Microplastics (MPs) have been frequently found in the aquatic ecosystems around the world and may constitute a serious threat to the environment. Only a few papers have examined their ecological consequences on freshwater animals, even though several studies have shown direct negative effects on marine organisms [1]. Toxic potential of waterborne pollutants must be accurately assessed to regulate and control pollution in aquatic environments. Fish are frequently used in aquatic ecotoxicology studies due to their ecological significance, adaptability, easy breeding, and ease of maintenance in laboratory settings [2]. The gills of fish are multipurpose organs that perform gas exchange, osmoregulation, trace metal transport, and waste excretion. It is also the primary place where contaminants are absorbed by fish [3]. Polystyrene (PS) is among the most extensively manufactured plastics in the world [4]. It is also one of the most commonly found MPs in aquatic environments, and because of its large molecular weight and structural stability, PS is commonly deemed as nonbiodegradable [5].
The aim of this study is to investigate chronic genotoxic and cytotoxic effects of PS MPs at environmentally relevant concentrations on erythrocytes in rainbow trout (Oncorhynchus mykiss) gills. Samples were collected by spreading freshly dissected gills on to a microscopic slide. Cytogenetic analysis was carried out using erythrocyte nuclear abnormalities assay with criteria described by Baršienė et al. [6], Fenech et al. [7] and Heddle et al. [8]. For this analysis 4000 intact erythrocytes were analyzed for each slide/fish. The formation of micronuclei (MN), nuclear buds (NB), nuclear buds on filament (NBf), blebbed nuclei (BL) cells were assessed as genotoxicity endpoints, as well as 8-shaped and bean-shaped nuclei, fragmented apoptotic (FA) and bi-nucleated (BN) cells as cytotoxicity endpoints. Data analysis was performed using R [9] software (version 4.2.2).
There were no significant differences in the frequencies of any observed nuclear abnormalities although mean differences between PS and control (CTRL) groups ranged from -0.0270 ‰ (NBf) to 0.0612 ‰ (BL). A standardized effect size (Cohen's d) for measuring the difference between two group means was also calculated. Effect size ranged from negligible ($|d| < 0.2$) to medium (for BL $|d| = 0.52$). The investigation of relationships between observed cytogenetic endpoints in rainbow trout using principal component analysis (PCA) yielded two principal components (PCs) which explained 47.98% of the total variance. In PC1 NB and 8-shaped nuclei had the highest contribution to variance (37.31% and 33.82% respectively). Afterwards, PCA was done in each group separately to observe group specific contributions to variance. In conclusion, environmentally realistic concentrations of PS MP did not induce genotoxic or cytotoxic effects in fish gill erythrocytes. This study in no way contradicts all the previous research on MP toxicity but highlights the use of environmentally realistic concentrations to assess the practical ecological impact of plastic pollution.