Evaluation of the Cytotoxic and Oxidative Stress Effects of Polyhydroxybutyrate (PHB) Microplastics Mixed with Copper on PLHC-1 (Poeciliopsis lucida) Cells

Abstract

In recent years, bioplastics have been developed as a biodegradable alternative to conventional plastics. Commonly used for single-use packaging, it is expected that bioplastics will follow a similar trajectory and enter the aquatic environment, yet in comparison to conventional plastics, little is known about the effect on aquatic species. Polyhydroxybutyrate (PHB), a popular bioplastic, has been reported to have similar effects to conventional plastics at an acute and chronic level. The objective of this study was to determine if PHB microplastics (micro-PHBs) (10–80 µm) alter cellular viability and reactive oxygen species (ROS) production in Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) cells, while using nano-polystyrene (nano-PS) (100 nm) as a control particle throughout. After 24 h exposure, micro-PHB (100–400 mg/L) and nano-PS (20–80 mg/L) had no effect on cellular viability in the alamarBlue (AB) assay, but a significant difference to the negative control was demonstrated with the Neutral Red (NR) assay. To date, limited data on the toxicological mixture effects of micro-PHB and environmental contaminants have been documented. Copper (II) chloride (CuCl2) was chosen as a traditional contaminant to investigate mixture effects. Although an essential element, copper can be toxic and an oxidative stress inducer at elevated concentration levels. The half maximal effective concentration (EC50) for CuCl2 (AB = 104.5 mg/L; NR = 90.5 mg/L) was higher when mixed with nano-PS with the AB assay (EC50 = 120.2 mg/L) and NR assay (EC50 = 115.7 mg/L), although the overlapping confidence intervals indicate that these differences should be interpreted cautiously. Micro-PHB had no effect on CuCl2 cellular viability toxic units. It was determined that micro-PHB (>100 mg/L) and nano-PS (20–80 mg/L) significantly increased oxidative stress with a dose-dependent response. While exposure to CuCl2 significantly induced ROS, a dose–response relationship was not found. Co-exposure of CuCl2 and each particle produced variable ROS responses across trials, dependent on polymer type and CuCl2 concentration. These results suggest that specific conventional and bioplastics may have the capacity to alter the toxicological effects of a contaminant such as copper, although this effect requires further investigation. Before bioplastics are adopted as an “environmentally friendly” alternative to conventional plastics, further toxicological evaluations are warranted.