In vitro toxicity assessment of polyethylene terephthalate and polyvinyl chloride microplastics using three cell lines from rainbow trout (Oncorhynchus mykiss)

Abstract

The RTgill-W1 (gill), RTG-2 (gonad), and RTL-W1 (liver) cell lines derived from a freshwater fish rainbow trout (Oncorhynchus mykiss), were used to assess the toxicity of polyethylene terephthalate (PET) and two forms of polyvinyl chloride (PVC). Two size fractions (25-μm and 90-μm particles) were tested for all materials. The highest tested concentration was 1 mg/ml, corresponding to from 70 000 ± 9000 to 620 000 ± 57 000 particles/ml for 25-μm particles and from 2300 ± 100 to 11 000 ± 1000 particles/ml for 90-μm particles (depending on the material). Toxicity differences between commercial PVC dry blend powder and secondary microplastics created from a processed PVC were newly described. After a 24-h exposure, the cells were analyzed for changes in viability, 7-ethoxyresorufin-O-deethylase (EROD) activity, and reactive oxygen species (ROS) generation. In addition to the microplastic suspensions, leachates and particles remaining after leaching resuspended in fresh exposure medium were tested. The particles were subjected to leaching for 1, 8, and 15 days. The PVC dry blend (25 μm and 90 μm) and processed PVC (25 μm) increased ROS generation, to which leached chemicals appeared to be the major contributor. PVC dry blend caused substantially higher ROS induction than processed PVC, showing that the former is not suitable for toxicity testing, as it can produce different results from those of secondary PVC. The 90-μm PVC dry blend increased ROS generation only after prolonged leaching. PET did not induce any changes in ROS generation, and none of the tested polymers had any effect on viability or EROD activity. The importance of choosing realistic extraction procedures for microplastic toxicity experiments was emphasized. Conducting long-term experiments is crucial to detect possible environmentally relevant effects. In conclusion, the tested materials showed no acute toxicity to the cell lines.