十溴聯苯醚與二氧化矽奈米顆粒共存對斑馬魚胚胎致畸胎性及生物累積性之研究

Abstract

環境中流佈的奈米顆粒除了自身就會對環境與生物造成危害，也因其具有較大的比表面積和吸附能力，易與環境中的其他污染物質反應，不僅影響自身在環境中的穩定性，同時也會干擾污染物的傳輸，提升其在生態環境的不確定性。十溴聯苯醚為(decabrominated diphenyl ethers, BDE-209)近年備受關注的新興化學物質，常作為電腦的主機殼或防火建材的阻燃劑，其脂溶性高且揮發性低，雖然在水中的含量不高，但易累積在河川沉積物，若與奈米顆粒共存，BDE-209可能會被奈米顆粒吸附，增加被水生生物攝入或接觸的機會，進而提升其生物可利用性。本研究利用斑馬魚胚胎來評估BDE-209與奈米顆粒共存對環境生物造成的影響，選用二氧化矽(nano-SiO2 particles, nSiO2)作為目標奈米顆粒，胚胎毒性效應評估包含急毒性與致畸胎性、長期暴露對幼魚的移動行為和生物累積性。 第一部分的研究主要探討BDE-209與nSiO2共存對斑馬魚胚胎造成的生物急毒性與致畸胎性評估。研究結果顯示單獨BDE-209對斑馬魚胚胎不具生物急毒性和致畸性；但BDE-209和nSiO2同時存在會使得胚胎在60小時會有短暫地延遲孵化的情形發生；在96小時也會造成幼魚的心跳速率減緩(28.3次/10秒)，且有45.8%的幼魚有心律不整(arrhythmia)的情形；同時也會提高斑馬魚幼魚畸形率，畸形型態包含脊椎彎曲(33.9%)、心包膜腫大(23.4%)和卵黃囊腫大(18%)，顯示出nSiO2的添加會增加BDE-209和斑馬魚胚胎接觸的機會進而造成胚胎發育的異常，提升BDE-209對環境生物的危害。此外，在每個胚胎發育的時期先行移除胚胎絨毛膜後進行暴露試驗，結果顯示在體節形成期(segmentation stage)的胚胎對BDE-209最為敏感，造成的畸形程度最高。 第二部分的研究主要探討在有nSiO2存在的情況下，BDE-209對斑馬魚胚胎吸附或吸收之影響，分別針對胚胎外殼的絨毛膜和內部胚胎的累積進行討論。結果顯示胚胎絨毛膜對單獨nSiO2的累積量顯著高於內部胚胎，絨毛膜對nSiO2的吸附量至48小時皆未達飽和(129-200 mg nSiO2/g)，吸附量隨著暴露時間的增加有顯著的上升；內部胚胎則在6小時候達吸附平衡(0.42-0.54 mg nSiO2/g)。同時暴露BDE-209和nSiO2之胚胎絨毛膜至48小時的BDE-209累積量(17.26±1.41 mg BDE-209/g)明顯高於單獨暴露的BDE-209 (6.13±1.55 mg/g)；內部胚胎之BDE-209累積量也有相同的趨勢，單獨暴露BDE-209的胚胎內部在第48小時才檢測出BDE-209的存在，而BDE-209和nSiO2共存在第3小時暴露後BDE-209的累積量即顯著的提升。此外，從SEM和EDS的分析結果在內部胚胎可發現矽的存在，特別是在胚胎卵黃囊相對有較高的分布量。 第三部分為斑馬魚胚胎在14天長期共暴露於BDE-209和nSiO2對幼魚的游泳行為和生物累積性之影響。結果顯示共暴露BDE-209和nSiO2之幼魚的魚鰾異常率(35.6-57.8%)較單獨暴露BDE-209的幼魚(< 8%)高，且幼魚魚鰾充氣面積也顯著小於控制組和單獨暴露的幼魚；此現象也和幼魚的游泳行為有相同的趨勢，隨著nSiO2的添加濃度增高，幼魚的游泳速度(1.55-1.91 mm/s)也顯著低於單獨暴露於BDE-209 (3.09-3.37 mm/s)，推測nSiO2會使BDE-209累積在幼魚魚鰾上，干擾其充氣進而減緩幼魚的游泳速度。累積性研究結果發現添加nSiO2會增加BDE-209在幼魚的累積量，且加速BDE-209在幼魚魚體代謝脫溴，在短時間即可發現毒性較高的BDE-183和BDE-153存在於斑馬魚體。上述結果顯示nSiO2的存在不僅會增加BDE-209在斑馬魚胚胎外層絨毛膜的累積和穿透至胚胎內部，而造成斑馬魚胚胎的致畸胎效應；長時間的暴露也會減緩其泳速，並增加BDE-209在幼魚魚體內的累積量和脫溴速率。因此，奈米顆粒若和水環境中有機污染物共存，如在半導體製程中的化學研磨廢水(chemical mechanical polishing, CMP)裡含有大量的nSiO2，它會吸附廢水中的其他污染物質，且可作為一個載體，不僅會提升污染物在生物體的累積量，增強污染物對生物的毒性效應，進而對環境與生態造成深遠的影響和衝擊，故考慮到對環境永續發展的問題，勢必更加重視這類的議題。

The rapidly growing and widespread application of nanotechnology in high-tech industries such as semiconductor manufacturing and biomedical engineering result in release of manufactured nanoparticles (NPs) into the aquatic environment, especially nano-SiO2 particle (nSiO2). Decabromodiphenyl ether (BDE-209), an emerging contaminant, is a flame retardant used in consumer electronic equipment. The interaction between NPs and BDE-209 in aquatic environment is getting much concern since the co-existence of NPs and BDE-209 can strengthen the transport of these contaminants into aquatic organisms, thus promoting potential toxic impacts. Therefore, we investigate the teratogenic effects and uptake mechanism of the interactions between BDE-209 and zebrafish in the presence of nSiO2. The first part of this thesis was to investigate the influence of nSiO2 on the teratogenic responses of zebrafish embryo to BDE-209. Zebrafish embryo was exposed to BDE-209 in the absence and presence of nSiO2 for 96 hours post fertilization (hpf). Results also show that embryo temporarily delayed hatching when co-exposure to BDE-209 and nSiO2 at 60 hpf. Furthermore, there was heartbeat decline (28.3 beats/10s) and increase in irregular heartbeat (45.8%) in zebrafish larvae at 96 hpf, compared to the sole exposure to BDE-209 (32.7 beats/10s and 0%). Malformation in terms of spinal curvature (SC), pericardial edema (PE) and yolk sac edema (YSE) were observed on zebrafish larvae at 33.9, 23.4, and 18%, respectively. Overall, abnormal development of zebrafish was apparent at the co-existence of BDE-209 and nSiO2. Besides, the segmentation stage of the embryo was the most sensitive to BDE-209. As a result, nSiO2 could facilitate the transport of BDE-209 towards zebrafish embryos and negatively impact the development of zebrafish. The second part of this thesis investigated the influence of nSiO2 on the uptake behavior of BDE-209 by zebrafish embryo. The amount of BDE-209 on the outside chorion and inside embryo (dechorionated embryo) were measured. For single exposure of nSiO2, results show that nSiO2 accumulation on the chorion surface was higher than that of the dechorionated embryo. Clearly, nSiO2 accumulation on the chorion surface increased (129-200 mg nSiO2/g) with increasing exposure time (48 hpf), while the equilibrium adsorption of nSiO2 on the dechorionated embryo was approximately 0.42-0.54 mg nSiO2/g at 6 hpf. Results show that the formation of nSiO2-BDE-209 associates promoted both extracellular and intracellular uptake of BDE-209 by zebrafish embryo, thereby increasing the bioconcentration of BDE-209 on the chorion surface and embryo. The results also reveal that the accumulative amount of BDE-209 on the chorion was remarkably greater than that on dechorionated embryo at 48 hpf. Clearly, the uptake of BDE-209 was 17.2±0.45 mg/g chorion (or 86 ng BDE-209/chorion/embryo) and 0.37±0.01 mg/g embryos (or 18.6 ng BDE-209/dechorionated embryo/embryo), respectively under the co-exposure of BDE-209 and nSiO2. Results from the SEM and EDS analysis revealed that nSiO2 already passed through the chorion and adhered to the inside embryo. The third part of this thesis was to evaluate the 14-day long-term exposure of BDE-209 and nSiO2 on swimming performance and bioaccumulation of zebrafish larvae. Results showed that the degree of swimming bladder disorder was 35.6-57.8% after co-exposure to BDE-209 and nSiO2 compared to single exposure (< 8%). Also, the area of swimming bladder was significantly smaller than that of BDE-209 alone. The above results were consistent with the change in swimming behavior. Exposure to BDE-209 and nSiO2 caused a significant effect on swimming speed (1.55-1.91 mm/s) in comparison with the control larvae (2.94-3.41 mm/s). Furthermore, BDE-209 accumulation under co-exposure of BDE-209 and nSiO2 was 3-7 folds that of single exposure at 7 dpf. Interestingly, both BDE-183 and BDE-153 was observed in larvae due to the metabolic debromination of BDE-209. Results suggested that the presence of nSiO2 facilitated the debromination of BDE-209 and produced more toxic lower-brominated PBDEs. All relevant evidences suggested that nSiO2 aggregate acted as a carrier of the accumulation of BDE-209 on the chorion surface and accelerate the penetration of BDE-209 into embryo, thereby the bioconcentration of BDE-209 was significantly enhanced in zebrafish, and increased the ecotoxicity of BDE-209. Moreover, the decline of swimming speed and enhancement bioaccumulation of BDE-209 in zebrafish larvae were induced after long-term exposure. The toxicity enhancement of BDE-209 may be due to the synergy effects of BDE-209 adsorption by nSiO2 aggregates. Interactions between nanoparticles and hazardous chemicals can have far-reaching implications on ecological health.