以斑馬魚胚胎評估半導體廢水之生物毒性

Abstract

半導體製程中常使用到許多含氮物質作為原物料，導致廢水含氮量過高，排放至水體會造成優養化及產生毒性。目前環保署公告的放流水管制標準為化學物質濃度管制，雖可有效控管各事業單位污染物的排放，卻無法反映水體生物可承受之濃度，因此目前許多國家亦使用生物毒性試驗來評估水體污染程度。相較於傳統生物毒性試驗單以死亡率為標的，斑馬魚胚胎具有繁殖容易、產量大與胚胎外觀透明等優勢，不僅可進行大量毒性暴露試驗，亦可直接使用顯微鏡觀察胚胎發育的狀態以獲得更多毒物暴露後的資訊，因此本研究選用斑馬魚(Danio rerio)胚胎來評估半導體廢水對水體生物的影響，此處使用模擬含氮廢水與半導體廢水進行胚胎毒性暴露試驗，所取用的半導體廢水為氟系與化學研磨製程混合廢水、製程酸鹼廢水以及經處理過的出流水，除觀察胚胎的死亡率，另選用不同的觀測終點如累積孵化率、心跳、體長與致畸率，分析含氮物質對胚胎發育產生的影響。 斑馬魚胚胎暴露於實驗室配置的氯化銨、硝酸鈉及氫氧化四甲基銨溶液，其96小時半數致死濃度值分別為111 mg NH3-N/L、1347.3 mg NO3--N/L及68 mg N/L，最低效應濃度(Low observed effect concentration, LOEC)為0.41 mg NH3-N/L、0.51 mg NO3--N/L及0.1 mg N/L。經含氮物質暴露後的斑馬魚，皆有累積孵化率下降、心跳次數降低、生長速度趨緩與畸形發育的現象，而畸形發育的型態有三種，暴露後出現的先後順序為脊椎彎曲，其次是心包膜腫大，最後為卵黃囊腫大。 三股半導體廢水毒性結果顯示，除了氟系與化學研磨製程混合廢水96小時死亡率為77%，其他兩股廢水的死亡率皆在20%以下。觀察尚未死亡的斑馬魚胚胎發現累積孵化率、心跳與生長體長有發育減緩的趨勢，其中又以心跳抑制的現象最為顯著，且有脊椎彎曲、心包膜腫大與卵黃囊腫大畸形發育的情形。半導體廢水毒性試驗結果雖為低死亡率，但在終點項目的觀察可發現廢水中的毒性物質對胚胎發育有產生影響，且與模擬含氮廢水有相似的畸形發育型態，表示此毒性試驗可用於評估不同類型的暴露溶液與濃度範圍，且可給予更多的毒性訊息，對於毒性評估有很大的潛力，未來可使用此毒性暴露試驗評估毒性物質對水體的潛在危害。

In semiconductor manufacturing processes nitrogenous substances are extensively used, resulting in production of very high nitrogen-containing wastewater. The high concentration of nitrogen-containing wastewater discharged to aquatic environment may cause eutrophication and toxicity. Although Taiwan EPA has regulated effluent standards for many pollutants, they are not sufficient to tell us if the discharge is toxic or not. Therefore, many countries are using animals to detect the toxicity of wastewater. Traditional toxicity tests usually use mortality as endpoint, but the sensitivity of this method is not adequate for low concentration pollutants. Zebrafish embryo assay has been regarded as a suitable alternative to the fish acute toxicity test which, apart from ethical reasons, provides very limited data (only LC50) and no ecotoxicological relevance. Due to transparency and extra-uterine development, zebrafish embryos can be directly observed the phenotypic changes during embryonic development. As a result, in this study zebrafish embryos were used to evaluate the toxicity and teratogenicity in nitrogen-containing wastewater. The purpose of this study is to examine the acute toxicity and developmental abnormalities in synthetic and real wastewater by using zebrafish embryos. The results show the 96h-LC50 of ammonium chloride, sodium nitrate and tetramethylammonium hydroxide were 111 mg NH3-N/L, 1347.3 mg NO3--N/L and 68 mg N/L, respectively; the low observed effect concentration were 0.41 mg NH3-N/L, 0.51 mg NO3--N/L及0.1 mg N/L, respectively. The endpoints such as hatching rate, heart rate and body length significantly declined in these three nitrogenous solutions. Abnormalities including spinal curvature, pericardial edema and yolk sac edema were also observed in sequence in zebrafish larvae. In real wastewater, the HF/CMP wastewater, acid-base wastewater and effluent from a semiconductor manufacturing factory were used. The mortality of zebrafish embryos in the acid-base wastewater and effluent was under 20% and 77% in HF/CMP wastewater in 96 hours. Although the mortality was low in the acid-base wastewater and effluent, the cumulative hatching rate, body length and especially heartbeat significantly decline. Abnormalities including spinal curvature, pericardial edema and yolk sac edema were also observed in real wastewater tests. The results indicate that the zebrafish embryos can be instrumental to detect the potential harm caused by wastewater on aquatic organisms at low concentrations. It suggests that embryo toxicity assay should be used to evaluate the toxicity of wastewater in the future.