rhizobiales (α-Proteobacteria)
(Liu et al., 2015). Some taxo-
nomic groups, such as Patescibacteria (16S dataset),
as well as Ich-
thyosporea and Haptophyta (18S-V9 dataset), were
present only in seasons with milder temperatures (winter
and autumn), corroborating that the occurrence of some
groups are susceptible to seasonal variation.
Another indication of this susceptibility was that the lowest diversity of taxonomic groups was found in spring and summer, when the water temperature is higher. Studies exploring the diversity and changes of communities adhered to different substrates at PLE are scarce, but for epiphytic diatoms on the salt-tolerant grass *Ruppia maritima*, highest richness was found during winter (da Rosa & Copertino, 2022), which is in accordance with our findings on plastics. Temperature and salinity variations (caused by rainfall, winds and El Ninño Southern Oscillation events) can influence seasonal variations in a variety of taxa in natural compartments of the PLE, from primary producers to predators (Pos-samai et al., 2018; Lanari et al., 2021).

PE and PP are the most common plastics found in coastal areas due to their improper management and disposal on land, and are therefore the most likely to occur in estuaries. Some authors suggest that plastisphere microorganisms are opportunistic colonizers, with no differences be-tween polymer types (Oberbeckmann et al., 2021; Wallbank et al., 2022). We observed a difference in OTU richness according to polymer type for the 16S and 18SV9 datasets, although the community compo-sition was not significantly varied between PE and PP for any dataset. A possible explanation for this could be linked to the organisms' physio-

logical characteristics. For example, Pinheiro et al. (2021a) demon-

strated that some algae preferred to settle on more transparent plastic materials, as these materials allow more light to pass, which is pivotal for photosynthesis. In accordance with this, we found that poly-propylene – more transparent and less opaque – was the polymer that showed the highest OTU richness throughout seasons, except for autumn.

The lack of differences in the community composition for some groups, such as fungi and general eukaryotes (18S-V4), among incu-bation times leads us to infer that these groups were already well

established at the first incubation period (30 days). Other studies assessing the plastisphere in different times have shown that community composition tends to vary mainly in the first stages of colonization, which occurs in the first few days; then, the community remains stable (Nauendorf et al., 2016; Fr`ere et al., 2018; Tagg et al., 2019). The duration of this ‘first stage of colonization’ varies according to the rate of

biofilm growth, which is determined by environmental factors, but in general it lasts just over one week (Erni-Cassola et al., 2020). It is important to note that while it is essential to understand how the initial processes of colonization take place, the mature plastisphere has a more significant ecological impact, since relationships such as symbiosis, parasitism and mutualism will have been established.

The PLE is a very heterogeneous estuary in terms of coastal popu-lation, input of industrial and urban waste, and agricultural activities (Seeliger et al., 2010; dos Santos et al., 2023). It has different habitats such as

saltmarshes, shoals, and channels (>15 m), thus providing diverse micro-habitats (Seeliger, 2001) with unique characteristics (e.g.

temperature, salinity, light penetration, permeable sediments). We can expect that the plastisphere would also be heterogenous and its micro-bial community would be different in different parts of the estuary.

### Ecological role of plastisphere organisms in the Patos Lagoon estuary

Putative pathogens associated with plastic debris have already been reported in previous plastisphere studies (Kirstein et al., 2016; Kesy et al., 2020; Pedrotti et al., 2022). We also found putative pathogenic Fungi, including *Meyerozyma guilliermondii* (Yan et al., 2021), *Lep-tosphaerulina australis* (Zhang & Zhong, 2021) as well as *Alternaria* and *Clasdodporium* species. Ichthyosporea, which is a small group of protists that are mostly parasites of fish and other animals (Shinn, 2023; Shields et al., 2022; Kocan, 2019) was also present. It is important to note that the occurrence of potentially harmful organisms in the plastisphere does not confirm their pathogenicity (Oberbeckmann & Labrenz, 2020), but their presence is of concern nonetheless. Paillard et al. (2004) recog-nized that the emergence of agents of diseases is favored by high tem-peratures, and will probably increase in the coming years due to global
warming.

It is also important to consider the role of the plastisphere in nutrient cycling. Previous studies have suggested that the microbial composition of the plastisphere can influence the carbon, nitrogen and phosphorus cycles (Chen et al., 2020; Galgani & Loiselle, 2021). For example, some benthic Cyanobacteria such as *Rivularia* sp. and *Cyanobium* sp. found in our samples, are known to fix atmospheric Nitrogen and transform it into other nitrogen compounds in aquatic systems (Livinstone et al., 1984; Ehrenfels et al., 2021).

Plastics can harbor organisms that degrade the plastic itself (Shah et al., 2008; Paço et al., 2017; Urbanek et al., 2018; Oberbeckmann & Labrenz, 2020). Species of Sphingomonadaceae (Proteobacteria) possess the ability to degrade hydrocarbons, as well as to synthesize carotenoids, pigments that protect them from degradation by ultraviolet rays, thus facilitating their survival on floating plastics in the ocean (Oberbeckmann and Labrenz, 2020). Our analyses identified several Sphingomo-nadaceae OTUs: for example, *Erythrobacter* sp. was present, which has already been described using Polycyclic Aromatic Hydrocarbons (PAH), an additive found in plastics (Lassen et al., 2015; Gao et al., 2015). Furthermore, we found several *Pseudomonas*, *Bacillus* and *Flavobacterium* OTUs, which are among hydrocarbon-degrading microorganisms asso-ciated with plastics (Quero & Luna, 2017; Nowak et al., 2011; Koutny et al., 2009). Moreover, *Aspergillus*, *Pleosporales* and *Cladosporium* spe-cies, fungal groups recognized as plastic-degrading organisms, were also present in our samples (Munir et al., 2018; Ekanayaka et al., 2022; Kale et al., 2015).

The presence of plastics in the PLE can intensify
changes in the composition of aquatic communities in
the estuary and its adjacent coast. Additionally, the
estuary is directly linked to the South Atlantic Ocean,
which could favor the transport of plastic debris and its
plasti-sphere between regions. It has been observed that
the increased urban waste pollution at PLE has modified
phytoplankton (Odebrecht et al., 2010a), zooplankton
(Muelbert et al., 2010) and macrobenthos (Bem-venutti
& Colling, 2010). In addition, Pinheiro et al. (2021b)
suggested that the inner part of the estuary is more
contaminated by solid waste - this greater availability of
artificial substrates may facilitate the colo-nization and
dispersal of organisms. It is also important to consider
the presence of benthic organisms found in our samples,
especially the more frequent eukaryotic taxa, such as
ascidians, crustaceans (classes: Max-illopoda,
Malacostraca and Ostracoda), and some pennate
diatoms. We also found OTUs of organisms that have a
benthic life stage, as for example hydrozoans (Cnidaria).
The presence of these organisms on plastic highlights
the threat that plastic pollution poses to ecosystems
sustainability by increasing the dispersal capacity for
many benthic taxa (Carlton et al., 2017).

This study demonstrates the importance of molecular
techniques to
the description of the plastisphere, highlighting the value
of using multi-marker DNA analysis for obtaining more
robust results, including a thorough identification of
eukaryotes. We suggest that future in-vestigations

should include the initial colonization of plastics,
especially to evaluate if it is the plastic surface itself or
the first stages of biofilm formation that drives the later
stages of colonization. We also encourage the evaluation
of different compartments (e.g. mouth and inner estuary)
in order to expand our knowledge on the plastisphere's
response to
different environmental characteristics. Furthermore, it
is necessary to
assess the plastisphere in other types of polymers
commonly present in coastal environments, such as
polystyrene, polyamide (nylon), polyvinyl chloride (PVC)
and polyethylene terephthalate (PET). It is also impor-
tant to determine the effects of the plastisphere on the
physical char-acteristics of polymers (e.g. density) and,
consequently, their behavior in aquatic systems.

Conclusion

The present work described well-established epiplastic
communities in the Patos Lagoon estuary, with
several groups of bacteria and
eukaryotes associated with polyethylene and polypropylene. The plas-tisphere composition varied between seasons, evidencing the strong effects of seasonality. No difference in community composition in relation to polymer types was observed for any molecular marker, and therefore, it is possible that plastisphere organisms are more generalists. There was also no difference in the composition of the plastisphere among the incubation times for bacteria and fungi, suggesting that the general eukaryotic communities remained stable in the evaluated periods. From an ecosystem point of view, studying a mature plastisphere is more significant, since it will have more permanent roles in ecosystem dynamics in terms of ecological relationships.

## Author statement

Tobias Sérvulo: conceived labwork, analysed the data and wrote the first draft of the paper, Joe D. Taylor: conceived labwork and analysed the data, Maíra C. Proietti: conceptualized the research, obtained funds for sampling and analysis, supervised the study, Lucas de S. Rodrigues: analysed the data, Igor P. Puertas: conceived labwork, analysed the data, Roberta A. Barutot: conducted sampling, Ana Luzia de F. Lacerda: conceptualized the research, conducted sampling, obtained funds for sampling and analysis, supervised the study and wrote the first draft of the paper, All authors contributed to discussing and editing the manuscript.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Data availability

Data will be made available on request.

## Acknowledgments

This study was conducted within the activities of the Brazilian Long-Term Ecological Research Program (PELD, CNPq Proc. 441492/2016-9, and FAPERGS - Proc. 16/2551-0000102-2). We thank the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES) that provided a scholarship to ALdFL and LdSR, and also provided access to the Portal de Periódicos and financial support through Programa de Excele nca Acadêmica – PROEX. The National Council for Research and Development (CNPq) provided a scholarship to ALdFL (SWE 206250/2017-7), as well as a research fellowship to MCP (PQ 312470/2018-5). The Research Support Foundation of the State of Rio Grande do Sul (FAPERGS) provided a scholarship to TS (PROA 21/2551-0000981-0) and IPP (PROA 20/2551-0000307-8). JDT was funded

by a University of Salford Career Development Fellowship. We acknowledge Marlí and Alfredo for supervising the experiment in the field. We thank Pedro Prata, Ileana Ortega and Andres M. Diaz for the assistance with field sampling. SEM images were taken at the Electron Microscopy Center (CEME-Sul, PROPESP-FURG) with the assistance of Rudmar Krumreick and Caroline P. Ruas. We thank Dr. Thorunn Helgason, Dr. Sally James and Dr. Peter Aston, of the University of York Genomics & Bioinformatics Laboratory, for assistance, support and technical expertise with DNA sequencing.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2023.121873.
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