Neurobehavioral and mechanistic sub-lethal studies in aquatic toxicology on potential micro-pollutants.
Creators: name: Walpitagama, Milanga
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 401609
subject: Polymers and plastics
classificationCode: 401705
subject: Microelectromechanical systems (MEMS)
classificationCode: 410402
subject: Environmental assessment and monitoring

description: Additive manufacturing (three-dimensional [3D] printing) is a rapidly developing industry that uses solid and liquid polymers during the production process. By 2027, the 3D printing industry is estimated to be a US$16 billion industry; however, the toxicity of 3D-printed polymers and chemical components leaching into aquatic systems remains largely unknown. The main objective of this research project was to evaluate the toxicity of 3D-printed polymer leachates as an emerging micropollutant. To achieve this objective, 3D-printing polymer extract acquired from Form 1 clear photoreactive resin, used in FormLabs SLA 3D printers, was studied. To determine its biological toxicity, a panel of standard ecotoxicological experiments was employed, using Danio rerio embryos, Daphnia magna, Brachionus calyciflorus, Paramecium caudatum, and Aliivibrio fischeri. The results confirmed the toxicity of the leachate, the main component of which was identified as the known cytotoxic photoinitiator compound 1- hydroxycyclohexyl phenyl ketone (1-HCHPK) using gas chromatography–mass spectrometry (GC-MS). Additionally, the leaching rate and stability of the compound were determined using GC-MS.<br><br>In further studies conducted using Danio rerio embryos, lower hatching rates and developmental abnormalities were observed. The development of oxidative stress caused by increased reactive oxygen species (ROS) levels was studied by exposing the abovementioned embryos to leachates and 1-HCHPK with a battery of bioassays. Superoxide dismutase (SOD), lipid peroxidation (MDA), protein carbonyl, and Glutathione S-transferase (GST) were used as oxidative stress markers, and the results obtained from all the bioindicators revealed elevated levels of target compounds in the presence of the leachate. Oxidative stress development caused by ROS formation is known to induce apoptosis. This study hypothesised that the developmental and behavioural abnormalities and oxidative stress observed were caused by the apoptosis of early embryo developing cells. The apoptosis of the embryos exposed to the leachate was studied using a caspase-3 whole-mount immunohistochemistry assay as well as through quantifying externalised phosphatidylserine residues with Annexin-V transgenic zebrafish embryos. The results revealed an increase in apoptotic cells in the exposed embryos. Furthermore, the Annexin-V transgenic fish embryos exposed to leachate with ROS inhibitor exhibited a reduced number of apoptotic cells, indicating apoptosis caused by ROS formation. The caspase-3 whole-mount immunohistochemistry assay conducted with pan-kinase inhibitor z-VAD-fmk confirmed that the cellular demise primarily occurred through caspase-mediated apoptosis. These results confirmed the relationship between oxidative stress and cell apoptosis caused by the leachate.<br><br>Neurotoxicity and neurobehavioural toxicity of the polymer leachate were studied with embryo (ESA assay) and larval (LPR assay) behavioural analysis, acetylcholinesterase (AChE) assay, AlamarBlue assay, and neurone and skeletal muscle development using transgenic embryos. Diminished behavioural capacity was observed in embryos and larvae in the presence of the leachate. Furthermore, zebrafish embryos exposed to the leachate showed decreases in neurotransmitter AChE. The AlamarBlue and AChE assays revealed a reduced metabolic rate, which in combination with the apoptosis explained the impact of the leachate on the organisms’ behaviour.<br><br>After observing the toxicity of the Form 1 clear photoreactive resin polymer leachate, three water treatment techniques (exposure to simulated sunlight, UV irradiation, and activated charcoal filtering) were conducted on the polymer leachate to remove itstoxicity. Among them, activated charcoal filtration was found to be the most effective approach. Polymer leachate filtered by activated charcoal exhibited diminished or reduced toxicity in all five standard ecotoxicological experiments conducted on Danio rerio embryos, Daphnia magna, Brachionus calyciflorus, Paramecium caudatum, and Aliivibrio fischeri. Furthermore, GC-MS chemical analysis revealed the absence of 1-HCHPK in the treated polymer leachate.<br><br>Furthermore, a high-throughput, semiautomated video capturing, and behavioural analysis protocol was developed using zebrafish early embryo spontaneous behaviour (ESA), which was used to identify the behavioural alterations of environmental pollutants using chorionated and dechorionated embryos. The results proved this newly developed protocol to be faster, more sensitive, and with a higher-throughput than conventional lethal toxicity tests. Therefore, it was used for analysing the behavioural alterations of zebrafish embryos in the presence of polymer leachate obtained from 3D-printed objects developed using Form 1 clear photoreactive resin.