# Toxicity assessment of DMSO extracts of environmental aged beached plastics using human cell lines

Florane Le Bihanic, Bettie Cormier, Emilie Dassié, Sophie Lecomte, Justine

Receveur, Stéphane Le Floch, Jérôme Cachot, Bénédicte Morin

► To cite this version:

Florane Le Bihanic, Bettie Cormier, Emilie Dassié, Sophie Lecomte, Justine Receveur, et al. Toxicity assessment of DMSO extracts of environmental aged beached plastics using human cell lines. Ecotoxicology and Environmental Safety, 2025, 289, pp.117604. 10.1016/j.ecoenv.2024.117604. hal-05038530

HAL Id: hal-05038530

https://hal.science/hal-05038530v1

Submitted on 17 Apr 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

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

# Toxicity assessment of DMSO extracts of environmental aged beached plastics using human cell lines

Florane Le Bihanic<sup>a, ●</sup>, Bettie Cormier<sup>a</sup>, Emilie Dassié<sup>a, ●</sup>, Sophie Lecomte<sup>c</sup>, Justine Receveur<sup>b</sup>, Stéphane Le Floch<sup>b</sup>, Jérôme Cachot<sup>a, ●</sup>, Bénédicte Morin<sup>a, *, ●</sup>

## ARTICLE INFO

Edited by Richard Handy

Keywords:

Environmental aged plastic

Additives

Cytotoxicity

Oxidative stress

Estrogenic effect

## ABSTRACT

Plastic products contain complex mixtures of chemical compounds that are incorporated into polymers to improve material properties. Besides the intentional chemical additives, other compounds including residual monomers and non-intentionnally added substances (NIAS) as well as sorbed pollutants are usually also present in aged plastic. Since most of these substances are only loosely bound to the polymer via non-covalently interactions, i.e., van der Waals forces, they may leach to the surrounding environment. Although there is increasing knowledge about toxicity of weathered plastic to aquatic organisms, only little is known about how plastic associated chemicals affect human health. Seafood consumption is one of the routes of human exposure to microplastics. The aim of this study was to evaluate the ability of naturally aged plastic associated chemicals to induce harmful effects to human health via the consumption of MP-contaminated seafood. Human colorectal adenocarcinoma Caco-2 and human hepatocyte carcinoma HepG2 cells were selected as model of the colon and liver cells respectively. They are known for their high capacity to metabolize organic contaminants. Both cell lines were exposed to DMSO extracts of different plastics to investigate the effects of chemicals on cell viability, oxidative stress induction and genotoxicity. In addition, the estrogenic effects of DMSO-extracts were evaluated using an estrogen-dependent reporter gene assay in T47D-Kbluc human breast cancer cells. Chemical profiles of the DMSO extracts were polymer-dependent, with polyvinyl chloride (PVC) highly contaminated with metals while polypropylene (PP) contained the lowest concentration of metals. Organic pollutants, including polycyclic aromatic hydrocarbons, were mainly found in PVC, high density polyethylene (HDPE) and PP extracts, whereas other extracted plastics had less (PP) to no organic contamination (polyethylene terephthalate PET). PVC was the most toxic plastic inducing cytotoxicity for both cell lines. DNA damage was observed for Caco-2 cells exposure to HDPE, PVC and nylon. Reactive oxygen species were induced only with nylon extracts in intestinal cells. No toxicity was observed for PP and PET and none of the tested plastics had any estrogenic effect. Our results demonstrate that some environmental aged plastic material released a variety of known and unknown chemical compounds some of which are toxic *in vitro* and contribute to the knowledge on adverse human health effects of plastics.

## 1. Introduction

Humans are exposed to plastic via ingestion, inhalation and dermal exposure (Bouwmeester et al., 2015; Gasperi et al., 2018; Prata et al., 2020). Ingestion is considered as the main entry point, with a predicted intake of 39–52 thousand microplastics per person annually (Galloway, 2015; Prata et al., 2020). Exposure to plastics can occurs from food,

drinking water, cigarettes, textile fibers and urban dust.

Plastic is a cheap material extremely polyvalent with very good properties of thermic or electric insulation. Plastics are composed of a polymer to whom chemical compounds are added to improve their physico-chemical characteristics and to reduce cost at the production and sell. There are different categories of additives including chemical compounds to limit degradation of plastics such as flame retardants

<footnote><sup>a</sup> Univ. Bordeaux, CNRS, Bordeaux INP, EPOC, UMR 5805, Pessac F-33600, France</footnote>

<footnote><sup>b</sup> CEDRE, Research Department, 715 rue Alain Colas, CS 41836, Cedex 2, Brest 29218, France</footnote>

<footnote><sup>c</sup> Univ. Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, Pessac F-33600, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: benedicte.morin@u-bordeaux.fr (B. Morin).</footnote>
(polybrominated diphenyl ethers PBDE, tetrabromobisphenol A TBBPA), antioxidants, stabilizers (organotin compounds), biocides. There also are functional additives such as antistatic agents, plasticizers (phthalates, alkylphenols), lubricants, foamers or hardeners; colorants (pigment); charge (minerals) or reinforcement agents (glass or carbon fiber) (Bouwmeester et al., 2015). Moreover, resins can contain chemicals considered as impurities during the plastic process such as monomers, residual solvents, catalyzers, etc. The concentration and nature of these chemical compounds vary a lot in the final product according to the plastic type, their uses and even with the producers.

To assess the toxicity of environmental microplastics, it is necessary to consider both the chemicals included during the plastic manufacturing as well as non-intentional products and the chemicals produced by polymers or additives oxidation and those that can adsorb to the plastic surface during aging in the environment. Indeed, plastic can act as a source of diffuse contamination of all chemicals accumulated. Heavy metals and persistent organic pollutants present in air, water and sediment can be adsorbed to atmospheric particles, water suspended particles, organic matter or sediments and can migrate to polymer surfaces. It includes organochloride pesticides (DDT, HCH) as well as polychlorinated biphenyls (PCBs), polycylic aromatic hydrocarbons (PAHs), polybrominated diphenylethers (PBDEs), or hexabromocyclododecane, per- and polyfluoroalkyl substances (PFAS). These plastic chemicals can be both already present in the environment and/or directly added to the polymer during processing. These compounds can be released into the environment at all stages of plastic life cycle: from plastic synthesis, the use, to the end of life after physicochemical degradation (Bouwmeester et al., 2015).

Most of the seafood and fish are contaminated with plastics (Van Cauwenberghe and Janssen, 2014; Danopoulos et al., 2020; Zitouni et al., 2020). Polymers, the most encountered in the seafood, are PET, PP, PE, PVC and nylon (Karami et al., 2018; Akoueson et al., 2020; Hantoro et al., 2019). Quantity of plastic particles ingested by human are still unclear as well as physical and chemical effects of plastics in the digestive system. It seems that retention time of plastics in organisms are quite short because particles are quickly excreted (Waring et al., 2018). However, it is important to consider that increasing exposure to plastic lead to increasing exposure to associated chemical contaminants. Plastic may represent therefore a threat for human health.

Toxic pathways of plastics can occur via translocation or chemical leaching, leading to developmental defects, endocrine disruption, disruption of energy and metabolism balance, disruption of the immune function and neurotoxicity (Prata et al., 2020). It is most likely that particles once entered a human organism, if translocated, will penetrate the intestinal barrier and will enter the intestinal tissue, and otherwise will be excreted. Particle could cause local injuries such as inflammation (Lu et al., 2016). However, the major damage to the organism might be from the toxicity of leachable plastic chemicals (Klein et al., 2021). Cytotoxicity and oxidative stress may be substantial toxic effect of plastic chemicals (Liu et al., 2020; Hosseinzadeh et al., 2024).

Most of the plastic chemicals are unknown. We know how to evaluate the toxicity of a single molecule but it can be problematic when it comes to a plastic considering the large cocktail of chemicals that compose it, including the degradation products. Moreover, the majority of the scientific literature study effects of industrial pristine plastic, which can be weakly representative of the environmental exposure. Therefore, in this study we focused on the chemical effects of naturally aged environmental plastics collected on the North Atlantic French coast. Chemical compounds associated to these plastics were extracted from the plastics using the organic solvent dimethyl sulfoxide (DMSO). Two human cell lines were exposed to the DMSO extracts to investigate the effects of plastic chemicals on cell viability, oxidative stress induction and genotoxicity. The human epithelial colorectal adenocarcinoma cells Caco-2 have been widely used as a model of the intestinal epithelial barrier (Lea, 2015). The human hepatocyte carcinoma cells HepG2 were selected for their suitability to model liver toxicity including a high

ability to metabolize organic contaminants. In addition, the estrogenic effect of DMSO-extracts was evaluated using an estrogen-dependent reporter gene assay in T47D-Kbluc breast cancer cells. Additionally, some plastic chemicals and pollutants putatively present in the DMSO extracts were analyzed.

## 2. Material and methods

### 2.1. Plastic debris

Old plastic items that appeared well weathered in the environment were collected at La Salie beach (44.528766, -1.256533, North Atlantic French coast, France) on November 12th 2019 (Table 1). Polymer composition of each plastic debris was determined by Attenuated Total Reflection Fourier Transform InfraRed (ATR-FTIR) spectroscopy using a Nicolet Nexus 870 equipped with diamond crystal. 100 scans were added, the spectral resolution was 4 cm⁻¹. The recorded spectra were compared to reference spectra through libraries supplied by Thermost Fisher, with a > 75 % similarity threshold (Supplementary Table S1). Plastic debris from the same polymer where mixed in 50/50 ratio (g/g) i. e. oyster cup and fishing line in PP and hard plastic container and fishing line in HDPE.

### 2.2. DMSO plastic extracts

Plastic sample were cut with clean stainless-steel scissors to obtain section smaller than 1 cm² and disposed in 8 mL amber vials. One g of plastic was extracted with 2 mL of dimethyl sulfoxide DMSO (1:2 w/v) (Biomolecular grade, Sigma-Aldrich). After 16 h shaking (175 rpm, 20 °C), DMSO extracts were sampled and kept at -20 °C prior toxicological analysis. Each plastic extraction was replicated three times for each corresponding replicated experiment.

### 2.3. Cell lines

HepG2 and Caco-2 cells (ATCC, USA) were cultured in minimum essential medium completed with 10 % and 20 % fetal bovine serum respectively, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 100 units/mL penicillin and 0.1 mg/mL streptomycin, and maintained at 37 °C in 5 % CO₂. Cells were seeded at a concentration of 2 × 10⁵ cell/ mL in 96-well, 48-well or 24-well plates and cultured for 48 h for all assays. Doubling time was verified prior performing the experiments in the test conditions and calculated 60 h for HepG-2 and 45 h for CaCo-2 cells approximately. Therefore sub-culture time was elongated to 48 h (data not shown). For cytotoxicity assay, cell lines were exposed to a serial of dilutions from 0.01 % to 1 % of plastic DMSO extracts (corresponding to 0.05 mg/mL and 5 mg/mL respectively). For the comet and the reactive oxygen species (ROS) assays, cell lines were only exposed to the highest non-cytotoxic dilution (0.3 %). A solvent control corresponding to the maximum DMSO concentration used (1 %) and a negative control (cell medium only) were tested in each microplate.

T47D-Kbluc cells were cultured in RPMI growth media supplemented with 10 % fetal bovine serum and maintained at 37 °C in 5 % CO₂. Cells were seeded at concentration of 10 × 10⁵ cell/mL in 96-well microplate. Cells were exposed to a serial of dilutions from 0.01 % to 1 %

<table><caption>**Table 1**
Aging plastic materials collected at La Salie beach (Biscay Bay, France) in 2019.</caption><thead><tr><td>Item</td><td>Polymer type</td><td>Reference spectrum matching (%)</td></tr></thead><tbody><tr><td>Juice bottle</td><td>PET</td><td>84</td></tr><tr><td>Oyster cup</td><td>PP</td><td>95</td></tr><tr><td>Hard plastic container</td><td>PVC</td><td>75</td></tr><tr><td>Hard plastic container</td><td>HDPE</td><td>98</td></tr><tr><td>Fishing line</td><td>Nylon</td><td>88</td></tr><tr><td>Trawl net</td><td>HDPE</td><td>85</td></tr><tr><td>Rope</td><td>PP</td><td>89</td></tr></tbody></table>
of plastic DMSO extracts. As for HepG2 and Caco-2, two different control treatments were used and consisted in medium only and DMSO 1 %.

## 2.4. Cytotoxicity

The cytotoxicity of the DMSO extracts was measured by fluorescence using the Alamar Blue reduction assay. After 24 h exposure in 96-well plate, cells were incubated during 2 h at 37 °C in a 10 % Alamar Blue solution (Invitrogen). The rate of increase of resorufin fluorescence was measured at 570 nm excitation and 590 nm emission with a Fluostar optima (BMG LABTECH) microplate reader. Results were expressed compared to the control medium only treatment.

## 2.5. ROS assay

Ability of the DMSO extracts to induce the production of ROS was measured using the fluorometric probe chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H2DCFDA, Molecular Probes TM, Life Technologies) (Pannetier et al., 2018). After 48 h seeding in 48-well microplate, cells were rinsed with HBSS buffer and incubated 20 min at 37 °C with 125 µL of 20 µM CM-H2DCFDA dissolved in DMSO and HBSS. Then cells were rinsed twice with white culture medium without phenol red. Thereafter, cells were exposed during 4 h at 37 °C to a sub-lethal concentration of 0.3 % (v/v) of plastic extracts or to 1 µM H₂O₂ as a positive control. Medium fluorescence was measured at 485/520 nm (exc./em.). Results were expressed in percentage of ROS production over a 240 min period.

## 2.6. Comet assay

The genotoxicity of the DMSO extract was measured by the comet assay (Singh et al., 1988; Pannetier et al., 2018, 2019). Cells were seed for 48 h in 24-well microplate and then exposed for 24 h. Briefly, after 40 min of DNA unwinding, electrophoresis was performed at 25 V, 300 mA, during 20 min. Slides were prepared and stained with 20 µL of ethidium bromide (BET, 20 µg/mL) and then read at x 200 with an epifluorescence microscope (Olympus BX51) coupled to the Comet assay IV software (Instrument Perspective Ltd). For each sample, 100 random nuclei were scored over 2 gels. DNA damage are expressed as percentage of DNA in the comet tail that represent the percentage of damaged DNA that have migrated from the head of the comet, the original nucleus.

## 2.7. Estrogenic activity

The estrogenic activity was measured by using T47D-Kbluc cells, naturally expressing Erα and Erβ and are transfected with an estrogen-responsive element promoter-luciferase reporter gene construct. Stock cells were maintained at 37 °C in 5 % CO₂ and cells were seeded 48 h in 96-well plate with withdrawal RPMI with media replacement at 24 h. Cells were then exposed to DMSO plastic extracts for 24 h. Luciferase activity was determined using a Fluostar optima (BMG LABTECH) microplate reader. Results are expressed compared to the control medium only treatment.

## 2.8. Chemical analysis

### 2.8.1. Organic contaminants

24 PAH, among which the 16 listed as priority pollutants by US EPA, 14 PCB, 29 pesticides and 7 PBDE were analyzed in DMSO extracts. Extracts were spiked with internal standards (perdeuterated PAHs). Analyses were performed by stir bar sorptive extraction-thermal desorption-gas chromatography-tandem mass spectrometry (SBSE-GCMS/MS) as described in (Lacroix et al., 2014). Briefly, each sample was extracted for 16 hours at 750 rpm using polydimethylsiloxane stir-bars (Twister 20 mm×0.5 mm, Gerstel). Bars were subsequently analyzed using a gas chromatography system Agilent 7890 A coupled to an

Agilent 7000 triple quadripole mass spectrometer (Agilent Technologies) and equipped with a Thermal Desorption Unit (TDU) combined with a Cooled Injection System (Gerstel). The interface temperature was 300°C. The GC temperature gradient was: from 50 °C (1 min) to 320°C (20 min) at 3 °C/min. The carrier gas was Helium at a constant flow of 1 mL/min. The capillary column used was a RXi 5-ms (Restek, Bellefonte, USA): 30 m x 0.25 mm ID x 0.25 µm film thickness. The quadripole was used in the Electronic Impact mode (Electronic Impact: 70 eV. voltage: 2000 V). Limits of quantification (LOQ) were calculated by the calibration curve method (Shrivastava, 2011) and detection limits were estimated by dividing quantification limits by 3.

### 2.8.2. Metal analysis

Prior to the analyses, 100 mL of concentrated DMSO extracts was diluted with 2000 mL of 1 % nitric acid, filtered with a 0.20 mm filter and stored into quartz tubes. Fifteen metals were analyzed simultaneously by Inductively Coupled Plasma Optical Emission Spectrometer (700 Series ICP-OES, Agilent), among them only four presented values above the limit of quantification at 10 % (Table 2): Aluminum (Al), Iron (Fe), Manganese (Mn), and Zinc (Zn). The validity of our analytical method has been verified by the analysis of a homemade quality control solution. For the duration of the analyses, the average yield of this quality control was 95.36 %, for N = 8 analyses. Sample results were expressed as blank-corrected concentrations in mg/L.

## 2.9. Statistics

Each experiment was repeated 3 times independently for each cell line. Significative differences between treatments and the solvent control were tested using the GraphPad PRISM software. Normality (Shapiro-Wilk test) and variance equality (Bartlett test) were checked. Then statistical analysis was realized using the parametric ANOVA test followed by a Dunett post-hoc test with $p < 0.05$.

# 3. Results

## 3.1. Plastic extracts toxicity on human cell lines

### 3.1.1. Cytotoxicity

Cytotoxicity assay has been validated with a cellular viability significantly decreased to 44.3 ± 12.7 % (Hep-G2) and 49.6 ± 13.0 % (Caco-2) after exposure to the positive control DMSO 5 % (v/v). No difference was noted between the negative (cell medium) and the solvent (1 % DMSO) controls. No cytotoxicity was observed for the DMSO extracts from HDPE, nylon, PP or PET at the tested concentrations. The highest tested PVC extract concentration of 1 % (v/v) decreased significantly cell viability of both cell lines to 35.6 ± 12.2 % (Hep-G2) and 57.6 ± 21.0 % (Caco-2) (Fig. 1).

### 3.1.2. Oxidative stress

Oxidative stress measurement assay was validated with a positive control containing 1 µM of H₂O₂ that induced a 6-fold and a 5-fold ROS production for HepG2 and Caco-2 respectively compared to negative controls (data not shown). Cells exposed to the DMSO extracts from HDPE, PVC, PP and PET did not induce a significant production of ROS. However, nylon extract increased ROS production in Caco-2 cells up to 1.32 ± 0.12 fold (Fig. 2).

### 3.1.3. Genotoxicity

Genotoxicity of plastic extracts was assessed using the comet assay. The assay was validated with a positive control (1 µM of H₂O₂) that induced 80.5 ± 13.1 % of Tail DNA damage in HepG2 cells and 77.4 ± 1.7 % in Caco-2 cells (data not shown). No genotoxicity was observed on HepG2 cells whatever the plastic extracts considered. HDPE, PVC and nylon but not PP and PET extracts induced genotoxicity on Caco-2 cells (Fig. 3).
<table><caption>**Table 2**
ICP-OES limit of quantification (LQ) for the day of the analyze in ppb (µg/L).</caption><thead><tr><th></th><th>Ag</th><th>Al</th><th>As</th><th>Cd</th><th>Co</th><th>Cr</th><th>Cu</th><th>Fe</th></tr></thead><tbody><tr><td>LQ (ppb)</td><td>22.0481</td><td>104.53</td><td>1034.97</td><td>22.1589</td><td>22.0259</td><td>103.375</td><td>104.843</td><td>104.843</td></tr><tr><td>Mn</td><td></td><td>Mo</td><td>Ni</td><td>Pb</td><td>Se</td><td>V</td><td>Zn</td><td></td></tr><tr><td>LQ (ppb)</td><td>22.0702</td><td>104.843</td><td>104.321</td><td>104.423</td><td>290.121</td><td>22.1589</td><td>22.1145</td><td></td></tr></tbody></table>

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Cytotoxicity of DMSO plastic extracts to Caco-2 (blue) and HepG2 (red) cells (% compared to control treatment mean ± SD, n = 3–8, Anova). Different letters indicate significant differences with p < 0.05.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Oxidative stress induction by DMSO plastic extracts at 0.3 % v/v concentration (% compared to control treatment, mean ± SD, N = 3, Anova (p < 0.05), asterisks refer to significant differences to the control DMSO treatment ** p < 0.01).</figcaption></figure>

### 3.1.4. Estrogenic activity

Estrogenic activity assay was validated with a positive control using 0.6 µM bisphenol-A that induced a 20-fold activity compared to solvent control. Cells exposed to the DMSO extracts from all tested plastics did not induce a significant estrogenic activity (Fig. 4).

### 3.2. Chemical analysis

Chemical analysis of DMSO extracts revealed the presence of both organic and metallic compounds. Analysis of PAHs established that PVC

was the highest contaminated plastic with various PAHs (total 357.1 ng of PAHs per g of plastic), mainly low molecular weight PAHs, up to 93.1 ± 13.4 ng.g⁻¹ of naphthalene and 72 ± 24.2 ng.g⁻¹ of phenanthrene. Hexachlorobenzene was found in HDPE (5.9 ± 2.2 ng.g⁻¹) and at a lower concentration in PVC (0.5 ± 0.3 ng.g⁻¹), while PP contained DDT, DDE and various PCBs (Table 3). The concentration of total PBDEs was under quantification limits for all of the samples. No organic pollutant was detected in PET. Table 4 shows the release of inorganic elements from collected plastics. Results showed that zinc is the metal present in greater quantities in the plastic extracts. In particular, the
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** DNA damage induction by DMSO plastic extracts at 0.3 %v/v concentration (mean ± SD, n = 3, Anova, asterisks refer to significant differences to the solvent control: \* p < 0.05; \*\* p < 0.01).</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Estrogenic activity (T47D-KBluc assay) of DMSO plastic extracts (% compared to control treatment, mean ± SD, N = 3, Anova, p < 0.05). Standard curve is also represented in the last graphic panel (right bottom) for concentration of E2 varying from 0 to 30 pM per well (mean ± SD, n = 3, Anova (p < 0.05), asterisks refer to significant differences to the control DMSO treatment: \* p < 0.05; \*\* p < 0.01; \*\*\* p < 0.001).</figcaption></figure>

highest Zn concentration was recorded in PVC extract with 4880.8 ± 1973.3 ng.g⁻¹. Considering the other metals, aluminum, iron, manganese and vanadium were also present in high concentrations in PVC (Table 4).

## 4. Discussion

In this study, the toxicity on human cells of chemicals associated with environmental aged plastics PVC, PET, PP, HDPE and nylon was assessed in terms of cytotoxicity, oxidative stress, genotoxicity and estrogenic activity. The mixture of chemicals was extracted with DMSO solvent and exposed to three different human cell lines. In the absence of

epidemiological evidence, human cell lines are one of the currently available sources of scientific evidence for human health effects, the other being animal in vivo studies (Danopoulos et al., 2022). However today, there are a number of economic, scientific and ethical reasons for supporting efforts to develop and apply *in vitro* assays in aquatic ecotoxicology as alternative tools to animal testing (Castaño et al., 2003). These assays aim to quickly obtain reliable data on the toxic and eco-toxic properties of chemicals, while considering ethical concerns about animal testing. *In vitro* models are usually not suitable for chronic studies because the duration of exposure is limited. To the best of our knowledge, only one study dealing with long term effect of plastic particles on human cultured cells (Caco-2) has been published (Domenech et al.,
<caption>**Table 3**</caption>

Organic compounds contents in the DMSO extracts of plastics (ng/g of plastic) of each plastic type, including blank (DMSO). Mean ± SD (n = 3), LD: limit of detection; LQ: limit of quantification; nc: non-calculable.

<table><thead><tr><th rowspan="2"></th><th rowspan="2">Compound</th><th rowspan="2">LD</th><th rowspan="2">LQ</th><th colspan="2">Blank</th><th colspan="2">HDPE</th><th colspan="2">PVC</th><th colspan="2">Nylon</th><th colspan="2">PP</th><th colspan="2">PET</th></tr><tr><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th></tr></thead><tbody><tr><td rowspan="20">PAHs</td><td>naphthalene</td><td>0,6</td><td>2,0</td><td>&lt;LD</td><td>nc</td><td>4,6</td><td>1,3</td><td>93,1</td><td>13,4</td><td>5,6</td><td>nc</td><td>4,1</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>1-methylnaphthalene</td><td>0,6</td><td>2,0</td><td>&lt;LD</td><td>nc</td><td>3,9</td><td>nc</td><td>14,0</td><td>4,6</td><td>&lt;LQ</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>2-methylnaphthalene</td><td>0,6</td><td>2,0</td><td>&lt;LQ</td><td>nc</td><td>7,5</td><td>nc</td><td>13,8</td><td>5,0</td><td>3,9</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td></tr><tr><td>benzothiophene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>0,7</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>biphenyl</td><td>0,6</td><td>2,0</td><td>&lt;LD</td><td>nc</td><td>4,9</td><td>1,2</td><td>49,6</td><td>13,0</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>acenaphthylene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>19,6</td><td>6,1</td><td>&lt;LQ</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>acenaphthene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>2,6</td><td>0,1</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>fluorene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>23,0</td><td>6,8</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>dibenzothiophene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>0,8</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>Phenanthrene</td><td>0,6</td><td>2,0</td><td>&lt;LD</td><td>nc</td><td>6,7</td><td>3,1</td><td>72,0</td><td>24,2</td><td>4,9</td><td>0,5</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>anthracene</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>0,7</td><td>nc</td><td>10,4</td><td>5,1</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>fluoranthene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>5,3</td><td>4,0</td><td>23,4</td><td>13,3</td><td>4,1</td><td>0,8</td><td>1,0</td><td>0,5</td><td>&lt;LD</td><td>nc</td></tr><tr><td>pyrene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>6,5</td><td>4,6</td><td>19,0</td><td>10,8</td><td>2,8</td><td>0,3</td><td>1,3</td><td>0,4</td><td>&lt;LD</td><td>nc</td></tr><tr><td>2-methylfluoranthene</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>1,0</td><td>nc</td><td>1,2</td><td>0,6</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>benzo[a]anthracene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>5,0</td><td>2,9</td><td>3,0</td><td>1,7</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>chrysene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>11,8</td><td>6,8</td><td>5,7</td><td>2,6</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>benzo[b]fluoranthene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>7,8</td><td>4,0</td><td>1,2</td><td>nc</td><td>1,9</td><td>0,6</td><td>0,6</td><td>0,3</td><td>&lt;LD</td><td>nc</td></tr><tr><td>benzo[k]fluoranthene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>10,3</td><td>5,1</td><td>1,8</td><td>nc</td><td>1,7</td><td>0,4</td><td>0,7</td><td>0,1</td><td>&lt;LD</td><td>nc</td></tr><tr><td>Benzo[e]pyrene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>2,1</td><td>1,8</td><td>1,1</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>benzo[a]pyrene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>6,2</td><td>3,6</td><td>1,3</td><td>nc</td><td>0,7</td><td>0,1</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>perylene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>1,1</td><td>0,6</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>Indeno[1,2,3-cd]pyrene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>4,4</td><td>2,8</td><td>&lt;LD</td><td>nc</td><td>1,2</td><td>0,6</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>Dibenzo[a,h]anthracene</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>1,3</td><td>0,9</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>Benzo[ghi]péryléne</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>3,7</td><td>3,3</td><td>&lt;LQ</td><td>nc</td><td>1,3</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td><strong>Total (PAHs)</strong></td><td></td><td></td><td>0,0</td><td>0,00</td><td>-</td><td>94,9</td><td>357,1</td><td>28,0</td><td>7,6</td><td>0,0</td><td>0,0</td><td>nc</td><td>nc</td><td>nc</td></tr><tr><td rowspan="20">PCBs</td><td>PCB-7</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>PCB 28</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>PCB-52</td><td>0,0</td><td>0,0</td><td>&lt;LD</td><td>nc</td><td>0,1</td><td>0,0</td><td>&lt;LD</td><td>nc</td><td>0,1</td><td>0,0</td><td>0,2</td><td>0,1</td><td>&lt;LD</td><td>nc</td></tr><tr><td>PCB-35</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>PCB 101</td><td>0,1</td><td>0,2</td><td>&lt;LQ</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>1,0</td><td>0,3</td><td>&lt;LD</td><td>nc</td></tr><tr><td>PCB 77</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>0,5</td><td>0,1</td><td>&lt;LD</td><td>nc</td></tr><tr><td>PCB 135</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>PCB 118</td><td>0,1</td><td>0,2</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>1,6</td><td>0,5</td><td>&lt;LD</td><td>nc</td></tr><tr><td>PCB 153</td><td>0,0</td><td>0,0</td><td>&lt;LD</td><td>nc</td><td>0,1</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>6,9</td><td>1,6</td><td>&lt;LD</td><td>nc</td></tr><tr><td>PCB 105</td><td>0,1</td><td>0,2</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>0,3</td><td>0,1</td><td>&lt;LD</td><td>nc</td></tr><tr><td>PCB 138</td><td>0,1</td><td>0,2</td><td>&lt;LQ</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>5,7</td><td>1,2</td><td>&lt;LD</td><td>nc</td></tr><tr><td>PCB 156</td><td>0,1</td><td>0,2</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>PCB 180</td><td>0,0</td><td>0,0</td><td>&lt;LQ</td><td>nc</td><td>0,1</td><td>nc</td><td>0,1</td><td>0,0</td><td>&lt;LQ</td><td>nc</td><td>2,4</td><td>0,4</td><td>&lt;LQ</td><td>nc</td></tr><tr><td>PCB 169</td><td>0,1</td><td>0,2</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td><strong>Total (PCBs)</strong></td><td></td><td></td><td>0,00</td><td>-</td><td>0,3</td><td>-</td><td>0,1</td><td>nc</td><td>0,1</td><td>18.6</td><td>0,0</td><td>nc</td><td>nc</td><td>nc</td></tr><tr><td rowspan="20">Pesticides</td><td>Alpha-BHC</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>hexachlorobenzene</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>5,9</td><td>2,2</td><td>0,5</td><td>0,3</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>atrazine</td><td>0,6</td><td>2,0</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>Beta-BHC</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>gama bhc</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>Delta-BHC</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>acetochlore</td><td>0,1</td><td>0,2</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>methylparathion</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>alachlore</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>aldrine</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>metolachlore</td><td>0,6</td><td>2,0</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>chlorpyrifos</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>ethylparathion</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>isodrine</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>metazachlore</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>pendimethalin</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>chlorfenvinphos</td><td>0,6</td><td>2,0</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>2-4-dde</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>endosulfan alpha</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>4-4-dde</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>dieldrine</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>2-4-ddd</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>endrine</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>endosulfan beta</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>4-4ddd</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>0,4</td><td>0,2</td><td>&lt;LD</td><td>nc</td></tr><tr><td>2,4-ddt</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LQ</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>endosulfan sulfate</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>4-4ddt</td><td>0,1</td><td>0,2</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>0,7</td><td>0,6</td><td>&lt;LD</td><td>nc</td></tr><tr><td><strong>Total (Pesticides)</strong></td><td></td><td></td><td>0,0</td><td>-</td><td>5,9</td><td>0,5</td><td>0,0</td><td>nc</td><td>10,1</td><td>0,0</td><td>nc</td><td>nc</td><td>nc</td><td>nc</td></tr><tr><td rowspan="3">PBDEs</td><td>BDE 28</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>BDE 47</td><td>0,6</td><td>2,0</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>BDE 100</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 3 (continued)**</caption><thead><tr><th rowspan="2">Compound</th><th rowspan="2">LD</th><th rowspan="2">LQ</th><th colspan="2">Blank</th><th colspan="2">HDPE</th><th colspan="2">PVC</th><th colspan="2">Nylon</th><th colspan="2">PP</th><th colspan="2">PET</th></tr><tr><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th></tr></thead><tbody><tr><td>BDE 99</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>BDE 154</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>BDE 153</td><td>0,1</td><td>0,4</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>BDE 183</td><td>0,6</td><td>2,0</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td><td>&lt;LD</td><td>nc</td></tr><tr><td>Total (PBDEs)</td><td></td><td></td><td>0,0</td><td></td><td>0,0</td><td></td><td>0,0</td><td></td><td>0,0</td><td></td><td>0,0</td><td></td><td>0,0</td><td></td></tr></tbody></table>

<caption>**Table 4**</caption>

Blank-corrected concentrations of inorganic elements measured in the DMSO extracts of plastics (ng/g of plastic). NS: Not significantly different from the blank.

<table><thead><tr><th></th><th>HDPE<sup>a</sup></th><th>PVC</th><th>Nylon</th><th>PP</th><th>PET</th></tr></thead><tbody><tr><td>Al</td><td>54.6<br/>± 209.2</td><td>1606.9<br/>± 394.1</td><td>128.8<br/>± 449.8</td><td>357.7<br/>± 70.8</td><td>708.5<br/>± 189.0</td></tr><tr><td>Fe</td><td>83.0 ± 0.8</td><td>845.6 ± 54.0</td><td>234.6<br/>± 75.3</td><td>77.3<br/>± 81.3</td><td>67.0<br/>± 21.7</td></tr><tr><td>Mn</td><td>55.9 ± 17.6</td><td>135.7 ± 20.5</td><td>368.0<br/>± 95.7</td><td>98.5<br/>± 46.8</td><td>36.0<br/>± 28.9</td></tr><tr><td>V</td><td>NS</td><td>NS</td><td>167.5<br/>± 82.5</td><td>NS</td><td>1.1 ± 5.3</td></tr><tr><td>Zn</td><td>1494.5<br/>± 279.3</td><td>4880.8<br/>± 1973.3</td><td>2120.2<br/>± 285.8</td><td>148.0<br/>± 85.8</td><td>965.0<br/>± 202.5</td></tr></tbody></table>

2021). Human Caco-2 and HepG2 cell lines were used in the current study, because ingestion is one of the main routes of MPs intake in humans. Numerous studies in mammalian cancerous cell lines reported modified cell cycle, reduced DNA repair capacities and pathway-specific differences compared to non-cancerous ones (Windhorfer et al., 2008). However, HepG2 cells are often used as a model system for studies of liver metabolism and toxicity of xenobiotics and Caco-2 cells is also a widely used and well-established *in vitro* model of the human intestinal absorptive epithelium for toxicological studies. Those two cell lines express most metabolic enzymes such as cytochrome P450 (CYP) enzymes (Sergent at al., 2009) and are of interest for substances requesting metabolic activation such as those detected in DMSO extracts (PAHs, PCBs, PBDEs.). In addition, the estrogenic effects of DMSO-extracts were evaluated using an estrogen-dependent reporter gene assay in T47D-Kbluc human breast cancer cells.

Although organic extracts do not correspond with the real process of chemicals release in the environment, they present a convenient way to assess “worst-scenario” exposure. The DMSO extraction performed in this study was used to mimic the potential desorption of microplastic-associated pollutants in the intestinal tract following ingestion. We previously showed that soft DMSO extraction was an efficient way to extract both organic and metallic compounds and to assess the toxic effects of pollutants sorbed at the surface of environmental microplastics in fish cell lines (Pannetier et al., 2019a) and Japanese medaka early life stages (Pannetier et al., 2019b). In this study, targeted analysis was performed for metals by ICP-OES and organic chemicals by GC/MS/MS. Environmental plastics contain a wide variety of chemicals including metal and organic additive compounds and non-intentionally added substances (NIAS). These NIAS are, impurities of the starting substances (monomers, catalysts, solvents, etc) and additives as well as intermediates and reaction and breakdown products during processing or weathering. Furthermore, plastics have the capacity to sorb environmental pollutants such as heavy metals and highly hydrophobic contaminants on their surface. Most of these chemicals are bound to the polymer only by weak van der Waals forces and then can leach during weathering into the surrounding environment. A vast and heterogeneous array of compounds could therefore be present in various quantity, depending on the life cycle stage of the material. Some of these chemicals have been identified as hazardous substances and may affect

human health. The heterogeneity of plastic and additives properties are reflected in our results, which showed that the toxicity of extracts to human cells is variable and material-dependent.

PVC was the most toxic tested polymer, inducing acute toxicity on both liver and colon cell lines and genotoxic effects on Caco-2 cells. Previous studies have shown that PVC product extracts are more toxic than other plastic products in aquatic organisms, such as marine harpacticoid copepods (Bejgarn et al., 2015), barnacle larvae (Li et al., 2016) or sea urchin larvae (Paganos et al., 2023). Methanol extract from PVC but not aqueous leachate induced acute toxicity in *Daphnia magna* (Zimmermann et al., 2020). These authors demonstrated that plastic chemicals were the main driver of PVC microplastic toxicity. A very recent study showed the leachate from 1 g/mL PVC dry blend did not cause any significant change in three different rainbow trout cell lines viability but increased ROS generation in both cell lines (Boháčková et al., 2023). These observations are not surprising, given the large quantities of additives used in PVC composition compared to other polymers (Groh et al., 2019; Hahladakis et al., 2018). In particular, phthalates are used in PVC as plasticizers to achieve greater product flexibility. Phthalates, bisphenol A and PBDEs are known to be endocrine-disruptive chemicals (Meeker et al., 2009). We were unable to identify phthalates in our extracts, as their analysis is often complicated by high contamination of laboratory blanks. PBDEs were not detected in PVC extracts or any other polymers. As no estrogenic activity was observed in T47D-Kbluc cells, it is possible that this PVC aged in the environment has lost endocrine-disruptive chemicals which, like many plastic additives, are non-covalently bound with the polymer matrix and are therefore easily leached into aquatic environments. Indeed, a recent study showed that PVC microbeads incubated for three months in the marine environment lost a third of their phthalates (Pedà et al., 2022). Nevertheless, different results were reported for plastic products made in PVC (Zimmermann et al., 2021). Consumer products made from PVC were identified as one of the most potent plastics that induced baseline toxicity, oxidative stress, estrogenic as well as antiestrogenic and antiandrogenic activities (Zimmermann et al., 2019; Klein et al., 2021). In total, four metallic elements were detected in the PVC extracts. The highest concentrations were found for zinc followed by high concentrations of aluminum and iron which confirm earlier findings reporting zinc as the most abundant trace metal in PVC (Cormier et al., 2021; Peters et al., 2022). The toxic effects observed in our study could be associated with the high concentration of zinc in PVC extracts. Zn is an additive used in a variety of plastics as slip agents (stearate), fillers and pigments (Hahladakis et al., 2018), heat stabilizer and antioxidant (Lithner et al., 2012). This compound, is cytotoxic in zebrafish (Ajitha et al., 2021) and carp gill cell lines (Taju et al., 2013) at 0.16 µg/mL and 15 µg/mL respectively. Zinc is able to induce oxidative stress and ROS production in different animal species and cell lines (Ajitha et al., 2021; Valko et al., 2005). The high zinc concentrations measured in PVC, nylon and HDPE extracts (respectively 4880.8 ± 1973.3, 2120.2 ± 285.8 and 1494.5 ± 279.3 ng Zn/g plastic) may explain ROS production and/or induction of genotoxicity in CaCO-2 cells. It has been shown that PVC litter may be particularly problematic due to its potential to leach zinc over a considerable period of ageing, continuing to maintain toxicity to ecosystems (Sarker et al., 2020). PAHs were more abundant in weathered PVC extracts than in other plastic items with total PAH concentrations of 357 ng.g⁻¹ and between 0 and 93 ng.g⁻¹ for

<footnote><sup>a</sup> n = 2</footnote>
individual PAHs. PAHs can have multiple sources because they can originate from the plastic as well as other sources such as environmental contamination or biofilms. Our PVC samples were moderately contaminated with PAHs and PCBs compared to previous reported concentrations. For instance, PAHs concentrations of the 16 EPA-PAHs determined on beached pellets, sampled along the Belgian coastline, varied between 1076 and 1664 ng.g⁻¹ (Gauquie et al., 2015), contrary to other studies in which the level of PAHs ranged from 2 ng.g⁻¹ to 70 ng.g⁻¹ (Pannetier et al., 2019).

Although polyamide (PA) or nylon represents about 44.7 % of polymers discharged in the marine environment (Hamidian et al., 2021) and is abundantly found in aquatic biota, both marine and freshwater (Cesarini et al., 2023; Corami et al., 2022; Salazar-Pérez et al., 2021), knowledge is largely lacking regarding the toxic effects of polyamides especially when considering toxicity of weathered plastics. PA along with PVC is classified as hazardous by the European Chemical Agency with warning or danger signals (Boskovic et al., 2023). Therefore, the few available reports investigating the toxicity of virgin or artificially weathered polyamide particles are not unanimous. Khosrovyan and Kahru (2022) reported no adverse effects of either virgin or UV-weathered polyamide MP on the survival and reproduction of *D. Magna*, nor induction of oxidative stress or an altered growth rate with Gilt-head seabream *Sparus aurata* exposed to increased concentration of PA (Jovanovic et al., 2018). In contrast, Hasan et al. (2023) found changes in hemato-biochemicals parameters, increase of erythrocytic abnormalities and histological damage of gills and intestine in Nile tilapia exposed to virgin PA microparticles. Similarly, intestine tissue lesions were observed in zebrafish *Danio rerio* after 10 days of exposure to 1 mg/L water of different MPs including PA (Lei et al., 2018). To the best of our knowledge, no toxicity evaluation of leachates or extracts obtained from environmentally aged nylon has been reported to date. In our study, nylon was the second most toxic plastic extract, inducing both increase of ROS production and DNA damage in Caco-2 cell line. As already discussed for PVC, the high Zn concentration found in PA extracts can partly explain the oxidative stress response in intestine cells. In addition, higher concentrations of manganese (368.0 ± 95.7 ng.g⁻¹) were found in nylon compared to the other studied polymers. Indeed, Mn can interfere with the mitochondrial electron transport chain, leading to a disruption of the inner mitochondrial membrane potential, reducing intracellular ATP production and elevating ROS production (Smith et al., 2017). PAHs were also detected in the chemical extracts of PA but at a low concentration of 28 ng/g. PAHs can activate aryl-hydrocarbon receptor (AhR) that can lead to the formation of reactive metabolites which can generate ROS and DNA lesions such as strand breaks (Wessel et al., 2010). A very recent study investigated the toxicity of virgin and reused polyamide microplastics on human peripheral blood monocyte cell line THP-1 (Alijagic et al., 2024). Neither particles at 100 µg/mL nor extracted chemicals were able to induce an acute inflammatory response with regards to cytokine release. Polyamide particles were able to activate p53, as a marker of genotoxic response. However, chemical leaching did not contribute to p53 activation nor estrogenic activity. There are still numerous unanswered questions regarding the toxicity mechanisms and health effects of polyamide MPs.

HDPE extracts did not affect Caco-2 or HepG2 cells viability and did not induce oxidative stress. Exposure to extracts increased genotoxicity in Caco-2 cells but no effects were observed in HepG2 cells. Zimmermann et al. (2021) reported that different type of plastics, including PE, release chemicals that induce *in vitro* toxicity, oxidative stress and endocrine activity. Similarly, chemicals extracted from PE with methanol caused adverse effects to the human colorectal adenocarcinoma Caco-2 and HT-29 cells (Herrala et al., 2023). However, in these studies, plastic products were newly purchased materials while plastic items used in our study have been weathered in real marine environment. One can assume that leachates from plastics weathered in the natural environment are less toxic because additives have been depleted compared

to new plastic. Compared to other studies, PAHs are present in none negligible quantities in HDPE extracts (total 95 ng.g⁻¹) (Pannetier et al., 2019). PE from marine debris is known to sorb greater amounts of PAHs than other plastics including PP and PVC (Rochman et al., 2013). But the assumption that the chemicals from the environment sorbed to plastics contribute to leachate toxicity is not verified here. Hexachlorobenzene is a legacy contaminant that has been banned since 1981. It can be classified as NIAS and was used as antimicrobials and pesticides products. This persistent insecticide as well as PCBs and DDTs may have been adsorbed to PP and HDPE during their time in the marine environment (Pedà et al., 2022).

No toxic effects were observed for PP and PET extracts. The absence of toxicity of these leachates are consistent with the results from Li et al. (2016) reporting low toxic effects on survival and settlement of barnacle larvae. PET is generally regarded as a non-harmful polymer, as it is often used as plastic food packaging. In our study, none of the organic compounds analyzed were found in PET, and metals were not present in large quantities either. When testing the effects of exposure to DMSO extract of PET, no significant changes in viability, ROS, comet and ER activity assays were detected, even at the highest concentration level, indicating that either the human cell lines are not sensitive to PET extracts or PET extracts do not contain enough toxic chemicals. Very recent study using three different rainbow trout cell lines showed that leachate from 1 mg/mL commercially available bottle-grade PET did not induce cytotoxicity, ROS generation or EROD activity (Boháčková et al., 2023). In fact, in our study, chemical analysis of PET extracts did not show any pollutants in PAHs, PCBs, PBDE, pesticides families. In our study, no additives, non-intentionally added substances or degradation products such as photooxidation compounds, which may contribute to plastic toxicity, were detected in PET extracts. While chemical screening did not enable to identify phthalates in our plastic leachates, the analysis of such compounds is often complicated by persistent and elevated laboratory blanks. As estrogenic activity was not induced, one can assume that PET do not initially contain additives or they were released during environmental plastic weathering. In other studies, the leachates of artificial UV-weathered PP, PET, PS and PVC plastics were more active in the AReC32 assay with the human breast cancer cell line MCF7 than non-irradiated controls indicating that photodegradation products resulting from intense UV A and B irradiation contribute to the oxidative stress induction (Rummel et al., 2019, Klein et al., 2021). However realistic weathering scenario using daylight at 40 °C induced no toxicity for PET (Klein et al., 2021).

The Caco-2 line appears to be more sensitive than the HepG2 line to contaminants extracted from various plastics. HepG2 cells did not show any sub-lethal effect including ROS activity or DNA damage. Caco-2 cells were previously shown to display stronger responses to microplastic methanol-extracts compared to another intestinal epithelium cell line (HT-29) (Herrala et al., 2023) and exhibited the highest association to MP effects, out of 10 different cell models used in cell viability experiments (Danopoulos et al., 2022). Although conventional 2D cell cultures grown in monolayers are convenient for screening purposes, more sophisticated cell models involving different cell types such as co-cultures or2 organoids could be more predictive of *in vivo* responses. Those advanced cellular models should be more studied in the future even if such technologies are just emerging in the field of *in vitro* toxicology and are still associated with technical challenges (Forest et Pourchez, 2023).

## 5. Conclusion

Our results demonstrate that naturally aged environmental plastics collected on beaches induced cytotoxicity, oxidative stress and genotoxicity and none of them induced estrogenic activity. Some plastics affected several *in vitro* endpoints whereas others caused little to no effects in human cell lines such as PET and PP. The overall toxicity of DMSO extracts from tested polymers was ranked
PVC>Nylon>HDPE>PET=PP. Our results support the scientific assessment of the effects of different types of plastic litter on hepatocyte and intestine toxicity and draw more attention to the issue of plastic pollution affecting human health. Despite the increasing literature on the omnipresence of plastic in the environment and food, the risk of plastic to human health is still difficult to apprehend. The effects from contaminants associated to plastics have to be distinguished from other environmental contaminants. Indeed, to quantify the risk of plastic contamination it is necessary to quantify human exposure and settle toxicity thresholds above whom risk is significant. Now, there is still a lack of information on both sides to fulfill this task. Actual legislation does not require risk evaluation for plastic components that are not in direct contact to food such as stabilizer or oligomers. It is necessary to legally urge plastic producers to inform customers about the composition not only of the polymer but also the exhaustive details of the additives. A standardization of the communication on plastics formulations would clearly improve their use, management, recycling and hazard identification.

## Funding

This study was conducted in the frame of the ARPLASTIC research project. This work was funded by the Nouvelle-Aquitaine French region. the Water council Agence de l'eau Adour-Garonne, the local inter-city board Syndicat Intercommunal du Bassin d'Arcachon and the local marine nature park Parc Naturel Marin du Bassin d'Arcachon (Office français de la biodiversité).

## CRediT authorship contribution statement

**Stéphane Le Floch:** Writing – review & editing. **Jérôme Cachot:** Writing – review & editing, Methodology, Funding acquisition, Conceptualization. **Bénédicte Morin:** Writing – original draft, Project administration, Methodology, Funding acquisition, Conceptualization. **Florane Le Bihanic:** Writing – original draft, Visualization, Validation, Methodology, Investigation, Funding acquisition, Conceptualization. **Bettie Cormier:** Writing – review & editing, Methodology, Investigation. **Emilie Dassié:** Writing – review & editing, Investigation. **Sophie Lecomte:** Writing – review & editing. **Justine Receveur:** Writing – review & editing, Investigation.

## Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Benedicte MORIN reports financial support was provided by SIBA. Benedicte MORIN reports financial support was provided by Nouvelle-Aquitaine Regional Council. Benedicte MORIN reports financial support was provided by French Biodiversity Office. Benedicte MORIN reports financial support was provided by French water agency Adour Garonne. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors would like to thank Christelle Clérandeau, University of Bordeaux, for her technical help.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2024.117604.

## Data Availability

Data will be made available on request.

## References

Ajitha, V., Sarasasan, M., Sreevidya, C.P., Aswathy, C., Kachiprath, B., Mohandas, A., Sarojini, I., Singh, B., Sahul Hameed, A.S., Schlenk, D., Magnuson, J.T., Puthumana, J., 2021. Cytotoxic impacts of treated electroplating industrial effluent and the comparative effect of their metal components (Zn, Hg, and Zn+Hg) on Danio rerio gill (DrG) cell line. Sci. Total Environ. 793, 148533–148543. https://doi.org/10.1016/j.scitotenv.2021.148533.

Akoueson, F., Sheldon, L.M., Danopoulos, E., Morris, S., Hotten, J., Chapman, E., Li, J., Rotchell, J.M., 2020. A preliminary analysis of microplastics in edible versus non-edible tissues from seafood samples. Environ. Pollut. 263, 114452–114462. https://doi.org/10.1016/j.envpol.2020.114452.

Alijagic, A., Kotlyar, O., Larsson, M., Salihovic, S., Hedbrant, A., Eriksson, U., Karlsson, P., Persson, A., Scherbak, N., Färllund, K., Engwall, M., Särdahl, E., 2024. Immunotoxic, genotoxic, and endocrine disrupting impacts of polyamide microplastic particles and chemicals. Environ. Inter 183, 108412–108427. https://doi.org/10.1016/j.envint.2023.108412.

Bejgarn, S., MacLeod, M., Bogdal, C., Breitholtz, M., 2015. Toxicity of leachate from weathering plastics: An exploratory screening study with Nitocra spinipes. Chemosphere 132, 114–119.

Boháčková, J., Havlíčková, L., Semerád, J., Titov, I., Trhlíková, O., Beneš, H., Cajthaml, T., 2023. In vitro toxicity assessment of polyethylene terephthalate and polyvinyl chloride microplastics using three cell lines from rainbow trout (Oncorhynchus mykiss). Chemosphere 312, 136996–137007. https://doi.org/10.1016/j.chemosphere.2022.136996.

Boskovic, N., Joksimovic, D., Bajt, O., 2023. Microplastics in mussels from the Boka Kotorska Bay (Adriatic Sea) and impact on human health. Food Chem. Toxicol. 173, 113641–113650.

Bouwmeester, H., Hollman, P.C.H., Peters, R.J.B., 2015. Potential health impact of environmentally released micro- and nanoplastics in the human food production chain: experiences from nanotoxicology. Environ. Sci. Technol. 49, 8932–8947. https://doi.org/10.1021/acs.est.5b01090.

Castano, A., Bols, N., Braunbeck, T., Dierickx, P., Halder, M., Kawahara, K., Lee, L.E.J., Mothersill, C., Pärt, P., Sintes, J.R., Rufli, H., Smith, R., Wood, C., 2003. The Use of Fish Cells in Ecotoxicology. Vitro 317–351.

Cesarini, G., Corami, F., Rosso, B., Scalici, M., 2023. Microplastics, additives, and plasticizers in freshwater bivalves: preliminary research of biomonitoring. Water 15 (14), 2647–2663. https://doi.org/10.3390/w15142647.

Corami, F., Rosso, B., Iannilli, V., Ciadamidaro, S., Bravo, B., Barbante, C., 2022. Occurrence and characterization of small microplastics (<100 MM), additives, and plasticizers in larvae of simuliidae. Toxics 10, 383–399. https://doi.org/10.3390/toxics10070383.

Cormier, B., Gambardella, C., Tato, T., Perdriat, Q., Costa, E., Veclin, C., Le Bihanic, F., Grassl, B., Dubocq, F., Kärrman, A., Van Arkel, K., Lemoine, S., Lagarde, F., Morin, B., Garaventa, F., Faimali, M., Cousin, X., Bégout, M.L., Beiras, R., Cachot, J., 2021. Chemicals sorbed to environmental microplastics are toxic to early life stages of aquatic organisms. Ecotoxicol. Environ. Saf. 208, 111665–111675. https://doi.org/10.1016/j.ecoenv.2020.111665.

Danopoulos, E., Jenner, L.C., Twiddy, M., Rotchell, J.M., 2020. Microplastic contamination of seafood intended for human consumption: a systematic review and meta-analysis. Environ. Health Perspect. 128, 126002-1-126002-32. https://doi.org/10.1289/EHP7171.

Danopoulos, E., Twiddy, M., West, R., Rotchell, J.M., 2022. A rapid review and meta-regression analyses of the toxicological impacts of microplastic exposure in human cells. J. Hazard Mat. 427, 127861–127883. https://doi.org/10.1016/j.jhazmat.2021.127861.

Domenech, J., de Britto, M., Velázquez, A., Pastor, S., Hernández, A., Marcos, R., Cortés, C., 2021. Long-term effects of polystyrene nanoplastics in human intestinal Caco-2 cells. Biomolecules 11, 1442–1458. https://doi.org/10.3390/biom1101442.

Forest, V., Pourchez, J., 2023. Can the impact of micro- and nanoplastics on human health really be assessed using in vitro models? A review of methodological issues. Environ. Inter 178, 108115–108125. https://doi.org/10.1016/j.envint.2023.108115.

Galloway, T.S., 2015. In: Bergmann, M., Gutow, L., Klages, M. (Eds.), Micro- And Nano-plastics And Human Health BT-marine Anthropogenic Litter. Springer International Pub-lishing, Cham, pp. 343–366. https://doi.org/10.1007/978-3-319-16510-3_13.

Gasperi, J., Wright, S.L., Dris, R., Collard, F., Mandin, C., Guerrouache, M., Langlois, V., Kelly, F.J., Tassin, B., 2018. Microplastics in air: are we breathing it in? Curr. Opin. Environ. Sci. Heal. 1, 1–5. https://doi.org/10.1016/j.coesh.2017.10.002.

Gauquie, J., Devriese, L., Robbens, J., Witte, B.De, 2015. A qualitative screening and quantitative measurement of organic contaminants on different types of marine plastic debris. Chemosphere 138, 348–356. https://doi.org/10.1016/j.chemosphere.2015.06.029.

Groh, K.J., Backhaus, T., Carney-Almroth, B., Geueke, B., Inostroza, P.A., Lennquist, A., Leslie, H.A., Maffini, M., Slunge, D., Trasande, L., Warhurst, A.M., Müncke, J., 2019. Overview of known packaging-associated chemicals and their hazards. Sci. Total Environ. 651, 3253–3268.
Hahladakis, J.N., Velis, C.A., Weber, R., Iacovidou, E., Purnell, P., 2018. An overview of chemical additives present in plastics: migration, release, fate and environmental impact during their use, disposal and recycling. *J. Hazard Mater.* 344, 179–199.

Hamidian, A.H., Ozumchelouei, E.J., Feizi, F., Wu, C., Zhang, Y., Yang, 2021. A review on the characteristics of microplastics in wastewater treatment plants: a source for toxic chemicals. *J. Clean. Prod.* 295, 126480–126502. https://doi.org/10.1016/j.jclepro.2021.126480.

Hantoro, I., Löhr, A.J., Van Belleghem, F.G.A.J., Widianarko, B., Ragas, A.M.J., 2019. Microplastics in coastal areas and seafood: implications for food safety. Food Addit. Contam. Part A 36 (5), 674–711. https://doi.org/10.1080/19440049.2019.1585581.

Hasan, J., Siddik, M.A., Ghosh, A.K., Mesbah, S.B., Sadat, M.A., Shahjahan, M., 2023. Increase in temperature increases ingestion and toxicity of polyamide microplastics in Nile tilapia. *Chemosphere* 327, 138502–138511. https://doi.org/10.1016/j.chemosphere.2023.138502.

Herrala, M., Huovinen, M., Järvelä, E., Hellman, J., Tolonen, P., Lahtela-Kakkonen, M., Rysä, J., 2023. Micro-sized polyethylene particles affect cell viability and oxidative stress responses in human colorectal adenocarcinoma Caco-2 and HT-29 cells. *Sci. Total Environ.* 867, 161512–161521. https://doi.org/10.1016/j.scitotenv.2023.161512.

Hosseinzadeh, M., Wang, T., Morales-Caselles, C., Callejas, S., Eljarrat, E., Porte, C., 2024. Comparative toxicity of beach mesoplastics from South Spain: an in vitro approach. *Chemosphere* 352, 141494–141503. https://doi.org/10.1016/j.chemosphere.2024.141494.

Jovanovic, B., Gökdag, K., Güven, O., Emre, Y., Whitley, E.M., Kideys, A.E., 2018. Virgin microplastics are not causing imminent harm to fish after dietary exposure. *Mar. Pollut. Bull.* 130, 123–131. https://doi.org/10.1016/j.marpolbul.2018.03.016.

Karami, A., Golieskardi, A., Choo, C.K., Larat, V., Karbalaei, S., Salamatinia, B., 2018. Microplastic and mesoplastic contamination in canned sardines and sprats. *Sci. Total Environ.* 612, 1380–1386. https://doi.org/10.1016/j.scitotenv.2017.09.005.

Khosrovyan, A., Kahru, A., 2022. Virgin and UV-weathered polyamide microplastics posed no effect on the survival and reproduction of Daphnia magna. *PeerJ* 10, e13533. https://doi.org/10.7717/peerj.13533.

Klein, K., Hof, D., Dombrowski, A., Schweyen, P., Dierkes, G., Ternes, T., Schulte-Oehlmann, U., Oehlmann, J., 2021. Enhances in vitro toxicity of plastic leachates after UV irradiation. *Water Res* 199, 117203–117213.

Lacroix, C., Le Cuff, N., Receveur, J., Moraga, D., Auffret, M., Guyomarch, J., 2014. Development of an innovative and "green" stir bar sorptive extraction–thermal desorption–gas chromatography–tandem mass spectrometry method for quantification of polycyclic aromatic hydrocarbons in marine biota. *J. Chroma.* 1349, 1–10. https://doi.org/10.1016/j.jchroma.2014.04.094.

Lea, T., 2015. Caco-2 cell line. In: Verhoecks, K., Cotter, P., López-Expósito, I., et al. (Eds.), The Impact of Food Bioactives on Health: In Vitro And Ex Vivo Models. Springer International Publishing, Switzerland, pp. 343–366. https://doi.org/10.1007/978-3-319-16104-4.

Lei, L., Wu, S., Lu, S., Liu, M., Song, Y., Fu, Z., Shi, H., Raley-Susman, K.M., He, D., 2018. Microplastic particles cause intestinal damage and other adverse effects in zebrafish Danio rerio and nematode Caenorhabditis elegans. *Sci. Total Environ.* 619–620, 1–8. https://doi.org/10.1016/j.scitotenv.2017.11.103.

Li, H.X., Getzinger, G.J., Ferguson, P.L., Orihuela, B., Zhu, M., Rittschof, D., 2016. Effects of toxic leachate from commercial plastics on larval survival and settlement of the barnacle Amphibalanus amphitrite. *Environ. Sci. Technol.* 50, 924–931.

Lithner, D., Halling, M., Dave, G., 2012. Toxicity of electronic waste leachates to Daphnia magna: screening and toxicity identification evaluation of different products, components and materials. *Arch. Environ. Contam. Toxicol.* 62, 579–588.

Liu, S., Wu, X., Gu, W., Yu, J., Wu, B., 2020. Influence of the digestive process on intestinal toxicity of polystyrene microplastics as determined by in vitro Caco-2 models. *Chemosphere* 256, 127204–127213. https://doi.org/10.1016/j.chemosphere.2020.127204.

Lu, Y., Zhang, Y., Deng, Y., Jiang, W., Zhao, Y., Geng, J., Ding, L., Ren, H., 2016. Uptake and accumulation of polystyrene microplastics in zebrafish (Danio rerio) and toxic effects in liver. *Environ. Sci. Technol.* 50 (7), 4054–4060.

Meeker, J.D., Sathyanarayana, S., Swan, S.H., 2009. Phthalates and other additives in plastics: human exposure and associated health outcomes. *Philos. Trans. R. Soc. B Biol. Sci.* 364, 2097–2113. https://doi.org/10.1098/rstb.2008.0268.

Paganos, P., Ullmann, C., Gaglio, D., Bonanomi, M., Salmistraro, N., Arnone, M., Jimenez-Guri, E., 2023. Plastic leachate-induced toxicity during sea urchin embryonic development: insights into the molecular pathways affected by PVC. *Sci. Total Environ.* 864, 160901–160917. https://doi.org/10.1016/j.scitotenv.2022.160901.

Pannetier, P., Cachot, J., Clérandeau, C., Faure, F., Van Arkel, K., de Alencastro, L.F., Levasseur, C., Sciacca, F., Bourgeois, J.-P., Morin, B., 2019. Toxicity assessment of pollutants sorbed on environmental sample microplastics collected on beaches: Part I-adverse effects on fish cell line. *Environ. Pollut.* 248, 1088–1097. https://doi.org/10.1016/J.ENVPOL.2018.12.091.

Pannetier, P., Fuster, L., Clérandeau, C., Lacroix, C., Gourves, P.Y., Cachot, J., Morin, B., 2018. Usefulness of RTL-W1 and OLCAB-e3 fish cell lines and multiple endpoint measurements for toxicity evaluation of unknown or complex mixture of chemicals. *Ecotoxicol. Environ. Saf.* 150, 40–48. https://doi.org/10.1016/j.ecoenv.2017.12.027.

Pedà, C., Romeo, T., Panti, C., Caliani, I., Casini, S., Marsili, L., Campani, T., Baini, M., Limonta, G., de Rysky, E., Caccamo, L., Perdichizzi, A., Gai, F., Maricchiolo, G., Consoli, P., Fossi, M.C., 2022. Integrated biomarker responses in European seabass Dicentrarchus labrax (Linnaeus, 1758) chronically exposed to PVC microplastics. *J. Hazard Mater.* 438, 129488–129500. https://doi.org/10.1016/j.jhazmat.2022.129488.

Peters, R., de Jong, N., de Haan, L., Wright, S., Bouwmeester, H., 2022. Release and intestinal translocation of chemicals associated with microplastics in an in vitro human gastrointestinal digestion model. *Micro Nanoplastics* 2, 3–24. https://doi.org/10.1186/s43591-021-00022-y.

Prata, J.C., da Costa, J.P., Lopes, I., Duarte, A.C., Rocha-Santos, T., 2020. Environmental exposure to microplastics: An overview on possible human health effects. *Sci. Total Environ.* 702, 134455. https://doi.org/10.1016/j.scitotenv.2019.134455.

Rochman, C.M., Hoh, E., Hentschel, B., Kaye, S., 2013. Long-term field measurement of sorption of organic contaminants to five types of plastic pellets: implications for plastic marine debris. *Environ. Sci. Technol.* 47 (3), 1646–1654.

Rummel, C.D., Escher, B.I., Sandblom, O., Plassmann, M.M., Arp, H.P.H., MacLeod, M., Jahnke, A., 2019. Effects of leachates from UV-weathered microplastic in cell-based bioassays. *Environ. Sci. Technol.* 53, 9214–9233.

Salazar-Pérez, C., Amezcuña, F., Rosales-Valencia, A., Green, L., Pollorena-Melendrez, J. E., Sarmiento-Martínez, M.A., Tomita Ramírez, I., Gil-Manrique, B.D., Hernandez-Lozano, M.Y., Muro-Torres, V.M., et al., 2021. First insight into plastics ingestion by fish in the Gulf of California, Mexico. *Mar. Pollut. Bull.* 171, 112705–112715. https://doi.org/10.1016/j.marpolbul.2021.112705.

Sarker, I., Moore, L.R., Paulsen, I.T., Tetu, S.G., 2020. Assessing the toxicity of leachates from weathered plastics on photosynthetic marine bacteria *Prochlorococcus*. *Front. Mar. Sci.* 7, 571929. https://doi.org/10.3389/fmsci.2020.571929.

Sergent, T., Dupont, I., Jassogne, C., et al., 2009. CYP1A1 induction and CYP3A4 inhibition by the fungicide imazalil in the human intestinal Caco-2 cells—comparison with other conazole pesticides. *Toxicol. Lett.* 184, 159–168.

Shrivastava, A., 2011. Methods for the determination of limit of detection and limit of quantitation of the analytical methods. *Chronicks Young Scientists* 2, 21–25. https://doi.org/10.4103/2229-5186.79345.

Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. *Exp. Cell Res* 175 (1), 184–191. https://doi.org/10.1016/0014-4827(88)90265-0.

Smith, M.R., Fernandes, J., Go, Y.M., Jones, D.P., 2017. Redox dynamics of manganese as a mitochondrial life-death switch. *Biochem Biophys. Res Commun.* 482, 388–398.

Taju, G., Abdul Majeed, S., Nambi, K.S.N., Sahul Hameed, A.S., 2013. Development and characterization of cell line from the gill tissue of Catla catla (Hamilton, 1822) for toxicological studies. *Chemosphere* 90, 2172–2180.

Valko, M., Morris, H., Cronin, M., 2005. Metals, toxicity and oxidative stress. *Curr. Med Chem* 12 1161–1208.

Van Cauwenberghie, L., Janssen, C.R., 2014. Microplastics in bivalves cultured for human consumption. *Environ. Pollut.* 193, 65–70. https://doi.org/10.1016/j.envpol.2014.06.010.

Waring, R.H., Harris, R.M., Mitchell, S.C., 2018. Plastic contamination of the food chain: A threat to human health? *Maturitas* 115, 64–68. https://doi.org/10.1016/j.maturitas.2018.06.010.

Wessel, N., Santos, R., Menard, D., Le Menach, K., Buchet, V., Lebayon, N., Loizeau, V., Burgeot, T., Budzinski, H., Akcha, F., 2010. Relationship between PAH biotransformation as measured by biliary metabolites and EROD activity, and genotoxicity in juveniles of sole (Solea solea). *Mar. Environ. Res* 69, S71eS73. https://doi.org/10.1016/j.marenvres.2010.03.004.

Windhorfer, F., Krause, S., Hader, C., Schulz, W., Florl, A., 2008. Distinctive differences in DNA double-strand break repair between normal urothelial and urothelial carcinoma cells. *Mutat. Res* 638, 56–65.

Zimmermann, Bartosova, Z., Braun, K., Völker, C., Wagner, M., 2021. Plastic products leach chemicals that induce in vitro toxicity under realistic use conditions. *Environ. Sci. Technol.* 55, 11814–11823.

Zimmermann, L., Göttlich, S., Oehlmann, J., Wagner, M., Völker, C., 2020. What are the drivers of microplastic toxicity? Comparing the toxicity of plastic chemicals and particles to Daphnia magna. *Environ. Pollut.* 267, 115392–115402. https://doi.org/10.1016/j.envpol.2020.115392.

Zitouni, N., Bouserrhine, N., Belbekhouche, S., Missawi, O., Alphonse, V., Boughtass, I., Banni, M., 2020. First report on the presence of small microplastics (≤ 3 µm) in tissue of the commercial fish Serranus scriba (Linnaeus, 1758) from Tunisian coasts and associated cellular alterations. *Environ. Pollut.* 263, 114576. https://doi.org/10.1016/j.envpol.2020.114576.