There has been a recent trend to replace petroleum-based plastics with greener and more sustainable alternatives, such as bio-based ones. During this transition, there is a high probability that both types of polymers will be encountered and interact in environmental matrices. An integrated ecotoxicological test, that is, covering standard physiological parameters, such as mortality or growth, with behavioural characteristics, can be extremely important to understand the potential environmental and human impacts of these mixtures. This study aimed to evaluate the effects on Danio rerio embryonic-larval stage of exposure to micro(nano)plastics of polyhydroxybutyrate (PHB-MNPLs) and polymethylmethacrylate (PMMA-MNPLs) at concentrations of 0.1, 1, 10, 100, and 1000 µg/L on a full cross design. A control group was also included. Generally following the OECD 236 Fish Embryo Toxicity (FET) adapted for a period of 120 hours, the following endpoints were observed: mortality, malformations, heartbeats (only at 48 hours), hatching rate (starting at 48 hours), and overall length (at 120 hours). At 120h of exposure, the total time active, the total distance swum, thigmotaxis behaviour, and trajectory angles were analyzed.

The individual exposure to PHB-MNPLs and PMMA-MNPLs had no impact on mortality, malformations, or hatching rates of D. rerio. The PMMA-MNPLs alone induced tachycardia at 100 and 1000 µg/L and reductions in larvae length at concentrations between 0.1 and 100 µg/L. The PHB-MNPLs induced effects on fish swimming-related parameters, similar to those induced by PMMA-MNPLs (decreased total time active under light periods; decreased high amplitude movements at 0.1-10 µg PHB-MNPLs/L. These effects were also observed at 100 and 1000 µg PMMA-MNPLs/L under light periods. The mixture of both polymers induced patterns of response different than those observed for both MNPLs alone (e.g., in heartbeat rates and swimming parameters). This study highlights the need to Assess the effects of combined nanoparticles of different polymers.