Zebrafish embryos as a biological model to study the effects of nanoplastics

Plastic waste is currently recognized as a critical factor for environmental health. This environmental problem is aggravated by fact that plastics fragment to smaller and smaller particles, reaching nanometric dimensions, increasing their bioavailability and potentially their toxic effects. There are, however, few studies of nanoplastics effects (NPs, less than 100 nm plastic size reduction), especially in freshwater vertebrates. Thus, this work aimed to study the potential effect of nanoparticles of two different polymers, polystyrene (PS) and polymethylmethacrylate (PMMA), ~ 30 nm, in a range of concentration between 0.001 and 100 mg / L. In order to ensure that NPs has desirable characteristics (size, shape, different analysis methods have been tested for reproducibility of the final product).After synthesis, the NPs were purified using dialysis sleeves and characterized in ultrapure water, in terms of hydrodynamic size (using dynamic light scattering (DLS), size and morphology (using electron microscopy). Following characterization, experimental assays with zebrafish embryos that involved exposure to NPs (in single exposures and combined with an environmental pollutant with a wide distribution in aquatic systems, pyrene, four rings polycyclic aromatic hydrocarbon). In the NPs single assays, parameters such as developmental changes, swimming behaviour and stress response and biochemical responses associated with neurotransmission (cholinesterase - ChE), energy metabolism (glycogen - Gly, isocitrate dehydrogenase- IDH and lactate dehydrogenase - LDH) and antioxidant response (glutathione) were evaluated. S-transferase - GST, glutathione peroxidase - GPx and catalase - CAT) and oxidative damage (lipid peroxidation - LPO). In trials where embryos were subjected to combined exposure to NPs and pyrene, only developmental changes, swimming behaviour and stress response were evaluated. In general, it was found that under the experimental conditions tested, that NPs (round, average size ~30 nm) had no significant impact on mortality or embryo development. However, changes in larval swimming behaviour and biochemical parameters were observed. Thus, in organisms exposed to PS NPs, the total swim distance increased in organisms exposed to 0.001, 1 and 100 mg/L. In terms of PMMA NPs, total swim distance increased significantly in organisms exposed to 0.001, 0.01 and 1 mg/L. However, the swimming distance and time in outside of the test area (expressed as a percentage) decreased in the exposed organisms at 0.001 and 10 mg/L. In terms of biochemical responses, it was found that PS NPs could affect neurotransmission (causing increased cholinesterase activity at 1 mg/L concentration and decreased at 0.01.0.1 mg/L (PS NPs) and 0.01 mg/L (PMMA). In terms of parameters associated with the antioxidant response, PS inhibited GST (0.01,10 and 100 mg / L) and CAT (0.001 and 10 mg / L), while GPx was induced to (0.001 mg / L) and inhibited at (0.1 and 1 mg / L). PMMA induced GPx (10 mg / L), increased CAT activity (0.001, 0.1 and 10 mg / L). Also, CAT was decreased by PMMA at 0.01 mg/L. There was lipid peroxidation in the exposure by PS NPs (1 mg / L) and PMMA NPs (0.001, 0.01 and 0.1 mg / L). In terms of energy metabolism, IDH was found decreased in organisms exposed to PS NPs (0.001, 0.01, 0.1 and 100 mg / L) and an increase for PMMA (0.001 mg / L). Results of simultaneous exposure to NPs and pyrene disassembled that the effect of pyrene toxicity (pericardial oedema and tail curvature) (0.01 and 1 μg / L) decreased. In addition, the combination of NPs and pyrene also reduced the effect of NPs (0.1 and 100 mg / L) on zebrafish swimming behaviour of both polymers. Thus, it is demonstrated that short-term exposure to NPs may cause biochemical and behavioural changes that may compromise animal welfare and have environmental consequences (e.g. ability to acquire food, breeding and escape from predation). Zebrafish embryos demonstrated sensitivity to low concentrations of NPs, justifying their application in ecotoxicological studies of emerging contaminants.