Nile red staining of polyethylene and polystyrene in Daphnia magna tests

Microplastics pollution represents a stringent global issue. Their accumulation in the aquatic environment remains complicated due to the abundance, distribution, and resulting environmental effects they induce. Recently, assessing the toxicity level of aquatic organisms has gained scientific interest. Due to the diverse types and sizes of polymers present in the environment, the detection and evaluation of their effects are still a challenging issue. Polyethylene (PE) and polystyrene (PS) are the most commonly used polymers and are therefore predominantly detected in both marine and freshwater aquatic environments. This paper aims to evaluate the effects of Red Nile-stained PE and PS, tested in different particle sizes (PE 40-48, 125, >125 µm, and PS 20, 200, 430 µm) on Daphnia magna, and highlight them through microscopic analysis. Acute toxicity tests conducted over a 48-hours exposure did not reveal significant toxicity effects in terms of mortalities compared to the controls. Red Nile staining allowed the visualization of the shapes and sizes of the tested microplastics and their entry pathways into the Daphnia bodies. PS of 20 µm size was detected in the digestive tract of Daphnia, indicating as primary pathway of entry into the body of aquatic organisms. No acute toxic effects were recorded as a result of direct exposure to PE and PS particles. However, sub-lethal effects such as feeding and growth disturbances, which could affect organisms in the long term, are suspected.