Nanotechnology has been applied to various industrial sectors (e.g. textiles, electronics, and semiconductors), and therefore engineered nanoparticles (ENPs) are likely to enter the environmental waters during the processes of industrial wastewater discharges. Consequently, they not only directly pollute the eco-environments, but also indirectly endanger human health through the food chain. For the time being, toxicity assessments of ENPs have been largely carried out under simplified and controlled environmental conditions. In reality, chemical transformation of ENPs, however, may take place upon their entrance into the natural environments, thus altering their physico-chemical properties and biological effects. In light of this, how to perform hazard analysis and risk assessment of the ENPs released into the environment has become a worldwide challenging and important issue. To address it, this commissioned project aims to work out a draft regarding the standard methodologies for assessing the toxicity of nanoparticles from environmental water sources, and further conducts a survey of the harmful effects of ENPs sampled from Erren River, Zengwun Reservoir, and Cheng-Kung Lake. At present, we have finished the literature review with respect to the methodologies for nanotoxicity assessment, which have been issued from several international organizations, such as ISO, OECD and NCL. We have also written an outline of the cell line- and zebrafish embryo-based standard methodologies applicable for nanotoxicity assessment. The items pertaining to cell-line based tests include viability assay, apoptosis and autophagy analysis, and oxidative stress measurement, and the tests carried out using zebrafish embryos include measurements of the survival rate, lethal dose 50 (LD50), malformation rate, body length, oxidative stress and apoptosis. Furthermore, the synthetic nanoparticles ZnO NPs and AgNPs as well as their bulk counterparts were also subjected to the above-mentioned assessments for the purpose of providing more practical guidance. We have proceeded to the second stage of this project, with completion of the work on the first and second seasons of environmental water sampling at Erren River, Zengwun Reservoir, and Cheng-Kung Lake; a total of six independent sampling works were done during this period. We measured the dimensions of potentially contaminating ENPs and characterized their size distribution. Transmission electron microscopy analysis revealed the existence of smaller-sized NPs in the environmental water samples regardless of whether they were gathered in the first or second seasons. However, when carrying out dynamic light scattering (DLS) analyses, we found the presence of nanoparticles in all samples was at a quantity less than the detection limit, except for that obtaining from the first-season Erren River. Our in vitro studies demonstrated that exposure to particulate matters, which were isolated from the unfiltered parts of each sample on the 0.22- and 0.1-μm filter papers respectively, didn’t show significant cytotoxicity no matter whether they were sampled in first or second seasons. Surprisingly, samples from Cheng-Kung Lake led to mild ROS generation in vitro. Regarding the in vivo experiments using Zebrafish embryos, we didn’t observe a significant reduction in their survival rates when receiving exposure to filtrates, through 1, 0.45, 0.22 and 0.1 μm filters, of the samples from Erren River, Zengwun Reservoir, and Cheng-Kung Lake. Interestingly, all of these samples contributed to body length elongation of the hatched fish, whereas they didn’t evoke/enhance oxidative stress and apoptosis in such system. Altogether, we’ve drawn up the practical methodological protocols, by examining the ultrapure water separately spiked with engineered ZnONPs and AgNPs, for assessing toxicity of nanoparticles from environmental water sources. The current data suggest that nanoparticles existing in the collected environmental waters are insufficiently capable of eliciting obvious toxicity towards the selected cultured cells and zebrafish embryos.

Nanoparticles, cytotoxicity, zebrafish embryo