With polystyrene nanoparticles being widely used in various applications, there is a great need for deeper knowledge on the safety, fate and biological effects of these particles on both individual living organisms and the whole ecosystems. Due to this, there is a growing interest in performing ecotoxicological studies using model plastic nanoparticles, and consequently it generates an increasing number of published papers describing the negative impact on wildlife caused by such nanoparticles. Polystyrene is the most studied nanosized plastic, therefore this review focuses on research conducted with manufactured polystyrene nanoparticles. The aim of the present article is to provide a critical methodological outline of the existing ecotoxicological studies on the effects of polystyrene nanoparticles on aquatic organisms. Going through the published articles, we noted that particle characterization especially in the test medium, can be improved. The analysis also highlights the importance of purifying the polystyrene nanoparticles before studying its toxicity. Furthermore, the size characterization of such nanoparticles is underemphasized, and in future studies, authors should consider including more techniques to achieve this goal. Finally, short-term or direct exposure scenarios do not add the most environmentally relevant knowledge in terms of the toxicity caused by polystyrene nanoparticles.