Additives migrating from 3D-printed plastic induce developmental toxicity and neuro-behavioural alterations in early life zebrafish (Danio rerio)

The environmental impact of exposure to 3D-printed plastics as well as potential migration of toxic chemicals from 3D-printed plastics remains largely unexplored. In this work we applied leachates from plastics fabricated using a stereolithography (SLA) process to early developmental stages of zebrafish (Danio rerio) to investigate developmental toxicity and neurotoxicity. Migration of unpolymerized photoinitiator, 1-hydroxycyclohexyl phenyl ketone (1 - HCHPK) from a plastic solid phase to aqueous media at up to 200 mg/L in the first 24 h was detected using gas chromatography-mass spectrometry. Both plastic extracts (LC50 22.25% v/v) and 1 - HCHPK (LC50 60 mg/L) induced mortality and teratogenicity within 48 h of exposure. Developmental toxicity correlated with in situ generation of reactive oxygen species (ROS), an increase in lipid peroxidation and protein carbonylation markers and enhanced activity of superoxide dismutase (SOD) and glutathione-S-transferase (GST) in embryos exposed to concentrations as low as 20% v/v for plastic extracts and 16 mg/L for 1 - HCHPK. ROS-induced cellular damage led to induction of caspase-dependent apoptosis which could be pharmacologically inhibited with both antioxidant ascorbic acid and a pan-caspase inhibitor. Neuro-behavioral analysis showed that exposure to plastic leachates reduced spontaneous embryonic movement in 24-36 hpf embryos. Plastic extracts in concentrations above 20% v/v induced rapid retardation of locomotion, changes in photomotor response and habituation to photic stimuli with progressive paralysis in 120 hpf larvae. Significantly decreased acetylcholinesterase (AChE) activity with lack of any CNS-specific apoptotic phenotypes as well as lack of changes in motor neuron density, axonal growth, muscle segment integrity or presence of myoseptal defects were detected upon exposure to plastic extracts during embryogenesis. Considering implications of the results for environmental risk assessment and the growing usage of 3D-printing technologies, we speculate that some 3D-printed plastic waste may represent a significant and yet very poorly uncharacterized environmental hazard that merits further investigation on a range of aquatic and terrestrial species.