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研究生：	施詠笙 Shih, Yung-Sheng
論文名稱：	天然水體之化學物質對奈米銀及氧化鋅微粒和斑馬魚胚胎行為的毒性影響 Effects of Natural Water Chemistry on Ag/ZnO Nanoparticles Behavior and Toxicity to Embryos of Zebrafish (Danio rerio)
指導教授：	王應然 Wang, Ying-Jan
學位類別：	碩士 Master
系所名稱：	醫學院 - 環境醫學研究所 Department of Environmental and Occupational Health
論文出版年：	2019
畢業學年度：	107
語文別：	中文
論文頁數：	98
中文關鍵詞：	奈米銀、奈米氧化鋅、奈米毒性、斑馬魚胚胎、水生毒性
外文關鍵詞：	zebrafish embryos, silver nanoparticles, zinc oxide nanoparticles, Nanotoxicity, aquatic toxicity
相關次數：	點閱：85 下載：2
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奈米微粒在日常生活中的應用越來越廣泛，例如醫療保健和工業產業，因此奈米微粒大量增加進入到環境水體中的可能性。現今奈米微粒的毒性暴露評估主要是在實驗室已知條件和純淨去離子水，然而，奈米微粒可能因周圍環境不同因而改變其物化特性及生物毒性，因此探討環境水體中奈米微粒之毒性效應及奈米微粒進入環境水體之生物毒性效性是個重要的議題。為探討奈米微粒之毒性效應，本研究使用斑馬魚胚胎探討奈米微粒在自然環境中的物理化學變化，及其所誘發之生物毒性效應。
為了萃取出環境水體之奈米微粒亦不改變其物化特性，我們使用物理不同孔徑過濾，針對過濾環境水體之奈米微粒結果顯示，環境水體之濾液對於存活率、氧化壓力及細胞凋亡試驗中，並不會導致明顯生物毒性，而暴露自然水體濾液斑馬魚的體長較長；另外，添加奈米微粒至環境水體結果證實，不論是成功湖、曾文水庫、二仁溪在添加奈米銀及奈米氧化鋅微粒後，奈米微粒會聚集為較大的顆粒，相較於純水而言，環境水體可減緩奈米銀及奈米氧化鋅微粒所誘發的毒性效應，其中曾文水庫相較成功湖和二仁溪，其斑馬魚胚胎存活率較低，，而於細胞毒性機轉上，添加於環境湖水中的奈米銀和奈米氧化鋅微粒會誘發氧化壓力、細胞自噬、細胞凋亡…等機轉的發生，但曾文水庫相較成功湖和二仁溪亦會誘發較顯著的氧化壓力、細胞自噬、細胞凋亡現象。
本研究證實過濾成功湖、二仁溪及曾文水庫水體中之奈米微粒，不會引發明顯毒性。而於添加奈米微粒至環境水體實驗結果證實，不論成功湖、曾文水庫、二仁溪在添加奈米銀及奈米氧化鋅微粒後，奈米微粒會導致斑馬魚胚胎死亡率增加，並且誘發氧化壓力、細胞自噬、細胞凋亡…等細胞毒性機轉，但相較於純水而言，奈米微粒進入環境水體後會與環境水體的元素發生化學反應，改變其物化特性並且聚集成較大的顆粒，因而減緩奈米銀及奈米氧化鋅微粒所誘發的毒性效應。

SUMMARY

The growing applications of nanoparticles have been in various aspects of daily life, such as healthcare and industrial sectors. The toxicity exposure assessment of nanoparticles mainly in the laboratory with pure Milli-Q water solvent. As a result, it is a big issue to study nanoparticles physico-chemical properties and biological effects in the natural environments. This research, we using zebrafish embryo as an in vivo modle to determine the toxic effects of nanoparticles in the natural waterZebrafish embryo did not observe a significant reduction in their survival rates when 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. AgNPs/ZnONPs changed their physicochemical characteristics and toxicity after spiking in Erren River, Zengwun Reservoir, and Cheng-Kung Lake water. The results also indicated zebrafish embryos exposed AgNPs/ZnONPs in Erren River, Zengwun Reservoir, and Cheng-Kung Lake lead to reduce the survival rate and led to mild ROS generation, autophagy, apoptosis. This study confirmed AgNPs/ZnONPs in three natural water zebrafish decrease survivial rate and mitigated cellular toxicity including oxidative stress , autophagy and apoptosis. AgNPs and ZnONPs spiked in the natural water changed the physicochemical properties and caused to aggregation. In conclusion, AgNPs/ZnONPs spiked in the natural water reduced nanoparticle triggered toxicity due to changed physicochemical properties.

Keywords: zebrafish embryos, silver nanoparticles, zinc oxide nanoparticles, Nanotoxicity, aquatic toxicity

第一章、序論	1
第二章、文獻回顧	2
2-1奈米材料	2
2-2奈米材料特性及潛在生物效應	3
2-3.奈米微粒進入環境交互作用及可能產生之毒性影響	5
2-3.1環境水體與奈米微粒之交互作用及可能產生之毒性影響	5
2-3.2工程奈米材料影響生態系統的證據:地面植物與水生動物	6
2-3.3奈米微粒於環境中流向與毒性影響	6
2-3.4奈米微粒於水體環境可能造成毒性原因	7
2-4. 奈米銀/氧化鋅的使用現況與生物不良反應	8
2-4.1 奈米銀之毒性	8
2-4.2 奈米氧化鋅之毒性	9
2-5奈米微粒所引發的毒性及細胞毒性機轉	9
2-5.1 氧化壓力(Oxidative Stress)	9
2-5.2細胞自噬(Autophagy)	10
2-5.3細胞凋亡(Apoptosis)	13
2-6斑馬魚	14
2-6.1斑馬魚基本介紹	14
2-6.2斑馬魚做為奈米物質毒性測試物種	15
2-6.3斑馬魚於環境物質監測應用	17
第三章、研究目的	19
第四章、研究材料與方法	20
4-1研究使用模式	20
4-2研究方法與實驗步驟	22
4-2.1湖水採樣及原水樣過濾	22
4-3.1奈米銀及奈米氧化鋅微粒合成	26
4-4.1奈米微粒物化特性分析	27
4-5.1奈米銀及奈米氧化鋅添加入湖水流程及策略	28
4-6.1斑馬魚養殖與實驗準備	28
4-7.1胚胎毒性測試參考準則	29
4-8.1斑馬魚胚胎之奈米銀微粒分布	31
4-9.1統計分析	31
第五章、研究架構	32
第六章、研究結果	33
6-1湖水水質基本檢測	33
6-2 原始奈米銀其奈米氧化鋅在去離子水物化特性分析	33
6-3湖水奈米微粒物化特性分析	33
6-4原水樣不經過濾暴露斑馬魚胚胎	33
6-5斑馬魚胚胎暴露不同孔徑之濾液湖水之存活率	34
6-6成功湖、曾文水庫、二仁溪濾液暴露斑馬魚胚胎之體長	34
6-7第一季及第二季成功湖、曾文水庫、二仁溪之濾液對於斑馬魚胚胎氧化壓力之影響	34
6-8第二季成功湖、曾文水庫、二仁溪之濾液對於斑馬魚胚胎細胞凋亡之影響	35
6-9不同濃度奈米銀及奈米氧化鋅微粒在去離子水和三種天然水中的變化	35
6-10奈米銀及奈米氧化鋅微粒在去離子水和三種天然水的型態變化	35
6-11奈米銀及奈米氧化鋅微粒在去離子水和三種天然湖水之光散射分析	36
6-12過濾後湖水添加奈米銀暴露班馬魚胚胎之存活率	36
6-13奈米銀在去離子水與添加入三種湖水過濾後暴露班馬魚胚胎之存活率	37
6-14奈米氧化鋅在去離子水與添加入三種湖水暴露班馬魚胚胎之存活率	37
6-15奈米氧化鋅在去離子水與添加入三種湖水過濾後暴露班馬魚胚胎之存活率	38
6-16奈米氧化鋅在去離子水與添加入三種湖水暴露班馬魚胚胎之孵化率	38
6-17過濾後湖水添加奈米銀所引發的氧化壓力	38
6-18過濾後湖水添加奈米氧化鋅所引發的氧化壓力	39
6-19過濾後湖水添加奈米銀微粒所引發的細胞自噬	39
6-20過濾後湖水添加奈米氧化鋅所引發的細胞自噬	39
6-21過濾後湖水添加奈米銀所引發的細胞凋亡	39
6-22過濾後湖水添加奈米氧化鋅所引發的細胞凋亡	40
6-23奈米銀微粒在三種天然水的結構成分	40
6-24奈米氧化鋅在三種天然水的結構成分	40
第七章、結論與討論	41
第八章、結論與建議	49
第九章、參考文獻	50
表目錄	57
Table-1 Water quality of first season NCKU lake, Zengwun Reservoir, Er-zen river and the monitoring station value.	57
Table-2 Water quality testing of second season NCKU lake, Zengwun Reservoir, Er-zen river and their monitoring station value.	57
Table-3. Dynamic light scattering analysis of AgNPs spiked in different natural water. Hydrodynamic diameters of AgNPs became larger and deposit on the bottom when nanoparticles spiked in natural water. (N.D.=Non detected)	58
Table-4. Dynamic light scattering analysis of ZnONPs spiked in different natural water. Hydrodynamic diameters of ZnONPs become larger and deposit on the bottom when nanoparticles spiked in natural water. (N.D.=Non detected)	58
圖目錄	59
Fig. 1. Transmission electron microscope images of natural water in first season (A) NCKU lake; (B) Zengwun Reservoir; (C) Er-zen river water. Scale bar (A) 2 µm, 200 nm, 50 nm ; (B) 0.2µm, 200 nm, 0.2µm; (C) 10 µm, 1 µm, 0.2 µm	59
Fig. 2. Energy-dispersive X-ray spectroscopy analysis of two points in natural water in first season (A) NCKU lake; (B) Zengwun Reservoir; (C) Er-zen river after passing 0.45 µm filter.	60
Fig. 3. Transmission electron microscope images of natural water in second season (A) NCKU lake; (B) Zengwun Reservoir; (C) Er-zen river water. Scale bar, (A) 10 µm, 0.5 µm, 200 nm; (B) 10 µm, 0.5 µm, 200 nm; (C) 10 µm, 0.5 µm, 200 nm	61
Fig. 4. Energy-dispersive X-ray spectroscopy analysis of two points in natural water in second season (A) NCKU lake; (B) Zengwun Reservoir; (C) Er-zen river after passing 0.45 µm filter.	62
Fig. 5. First season survival rate of zebrafish embryos exposed to original water (A) survival rate of zebrafish embryos exposed to original. Survival rate of each natural water groups are high. 4 mg/L 3,4 Dichloroaniline (DCA) is positive control for the survival rate of zebrafish embryos. Pictures of exposing in 48 hpf is (B) NCKU lake ; (C) Zengwun Reservoir ; (D) Er-zen river 0, 24, 48, 72, 96 hpf with different natural water. (N=30)	63
Fig. 6. First season survival rate of zebrafish embryos exposed to original after passing 1 µm, 0.45 µm, 0.22 µm, 0.1 µm filter (A) NCKU lake (B) Zengwun Reservoir (C) Er-zen river 0 ,24, 48, 72, 96 hpf with different natural water. Survival rate is high in each natural water. (N=30)	64
Fig. 7. Second season survival rate of zebrafish embryos exposed to original after passing 1 µm, 0.45 µm, 0.22 µm, 0.1 µm filter (A) NCKU lake (B) Zengwun Reservoir (C) Er-zen river 0 ,24, 48, 72, 96 hpf with different natural water. Survival rate is high in each natural water. (N=30)	65
Fig. 8. First season body length of zebrafish embryos exposed to original after passing 1 µm, 0.45 µm, 0.22 µm, 0.1 µm filter (A) NCKU lake (B) Zengwun Reservoir (C) Er-zen river 72, hpf with different natural water. Body length is significantly longer than MQ water in each natural water (*p≤0.05). (N=30) Body length of zebrafish embryos were measured by view 7 software.	66
Fig. 9. Second season body length of zebrafish embryos exposed to original after passing 1 µm, 0.45 µm, 0.22 µm, 0.1 µm filter (A) NCKU lake (B) Zengwun Reservoir (C) Er-zen river 72, hpf with different natural water. Body length is significantly longer than MQ water in each natural water (*p≤0.05). (N=30). Body length of zebrafish embryos were measured by view 7 software.	67
Fig. 11. Zebrafish embryos exposed to first season Zengwun Reservoir water after passing 1 µm, 0.45 µm, 0.22 µm, 0.1 µm filter. After 72 hpf, zebrafish embryos were stained with DCFH-DA. ROS stained are rare in zebrafish embryos	69
Fig. 12. Zebrafish embryos exposed to first season Er-zen river water after passing 1 µm, 0.45 µm, 0.22 µm, 0.1 µm filter. After 72 hpf, zebrafish embryos were stained with DCFH-DA. ROS stained are rare in zebrafish embryos.	70
Fig. 13. Zebrafish embryos exposed to second season NCKU lake water after passing 1 µm, 0.45 µm, 0.22 µm, 0.1 µm filter. After 72 hpf, zebrafish embryos were stained with DCFH-DA. ROS stained are rare in zebrafish embryos.	71
Fig. 14. Zebrafish embryos exposed to second season Zengwun Reservoir water after passing 1 µm, 0.45 µm, 0.22 µm, 0.1 µm filter. After 72 hpf, zebrafish embryos were stained with DCFH-DA. ROS stained are rare in zebrafish embryos.	72
Fig. 15. Zebrafish embryos exposed to second season Er-zen river water after passing 1 µm, 0.45 µm, 0.22 µm, 0.1 µm filter. After 72 hpf, zebrafish embryos were stained with DCFH-DA. ROS stained are rare in zebrafish embryos.	73
Fig. 16. TUNEL assay at 4 hpf zebrafish embryos exposed to second season NCKU lake water after passing 1 µm, 0.45 µm, 0.22 µm, 0.1 µm filter until 72 hpf. Red color represents apoptotic cells.	74
Fig. 17. TUNEL assay at 4 hpf zebrafish embryos exposed to second season Zengwun Reservoir water after passing 1 µm, 0.45 µm, 0.22 µm, 0.1 µm filter until 72 hpf. Red color represents apoptotic cells.	75
Fig. 18. TUNEL assay at 4 hpf zebrafish embryos exposed to second season Er-zen river water after passing 1 µm, 0.45 µm, 0.22 µm, 0.1 µm filter until 72 hpf. Red color represents apoptotic cells.	76
Fig. 19. 5, 10, 15, 20, 25, 50, 75 and 100 µg/ml silver nanoparticles (AgNPs) and zinc oxide particles (ZnONPs) spiked in NCKU lake, Zengwun Reservoir, Er-zen river natural water. AgNPs and ZnONPs are aggregated and deposited on the bottom.	77
Fig. 20. Transmission electron microscope images of silver nanoparticles (AgNPs) spiked in (A) Milli-Q water(MQ); (B) NCKU lake; (C) Zengwun Reservoir (Zengwun); (D) Er-zen river natural water. One sample had two images. AgNPs aggregate and change shape in the natural water.	78
Fig. 21. Energy-dispersive X-ray spectroscopy analysis of AgNPs spiked indifferent water. One smaple had two images. (A) Milli-Q water; (B) NCKU lake; (C) Zengwun Reservoir; (D) Er-zen river.	79
Fig. 22. Transmission electron microscope images of zinc oxide nanoparticles (ZnONPs) spiked in (A) Milli-Q water (MQ); (B) NCKU lake; (C) Zengwun Reservoir (Zengwun); Er-zen river natural water. AgNPs aggregate and change shape in the natural water.	80
Fig. 23. Energy-dispersive X-ray spectroscopy analysis of ZnONPs spiked indifferent water. One smaple had two images. (A) Milli-Q water; (B) NCKU lake; (C) Zengwun Reservoir; (D) Er-zen river	81
Fig. 24. Two different concentrations (1, 10 μg/ml) survival rate (A) (B) of zebrafish embryos exposed to AgNPs spiked in (C) NCKU lake; (D) Zengwun Reservoir (Zengwun); (E) Er-zen river water passing 0.45 μm filter. 0 ,24, 48, 72, 96 hpf with different natural water. Nanoparticles aggregated/agglomerated in natural water (F). AgNPs in Milli-Q water is more toxicity than natural water. (N=30)	82
Fig. 25. Two different concentrations (1, 10 μg/ml) survival rate (A) (B) of zebrafish embryos exposed to AgNPs spiked in (C) NCKU lake; (D) Zengwun Reservoir (Zengwun); (E) Er-zen river water and then passed 0.45 μm filter. 0 ,24, 48, 72, 96 hpf with different natural water. AgNPs in Milli-Q water is more toxicity than natural water. (N=30)	83
Fig. 26. Two different concentrations (1, 10 μg/ml) survival rate (A) (B) of zebrafish embryos exposed to ZnONPs spiked in (C) NCKU lake; (D) Zengwun Reservoir (Zengwun); (E) Er-zen river water passing 0.45 μm filter. 0 ,24, 48, 72, 96 hpf with different natural water. 10μg/ml ZnONPs in Milli-Q water is more toxicity than natural water. (N=30)	84
Fig. 27 Two different concentrations (10, 50 μg/ml) survival rate (A) (B) of zebrafish embryos exposed to ZnONPs spiked in NCKU lake ; (C) Zengwun Reservoir ; Er-zen river water and then passed 0.45 μm filter. 0 ,24, 48, 72, 96 hpf with different natural water. ZnONPs in Milli-Q water is more toxicity than natural water but toxicity is lower after passing 0.45 μm filter. (N=30)	85
Fig. 28. Hatching rate of zebrafish embryos exposed to ZnONPs spiked in NCKU lake ; Zengwun Reservoir ; Er-zen river water and then passed 0.45 μm filter. (A) 48, (B) 72, (C) 96 hpf with different natural water. ZnONPs in different water have show (D) late haching time in all groups. (N=30)	86
Fig. 29. Zebrafish embryos exposed to second season natural water after passing 0.45 µm filter and then spiked AgNPs. NCKU lake+1 µg/ml/ 10 µg/ml AgNPs (NCKU 1/NCKU 10); Zengwun Reservoir +1 µg/ml/ 10 µg/ml AgNPs (Zen 1/Zen 10); Er-zen river water+1 µg/ml/ 10 µg/ml AgNPs (Er 1/Er 10). After 72 hpf, zebrafish embryos were stained with DCFH-DA. High toxicity group show more ROS stained in zebrafish embryos.	87
Fig. 30. Zebrafish embryos exposed to second season natural water after passing 0.45 µm filter and then spiked ZnONPs. NCKU lake+1 µg/ml/ 10 µg/ml ZnONPs (NCKU 1/NCKU 10); Zengwun Reservoir +1 µg/ml/ 10 µg/ml ZnONPs (Zen 1/Zen 10); Er-zen river water+1 µg/ml/ 10 µg/ml ZnONPs (Er 1/Er 10). After 72 hpf, zebrafish embryos were stained with DCFH-DA. All groups show high ROS stained in zebrafish embryos.	88
Fig. 31. Zebrafish embryos exposed to second season natural water after passing 0.45 µm filter and then spiked AgNPs. After 72 hpf, zebrafish embryos were stained with Lyso-Tracker Red. High toxicity group show more acidic vesicles stained in zebrafish embryos.	89
Fig. 32. Zebrafish embryos exposed to second season natural water after passing 0.45 µm filter and then spiked ZnONPs. After 72 hpf, zebrafish embryos were stained with Lyso-Tracker Red. High toxicity group show more acidic vesicles stained in zebrafish embryos.	90
Fig. 33. TUNEL assay at zebrafish embryos exposed to second season natural water after passing 0.45 µm filter and then spiked AgNPs. NCKU lake+1 µg/ml/ 10 µg/ml AgNPs (NCKU 1/NCKU 10); Zengwun Reservoir +1 µg/ml/ 10 µg/ml AgNPs (Zen 1/Zen 10); Er-zen river water+1 µg/ml/ 10 µg/ml AgNPs (Er 1/Er 10). Red color represents apoptotic cells.	91
Fig. 34. TUNEL assay at zebrafish embryos exposed to second season natural water after passing 0.45 µm filter and then spiked ZnONPs. NCKU lake+1 µg/ml/ 10 µg/ml ZnONPs (NCKU 1/NCKU 10); Zengwun Reservoir +1 µg/ml/ 10 µg/ml ZnONPs (Zen 1/Zen 10); Er-zen river water+1 µg/ml/ 10 µg/ml ZnONPs (Er 1/Er 10).Red color represents apoptotic cells.	92
Fig. 35. Elemental Mapping analysis of AgNPs spiked in NCKU lake water. Element of Sulphur (S) and Chlorine (Cl) attached on AgNPs.	93
Fig. 36. Elemental Mapping analysis of AgNPs spiked in Zengwun Reservoir water. Element of (Sulphur) S and Chlorine (Cl) attached on AgNPs.	94
Fig. 37. Elemental Mapping analysis of AgNPs spiked in Er-zen water. Element of Sulphur (S) and Chlorine (Cl) attached on AgNPs.	95
Fig. 38. Elemental Mapping analysis of ZnONPs spiked in NCKU lake water. Element of Sulphur (S), Chlorine (Cl), Phosphorum (P) and a little Ferrum (Fe) attached on ZnONPs.	96
Fig. 39. Elemental Mapping analysis of ZnONPs spiked in Zengwun Reservoir water. Element of Sulphur (S), Chlorine (Cl), Ferrum (Fe) and fewer Phosphorum (P) attached on ZnONPs.	97
Fig. 40. Elemental Mapping analysis of ZnONPs spiked in Er-zen river water. Element of Sulphur (S), Chlorine (Cl), Ferrum (Fe) and Phosphorum (P) attached on ZnONPs.	98
                                    

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