Plastic pollution is a fast-rising environmental threat. Due to plastics’ low cost, durability, and flexibility, their use has increased worldwide, leading to an augmentation of their release into the marine environment. Most of the plastic debris found in the seas originates from land-based sources [1]. Once in the natural environment, plastic can be degraded into micro- (MPs, <5 mm) and nanoscale sizes (NPs, <1 μm) ^[2][3][2,3] by weathering, sunlight radiation, and biodegradation processes ^{[4][5][6][7][8]}[4,5,6,7,8]. MPs and NPs (MNPs) can be also classified into primary and secondary based on their sources. Primary MNPs are those that enter the environment in their original small size associated with specific applications and consumer products (e.g., cosmetics, clothing fibers, drug delivery, ink for 3D printers), whilst secondary MNPs are a consequence of macro/microplastics degradation ^{[4][9][10][11]}[4,9,10,11]. The formation of smaller particles leads to alterations in the physical-chemical proprieties, surface area, and size of MNPs, wherein, once the nanoscale is reached, the strength, conductivity, and reactivity will differ substantially from those of macro-/micro-sized particles ^{[12][13][14][15]}[12,13,14,15]. Obviously, as the size of the plastic particle decreases, biological reactivity, on the other hand, increases. Thus, it is crucial to comprehend the burden of MNPs’ availability and their biological impact on aquatic biota ^[14][16][14,16].

Up to now, polystyrene (PS) ha sbeen chosen as a proxy for MNPs due to the fact that it is one of the most largely used non-biodegradable plastics worldwide and, unlike other polymers, it shows a greater stability in sea water suspension with low styrene release [17]. Several studies have been conducted to evaluate the lethal and sublethal effects of PS MNPs on aquatic biota, reporting fertility, growth, and reproduction abnormalities ^{[18][19][20][21][22][23][24][25][26][27][28]}[18,19,20,21,22,23,24,25,26,27,28], as well as metabolism disorders, oxidative stress, and immune and nervous system dysfunction ^{[13][29][30][31][32][33][34][35][36]}[13,29,30,31,32,33,34,35,36]. Consequently, one of the main challenges today is to understand the mechanism of the toxicity of MNPs correlated to the lethal/sublethal effects studied so far. With this aim, the aquatic ecotoxicology field can benefit significantly from using the -omics approaches, which are emerging systemic and holistic tools for the global identification of the processes and pathways involved in the normal and abnormal physiological states, that allow not only the study of the mode of action of chemicals, but also the prediction of their toxicological effects on a given biological system [37]. -Omics approaches permit the production of large-scale datasets, measuring simultaneously the changes in gene expressions, proteins, and metabolites (by application of transcriptomics, proteomics, and metabolomics, respectively) occurring at the molecular, cell, tissue, and whole-organism levels ^[38][39][38,39]. These approaches allow the characterization of complex signal pathways and correlation of gene/protein expression, rather than focusing on the modulation of individual genes/proteins. Among others, -omics technologies include: (i) transcriptomics, which is used to study the whole set of RNA transcripts and to identify general and specific transcript biomarkers as transcriptional consequences related to natural environmental factors or the mode of action of environmental pollutants in an organism ^[38][40][38,40]; (ii) proteomics, which is used to study the whole set of proteins in order to evaluate any alterations in their function and/or structure in an organism after changes in the environmental conditions [41]; and (iii) metabolomics, which is used to study the whole set of cell metabolites, and has been employed in the past several years with the purpose of unveiling the molecular and biochemical mechanisms underlying the response, sensitivity, tolerance, and adaptation of aquatic organisms to environmental challenges or pollution ^[42][43][42,43]. Transcriptomic studies dominated until 2016, whereas a shift towards proteomics, and mostly metabolomics, including multi-omics studies, is now apparent [44].

2. -Omics Approaches in Studies of PS MNP Toxicity

2.1. Transcriptomics

Over the past decades, transcriptomics has predominantly been applied for environmental risk assessment by evaluation of the health status of aquatic animals ^[45][48]. It determines the changes in gene expression by measuring the level of mRNA after studying the whole set of transcripts, also named the transcriptome, present in an organism. Indeed, the quantitative real-time polymerase chain reaction (qRT-PCR) is the simplest and most widely used technique to conduct a transcriptomic analysis. Although the relative expression of selected genes is easy to undertake, as the amount of the gene studied is compared to the amount of a control reference gene, qRT-PCR can quantify only a limited number of genes, with the requirement for prior knowledge of target genes. To cope with these limitations and to target thousands of single mRNAs in a single run, microarrays and RNA-sequencing (RNA-seq) have therefore been used lately. In particular, the last mentioned technique uses high-throughput sequencing methodologies to detect the presence and quantity of RNA in a biological sample with the aim of analyzing the whole cellular transcriptome. In brief, the method consists of isolating total RNAs from biological samples and then performing its reverse transcription to obtain double-stranded cDNA. After that, cDNAs are sequenced as short reads, aligned, and mapped against a known genomic reference sequence. In recent years, RNA-seq has been successfully used to assess differential responses in a variety of aquatic species since it is effectively able to analyse whole transcriptomes, generating data on more differentially expressed genes (DEGs), which, through bioinformatics, will give information about the major pathways affected following a stress condition [44]. A description of the effects of PS MNPs at the transcription level in fishes and aquatic invertebrates is reported in Table 1.