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環境奈米評析與綠色化學檢測技術研究(3/3)-環境水體奈米物質之細胞毒性研究

中文摘要 奈米技術已應用在許多工業(如紡織、電子、半導體),因此人造奈米微粒有可能在工業廢水的排放過程中進入環境水體,不僅污染生態環境,且有可能經由食物鏈危害人體健康。目前奈米微粒之毒性試驗大多是在成分簡化的實驗條件下進行,然而當奈米微粒進入自然環境中,可能發生化學轉化而改變其物理化學特性,進一步影響其生物效應。因此如何評估環境中奈米微粒之風險與危害效應是各國都在面對的重要議題。為了解決這個議題,本委託計畫案將嘗試建立水體奈米微粒毒性檢測的標準方法草案,並依此測試環境水體中(包括二仁溪、曾文水庫與成功湖)採樣到的奈米物質的生物危害性。目前我們已針對國際標準組織(International organization for standardization, ISO)、經濟合作暨發展組織(the organization for economic cooperation and development, OECD)與奈米技術檢測實驗室NCL (nanotechnology characterization laboratory)等國際單位公告之奈米微粒毒性檢測方法學進行文獻回顧,並撰寫適於檢測環境水體之奈米微粒的毒性之細胞株培養與斑馬魚胚胎模式標準方法草案,其細胞培養模式包括存活率、細胞凋亡、自噬作用與氧化壓力;斑馬魚胚胎包含存活率與半致死濃度、外觀型態畸形率、體長量測、氧化壓力測試和細胞凋亡,我們也利用人造奈米氧化鋅及奈米銀及相對應之大分子材料進行測試,並提出可行之標準方法草案,而第二階段我們已採集第一、第二季節之成功湖、二仁溪與曾文水庫環境水體,共計六件環境水體,並且檢測奈米微粒之粒徑及分散情形,穿透式電子顯微鏡檢分析結果發現不論第一季及第二季之環境水樣皆有較小粒徑之微粒,但進行動態光散射分析時,卻發現除第一季節二仁溪水樣以外,其餘樣本的微粒含量皆低於偵測極限。而在細胞暴露模式中,分別暴露0.22與0.1μm濾紙上之顆粒物質,其結果顯示不論是第一季或者第二季的三個水體樣品皆無顯著的細胞毒性效應,且只有在成功湖的樣品中偵測到些許的ROS生成。此外在斑馬魚胚胎模式中可發現,不論是成功湖、二仁溪或曾文水庫的水體,經過1、0.45、0.22與0.1 μm濾膜過濾之濾液都不會讓其存活率明顯下降,而於體長量測上可發現第一、二季節之成功湖、二仁溪及曾文水庫皆會使體長增加,並且環境水樣中皆不會誘發氧化壓力及細胞凋亡之增加,綜上所述,我們藉由以超純水分別添加人造奈米氧化鋅及奈米銀的實驗建立可行之標準方法草案,而六件環境水體的分析結果顯示,其存在環境水體之奈米微粒並不足以對選定細胞株培養與斑馬魚胚胎模式產生顯著毒性。
中文關鍵字 奈米微粒、細胞毒性、斑馬魚胚胎模式

基本資訊

專案計畫編號 EPA-106-13S5-02-01 經費年度 106 計畫經費 1500 千元
專案開始日期 2017/02/23 專案結束日期 2017/12/31 專案主持人 王應然
主辦單位 環檢所 承辦人 許令宜 執行單位 國立成功大學

成果下載

類型 檔名 檔案大小 說明
期末報告 EPA10613S50201.pdf 17MB

Development of green chemstry techniques for environmental nanoparticle exposure asssessmnent (3/3)-A study assessing cytotoxicity of nanoparticles from environmental water sources

英文摘要 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.
英文關鍵字 Nanoparticles, cytotoxicity, zebrafish embryo