**1. Introduction**

The observation that plastic materials are found at a global scale represents a major contamination problem, thus raising concerns about their long-term impacts on ecosystems. Indeed, plastic wastes reach millions of tons per year, and not all are recycled back into our economy, contaminating both terrestrial and aquatic environments. Microplastics (5 mm to 1 μm) are found in consumer products such as cosmetics, find their way into wastewater treatment plants—ill equipped to remove them—and are released into the aquatic environment [1,2]. To make this situation worse, the environment breakdown of plastics materials by abiotic and biotic processes will release an exponential number of particles as the size decreases, reaching the nanoscales (<100 nm). Plastic nanoparticles (NPs) are defined as particles with sizes generally <100 nm but some include the 100–1000 nm range as well. Small NPs (<100 nm) can become readily bioavailable towards organisms and are absorbed in cells [3,4].

Plastic NPs are considered toxic for organisms due to their ability to bioaccumulate in tissues, even passing the blood–brain barrier, leading to oxidative stress and tissue damage [5,6]. A recent meta-analysis on plastic NPs revealed that these NPs significantly decrease survival, and disrupt the behavior and reproduction of fish and invertebrates [7]. NPs are also known to produce oxidative stress and series of biophysical changes (viscosity and altered fractal organization of proteins/enzymes) in aquatic organisms, leading to toxicity [8,9]. Plastics NPs could also act as vectors by absorbing existing toxic compounds such as bisphenol A, polyaromatic hydrocarbons, and heavy metals [10]. This could modify the bioavailability and exacerbate the toxic impacts of these existing environmental

**Citation:** Gagné, F. Isolation and Quantification of Polystyrene Nanoplastics in Tissues by Low Pressure Size Exclusion Chromatography. *J. Xenobiot.* **2022**, *12*, 109–121. https://doi.org/ 10.3390/jox12020010

Academic Editors: Tiziana Cappello and Eider Bilbao

Received: 1 March 2022 Accepted: 5 May 2022 Published: 9 May 2022

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**Copyright:** © 2022 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

pollutants. In addition to oxidative stress, polystyrene NPs were also genotoxic to *Mytilus galloprovincialis* mussels, most notably in gills [11], suggesting long-term negative effects. Methods to quantify plastic NPs in tissues are scarce, which limits our understanding about their occurrence in the environment, distribution in tissues, and health impacts. Plastics are readily stained by the solvatochromic dye, neutral red (NR), often used to detect the presence of both micro and plastic NPs [6,11]. However, NR also reacts to nonpolar lipids in cells, which could interfere with the assay. Hence, there is a need to find other means to determine plastic NPs in biological tissues involving hyphenated procedures. More recently, a more specific fluorescent assay for polystyrene NPs in tissues was proposed using molecular rotor probes [12,13]. The presence of plastic NPs in the crowded environment of cells could disrupt the protein networks and lipid dynamics producing organized (nematic-like) liquid crystals, protein condensation/denaturation, and changes in metabolic pathways [8]. A methodology to study the interactions of plastic NPs in cells with respect to proteins and lipids would be valuable to help us better understand the uptake and toxicity of NP at the molecular level.

The purpose of this study was therefore to develop a low-pressure size exclusion chromatography (SEC) methodology for polystyrene NPs in tissue homogenate fraction. The method should be available to resolve NPs from the protein and lipids pool in the cytoplasm and permit some degree of quantification. The proposed method was first developed with fluorescently labeled polystyrene NPs, albumin, and in spiked homogenate fractions. The resulting method was optimized to analyze plastic NPs directly in marine and freshwater bivalve tissues in two real-life case studies.
