# Bioaccumulation of trace metals in the plastisphere:
Awareness of environmental risk from a European
perspective

Véronique Lenoble, Ana-Marija Cindrić, Jean-François Briand, Maria Luiza

Pedrotti, Ana Luzia Lacerda, Soledad Muniategui-Lorenzo, Veronica

Fernández-González, Carmen Ma Moscoso-Pérez, José Andrade-Garda,
Raffaella Casotti, et al.

► To cite this version:

Véronique Lenoble, Ana-Marija Cindrić, Jean-François Briand, Maria Luiza Pedrotti, Ana Luzia Lacerda, et al. Bioaccumulation of trace metals in the plastisphere: Awareness of environmental risk from a European perspective. Environmental Pollution, 2024, 348, pp.123808. 10.1016/j.envpol.2024.123808. hal-05007014

HAL Id: hal-05007014

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Distributed under a Creative Commons Attribution 4.0 International License
1 BIOACCUMULATION OF TRACE METALS IN THE PLASTISPHERE: AWARENESS

2 OF ENVIRONMENTAL RISK FROM A EUROPEAN PERSPECTIVE

3

4 Authors

5 Véronique Lenoble 1,\*; Ana-Marija Cindrić², Jean-François Briand ³, Maria Luiza Pedrotti ⁴;

6 Ana Luzia Lacerda⁴, Soledad Muniategui-Lorenzo⁵, Veronica Fernández-González⁵, Carmen

7 Mª. Moscoso-Pérez⁵, José M. Andrade-Garda⁵, Raffaella Casotti⁶, Carola Murano⁶,⁷, Vincenzo

8 Donnarumma⁸, Sébastien Frizzi¹, Colin Hannon⁹, Haleigh Joyce⁹, Róisín Nash⁹, João Frias⁹

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10 Affiliation

11 ¹Université de Toulon, Aix Marseille Univ., CNRS, IRD, MIO, Toulon, France lenoble@univ-tln.fr

12 ²Ruder Bošković Institute, Center for Marine and Environmental Research, Zagreb, Croatia ana-marija.cindric@irb.hr

13 ³Université de Toulon, Laboratoire MAPIEM, Toulon, France

14 ⁴Laboratoire d'Océanographie de Villefranche sur mer (LOV), UPMC Université Paris 06, CNRS UMR 7093, Sorbonne Université, Villefranche sur Mer, France. maria-luiza.pedrotti@imev-mer.fr; ana-luzia.lacerda@imev-mer.fr

15 ⁵University of A Coruña. Grupo Química Analítica Aplicada (QANAP), Instituto Universitario de Medio Ambiente (IUMA), Department of Chemistry. Faculty of Sciences. A Coruña 15071, Spain soledad.muniategui@udc.es, jose.manuel.andrade@udc.es; v.fernandez@udc.es; c.moscoso@udc.es

16 ⁶Stazione Zoologica Anton Dohrn, Naples, Italy raffaella.casotti@szn.it; carola.murano@szn.it

17 ⁷NBFC, National Biodiversity Future Center, Piazza Marina 61, 90133 Palermo, Italy carola.murano@szn.it

18 ⁸Institute of Marine Sciences - National Research Council ISMAR-CNR. Forte Santa Teresa Pozzuolo di Lerici, 19032 La Spezia, Italy vincenzo.donnarumma@sp.ismar.cnr.it

19 ⁹Marine & Freshwater Research Centre, Atlantic Technological University, Dublin Road, H91 T8NW, Galway, Ireland roisin.nash@atu.ie
20 joao.frias@atu.ie

21 *Corresponding author lenoble@univ-tln.fr

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24 Funding information

25 This research was funded by JPI Oceans *MicroplastiX* project https://www.microplastix.org/ in

26 France, Ireland, Spain and Italy

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GRAPHICAL ABSTRACT

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ABSTRACT

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The term "Plastisphere" refers to the biofilm layer naturally formed by microorganisms attaching to plastic surfaces. This layer possesses the capability to adsorb persistent organic and inorganic pollutants, particularly trace metals, which are the focus of this research study. Immersion experiments were concurrently conducted in five locations spanning four European countries (France, Ireland, Spain, and Italy) utilising eight distinct polymers. These immersions, repeated every three months over a one-year period, aimed to evaluate the baseline bioaccumulation of 12 trace metals. The study underscores the intricate nature of metal bioaccumulation, influenced by both micro-scale factors (such as polymer composition) and macro-scale factors (including geographical site and seasonal variations). Villefranche Bay in France exhibited the lowest metals bioaccumulation, whereas Naples in Italy emerged as the site where bioaccumulation was often the highest for the considered metals. Environmental risk assessment was also conducted in the study.

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The lightweight nature of certain plastics allows them to be transported across significant distances in the ocean. Consequently, evaluating trace metal concentrations in the plastisphere is imperative for assessing potential environmental repercussions that plastics, along with their associated biota, may exert even in locations distant from their point of emission.

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KEYWORDS

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plastisphere, metal bioaccumulation, plastic pollution, environmental risk

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1. INTRODUCTION
The unprecedented challenges that the marine environment is facing as a result of human activities, particularly climate change and chemical pollution, have placed humankind outside of the safe operating space (Rockström et al., 2009). Chemical pollution, which includes emerging contaminants such as large plastics (> 5 mm), microplastics (1-5 mm) and their chemical additives, is of particular concern (da Costa et al., 2022).

The widespread distribution and accumulation of plastic in aquatic environments has been reported since the early 1970s (Carpenter and Smith, 1972), with an initial focus on baseline concentrations, assessment of sources, pathways and distribution patterns, in both freshwater and marine environments (Galgani et al., 2015; Lambert and Wagner, 2018). Over the last decade, with growing evidence of weathering, degradation and fragmentation of plastics into micro- and nanoplastics, particular attention has focused on ecotoxicology assessments, including leaching of chemicals and additives (Yamashita et al., 2021; Ragusa et al., 2021). Such concern led to many regulatory approaches in the world (da Costa et al., 2020). Indeed, the European Parliament created, in 2019, a directive to reduce plastic products in the environment, known as the Single-Use Plastics Directive (SUP) (EU Directive, 2019). This Directive points out the need for circular approaches to give priority to sustainable and reusable products; to set up appropriate and adequate waste management; and to promote sustainable consumption. Similarly, at the UN Environment Programme (UNEP) a legally binding market based instrument (Plastics Treaty) is currently under negotiations (UNEP, 2020), and it is envisioned to be a relevant document in the plastic value chain. In parallel, solution-oriented research into market-based instruments, transition to circular economy models, and mitigation of environmental impacts are getting more prevalent, as the G20 (G20, 2021) and the United Nations (UNEP, 2022) started devising strategies and treaties to minimise impacts caused by plastics.

Plastics are synthetic materials that can be moulded into rigid or mildly elastic forms from a wide range of organic polymers, and can be divided into thermoplastics or thermosets, depending on their elastic properties (Shashoua, 2008). To enhance such physicochemical properties, plastic additives, including pigments, dyes, surfactants, antioxidants, UV stabilisers, flame retardants, plasticisers, lubricants, etc., (Hahladakis et al., 2018; Slama et al, 2021, Wiesinger et al., 2021) are incorporated during manufacturing processes. These additives can potentially leach into aquatic ecosystems (Ngoc Do et al., 2022). Additionally, plastics also have the ability to adsorb hazardous substances such as persistent organic pollutants and trace metals (Liu et al., 2021, Wang et al., 2021). These sorption processes in aquatic conditions are enhanced by the plastisphere, a biofilm layer developing on the surface of immersed plastics, that serves as a hotspot for microorganisms (Pete et al., 2023). The increasing number of scientific papers dedicated to the interactions between plastics and attached biofilm (Lobelle and Cunliffe, 2011; Wang et al., 2021; Yu et al., 2023) have raised environmental concerns on their potential to (bio)accumulate metals (Liu et al., 2021) or host various species, including potentially pathogenic bacteria such as Vibrio spp. (Kirstein et al., 2016; Pedrotti et al., 2022). The
plastisphere can harbour fungi (Lacerda et al., 2020) and viruses (Moresco et al., 2022), as well as potentially pathogenic bacteria (Pedrotti et al., 2022; Sababadichetty et al., 2024). It can also host antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) (Yang et al., 2019). In fact, even though the organisms associated with plastics in the ocean had been described in the early 1970s (Carpenter and Smith, 1972), their evaluation only gained attention in the last decade, after the term ‘plastisphere’ was created (Zettler et al., 2013).

The co-occurence of plastics and pollutants poses significant risks to marine ecosystems, particularly when combined with degradation and fragmentation processes that increase the plastics' surface-to-volume (S:V) ratio (Khalid et al., 2021). Metals and metalloids represent 85 % of the elements in the periodic table and are divided into trace metals, which are toxic even at low levels, and oligo-elements, which are essential for physiological processes (Chandrapalan and Kwong, 2021). For example, cadmium (Cd) and arsenic (As) are toxic even at µg/L levels (Rebelo and Caldas, 2016), while trace amounts of copper (Cu), zinc (Zn), selenium (Se) and manganese (Mn) are required for cell growth and reproduction (Chouvelon et al., 2022). Trace metal poisoning can impair growth and development of fish and shrimps, also causing reproductive abnormalities or compromising immune functions (Hossain et al., 2022). As such, the combination of trace metals and persistent organic pollutants in biofouled plastics represents a novel human health concern, due to the toxic potential associated with bioaccumulation, biomagnification, bioleaching, biomineralization, and biotransformation (Syed et al, 2022).

Bioaccumulation of metals in the plastisphere is a biofilm-induced process that has been previously described in the literature (Richard et al., 2019; Djaoudi et al., 2022; Sun et al., 2022), with more studies conducted in laboratory settings than *in situ*, and more in freshwater than in seawater (Pan et al., 2023). Yet, as underlined by Yu et al. (2023) in their review presenting the interactions between plastics and biofilms, there are still knowledge gaps, especially concerning the quantification of the contaminants accumulated in the biofilm, as well as exploring spatio-temporal variations. The presented work aims to fill some of these gaps. Indeed, the study presented in this paper was designed to assess the bioaccumulation of 12 trace metals by the biofilms developed on immersed plastics deployed at five sites across four European countries (France, Ireland, Italy and Spain). This *in-situ* experiment included eight polymers that were incubated for periods of three months over the course of one year, i.e. repeated every four months, to account for seasonal variations. The main objective of this study was to assess trace metals bioaccumulation in the plastisphere. Therefore, the investigation addressed the following research questions:

1. Are there seasonal variations influencing trace metals bioaccumulation in the plastisphere?

2. Are there relationships between the characteristics of the immersion sites and metals bioaccumulation?
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3. Are there trace metals bioaccumulation differences between fossil-fuel based and bio-based plastics?

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4. Are there trace metals bioaccumulation levels that pose a potential risk to the ecosystem?

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134 2. MATERIALS AND METHODS

135 2.1 Immersion sites

136 The sampling sites were: Port of Galway, Ireland; Port of Ares (A Coruña), Spain; Toulon Bay,
137 France; Villefranche Bay, France; and Bay of Naples, Italy (Figure 1).

<figure><img src="image_3.png" /></figure>

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<caption>Figure 1 - European *in-situ* cage deployment sites (map produced using ArcGIS Pro, Source: ESRI).</caption>

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The Port of Galway (53°16′08″N; 9°02′55″W) is located in the inner part of Galway Bay, in the west coast of Ireland. The port and the bay are protected from the strong Atlantic Ocean swells by the Aran islands (Frias et al., 2020). Galway city has a population of approximately 83,460 inhabitants (CSO, 2023) and in 2022 about 350 vessels passed through the city's port carrying approximately 500,260 t of cargo (Port of Galway, 2023). The immersion site was chosen inside the port of Galway, as it is protected from anthropogenic activities at sea.

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The Port of Ares (43°25′20″N; 8°14′22″W) is located in the village of Ares (Northwest of Spain), about 50 km north from the city of A Coruña. Ares hosts almost 5,800 inhabitants. The port is located at the end of an inlet or ría which is 800 m deep and 1.6 km wide, protected from open sea and Northern winds. Its main activity is recreational, so there is a high number of
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sailing boats that are docked at some pontoons (Port of Ares, 2023). The immersion site was chosen so as to be protected from any direct anthropogenic activity.

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Toulon Bay (43°04'60''N; 5°54'00''W) is located in Southeast France, close to the city of Toulon, a large urban area with a population of approximately 180,000 inhabitants (~600,000 in the metropolitan area). Toulon Bay hosts various anthropogenic activities (military/civil operations, aquaculture, urban sewage release, industrial shipping), which have been highlighted for their multi contamination ecosystem effects (Dang et al., 2015; Coclet et al., 2018; Layglon et al., 2020). The immersion site is a regular environmental monitoring site within the bay.

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Villefranche Bay (43°41'47''N; 7°18'33''W) is also located in Southeast France, close to the city of Villefranche-sur-mer. This city hosts approximately 5,000 residents, and is located between the urban centers of Nice and the principality of Monaco. Notably, it serves as a prominent tourist destination, attracting many visitors. Villefranche Bay experiences maritime traffic mostly due to luxury yachts, sailboats, and large cruise vessels, particularly during summer, and it is also a place of shipyards and nautical maintenance (Coglievina et al., 2016). However, distinct from the other sites investigated in this study, fishing and industrial activities are not relevant within this region. This site was chosen as a reference site given its unique conditions.

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Naples harbour (40°49'59''N; 14°15'11''W), located in the Southeastern Tyrrhenian Sea, hosts the city of Naples (Italy), which has a population of about 4 million people (with ~2 million in the metropolitan area) and presents diverse urban and industrial activities, which strongly impact its water quality (Cianelli et al., 2012; Esposito et al., 2020). With an annual traffic capacity of around 25 million tons of cargo and 500,000 TEUs capacity, the port of Naples is one of the main ports in Europe. It also serves as a tourist hub, servicing around 10 million people annually. The immersion site was situated in a marina very close to the commercial port, where three yacht clubs are located, so as to be protected from the commercial routes and other activities, but still representative of an urban-influenced coastal area.

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## 2.2 Experimental design

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Stainless steel cages (81.3 L x 27.9 W x 33 H cm) were used as a holding structure to harbour plastic containers that were drilled to allow water circulation, where plastics were introduced. One cage was deployed in each immersion site, with eight containers, each carrying one polymer. The polymers used in this work were: 1) High-Density PolyEthylene (HDPE), 2) Low Density PolyEthylene (LDPE), 3) Polyethylene Terephthalate (PET), 4) recycled Polyethylene Terephthalate (PET-Post), 5) PolyPropylene (PP), 6) recycled PolyPropylene (PP-Post), 7)
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PolyStyrene (PS) and the 8) bioplastic PolyLacticAcid (PLA). Polymers were chosen based on their relevance and use in global markets. All polymers were acquired from the same batch and supplier (Carat-lab GmbH, Germany), and were provided to partners in 6 x 6 cm sheets (1 mm thickness, n = 50). Each laboratory cut the sheets into 1 cm² squares, to have 100 pieces in each container, for several analyses in the MicroplastiX project. For the trace metals analysis, 5 pieces at the start of the project and 5 pieces after incubation for three months were sampled (Figure 2a).

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After 3 months of immersion, the bottles were retrieved and replaced by new ones, containing pristine polymer pieces, for another trimester. This was performed 4 times, to cover a one-year period, reducing the effect of overlapping seasons. Furthermore, throughout the immersion periods, the sea temperature was regularly monitored to assess whether it influenced trace metal accumulation.

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At each sampling time, the individual pieces of each polymer were retrieved into sterilised trace metal grade conical tubes (VWR) where the plastisphere was digested using 5 mL of a 0.1 M sodium hydroxide (NaOH) solution (Suprapur quality, 99.99%). After 24 h, the plastic pieces were removed from the solution, using plastic forceps in a fume hood. Then, 50 µL of Suprapur nitric acid (HNO₃) were added (to reach final 10 % concentration) to the remaining solutions and kept in dark until shipping (Figure 2b) and further analysis. Pristine pieces were also digested following the same protocol, to ensure that no metal leaching occurred from the various polymers. All solutions used in processing (including NaOH), were analysed as blanks and controls to quantify the baseline trace metal content.

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a.

<figure><img src="image_4.png" /></figure>

b.

<figure><img src="image_5.png" /><figcaption>Figure 2 - Experimental design. a) *In-situ* immersion deployment, b) Digestion of plastisphere and ICP-MS processing (image produced in Adobe Photoshop. Source: Biorender and Canva).</figcaption></figure>

2.3 Trace metals analysis
The concentrations of 12 trace metals (arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), tin (Sn), titanium (Ti) and vanadium (V)) were assessed in two matrices: a) the digested biofilm from the eight polymers and b) the surrounding seawater. Processing was conducted at the Ruđer Bosković Institute (Croatia) using an Inductively Coupled Plasma Mass Spectrometer (ICP-MS, Agilent 8900 qqq), with a 20-fold dilution for seawater samples to reduce salt-matrix effect. All samples were spiked with an Internal Standard (Indium). A certified reference material (CASS-5, Nearshore seawater reference material for metals, National Research Council Canada) was used as a quality control of ICP-MS measurements. Two separate CASS-5 control samples were measured after every 10-15 samples. Matrix matching calibration (in 20-fold diluted CASS-5 sample) was used for concentration quantification. Determined concentrations of trace metals in the CASS-5 sample were within 15 % of the certified reference material.

The measured concentrations were expressed in µg of metal/L of digestion solution (according to the volume of NaOH used). The obtained values correspond to the metals bioaccumulated in the biofilm developing over the immersed plastic pieces. Throughout the text, the term "bioaccumulation" refers to this process of metals assimilation by the biofilm, by comparison with the surrounding seawater.

## 2.4 Environmental risk assessment

To assess the environmental risk, the so-called Risk Quotient (RQ) was calculated. This parameter is obtained by calculating the Measured Environmental Concentration (MEC) divided by the Predicted No-Effect Concentration (PNEC) (Liu et al., 2015, Eq. 2). The MEC values were obtained from the plastisphere analysis and the PNEC concentrations (Table S1) were obtained from the NORMAN Ecotoxicology Database (NORMAN, 2023), which is based on the European Directive (EU, 2013) and on the REACH registration Process (ECHA, 2023). The RQ was calculated for all considered metals, for all immersion sites and all polymers. The risk quotient is classified as follows: when $RQ<0.01$, there is no environmental risk; when $0.01<RQ<0.1$, the risk is considered low; when $0.1<RQ<1$, there is medium risk; when $RQ>1$, the risk is high to very high (Nika et al., 2020).
245 **2.5 Quality Assurance and Quality Control**

246 Through all steps, a rigorous protocol was followed to prevent any metal contamination. This
247 protocol included the use of metal-free sterile polypropylene conical tubes to store the plastic
248 pieces, the use of plastic tweezers to manipulate them, and other volumetric containers that were
249 also made of plastic to prevent trace metal cross-contamination. To guarantee quality control, the
250 whole steps of retrieving, digestion and further processing were carried out in a cleaned and
251 reserved room, or inside a fume hood, depending on the country. The flow of people which
252 manipulated and processed the pieces was voluntarily kept low, to avoid cross-contamination
253 and operation errors. All the used solutions were Pure grade chemicals and were analysed for
254 metal content parallelly. Procedural blanks were used throughout the experiments. Quality
255 control standards were also performed during the analysis (see section 2.3).

256 **2.6 Statistical analysis**

257 All statistical analyses were performed with RStudio 2022.12.0.353 software (RStudio Core
258 Team, 2023) under R 4.2.2 environment. After data normalization and standardization (to give
259 each data the same weight), the variability of the bioaccumulation of metals according to seasons
260 and polymers for each immersion site was studied using Principal Component Analysis (PCA)
261 on standardized data (for all seasons and locations) with *FactoMiner* and *ggplot2* packages. The
262 significance of the experimental variables (the metals) was verified with the *factoextra* package.

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264 3. RESULTS AND DISCUSSION

265 **3.1 Seawater temperature**

266 Seawater temperature at each immersion site was measured to account for seasonal variations
267 within and between sites (Figure S1). Galway was the coldest site, with an average water
268 temperature in summer around 20 °C, which is the equivalent temperature experienced in the
269 Mediterranean during late spring and early autumn. The site with the smallest variation was A
270 Coruña, where the range went from 12 °C in winter to 18 °C in summer.

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272 **3.2 Bioaccumulation of trace metals**

273 The bioaccumulation of trace metals, expressed as µg/L, varied across seasons and sites.
274 Villefranche Bay frequently presented the lowest bioaccumulation, while Naples consistently
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presented amongst the higher bioaccumulation (Figure 3). Such higher bioaccumulation ratios (when comparing the value obtained in Naples to the maxima obtained in another site for the considered metal) were particularly apparent for Ti (181-fold increase), Ni (15-fold increase), Cr (10-fold increase), V (8-fold increase), Pb (5-fold increase) and Mn (2-fold increase).

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In Villefranche Bay, bioaccumulation was generally low (Figure 3). Small bioaccumulation of As, Cd and Cr was found at this site (below 15, 0.04 and 0.3 µg/L, respectively). Bioaccumulation of Ti was relatively high in spring for all polymers (around 45 µg/L), and also in autumn, although to a lesser extent (around 30 µg/L). High bioaccumulation in winter and spring was shown for V, and Sn had high levels in the PLA biopolymer. For Cu and Mn, oligo-elements, the bioaccumulation was very low (<10 µg/L for both), and for Co, a very toxic element, bioaccumulation was the lowest (< 0.3 µg/L) when compared to the other sites.

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The Port of Galway presented low levels of As, Co, Cd, Ni and Sn throughout all seasons (Figure 3), except for Sn on PP-post in summer. Bioaccumulation of Mn, Pb and V were low at this site (< 50, 6 and 2.5 µg/L, respectively), except in spring. Galway has low bioaccumulation of Cu (< 25 µg/L) all year around. Similarly, Mo and Ti were also bioaccumulated in low levels throughout the year (< 1 and 15 µg/L) except for summer.

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In Toulon, Co, Cu, Mn and Ni were more bioaccumulated in summer (71, 2.1, 250, 241 and 9.5 µg/L, respectively) (Figure 3). As, Cr, Mo, Pb, Ti and V were bioaccumulated all year except in winter, with a maximum in summer for Pb and Ti (200 and 51 µg/L, respectively). Cd and Sn bioaccumulation was very low all year (< 0.1 µg/L for Cd and < 1.5 µg/L for Sn, except for the bioaccumulation measured for PS and PLA which was higher).

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In A Coruña, winter was also the season promoting the lowest bioaccumulation of metals (Figure 3). As, Cd, Co, Ni and V were bioaccumulated all year except in winter, with a maximum in summer (110, 0.8, 16.5, 53 and 93 µg/L, respectively). Cr, Mn, Pb and Ti were more bioaccumulated in autumn (35, 955, 73 and 103 µg/L, respectively). Cu and Mo were more bioaccumulated in summer (323 and 11 µg/L, respectively). Sn was always low (< 0.8 µg/L, except for the bioaccumulation shown by PLA which was higher).

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Up to 10 metals out of 12 got bioaccumulated at the highest levels in samples immersed in Naples. In this site, 4 trends can be observed. As, Cd and Cu were more accumulated in summer and spring (at maximum 260, 0.95 and 1560 µg/L, respectively) (Figure 3). Cr, Pb and Sn were more accumulated in spring and autumn, with higher values in spring (402 and 1075 µg/L for Cr and Pb, respectively) or autumn (36 µg/L for Sn). Ni and Ti were mainly accumulated in autumn (603 and 19455 µg/L, respectively). Co, Mn, Mo and V were accumulated all year, except in winter, with maxima in summer for Co and Mn (8 and 1980 µg/L, respectively) or spring for V (391 µg/L at maximum).
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As

<figure><img src="image_6.png" /></figure>

Cd

<figure><img src="image_7.png" /></figure>

Co

<figure><img src="image_8.png" /></figure>

Cr

<figure><img src="image_9.png" /></figure>

Cu

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Mn

<figure><img src="image_11.png" /></figure>

Mo

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Ni

<figure><img src="image_13.png" /></figure>

Pb

<figure><img src="image_14.png" /></figure>

Ti

<figure><img src="image_15.png" /></figure>
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342 Figure 3: Bioaccumulation (µg/L) of trace metals in the plastisphere. When required, results for Naples are shown in separate plots due to changes in bioaccumulation scale.

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345 To ascertain the major trends that can be derived from the evolution of the metals on the polymers throughout the different seasons, a Principal Component Analysis (PCA) was performed using the autoscaled dataset for each site (Figure 4).

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<figure><img src="image_16.png" /><figcaption>Figure 4: Principal Component Analysis of the bioaccumulation of metals as a function of polymer, in each immersion site. Dim1 and Dim2 stand for principal component 1 and 2, respectively. The black dots representing the seasons were added to the biplots as the barycentre of the individuals corresponding to each of them.</figcaption></figure>
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The two first principal components represented between 70 and 80 % of the experimental variance of the data. Dim1 (principal component 1) represents the main data variance, which is noticeable due to the distribution of the contaminants. Dim2 (principal component 2) represents the second largest variance, and revealed some more definite patterns and, so, it will be discussed further, in relation with the results from the previous section. General conclusions seem that no clustering could be seen among polymers regardless of site in terms of metals composition. Conversely, seasons partially drive metal composition but without general trend. Finally, arrows in the biplot appear to point at max two seasons, indicating when metals were the highest. But they did not really point in the same direction, that means that metals did not bioaccumulate in the same way on polymers. This will be further discussed in detail. In addition, three metals showed opposing trends with others, Ni in Villefranche, Sn in Toulon and Ti in Naples.

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Concerning seasonal variation in Villefranche (Figure 4), metals bioaccumulation were correlated to spring and autumn. Two subsets of metals were evident: one with Cd, Cu, Pb, Ti on one side, and another with As, Co, Cr, Mn, Mo and V. This is in agreement with the results shown in Figure 3, where Cd, Cu, Pb and Ti had higher bioaccumulation in spring and in PET-Post. For the second group (As, Co, Cr, Mn, Mo and V), the bioaccumulation was higher in autumn and spring, and PS and PLA were the polymers with the highest bioaccumulation in autumn, while PP-Post had relatively higher bioaccumulation in spring.

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In Galway, winter and spring promoted the highest bioaccumulation for the considered trace metals, with one main group (As, Co, Cu, Mn and V) associated with PET and PET-Post (Figure 4).

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In Toulon, bioaccumulation of Co, Mn, Ni and Pb was strongly correlated with summer (Figure 4). This is in agreement with results presented in Figure 3, which also highlight spring and autumn as the seasons leading to high bioaccumulation, even though this trend was not specifically evidenced in the PCA results. Two polymers (PLA and PS) had systematically higher metals bioaccumulation (Figure 3 and Figure 4).

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In A Coruña, bioaccumulation was associated with summer and autumn (Figure 4). Two groups were observed depending on the season: Cr, Mn, Pb and Ti in autumn, and Co, Cu, Ni, Sn and V in summer. For the former, the highest bioaccumulation occurred on PET and/or PET-Post in autumn. For the second group, the highest metals bioaccumulation occurred for PLA in summer.

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In Naples, metals (As, Co, Cd, Cu and Mn) bioaccumulation was correlated with summer (Figure 4), and showed higher bioaccumulation in LDPE and PS.

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387 **3.3 Polymer type as a substrate and its influence on metals bioaccumulation in biofilms**

388 When considering metals bioaccumulation as a function of polymer type (Figure 3), in
389 Villefranche, the polymers promoting higher metal bioaccumulation were PS, PLA, PET and
390 PET-Post. In spring, both PET-Post and PP-Post promoted higher metals bioaccumulation,
391 except for As, Co, Cr, Mn on PET-Post, and for Ni in PP-Post. One objective of this work was to
392 compare the bioaccumulation on recycled plastics to their corresponding raw polymer. In
393 summer, both recycled plastics promoted a higher load in As, Cu, Ni, Pb and Sn compared to
394 their corresponding raw polymer. For the other trace metals, the recycled plastics did not seem to
395 have an effect, except for a lower load in V bioaccumulation. In autumn, both recycled plastics
396 promoted a lower load in all metals bioaccumulation, except for Cd and Cu (on PET-Post) and
397 Cr, Cu, Pb Sn on PP-Post. In winter, the trends between recycled plastics were completely
398 opposite: PET-Post promoted a lower load for all metals (except for Pb and Cu) whereas PP-Post
399 promoted a higher load (except for Ni).

400 In Galway, the polymers promoting the higher metals bioaccumulation were PET, PET-Post,
401 LDPE and PLA (Figure 3). When comparing the recycled plastics to their raw counterparts
402 during the seasons where the highest bioaccumulation was observed (winter and spring), both
403 PET-post and PP-post were mainly promoting a lower load in metals bioaccumulation. In
404 summer and autumn, recycled plastics presented opposite trends. PET-Post led to a higher load
405 of metals (except for Cd, Mn, Ti) in summer and a lower load (except for V) in autumn, whereas
406 it was the contrary for PP-Post.

407 In A Coruña, the polymers promoting the higher metals bioaccumulation were PET, PET-Post
408 and PLA (Figure 3). For these polymers, the highest bioaccumulation was measured in summer
409 and autumn. In summer, PET-Post mainly promoted an increase in metals bioaccumulation
410 (except for Mn, Pb), when no significant trend was observed for PP-Post. In autumn, both
411 recycled plastics promoted a decrease in metals bioaccumulation (except for Ni on PET-Post).
412 PP-Post promoted a decrease in metals bioaccumulation in autumn, winter and spring (except for
413 Sn). In winter, PET-Post promoted an increase in metals bioaccumulation (except for Co, Mn Ni)
414 as opposed to spring, where PET-Post promotes a decrease for all metals.

415 In Toulon, PS and PLA were systematically promoting the higher metals bioaccumulation
416 (Figure 3). In spring, both PET-Post and PP-Post promoted an increase in metals
417 bioaccumulation (except for Sn and Ti). In summer, no trend could be drawn out. In autumn,
418 both recycled plastics were promoting a decrease in metals bioaccumulation. In winter, the effect
419 was opposite, PP-post promoted an increase (except for As, Cd and Co), whereas PET-Post
420 promoted a decrease (except for Cd, Mo, Pb and V).
In Naples, the polymers promoting the higher metals bioaccumulation were PET-Post, PS and PLA (Figure 3). In spring, both PET-Post and PP-Post promoted an increase in metals bioaccumulation (except for Mn and Ni on PP-Post). In summer and winter, on the contrary, PET-Post promoted a decrease in metals bioaccumulation. In summer and in autumn, PP-Post did not present any trends.

The cumulative trace metals bioaccumulation at all sites in all seasons were plotted to identify which polymers promoted the higher metal bioaccumulation (Figure S2).

Generally, cumulative metal bioaccumulation was within the same order of magnitude for each polymer type, with some exceptions for PET, PET-Post, PP, PS and PLA. This was calculated by the sum of individual values per polymer type, and expressed using a box and whisker plot in Figure S2, as there were standardized volumes (5 mL NaOH) used for each polymer. Bioaccumulation of As, Cu, Cr, Mn, Mo, Ni, Pb and Ti, also had a different order of magnitude when compared to Cd, Co or Sn. Nonetheless, the highest bioaccumulation of Sn was recorded from PLA. If focusing on PLA, results here show that this polymer was often promoting a high metals bioaccumulation at all sites (Figure S2). In Villefranche, it promoted high bioaccumulation of Cd, Cr, Cu, Mo and Pb in autumn and spring (Figure 3). In Galway, PLA promoted high bioaccumulation of Pb and Ni in winter, especially Pb. In this site, PLA also promoted the highest Cd, Cr, Sn and Ti bioaccumulation among all polymers. In winter, which was a season promoting metals bioaccumulation in Galway, PLA was often among the polymers reflecting the highest bioaccumulation. In Naples, PLA promoted one of the highest bioaccumulation in spring, for all the measured metals (Figure 3). In Toulon, PLA promoted one of the highest bioaccumulation in summer for all the measured metals, in addition it promoted the highest Sn bioaccumulation in spring. In A Coruña, PLA promoted among the highest bioaccumulation in autumn and winter for all measured metals. It also promoted high bioaccumulation in summer for Cu, Mn, Ni, Sn and V.

## 3.4 Ecosystem Risk Assessment

Risk Quotients (RQ) were calculated for all metals (Table S2). For all immersion sites, RQ values for Arsenic were high and this will be further discussed. RQ values for three other trace metals (Cr, Cu and Pb), were selected according to their toxicity or variations and are presented in Figure 5.
<figure><img src="image_17.png" /><figcaption>Figure 5: Risk quotients (RQ) for bioaccumulation of metals at all immersion sites. The red dotted lines represent RQ value of 1, above which the environment is considered to be at high risk (Nika et al., 2020). On each graph, the season is evidenced by a symbol.</figcaption></figure>

In Villefranche, RQ for Cd, Mo and Ti were always below 0.01 (Table S2), which means that these trace metals did not represent environmental risk. RQ values for Co, Cu, Ni, Sn and Pb were always below 1 (except on some occasions), showing medium risk for these metals (Figure 5). RQ values for Mn were above 1 in autumn and spring only. RQ values for Cr were below 1 in summer only. Finally, RQ values for V were always above 1 (reaching 13 at maximum in autumn), demonstrating high risk for this contaminant. RQ for As in Villefranche ranged between 55 and 315, indicating a high risk for this element.
In Galway, RQ values for Mo and Ti were always below or close to 0.01 (Table S2), meaning that these trace metals did not represent an environmental risk. RQ values for Cd, Co, Ni, Sn and V were below 1, representing a medium risk, except in winter (and spring for V; Figure 5). RQ values for Cr, Cu and Pb were always above 1 (maximum value around 16), demonstrating a high risk for these trace metals, especially in winter and spring (Figure 5). RQ values for Mn and As were even higher, reaching values as high as 130 (in spring), showing that these contaminants are linked with high environmental risk, especially in winter and spring.

In A Coruña, RQ values for Mo, Sn and Ti were always below or close to 0.01 (Table S2), meaning that these trace metals did not represent an environmental risk. RQ values for Cd and Ni were always around 1, whereas those for Co, Cr, Cu, Pb and V were much higher, with a general decrease in winter (Figure 5). RQ values for Mn and As were really high.

In Toulon, RQ values for Mo and Ti were always below or close to 0.01 (Table S2), meaning that these trace metals did not represent an environmental risk. RQ values for Cd, Co, Ni and Sn were always low, proving that these trace metals did not impact on the environment. RQ values for Cr, Cu, Mn, Pb and V corresponded to medium to high environmental risk (Figure 5), with a general decrease in winter for all, especially for Mn, which turned to low environmental risk at that season. Like all other immersion sites, RQ values for As were particularly high.

In Naples, all RQ values were above the range of low environmental risk, except for Mo. The RQ values for Cd, Sn and Ti were in the medium environmental risk, except for summer and spring in the case of Cd, and in autumn in the case of Ti (Figure 5 and Table S2). RQ values for Co and Ni were always above 1, demonstrating the risk associated with these trace metals. RQ values for Cr, Cu, Mn, Pb and V were really high, though not reaching RQ values for As (Table S2).
487 **4. Discussion**

488 This work explores the bioaccumulation of trace metals in biofilm growing on immersed plastic
489 pieces from eight polymers, at five European coastal sites over a one-year period. The findings
490 presented herein significantly contribute to the understanding of bioaccumulation of trace metals
491 within the plastisphere, a rather understudied research topic, as they cover both Atlantic and
492 Mediterranean sites. Our results showed a high variability depending on polymer type and
493 associated trace metals. In this section, we advance hypotheses for the high metals
494 bioaccumulation observed at certain sites, when possible.

495 As previously mentioned, Villefranche Bay is a protected area with low fishing, industrial and
496 anthropogenic impacts, where we hypothesised that bioaccumulation of trace metals would be
497 generally low, the only potential source being natural (sediments for example) and this was
498 confirmed by our results. In contrast, due to its proximity to many anthropogenic activities,
499 Toulon harbour is known for a wide-range metallic contamination in water and seafloor
500 sediments (Tessier et al., 2011; Layglon et al., 2021). These sources seem to be linked to the high
501 bioaccumulation of trace metals in the biofilm developed on the plastic pieces immersed at this
502 site. Similarly, Naples, the site with the larger volume of cargo and anthropogenic activities, has
503 a multiple range of contamination sources, which are thought to contribute to the highest metals
504 bioaccumulation observed. The results of this study show that –qualitatively– the scale of nearby
505 anthropogenic activities might be the factor with the highest influence on the metal
506 bioaccumulation by the plastisphere. In A Coruña, despite the main activities being recreational
507 and sailing, bioaccumulation of trace metals was relatively high, potentially reflecting historical
508 and present anthropogenic activities still reflected in high bioaccumulation.

509 As already mentioned, a wide range of polymers, including both pristine and post-consumer
510 fossil fuel based, as well as biopolymers, were used in this study to understand whether polymer
511 type is one of the factors influencing trace metal accumulation onto the plastisphere. Plastic is a
512 known substrate for the growth of micro- but also macroorganisms, including bryozoans and
513 barnacles (Li et al., 2016), and is described as a potential vector for invasive species and/or
514 potential pathogenic bacteria and viruses, hosting antibiotic resistance genes (ARGs) and metal
515 resistance genes (MRGs) (Pedrotti et al., 2022; Li et al., 2022; Sababadichetty et al., 2024). The
516 accumulation of both organic and inorganic pollutants is expected to increase the resistance
517 genes within the plastisphere (Li et al., 2022); this, in turn, increases the exogenous pressures of
518 metals, triggering a cause-effect pattern which is the rationale behind this study. Separate papers
519 assessing persistent organic pollutants (Concha-Graña et al., 2022) and the genetic diversity
520 (Pedrotti et al., 2022; Lacerda et al., 2023) of the plastisphere from the same incubation
521 experiment were already published, and will be published in parallel.
In scientific literature, *in situ* immersion tests assessing pristine and recycled plastics and relating these to trace metals bioaccumulation are scarce, if nonexistent. Therefore, this study provides a valuable dataset and provides insights into a problem that deserves more attention. Trace metal bioaccumulation is incredibly complex, and as seen in this work, where geographical location, seasonality and polymer type were assessed, it depends on many variables. Regarding polymer type, PET, PET-Post, PS and PLA showed the highest bioaccumulation of metals by the plastisphere. This widens the toxicity linked to PET, PS and PLA, as they were already demonstrated to affect the gut microbiota of marine fish, even in their pristine form (Zhang et al, 2023). Understanding the physico-chemical properties of these polymers (hydrophobicity, surface charge, crystallinity, tensile structure, surface-area ratios, etc.) under natural environmental conditions will allow a better understanding of how weathering, degradation and fragmentation and/or inclusion of chemical additives contribute to the formation of biofilm layers. This will also allow us to understand how biofilm genetic diversity is linked to the adsorption and/or absorption of persistent organic and inorganic pollutants. The complexity of this assessment requires a transdisciplinary approach where microbiologists, chemists and material scientists collaborate to mitigate potential risks, as highlighted by Pedrotti et al. (2022), particularly with potentially pathogenic bacteria and viruses hitchhiking in plastics and representing vectors for invasive species and potential spread of diseases.

This work also compared pristine and recycled forms of common plastics. A recent study on the impact of recycling on PLA physical properties concluded that a succession of recycling operations would not affect the polymer properties (Davies et al., 2023). Here, from a microbiological and a chemical point of view, recycling had an effect as the metals load changed when compared to the raw polymers, but it was not possible to set a generalised trend as it was dependent from site, season and metal. Yet, it has to be underlined that PET-Post (and PP-Post in Villefranche) is on average promoting higher metal bioaccumulation than its pristine counterpart.

PolyLacticAcid (PLA), a popular commercial biopolymer used in this study, has become a popular alternative to fossil fuel based polymers, as its synthesis uses bioproducts that can be easily degraded in recycling plants (Park et al., 2020; Sun et al., 2022). PLA is a brittle and stiff polymer that requires chemical additives such as plasticizers to be added during production to enhance flexibility and toughness (Park et al., 2020). This polymer has similar fragmentation rates to other fossil-fuel based polymers (Sun et al., 2022), making it ideal for comparative studies. Overall, PLA promoted higher metals bioaccumulation at all immersion sites of this study, which could potentially be linked to its structural composition. In Toulon, PLA always led to the higher bioaccumulation for all evaluated trace metals (Figure S2). In all immersion sites and seasons cumulated, PLA presented significantly higher Sn bioaccumulation (Figure S2). It can therefore be concluded that PLA, at least in our tested *in situ* conditions, represents a threat as important as fossil-fuel based polymers. It should also be underlined that the term "biodegradable" should be supported by the conditions used for the test (Paul-Pont et al., 2023).
This study assessed environmental risk (using RQ values), to understand ecotoxicological impacts. Arsenic (As) was shown to be a metal with very high risk across all sampled sites. This could be explained by the chemistry of arsenic species, as it was mainly found in its oxic phase. As a matter of fact, in oxic conditions like the ones encountered at all immersion sites, arsenic is mainly present as arseniate, an oxyanion whose size, with charge and structure are really close to that of phosphate (Willsky and Malamy, 1980). These arsenic species could therefore use phosphate pumps that are present within the bacteria and the biofilm (Pothier et al., 2018). This could explain the high bioaccumulation at all immersion sites. The environmental risk for Cadmium (Cd), molybdenum (Mb) and titanium (Ti), on the other hand, were low across all sites.

Apart from Villefranche (which can be considered our reference site), all immersion sites showed potential ecological risk. This is particularly relevant because colonized microplastics are more likely to be ingested by marine organisms (Yu et al., 2023), and this would result in bioaccumulation of metals across marine food webs, potentially representing a human and environmental threat. However, to our knowledge, there are no studies that reflect this causal relationship. Nonetheless, given the fishing (e.g. Naples) and aquaculture (e.g. Toulon, and Galway) activities occurring at some of the sites, precaution should be taken.

In the literature, there appears to be a positive relationship between the plastisphere and metals bioaccumulation, as studies where biofilms were characterised report that trace metals bioaccumulation globally increased with growth of the plastisphere (Richard et al., 2019; Forero-Lopez et al., 2022). Once again, our results show that this is a complex matter, as metal bioaccumulation was not systematically higher in areas with higher biofilm growth. Galway is a typical counter-example of this statement, as for some metals, the highest bioaccumulation occurred in winter, when the temperature was the lowest, and biofilm growth was relatively slower. This highlights the relevance of characterising both biofilm microbial community and associated EPS production over its growth. Biofilm chemical functions, even with small growth, might be affecting the concentration of bioaccumulated trace metals, for instance because of the production of different kinds of EPS and/or the recruiting of metal resistance genes (MRGs) within the plastisphere. Even though it was reported that all polymer types (without biofilm) tend to adsorb similar concentrations of metals, as shown for San Diego Bay (Rochmann et al., 2014), our results show a different behaviour when biofilm is taken into account. Although it is thought that metal bioaccumulation is higher in areas with extensive anthropogenic activities, the opposite situation, for example in Villefranche Bay, might also occur, as the bioaccumulation of Ti and V were both high, potentially due to natural sources.

The results presented here demonstrate that the nature, rather than solely the biomass, of the plastisphere play an important role in accumulating trace metals from the surrounding environment. Further investigation into the composition of biofilms and EPS, known to be
controlled by variation of environmental parameters (e.g. hydrodynamics, oceanographic conditions, etc.) should be performed to unravel their impacts on bioaccumulation of trace metals. In this work, regular monitoring of parameters like temperature, salinity or pH were performed. Temperature was the only parameter presented due to the variations along seasons, which might have an impact on biofilm development, rather than other environmental parameters that remained relatively constant throughout the experiment. One limitation of this work is that no long-term monitoring of a cluster of environmental parameters, including hydrodynamics or nutrients, was performed. A further research effort will surely include and address such limitations and contribute to monitoring in partner countries.

606

## 5. Conclusion

The results presented in this study established a discernible correlation between seasonal variations and the bioaccumulation of trace metals in the plastisphere. The seasons leading to the higher bioaccumulation were not the same, each site exhibiting unique characteristics. Notably, Galway displayed a sustained peak in bioaccumulation during the winter season. The site with the lower anthropogenic activities, namely Villefranche, demonstrated the lowest metals bioaccumulation. Conversely, sites characterized by extensive anthropogenic influences consistently exhibited elevated metals bioaccumulation in the plastisphere. Villefranche therefore emerged as a reference area in the Mediterranean Sea for evaluating trace metals bioaccumulation in the plastisphere. The polymers promoting biofilm development with higher metals bioaccumulation were PET, PET-Post, PP, PS and PLA. Polylactic Acid (PLA), a biopolymer usually used as a replacement of fossil fuel based polymers, was shown here to promote similar or higher bioaccumulation of trace metals by the plastisphere. RQ values highlight that trace metals bioaccumulation can pose ecological risk, however, the complexity of this process requires further investigation. The data provided here provides new insights on how bioaccumulation relates to seasonality and polymer type. Further research into the impacts or interactions between the plastisphere and organic and inorganic pollutants is required to understand and potentially mitigate their effects to marine and human health.

625

## Acknowledgements

The authors acknowledge JPI Oceans for their financial support to the national funding agencies in each of the consortium countries, which allowed for this work to be possible. We also want to thank Harbour Master Captain Brian Sheridan (Port of Galway), Cyril Debost, Marie Emmanuelle Kerros (Sorbonne University), Ángel Barreiro, Ignacio Palmero and the staff of the
631 Club Náutico Ría de Ares (Port of Ares), Giovanni de Martino and Pasquale De Angelis (Lega

632 Navale Sezione di Napoli) for their support throughout the project, particularly under the

633 challenging circumstance of the COVID pandemic. V. Donnarumma was supported by a SZN-

634 Open University PhD fellowship.
635 References

636 Carpenter EJ, Smith KL Jr. Plastics on the Sargasso sea surface. Science. 1972 Mar 17;175(4027):1240-1.
637 https://doi.org/10.1126/science.175.4027.1240

638 Chandrapalan and Kwong, (2021). Functional significance and physiological regulation of essential trace metals in fish. J. Exp. Biol. 224,
639 https://doi.org/10.1242/jeb.238790

640 Chouvelon, T., Gilbert, L., Caurant, F., Méndez-Fernandez, P., Bustamante, P., Brault-Favrou, M. and Spitz, J. (2022), Nutritional grouping of marine forage species reveals contrasted exposure of high trophic levels to essential micro-nutrients. Oikos, 2022: e08844.
641 https://doi.org/10.1111/oik.08844

642 Cianelli, D., Uttieri M., Buonocore B., Falco P., Zambardino G., Zambianchi E. (2012). Dynamics of a Very Special Mediterranean Coastal Area: The Gulf of Naples, in: G.S. Williams Editor, Mediterranean ecosystems: Dynamics, Management and Conservation, pp 129-150.
643 https://novapublishers.com/shop/mediterranean-ecosystems-dynamics-management-and-conservation/

644 Coclet, C., Garnier C., Delpy F., Jamet D., Durrieu G., Le Poupon C., Mayer M., Misson B. (2018). Trace metal contamination as a toxic and
645 structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area. Progress in Oceanography,
646 163, https://doi.org/10.1016/j-pocean.2017.06.006

647 Coglievina C., Massiera B., Morales V., Mahmoud I.B Territorial dynamics of the bay of Villefrance-sur-Mer (FRENCH RIVIERA): From commercial and military vocation to tourism and sporting activities. (2016). Rev. Anais Bras. de Est. Tur., v.6, n.3, pp.76-84. https://periodicos.ufjf.br/index.php/abet/article/view/3152

648 Concha-Graña, E. Moscoso-Perez, C., Lopez-Mahia, P., Muniategui-Lorenzo, S., (2022). Adsorption of pesticides and personal care products on pristine and weathered microplastics in the marine environment. Comparison between bio-based and conventional plastics. Science of the Total Environment, 848, 157703

650 CSO (2023). Census of Population 2022. Table 2.2 Population by local authority, 2022. https://www.cso.ie/en/releasesandpublications/ep/p-cpr/censusofpopulation2022-preliminaryresults/geographicchanges/ (Consulted in November 2023)

651 da Costa, J.P., Mouneyrac, C., Costa, M., Duarte, A. C., Rocha-Santos, T., 2020. The Role of Legislation, Regulatory Initiatives and Guidelines on the Control of Plastic Pollution. Frontiers in Environmental Science, 24. https://doi.org/10.3389/fenvs.2020.00104

652 da Costa, J.P., Avellan, A., Mouneyrac, C., Duarte, A. and Rocha-Santos, T., 2022. Plastic additives and microplastics as emerging contaminants: Mechanisms and analytical assessment. TrAC Trends in Analytical Chemistry, p.116898.
660 https://doi.org/10.1016/j.trac.2022.116898

661 Dang H.D., Schäfer J., Brach-Papa C., Lenoble V., Durrieu G., Dutruch L., Mullot J.-U., Mounier S., Garnier C. (2015) Evidencing the impact of coastal polluted sediments onto mussels through Pb stable isotopes composition. Environmental Science and Technology 49, 11438-11448. DOI: 10.1021/acs.est.5b01893

662 Davies, P., Le Gall, M., Niu, Z., Catarino, A.I., De Witte, Y., Everaert, G., Dhakal, H., Park, C. H., Demeyer, E., (2023). Recycling and ecotoxicity of flax/PLA composites: Influence of seawater aging. Composites Part C: Open Access, 12, 100379.
667 https://doi.org/10.1016/j.jcomc.2023.100379

668 Djaoudi K., Tesán Onrubia J.A., Boukra A., Guesnay L., Portas A., Barry-Martinet R., Angeletti B., Mounier S., Lenoble V., Briand J.-F. (2022) Seawater copper content controls biofilm bioaccumulation and microbial community on microplastics. Sci. Total Env. 814, 152278.
669 http://dx.doi.org/10.1016/j.scitotenv.2021.152278

670 ECHA (2023). Information on Chemicals. https://echa.europa.eu/information-on-chemicals

671 Esposito, M., Canzanella S., Lambiase S., Scaramuzzo A., La Nucara R., Bruno T., Picazio G., Colarusso G., Brunetti R., Gallo P. (2020). Organic pollutants (PCBs, PCDD/Fs, PAHs) and toxic metals in farmed mussels from the Gulf of Naples (Italy): Monitoring and human exposure. Regional Studies in Marine Science, 40, 101497. https://doi.org/10.1016/j.rsma.2020.101497

672 EU (2013). Directive 2013/39/EU of the European Parliament and of the Council of 12 August 2013 amending Directives 2000/60/EC and 2008/105/EC as regards priority substances in the field of water policy Text with EEA relevance. https://eur-lex.europa.eu/legal-content/EN/ALL/?uri=celex:32019L0904

673 EU (2019). Directive 2019/904 of the European Parliament and of the Council of 5 June 2019 on the reduction of the impact of certain plastic products on the environment, text with EEA relevance. https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32019L0904

674 Forero-López A.D., Brugnoni L.I., Abasto B., Rimondino G.N., Lassalle V.L., Ardusso M.G., Nazzarro M.S., Martinez A.M., Spetter C.V., Biancalana F., Fernández-Severini, (2022). Plastisphere on microplastics: In situ assays in an estuarine environment, Journal of Hazardous Materials, https://doi.org/10.1016/j.jhazmat.2022.129737

675 Fernández-González, V., J. Andrade-Garda, López-Mahía, P., Muniategui-Lorenzo, S., (2022). Misidentification of PVC microplastics in marine environmental samples. TrAC Trends in Analytical Chemistry, 153. 116649. https://doi.org/10.1016/j.trac.2022.116649

676 Frias J., Lyashevska O., Joyce H., Pagter E., Nash R. (2020). Floating microplastics in a coastal embayment: A multifaceted issue. Marine Pollution Bulletin, 158. https://doi.org/10.1016/j.marpolbul.2020.111361

677 G20, (2021). G20 Report on Actions against Marine Plastic Litter - Third Information Sharing based on the G20 Implementation Framework. Ministry of the Environment, Japan, 2021. 2nd Edition https://g20mpl.org/wp-content/uploads/2021/07/G20MPL-report-2021_final-edition1119.pdf

678 Galgani, F., Hanke, G., Maes, T. (2015). Global Distribution, Composition and Abundance of Marine Litter. In: Bergmann, M., Gutow, L., Klages, M. (eds) Marine Anthropogenic Litter. Springer, Cham. https://doi.org/10.1007/978-3-319-16510-3_2
<table>
  <tr>
    <td>692</td>
    <td>Geetika Bhagwat, Thi Kim Anh Tran, Dane Lamb, Kala Senathirajah, Ian Grainge, Wayne O'Connor, Albert Juhasz, and Thava Palanisami (2021). Biofilms Enhance the Adsorption of Toxic Contaminants on Plastic Microfibers under Environmentally Relevant Conditions. Environmental Science & Technology 2021 55 (13), 8877-8887. https://doi.org/10.1021/acs.est.1c02012</td>
  </tr>
  <tr>
    <td>693</td>
    <td>Hahladakis, J., Velis, C., Weber, R., Iacovidou, E., Prunell, P., (2018). An overview of chemical additives present in plastics: Migration, release, fate and environmental impact during their use, disposal and recycling. Journal of Hazardous Materials, 344, 179-199. https://doi.org/10.1016/j.jhazmat.2017.10.014</td>
  </tr>
  <tr>
    <td>694</td>
    <td>Hossain MB et al (2022). Heavy Metals in Four Marine Fish and Shrimp Species from a Subtropical Coastal Area: Accumulation and Consumer Health Risk Assessment. PMC Biology (Basel) 11(12):1780. https://doi.org/10.3390/biology11121780</td>
  </tr>
  <tr>
    <td>700</td>
    <td>Kaur, R., Garkal, A., Sarode, L., Bangar, P., Mehta, T., Singh, D., Rawal, R., (2024). Understanding arsenic toxicity: Implications for environmental exposure and human health. Journal of Hazardous Materials Letters, 5, 100090. https://doi.org/10.1016/j.hazl.2023.100090</td>
  </tr>
  <tr>
    <td>702</td>
    <td>Khalid, N., Aqeel M., Noman A., Mulk Khan S., Akhter N. (2021). Interactions and effects of microplastics with heavy metals in aquatic and terrestrial environments. Environmental Pollution, 290, 118104. https://doi.org/10.1016/j.envpol.2021.118104</td>
  </tr>
  <tr>
    <td>704</td>
    <td>Kristein, I., Kirmizi, S., Wichels, A., Garin-Fernandez, A., Erler, R., Loder, M., Gerdts, G., (2016). Dangerous hitchhikers? Evidence for potentially pathogenic Vibrio spp. on microplastic particles. Marine Environmental Research, 120, 1-8. https://doi.org/10.1016/j.marenvres.2016.07.004</td>
  </tr>
  <tr>
    <td>707</td>
    <td>Lacerda, Ana Luzia, Frías, João, Pedrotti, Maria Luíza, Tardigrades in the Marine Plastisphere: New Hitchhikers Surfing Plastics. Available at SSRN: https://ssrn.com/abstract=4627737 or http://dx.doi.org/10.2139/ssrn.4627737</td>
  </tr>
  <tr>
    <td>708</td>
    <td>Lambert, S., Wagner, M. (2018). Microplastics Are Contaminants of Emerging Concern in Freshwater Environments: An Overview. In: Wagner, M., Lambert, S. (eds) Freshwater Microplastics. The Handbook of Environmental Chemistry, vol 58. Springer, Cham. https://doi.org/10.1007/978-3-319-61615-5_1</td>
  </tr>
  <tr>
    <td>712</td>
    <td>Layglon N., Misson B., Durrieu G., Colcet C., D'Onofrio S., Dang H.D., François D., Mullot J.-U., Mounier S., Lenobke V., Omanovic D., Garnier C. (2020). Long-term monitoring emphasizes impacts of the dredging on dissolved Cu and Pb contamination along with ultraplankton distribution and structure in Toulon Bay (NW Mediterranean Sea, France). Marine Pollution Bulletin. 156, 111196. https://doi.org/10.1016/j.marpolbul.2020.111196</td>
  </tr>
  <tr>
    <td>716</td>
    <td>Laygon, N., Misson, B., Gallois, B., D'Onofrio, S., Lenoble, V., Mounier, S., Omanović, D., Garnier, C., (2021). Negligible microbial heterotrophic quantitative contribution onto trace metals remobilization during marine sediment resuspension - insights from a Mediterranean urbanized bay. Marine Chemistry, 234, 103981. https://doi.org/10.1016/j.marchem.2021.103981</td>
  </tr>
  <tr>
    <td>719</td>
    <td>Li H.-X., Orihuela B., Zhu M., Rittschof D. Recyclable plastics as substrata for settlement and growth of bryozoans Bugula neritina and barnacles Amphibalanus Amphitrite. Environmental Pollution 218, 973-980. http://dx.doi.org/10.1016/j.envpol.2016.08.047</td>
  </tr>
  <tr>
    <td>721</td>
    <td>Li, Q., Tian, L., Cai, X., Wang, Y., & Mao, Y. (2022). Plastisphere showing unique microbiome and resistome different from activated sludge. Science of The Total Environment, 851, 158330. https://doi.org/10.1016/j.scitotenv.2022.158330</td>
  </tr>
  <tr>
    <td>723</td>
    <td>Liu, J., Lu, G., Xie, Z., Zhang, Z., Li, S., Yan, Z., (2015). Occurrence, bioaccumulation and risk assessment of lipophilic pharmaceutically active compounds in the downstream rivers of sewage treatment plants. Science of the Total Environment, 511, 54-62. https://doi.org/10.1016/j.scitotenv.2014.12.033</td>
  </tr>
  <tr>
    <td>726</td>
    <td>Liu S., Shi J., Wang J., Dai Y., Li H., Li J., Liu X., Chen X., Wang Z., Zhang P. (2021). Interactions between microplastics and heavy metals in aquatic environments: A review. Front. Microbiol. 12, https://doi.org/10.3389/fmicb.2021.652520</td>
  </tr>
  <tr>
    <td>728</td>
    <td>Lobelle D. and Cunliffe M. (2011). Early microbial biofilm formation on marine plastic debris. Mar Pollut Bull 62, 197-200.</td>
  </tr>
  <tr>
    <td>729</td>
    <td>Moresco V., Charatzidou A., Oliver D.M., Weidmann M., Matallana-Surget S., Quilliam R.S. (2022) Binding, recovery, and infectiousness of enveloped and non-enveloped viruses associated with plastic pollution in surface water. Environmental Pollution 308, 119594. https://doi.org/10.1016/j.envpol.2022.119594</td>
  </tr>
  <tr>
    <td>732</td>
    <td>Ngoc Do A.T., Ha Y., Kwon J.-H. (2022) Leaching of microplastic-associated additives in aquatic environments: a critical review. Environmental Pollution, 305, 119258. https://doi.org/10.1016/j.envpol.2022.119258</td>
  </tr>
  <tr>
    <td>734</td>
    <td>Nika, M.C., Ntaiou, K., Elytis, K., Thomaidi, V.S., Gatidou, G., Kalantzi, O.I., Thomaidis, N.S., Stasinakis, A.S. (2020). Wide-scope target analysis of emerging contaminants in landfill leachates and risk assessment using Risk Quotient methodology. Journal of Hazardous Materials, 394, 122493. https://doi.org/10.1016/j.jhazmat.2020.122493</td>
  </tr>
  <tr>
    <td>737</td>
    <td>NORMAN (2023). Ecotoxicology Database - Lowest PNECs. Available at: https://www.norman-network.com/nds/ecotox/lowestPnecsIndex.php</td>
  </tr>
  <tr>
    <td>739</td>
    <td>Pan, H., Zhao, X., Zhou, X., Yan, H., Han, X., Wu, M., Chen, F., (2023). Research progress on the role of biofilm in heavy metals adsorption-desorption characteristics of microplastics: A review. Environmental Pollution, 336, 122448. https://doi.org/10.1016/j.envpol.2023.122448</td>
  </tr>
  <tr>
    <td>741</td>
    <td>Park, M., Choi, I., Lee, S., Hong, S., Kim, A., Shin, J., Kang, H., Kim, Y., (2020). Renewable malic acid-based plasticizers for both PVC and PLA polymers. Journal of Industrial and Engineering Chemistry, 88, https://doi.org/10.1016/j.jiec.2020.04.007</td>
  </tr>
  <tr>
    <td>743</td>
    <td>Paul-Pont, I., Ghiglione, J.-F., Gastaldi, E., Ter Halle, A., Huvet, A., Bruzaud, S. et al. (2023) Discussion about suitable applications for biodegradable plastics regarding their sources, uses and end of life. Waste Management 157: 242-248. https://doi.org/10.1016/j.wasman.2022.12.022</td>
  </tr>
  <tr>
    <td>746</td>
    <td>Pedrotti, M., Lacerda, A., Petit, S., Ghiglione, J., Gorsky, G., (2022). Vibrio spp and other potential pathogenic bacteria associated to microfibers in the North-Western Mediterranean Sea. Plos One, https://doi.org/10.1371/journal.pone.0275284</td>
  </tr>
  <tr>
    <td>748</td>
    <td>Pete et al., (2023). Biofilm formation influences wettability and settling of microplastics. Environ. Sci. Technol. 10, 2, 159-164. https://doi.org/10.1021/acs.estlett.2c00728</td>
  </tr>
  <tr>
    <td>750</td>
    <td>Port of Ares (2023) General information about the Port. https://portosdegalicia.gal/es/ (Consulted in December, 2023)</td>
  </tr>
</table>
751 Port of Galway (2023). https://theportofgalway.ie/port-of-galway-general-information/ (Consulted in November 2023).

752 Pothier Martin P., Hinz Aaron J., Poulain Alexandre J. (2018). Insights Into Arsenite and Arsenate Uptake Pathways Using a Whole Cell

753 Biosensor. Frontiers in Microbiology. https://www.frontiersin.org/articles/10.3389/fmicb.2018.02310

754 RStudio Core Team (2023). R: A Language and Environment for Statistical Computing R Foundation for Statistical Computing, Vienna, Austria

755 (2023). https://www.R-project.org/

756 Ragusa, A., Svelato A., Santacroce C., Catalano P., Notarstefano V., Carnevali O., Papa F., Rongioletti M.C.A., Baiocco F., Draghi S., D'Amore

757 E., Rinaldo D., Matta M., Giorgini E. (2021). Plasticicenta: First evidence of microplastics in human placenta. Environment International, 146,

758 106274. https://doi.org/10.1016/j.envint.2020.106274

759 Rebelo, F, Caldas, E., (2016). Arsenic, lead, mercury and cadmium: Toxicity, levels in breast milk and the risks for breastfed infants.

760 Environmental Research 151, https://doi.org/10.1016/j.envres.2016.08.027

761 Richard, H., Carpenter, E., Komada, T., Palmer, P., Rochman, C. (2019). Biofilm facilitates metal accumulation onto microplastics in estuarine

762 waters. Science of the Total Environment, 683. https://doi.org/10.1016/j.scitotenv.2019.04.331

763 Rochman CM, Hentschel BT, Teh SJ (2014) Long-Term Sorption of Metals Is Similar among Plastic Types: Implications for Plastic Debris in

764 Aquatic Environments. PLoS ONE 9(1): e85433. https://doi.org/10.1371/journal.pone.0085433

765 Rockström, J., Steffen, W., Noone, K. et al. A safe operating space for humanity. Nature 461, 472–475 (2009). https://doi.org/10.1038/461472a

766 Sabadadichetty, L., Miltgen, G., Vincent, B., Guilhaumon, F., Lenoble, V., Thibault, M., et al. (2024). Microplastics in the insular marine

767 environment of the Southwest Indian Ocean carry a microbiome including antimicrobial resistant (AMR) bacteria: A case study from

768 Reunion Island. Marine Pollution Bulletin, 198, 115911. https://doi.org/10.1016/j.marpolbul.2023.115911

769 Sessini, V., Planzuela, M., Damian, J., Mosquera, M., (2020). Bio-based polyether from limonene oxide catalytic ROP as green polymeric

770 plasticizer for PLA. Polymers, 210, 123003. https://doi.org/10.1016/j.polymer.2020.123003

771 Shashoua, Y. (2008) Conservation of Plastics, - materials science, degradation, and preservation. Amsterdam: Elsevier/Butterworth-Heinemann.

772 ISBN: 978-0-7506-6495-0

773 Slama H.B., Bouket A.C., Pourhassan Z., Alenezi F.N., Silini A., Cherif-Silini H., Oszako T., Luptakova L., Golinska P., Belbahri L. (2021).

774 Diversity of synthetic dyes from textile industries, discharge impacts and treatment methods. Appl. Sci. 2021, 11(14), 6255;

775 https://doi.org/10.3390/app11146255

776 Sun, Y., Wang, X., Xia, S., Zhao, J., (2022). Cu(II) adsorption on Poly(Lactic Acid) Microplastics: Significance of microbial colonization and

777 degradation. Chemical Engineering Journal, 429, 132306. https://doi.org/10.1016/j.cej.2021.132306

778 Syed, Z., Sogani, M., Rajvanshi, J. et al. Microbial Biofilms for Environmental Bioremediation of Heavy Metals: a Review. Appl Biochem

779 Biotechnol (2022). https://doi.org/10.1007/s12010-022-04276-x

780 Tchounwou, P.B., Yedjou, C.G., Patlolla, A.K., Sutton, D.J. (2012). Heavy Metal Toxicity and the Environment. In: Luch, A. (eds) Molecular,

781 Clinical and Environmental Toxicology. Experientia Supplementum, vol 101. Springer, Basel. https://doi.org/10.1007/978-3-7643-8340-4_6

782 Tessier, E., Garnier, C., Mullot, J., Lenoble, V., Arnaud, M., Raynaud, M., Mounier, S., (2011). Study of the spatial and historical distribution of

783 sediment inorganic contamination in the Toulon bay (France). Marine Pollution Bulletin, 62. https://doi.org/10.1016/j.marpolbul.2011.07.022

784 UNEP, 2020. TACKLING PLASTIC POLLUTION: Legislative Guide for the Regulation of Single-Use Plastic Products. 83p. ISBN No: 978-

785 92-807-3853-7.

786 UNEP, 2022. Resolution UNEP/EA.5/Res.14. End plastic pollution: towards an international legally binding instrument.

787 https://www.unep.org/news-and-stories/press-release/historic-day-campaign-beat-plastic-pollution-nations-commit-develop

788 Yamashita et al., (2021). Plastic additives and legacy persistent organic pollutants in the preen gland oil of seabirds sampled across the globe,

789 Environmental Monitoring and Contaminants Research, 2021, Volume 1, Pages 97-112, Released on J-STAGE October 11, 2021, Online

790 ISSN 2435-7685, https://doi.org/10.5985/emcr.20210009

791 Yang, Y., Liu, G., Song, W., Ye, C., Lin, H., Li, Z., Liu, W., (2019). Plastics in the marine environment are reservoirs for antibiotic and metal

792 resistance genes. Environmental International, 123, 79-86. https://doi.org/10.1016/j.envint.2018.11.061

793 Yu Y., Miao L., Adyel T.M., Waldschläger K., Wu J., Hou J. (2023) Aquatic plastisphere: interactions between plastics and biofilms. Env. Poll.

794 322, 121196.

795 Wang, L., Lin, J., Dong, C., Chen, C., Liu, T., (2021). The sorption of persistent organic pollutants in microplastics from the coastal

796 environment. Journal of Hazardous Materials, 420, 126658. https://doi.org/10.1016/j.jhazmat.2021.126658

797 Wang, J., Guo, X., Xue, J., (2021). Biofilm-Developed Microplastics As Vectors of Pollutants in Aquatic Environments. E

798 https://doi.org/10.1021/acs.est.1c04466

799 Wiesinger, H., Wang, Z., Hellweg, S., (2021). Deep Dive into Plastic Monomers, Additives, and Processing Aids. Environ. Sci. Technol. 2021,

800 55, 13, 9339–9351. https://doi.org/10.1021/acs.est.1c00976

801 Willsky, G. R., and Malamy, M. H. (1980). Effect of arsenate on inorganic phosphate transport in Escherichia coli. J. Bacteriol. 144, 366–374.

802 Zhang, C., Li, F., Xie, L., Zhang, Y., Mu, J., (2023). Polylactic acid (PLA), polyethylene terephthalate (PET), and polystyrene (PS)

803 microplastics differently affect the gut microbiota of marine medaka (Oryzias melastigma) after individual and combined exposure with

804 sulfamethazine. Aquatic toxicology, 259, 106522. https://doi.org/10.1016/j.aquatox.2023.106522

805 Zettler E.R., Mincer T.J., Amaral-Zettler L. (2013) Life in the “Plastisphere”: Microbial Communities on Plastic Marine Debris. Environmental

806 Science and technology 47, 13, 7137-7146. https://doi.org/10.1021/es401288x

807 Zhen, Y., Hamed, M., De-la-Torre, G., Frias, J., Jong, M., Kolandhasamy, P., Chavanich, S., Su, L., Deng, H., Zhao, W., Shi, H., (2023). Holes

808 on surfaces of the weathered plastic fragments from coastal beaches. Marine Pollution Bulletin, 193, 115180.

809 https://doi.org/10.1016/j.marpolbul.2023.115180
810
811
812

813 **Figure Captions**

814 Figure 1 - European *in-situ* cage deployment sites (map produced using ArcGIS Pro, Source: ESRI)

815

816 Figure 2 - Experimental design. a) *In-situ* immersion deployment, b) Digestion of plastisphere and ICP-MS

817 processing (image produced in Adobe Photoshop. Source: Biorender and Canva).

818 Figure 3 - Bioaccumulation (µg/L) of trace metals in the plastisphere. When required, Naples results are shown in

819 separate plots due to changes in bioaccumulation scale.

820 Figure 4 -Principal Component Analysis of the metals bioaccumulation as a function of season and polymer, in each

821 immersion site.

822 Figure 5: Risk quotients (RQ) for metal bioaccumulation at all immersion sites. The red dotted lines represent RQ

823 value of 1, above which the environment is considered to be at high risk (Nika et al., 2020).