離子强度對氧化銅奈米顆粒特性改變與斑馬魚胚胎毒性與累積性研究

Transformation of CuO nanoparticles by ions in the solution and their effects on copper toxicity and bioaccumulation in zebrafish embryo

林志强(Chi-Cuong Lam)

指導教授 ： 黃志彬

國立交通大學/工學院/環境工程系所/碩士(2019年)

摘要

氧化銅奈米顆粒(CuO nanoparticles, CuO NPs)被廣泛應用在工業和農業，使這些CuO NPs的使用量大增，且因其具有較大的比表面積、高吸附能力及其不易被生物降解等特性，使它們能夠快速的累積在環境中。然因水體的pH、天然有機物和離子濃度(即離子強度) ionic strength, IS)等環境會改變其顆粒尺寸或溶解度，造成顆粒的轉化，進而影響到顆粒對環境和生態的毒性。水體環境種類繁多，如淡水、地下水和工業廢水等，其離子強度的差異，致使CuO NPs的轉化與在水環境傳輸也不盡相同。因此，本研究以斑馬魚胚胎來評估不同離子強度的懸浮溶液對CuO NPs的毒性和生物累積的影響。本研究自行配製三種不同離子強度的含CuO NPs懸浮溶液進行斑馬魚胚胎的致畸胎和生物累積性評估，分別為模擬淡水(SW, IS: 1.5 mM)、地下水(GW, IS: 15 mM)與廢水(WW, IS: 54 mM)。另為釐清CuO NPs在不同模擬水體條件所溶出的銅離子對斑馬魚胚胎的毒性影響，本試驗選用三種銅離子溶液(CuSO4、CuCl2、Cu(NO3)2)進行暴露試驗。
結果顯示相較於GW和WW，SW中會有較小粒徑的CuO NPs和較高的溶解度，使得其對斑馬魚胚胎具有較大的致畸胎性，顯著地延遲胚胎孵化時間和增加畸胎幼魚的比例。在SW中，10 mg CuO/L對斑馬魚的致死率有20%，僅有18.7%的胚胎成功孵化；而在GW和WW中，其胚胎成功孵化分別為25.6%和30.3%，顯示較低的IS對胚胎孵化具有較高的抑制性。此外，幼魚畸胎比例也有相同的趨勢，在SW、GW和WW中的致畸胎幼魚的比例依序為48.6%、34%和27%，其畸形型態以卵黃不透明(opaque yolk)為主；幼魚心跳次數也隨著IS降低而顯著減緩，SW、GW和WW中的平均心跳次數依序為27.8 ± 2.9、28 ± 3.4和28.9 ± 2.4次/10秒。在SW中，大約15％的斑馬魚胚胎在10mg CuO / L的濃度下心跳低於25跳/ 10s，而在WW中這個值僅為5％左右在48小時的生物累積性部分則顯示在SW中，有35%的銅會進入累積到胚胎內部；在WW中則僅有15%，意味在較低的IS介質中，會提升CuO NPs在胚胎內的累積量。此外，SW中的CuO NPs主要累積在胚胎的心臟；而GW和WW中CuO NPs則大都累積在體軸和尾部。綜上所述，較低的IS介質中(如SW)的CuNPs會有較小的粒徑和較高的溶解性，進而對胚胎具有較大的毒性與累積性，意味CuO NPs釋放在地表水或地下水體後，對環境或生態所造成的衝擊不容忽視。

關鍵字

毒性 ； 斑馬魚 ； 氧化銅奈米 ； 離子强度

並列摘要

With the rapid development of nanotechnology in the last few decades, there have been increasing concerns over the impact of nanoparticles (NPs) to human health, when they are released into aquatic environment, especially for copper oxide NPs (CuO NPs). CuO NPs is one of the common NPs, which is widely used in many industrial and agricultural applications. Once CuO NPs are released into the aquatic environment, the transformation of CuO NPs (i.e., hydrodynamic size and solubility) may occur, leading to change in their toxicity. Apart from the pH and natural organic matter (NOM), ion concentrations (i.e., ionic strength) is one of the typical parameters that directly affect the fate and transport of CuO NPs in aquatic environment. Therefore, this study investigated transformation of CuO nanoparticles by ions in the solution and their effects on copper toxicity and bioaccumulation by monitoring the responses and uptake behaviors of zebrafish embryo.
In this study, two copper sources were used, including copper nanoparticles (copper oxide nanoparticle, CuO NPs) and Cu ions. Besides, the simulated waters were prepared according to the ionic strength (IS) of surface water (SW), groundwater (GW), and wastewater (WW), which was 1.5, 15 and 54 mM, respectively. Acute toxicity & teratogenicity and bioaccumulation of CuO NPs and Cu ion were investigated using zebrafish embryo.
The results showed that SW caused the transformation of CuO NPs by reducing the hydrodynamic size and increasing the solubility, and led to the changes in toxic potential. The effect of CuO-NPs was mainly contributed to delay zebrafish hatching time and increase malformation instead of the mortality. In SW, for example, the mortality of zebrafish was 20% at the concentration of 10 mg CuO/L and only 18.7% of embryos had successful hatching. At the same concentration of CuO NPs, the rise of IS enhanced the hatched embryos (18.7%, 25.6% and 30.3% for SW, GW, and WW, respectively). Similarly, the total abnormal development of embryo was reduced by the increase of IS (48.6%, 34% and 27% for SW, GW, and WW, respectively). Furthermore, the heartbeat of larvae in SW was lowered than those obtained in GW and WW with the exposure 10 mg CuO/L (27.8 ± 2.9, 28 ± 3.4 and 28.9 ± 2.4 beats/10s). In SW, approximately 15% of larvae had the heartbeat below 25 beats/10s in the concentration of 10 mg CuO/L, while this value was only around 5% in WW. Moreover, in SW, 35% of uptake copper was found in inside zebrafish embryo. In contrast, there was only 15% in WW. Besides, the small diameter of CuO NPs in SW mainly accumulated in the heart of zebrafish larvae, whereas the big size of CuO NPs in GW and WW was found abundantly in body axis and tail.
In summary, the low IS solution (e.g., SW) could drop the hydrodynamic size and raise the solubility of CuO NPs. Thereby, the toxic potential of CuO NPs was enhanced in the low IS solution. The SW not only induced the reduction of hatching rate and heartbeat but also resulted in the increase of mortality and malformation. Therefore, the release of CuO NPs into low IS solutions such as surface water or groundwater would raise many concerns about their toxicity and bioaccumulation.

並列關鍵字

Toxicity ； Zebrafish ； CuO NPs ； Ionic Strength

參考文獻

Alsop, D., & Wood, C. M. (2011). Metal uptake and acute toxicity in zebrafish: Common mechanisms across multiple metals. Aquatic Toxicology, 105, 385–393.

Alstad, N. E. W., Kjelsberg, B. M., Vøllestad, L. A., Lydersen, E., & Poléo, A. B. S. (2005). The significance of water ionic strength on aluminium toxicity in brown trout (Salmo trutta L.). Environmental Pollution, 133, 333–342.

Applerot, G., Lellouche, J., Lipovsky, A., Nitzan, Y., Lubart, R., Gedanken, A., & Banin, E. (2012). Understanding the antibacterial mechanism of CuO nanoparticles: Revealing the route of induced oxidative stress. Small, 8, 3326–3337.

Bagatto, B. (2005). Ontogeny of cardiovascular control in zebrafish (Danio rerio): Effects of developmental environment. Comparative Biochemistry and Physiology - A Molecular and Integrative Physiology, 141, 391–400.

Bai, W., Zhang, Z., Tian, W., He, X., Ma, Y., Zhao, Y., & Chai, Z. (2010). Toxicity of zinc oxide nanoparticles to zebrafish embryo: A physicochemical study of toxicity mechanism. Journal of Nanoparticle Research, 12, 1645–1654.