人造氧化鋅奈米顆粒於水中及底泥之毒性與攝食傳輸分析

Abstract

人造奈米顆粒 (Engineered nanoparticles, ENPs) 於工業之應用及相關消費性、醫療性商品持續增加，使其生態環境釋放量亦隨之增加。奈米氧化鋅 (ZnO-NPs) 屬於高使用量及廣泛應用性之ENPs之一，在水環境中可能產生聚集及沉降而累積人造奈米顆粒 (Engineered nanoparticles, ENPs) 於工業之應用及相關消費性、醫療性商品持續增加，使其生態環境釋放量亦隨之增加。奈米氧化鋅 (ZnO-NPs) 屬於高使用量及廣泛應用性之ENPs之一，在水環境中可能產生聚集及沉降而累積於底泥中。，同時亦有可能隨食物鏈傳輸造成攝食傳輸效應。 有鑑於ZnO-NPs於水域環境的重要性，本論文旨在探討ZnO-NPs於水中、底泥及攝食傳輸中造成之毒性。本研究使用兩種淡水底棲無脊椎生物：秀麗隱桿線蟲 (Caenorhabditis elegans) 及亞洲蜆 (Corbicula fluminea)，並完成三項主要目標：(1) 了解ZnO-NPs在環境相關濃度之慢性暴露所造成之毒性；(2) 探討底泥中ZnO-NPs之毒性；(3) 評估ZnO-NPs之攝食傳輸及其毒性效應。 在主要目標一，利用C. elegans評估環境相關濃度之ZnO-NPs之慢性毒性。結果顯示在模擬表面水 (EPA water) 中，環境濃度之ZnO-NPs導致C. elegans行為模式毒性，並顯示顆粒造成之毒性效應。此外亦顯著造成ATP levels下降及ROS (Reactive oxygen species) 上升。 在主要目標二，利用C. elegans評估ZnO-NPs在底泥中之慢性毒性，結果顯示在長期底泥暴露ZnO-NPs後， C. elegans生長及繁殖能力並未受到影響，然而在行為模式及ATP levels則被顯著干擾。此外，C. elegans體內ROS及脂質過氧化皆顯著上升。結果亦發現轉錄因子DAF-16/FOXO入核，進而啟動下游壓力反應相關基因的表達 (mtl-1及sod-3)。 在主要目標三，利用C. fluminea及C. elegans探討ZnO-NPs攝食傳輸效應。結果指出淡水底泥microcosm中覆蓋水、孔隙水及底泥之Zn濃度在28天培養後，底泥累積濃度最為顯著。並且waterborne及foodborne 共同暴露ZnO-NPs之C. fluminea，其死亡率、壓力基因表達量、DNA damage，及體內Zn濃度皆顯著上升，顯示C. fluminea之健康狀況及金屬平衡受到嚴重干擾。此外，結果亦證明ZnO-NPs累積於E. coli OP50並進一步透過攝食傳輸至C. elegans，造成C. elegans行為模式毒性及D-type GABAergic運動神經損傷。然而，和ZnCl2結果比較，ZnO-NPs攝食傳輸造成之行為模式毒性及神經損傷較顯著，顯示ZnO-NPs攝食傳輸造成之神經毒性主要來自於顆粒型態。 綜合而論，本博士論文研究成果顯示ZnO-NPs顆粒毒性效應之重要性，結果亦有助於了解環境中ZnO-NPs之行為以及對底棲生物之衝擊，並能協助相關施政單位決策及評估ENPs於水體及底泥中的環境風險。

Engineered nanoparticles (ENPs) have been increasingly used in industrial applications as well as consumer and medical products, leading to potentially increased release of ENPs into the ecosystems. Among ENPs, ZnO nanoparticles (ZnO-NPs) are among the most significant and widespread particles. They might end up in sediment due to aggregation and sedimentation processes of these nanoparticles in aquatic environment. In addition, ZnO-NPs in the environment were suggested to be transferred through food chain, causing dietary transfer effect. Given the importance of released ZnO-NPs in aquatic environment, I investigated the toxicity of ZnO-NPs in water, sediment, and dietary transfer. Two benthic freshwater invertebrates, nematodes (Caenorhabditis elegans) and bivalves (Corbicula fluminea), were used in the present study. The three specific aims that I aimed to achieve are: (1) to understand the chronic toxicity of ZnO-NPs at environmentally relevant concentrations; (2) to investigate the toxicity of ZnO-NPs in sediment; and (3) to determine the dietary transfer effect of ZnO-NPs and its toxic effects. In specific aim 1, the aquatic toxicity of ZnO-NPs with chronic exposure at environmentally relevant concentrations using the nematode C. elegans was evaluated. The results showed that predicted environmentally relevant concentrations of ZnO-NPs exposure in simulated surface water (EPA water) caused locomotive toxicities in C. elegans, indicating particle-specific toxic effect. Moreover, metabolic toxicities (decreased ATP levels) and reactive oxygen species (ROS) was induced after ZnO-NPs exposure in EPA water. In specific aim 2, the nematode C. elegans was used to evaluate the chronic toxicity of ZnO-NPs in sediment. Following long-term sediment exposure to ZnO-NPs, growth and reproduction defects were not observed in C. elegans at the examined concentrations, whereas locomotive behaviours and ATP levels were adversely affected. In addition, significant increases in intracellular ROS and lipid peroxidation were induced by long-term sediment exposure to ZnO-NPs. Furthermore, long-term sediment exposure to ZnO-NPs triggered nuclear translocation of the transcription factor DAF-16/FOXO and further activated the targeted expression of the corresponding stress-responsive genes (mtl-1 and sod-3). In specific aim 3, the dietary transfer effects of ZnO-NPs on C. fluminea and C. elegans were studied. Analysis of ionic Zn in aquatic sediment microcosms showed that Zn released into overlaying water, pore water, and sediment, with the highest amount of Zn accumulation in sediment over 28 days. Moreover, co-exposure to foodborne and waterborne ZnO-NPs (“ZnO-NPs pretreated algae + ZnO-NPs”) caused significantly increased mortality, stress genes expression, DNA damage, and Zn body burden in Asian clam C. fluminea, indicating a disrupted health state and metal homeostasis C. fluminea in sediment microcosm. In addition, dietary effects of ZnO-NPs were investigated in C. elegans. The results showed that ZnO-NPs accumulated in E. coli OP50 further transfer to C. elegans through dietary exposure, which impairs locomotive behaviors and D-type GABAergic motor neurons. However, the toxic effects on locomotive behaviors and GABAergic neurons were not apparent in C. elegans fed with ZnCl2 pretreated E. coli, indicating particulate effect on the neurotoxicity caused by dietary transfer of ZnO-NPs. In conclusion, results from this doctoral dissertation highlight the particle-specific toxic effects of ZnO-NPs and improve our understanding in ZnO-NPs environmental behavior and their impact to benthic organisms. This information was helpful for government practitioners and regulators while evaluating environmental risks and making decisions for water bodies and sediment in the environment.