Micro/nano-plastics (MNPs) have been found within many environments and organisms including humans, making them a significant and growing concern. Initial research into the potential detrimental effects these MNPs both from acute and chronic exposure has been ongoing but still requires substantially more data to clarify. This research presents the response of nano-polystyrene (NPS) on Raphidocelis subcapitata, a freshwater alga, under an existing acute toxicity test along with additional analytical techniques to try identifying possible sources of toxicity. R. subcapitata cells were exposed for 72 h to a concentration range of 0-100 mg/l NPS. Growth Inhibition (GI) testing showed the R. subcapitata demonstrated statistically distinct reductions in growth over 72 h at all NPS exposure concentrations while not suffering culture collapse. By the 100 mg/l NPS exposure the R. subcapitata has suffered almost a 33.7% reduction in cell concentration after 72 h compared to control samples. Confocal imaging showed the NPS wasn't permeating into the algal plasma membrane or individual organelles but agglomerated onto the algal cell wall. The agglomeration was irregular but increased in total surface area covered as NPS concentration increases. UV-Vis fluorimetry testing produced a linear response of emission intensities to algae exposed to the 0-100 mg/l range of NPS. However, comparisons of emission intensity values of algae exposed to NPS to emission intensities of pure NPS at identical concentrations showed consistent intensity reduction. This response further indicated NPS agglomerating within the media and onto the alga cells seen from confocal imaging. Finally, Raman spectroscopy on R. subcapitata attempted to distinguish the key 1001 cm^-1 peak or other crucial identifier peaks of polystyrene from overall Raman spectra. This was not successful as emissions from algal component (e.g. phenylaniline) completely suppressed the signal region.

The presence of sub-micron-sized plastics in the environment has been increasing, with the possible risks of these particles remaining relatively unknown. In order to assess the toxicity of these particles, 100 nm diameter green fluorescent nano-polystyrene spheres (NPS) (20–60 mg/L) were exposed to zebra mussels (Dreissena polymorpha) to investigate the mortality, clearance rate and stress-related biomarker responses. D. polymorpha were collected and analysed with standard OECD toxicological tests and biomarker analysis to detect both physical and biochemical responses after exposure to NPS. The toxicity of the NPS to D. polymorpha was low, with 60 mg/L NPS causing a mortality rate of 11.1% at 96 h which was statistically significant compared to the 4.2% control. No statistical change could be found for the condition factor (k_c) of D. polymorpha after NPS exposure. Clearance rates in D. polymorpha using R. subcapitata algae showed NPS-exposed mussels had a reduction of filtering efficiency of up to 30.5%. Bioassay testing shows a mixed but undeniably negative response from the D. polymorpha to the NPS, notably a significant rise in DNA Strand Breaks (DSB) and Metallothionein (MT) responses for high NPS concentrations. Additionally, Lipid Peroxidation (LPO) and Ferric Reducing Antioxidant Power (FRAP) assay tests showed a significant increase in response from the higher (>40 mg/L) concentrations of NPS exposure. Although Glutathione S-Transferase (GST) assay showed no statistical change from the control for all NPS-exposed samples, an increase of 20% had occurred for 60 mg/L NPS. Overall, a minimal toxic response from D. polymorpha to the NPS exposure below 40 mg/L was seen. After 40 mg/L NPS, mussels presented more acute toxicity in terms of mortality, along with reduced algal clearance rates and anincrease in biomarker response. This study revealed a clear induction of oxidative stress and DSB in the digestive gland of zebra mussels following exposure to nano-polystyrene. While these findings provide valuable insights into the potential harmful effects of nanoplastics in freshwater bivalves, further studies are necessary to help understand the level of threat plastic pollution may pose to the health of freshwater ecosystems.

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.