本研究主要針對工業放流水為目標，區分工業園區廠商放流水（潛在性）化學物質評估、生物急毒性模式生物檢測方法建立、工業放流水毒性鑑定方法建立及污水處理廠放流水（潛在性毒物）的環境宿命等5大研究主軸。
工業放流水評估：重金屬選擇硫酸銅、硫酸鋅、硫酸鎘、硫酸鎵、硫酸銦、硝酸銅、硝酸鎘、硝酸鉛、三氧化鉬等金屬鹽類代表放流水主要毒物，作為參考毒物對水蚤、羅漢魚、斑馬魚胚胎等進行生物毒性試驗。結果顯示硝酸銅的生物毒性最嚴重。有機污染物（四甲基氫氧化銨及4-氯酚）及重金屬污染物（硫酸銅），製備具生物毒性之人工水樣，三種毒物對水蚤及羅漢魚毒性大小依序為：硫酸銅 ＞ 4-氯酚 ＞ 氫氧化四甲基銨。分別用（1）氫氧化四甲基銨/ 4-氯酚、（2）氫氧化四甲基銨/ 硫酸銅、（3）4-氯酚 / 硫酸銅、及（4）氫氧化四甲基銨 / 4-氯酚 / 硫酸銅等進行毒物混合毒性試驗。對水蚤及羅漢魚，皆以4-氯酚 / 硫酸銅之毒性為最強，且出現協同效應。含氧化物（次氯酸鈉、過氧化氫）對水蚤及羅漢魚之毒性高低依序為：次氯酸鈉＞過氧化氫；水蚤及羅漢魚的耐酸鹼範圍分別為pH 5.4~10.2及pH 4.2~10.8；二物種對二氧化矽及氟化鈣產生之濁度，皆具有極高的耐受性（＞18000 NTU）。
模式生物檢測方法建立研究：（1）建立斑馬魚胚胎毒性檢測方法；（2） 建立細菌螢光急毒性檢測方法；（3）建立短角異劍水蚤無脊椎海水生物急毒性檢測方法；（4） 建立日本青鱂魚淡水及海水水質生物急毒性檢測方法。後續進行參考毒物試驗及實際放流水檢測，結果顯示具有好的生物毒性檢測反應。
工業放流水毒性鑑定方法建立研究：我們參考國外研究建立適用於台灣工業放流水毒性鑑定程序。表明運用於水體毒性成因鑑定及評估的潛力。結果證實利用化學分析及生物毒性分析水樣，雙向評估鑑定毒化物之可行性，可改善及彌補台灣現在的工業廢水毒性鑑定方法及生物輔助檢測方式，並強化管制河川放流水監測機制。
放流水（潛在性毒物）的環境宿命研究：利用毒性鑑定評估程序與歐盟毒性效應導向分析方法結合，並以斑馬魚胚胎毒性檢測方法為輔助，有效鑑定及評估水體及沉積物中潛藏毒化物。
最後建議：（1）慎選生物毒性試驗測試生物，靈敏度須有所差異。（2）研發結合毒性鑑定評估流程及效應導向分析方法，鑑定工業廢水生物毒性及毒物。（3）研發自動化和智慧化的生物毒性方法，包括急毒性和慢毒性，以持續提升方法績效和數據品質。（4）研發生物化學和細胞基礎的高通量快篩測試方法及建置相關核心技術。（5）研發計算毒理技術及建置相關軟體設備，朝向預測和預防毒理學目標邁進。

This study is aimed at industrial wastewater, and distinguishes industrial park manufacturers' discharge water（potential chemical substances）assessment, Establishment of model bioassay method, industrial discharge water toxicity identification, and environmental fate of sewage treatment plant discharge water （potential poisons）. 5 major research spindles.
Industrial discharge water evaluation: heavy metal selection of copper sulfate, zinc sulfate, cadmium sulfate, gallium sulfate, indium sulfate, copper nitrate, cadmium nitrate, lead nitrate, molybdenum trioxide and other metal salts represent the main poison of the discharged water, as a reference poison to the water Biotoxicity tests were carried out on D.magna and Pseudorasbora parva and zebrafish embryos. The results show that copper nitrate is the most serious biological toxicity. Organic pollutants （tetramethylammonium hydroxide and 4-chlorophenol）and heavy metal pollutants （copper sulfate）to prepare bio-toxic artificial water samples. The toxicity of three poisons to D.magna and Pseudorasbora parva is: copper sulfate > 4-Chlorophenol > Tetramethylammonium hydroxide. Use（1）tetramethylammonium hydroxide / 4-chlorophenol,（2）tetramethylammonium hydroxide / copper sulfate,（3）4-chlorophenol / copper sulfate, and（4）tetramethylammonium hydroxide / 4-Chlorophenol / copper sulfate for mixed toxicity test. For D.magna and Pseudorasbora parva , the toxicity of 4-chlorophenol/copper sulfate is the strongest and synergistic effect occurs. The toxicity of sulphur（sodium hypochlorite, hydrogen peroxide）to D.magna and Pseudorasbora parva is: sodium hypochlorite > hydrogen peroxide; the acid and alkali resistance of D.magna and Pseudorasbora parva are pH 5.4 ~ 10.2 and pH 4.2 ~ 10.8, respectively. Both species have extremely high tolerance（>18000 NTU）for the turbidity produced by Silicon dioxide and calcium fluoride.
Establishment of model bioassay method:（1）Zebrafish embryo toxicity detection method;（2）Bacterial fluorescence acute toxicity detection method;（3）Apocyclops royi invertebrates marine acute toxicity detection method;（4）Method for detecting acute toxicity of freshwater and seawater in medaka. Subsequent reference poison tests and real industrial drain water tests showed good biotoxicity detection reactions.
Study on the method for the identification of industrial discharge water toxicity: We have established a procedure for the identification of industrial discharge water toxicity in Taiwan. Indicates the potential for identification and assessment of the cause of water toxicity. The results confirmed that chemical analysis and biological toxicity analysis of water samples, two-way evaluation of the feasibility of identifying poisons, can improve and make up for Taiwan's industrial wastewater toxicity identification methods and biological detection methods, and control the river discharge water monitoring mechanism.
Environmental fate study of released water（potential poisons）: The toxicity assessment procedure is combined with the Effect-Directed Analysis method, and the zebrafish embryo toxicity test method is used as a supplement to effectively identify and evaluate potential poisons in water bodies and sediments.
Final recommendations:（1）Careful selection of biological toxicity test test organisms, sensitivity must be different.（2）Research and development combined with toxicity identification evaluation process and effect-oriented analysis method to identify industrial wastewater biological toxicity and poison.（3）Automation and intelligent biotoxic methods, including acute toxicity and chronic toxicity, to continuously improve method performance and data quality.（4）Research and development of biochemical and cell-based high-throughput fast screening test methods and related core technologies.（5）Research and development of toxicology technology and the establishment of related software equipment, towards the goal of predicting and preventing toxicology.

第一章 緒論
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第二章 工業園區廠商放流水（潛在性化學物質）評估
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第三章 放流水生物毒性檢測方法建立研究
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14.USEPA, 1975. Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians. EPA-660/3-75-009
15.ASTME, 2014.Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians. 729 – 96.
16.Guido, P., Blahoslav, M., Irina, B., Andrea, T., Dzidra, Z., Levonas, M., Grzegorz, N.-J., Lucica, T., Nadejda, S., Livia, T., Boris, K., 2003. A practical and user-friendly toxicity classification system with microbiotests for natural waters and wastewaters. Environmental Toxicology 18, 395-402.
17.財團法人生物開發技術中心,1992. “生物急毒性標準試驗法建立研究”，第1-41頁。
18.行政院環境保護署，生物急毒性檢測方法－鯉魚靜水式法(NIEA B904)
19.行政院環境保護署，生物急毒性檢測方法－羅漢魚靜水式法(NIEA B902)
20.行政院環境保護署，生物急毒性檢測方法－粗首鱲靜水式法(NIEA B903)
21.行政院環境保護署，生物急毒性檢測方法－米蝦靜水式法(NIEA B905)
22.行政院環境保護署，生物急毒性檢測方法－水蚤靜水式法(NIEA B901)
23.行政院環境保護署，水樣急毒性檢測方法－藻類靜水式法(NIEA B906)
24.Nagel, R., 2002. DarT: The embryo test with the Zebrafish Danio rerio - A general model in ecotoxicology and toxicology.
25.Strähle, U., Scholz, S., Geisler, R., Greiner, P., Hollert, H., Rastegar, S., Schumacher, A., Selderslaghs, I., Weiss, C., Witters, H., Braunbeck, T., 2012. Zebrafish embryos as an alternative to animal experiments—A commentary on the definition of the onset of protected life stages in animal welfare regulations. Reproductive Toxicology 33, 128-132.
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27.OECD Guideline for the Terting of Chemicals, 2012.Draft Proposal for a New Guideline Fish Embryo Acute Aquatic Toxicity (FET) Test No.203.
28.BS-EN-ISO-15088-2008,Water quality- Determination of Acute Toxicity of Waste Water to Zebrafish Eggs(Danio rerio).
29.Weigt, S., Huebler, N., Strecker, R., Braunbeck, T., Broschard, T.H., 2011. Zebrafish (Danio rerio) embryos as a model for testing proteratogens. Toxicology 281, 25-36.
30.Incardona, J.P., Linbo, T.L., Scholz, N.L., 2011. Cardiac toxicity of 5-ring polycyclic aromatic hydrocarbons is differentially dependent on the aryl hydrocarbon receptor 2 isoform during zebrafish development. Toxicology and Applied Pharmacology 257, 242-249.
31.Kubota, A., Stegeman, J.J., Woodin, B.R., Iwanaga, T., Harano, R., Peterson, R.E., Hiraga, T., Teraoka, H., 2011. Role of zebrafish cytochrome P450 CYP1C genes in the reduced mesencephalic vein blood flow caused by activation of AHR2. Toxicology and Applied Pharmacology 253, 244-252.
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34.Tchan, Y.T., Roseby, J.E., Funnell, G.R., 1975. A new rapid specific bioassay method for photosynthesis inhibiting herbicides. Soil Biology and Biochemistry 7, 39-44.
35.ASTM STP 667, 1979.Use of Luminescent Bacteria for Determining Toxicity in Aquatic Environments.Aquatic Toxicology, 98-106.
36.U.S,2003.Environmental Protection Agency. Environmental Technology Verification Report.
37.UNE EN ISO 11348-3,2009 .Water Quality - Determination Of The Inhibitory Effect Of Water Samples On The Light Emission Of Vibrio Fischeri (luminescent Bacteria Test) - Part 3: Method Using Freeze-dried Bacteria.
38.行政院環境保護署，1994，「生物毒性篩選試驗研究」期末報告。
39.邱舜稜，2002，碩士論文「以Microtox檢測方法評估實際廢水生物毒性之研究」，國 立中央大學。
40.Shahidul Islam, M., Tanaka, M., 2004. Impacts of pollution on coastal and marine ecosystems including coastal and marine fisheries and approach for management: a review and synthesis. Marine Pollution Bulletin 48, 624-649.
41.Wang, S., Cao, Z., Lan, D., Zheng, Z., Li, G., 2008. Concentration distribution and assessment of several heavy metals in sediments of west-four Pearl River Estuary. Environmental Geology 55, 963-975.
42.Molisani, M.M., Marins, R.V., Machado, W., Paraquetti, H.H.M., Bidone, E.D., Lacerda, L.D., 2004. Environmental changes in Sepetiba Bay, SE Brazil. Regional Environmental Change 4, 17-27.
43.K.-W., L., S., R., D.-S., H., G., P.H., J.-S., L., 2007. Acute toxicities of trace metals and common xenobiotics to the marine copepod Tigriopus japonicus: Evaluation of its use as a benchmark species for routine ecotoxicity tests in Western Pacific coastal regions. Environmental Toxicology 22, 532-538.
44.Smit, M., Kater, B., Jak, R., Heuvel-Greve, M., 2006. Trandating bioassay results to field population responses using a Leslie-matrix model for the marine amphipod Corophium volutator.
45.Mauchline J, 1998. The Biology of Calanoid Copepods. Adv. Mar. Biol. 33, 1-710.
46.Nelson, J.D., Eckert, S.A., 2007. Foraging ecology of whale sharks (Rhincodon typus) within Bahía de Los Angeles, Baja California Norte, México. Fisheries Research 84, 47-64.
47.Rakhesh, M., Raman, A.V., Sudarsan, D., 2006. Discriminating zooplankton assemblages in neritic and oceanic waters: A case for the northeast coast of India, Bay of Bengal. Marine Environmental Research 61, 93-109.
48.Jürgen, J., 1961. LABORATORY CULTIVATION OF THE MARINE COPEPOD PSEUDODIAPTOMUS CORONATUS WILLIAMS1, 2. Limnology and Oceanography 6, 443-446.
49.Heinle, D.R., 1966. Production of a calanoid copepod,Acartia tonsa, in the Patuxent River estuary. Chesapeake Science 7, 59-74.
50.Su, H.M., Su, M.S., Liao, I.C., 1997. Collection and culture of live foods for aquaculture in Taiwan. Hydrobiologia 358, 37-40.
51.Kyun-Woo, L., Hans-U., D., Gi, P.H., Jung-Hoon, K., 2013. Population growth and productivity of the cyclopoid copepods Paracyclopina nana, Apocyclops royi and the harpacticoid copepod Tigriopus japonicus in mono and polyculture conditions: a laboratory study. Aquaculture Research 44, 836-840.
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53.鄭新鴻、陳凰琴、陳紫媖，2010，優質餌料生物(橈足類)量產及在種苗生產之應用研究, 東港生技研究中心, 99農科-10.3.1-水-A3(5).
54.Yuji, I., 2000. Medakafish as a model system for vertebrate developmental genetics. BioEssays 22, 487-495.
55.Jozuka K, 1976. Adaptation of gill adenosinetriphosphatase in the euryhaline teleost, medaka (Oryzias latipes). Ann. Sci. Kanazawa Univ. 13, 81-90.
56.Miyamoto T, Machida T, Kawashima S, 1986. Influence of environmental salinity on the development of chloride cells of freshwater and brackish-water medaka, Oryzias latipes. Zool. Sci. 3, 859-865.
57.Haruta, K., Yamashita, T., Kawashima, S., 1991. Changes in arginine vasotocin content in the pituitary of the medaka (Oryzia latipes) during osmotic stress. General and Comparative Endocrinology 83, 327-336.
58.Sakamoto, T., Kozaka, T., Takahashi, A., Kawauchi, H., Ando, M., 2001. Medaka (Oryzias latipes) as a model for hypoosmoregulation of euryhaline fishes. Aquaculture 193, 347-354.
59.孟振、王國棟、劉新富、劉濱、賈玉東、鲍鹰、張和森，2013.環境因素對短角異劍水蚤攝食的影響，海洋科学.，37( 1), 81-86.
60.許登瑋，2008，不同食物對橈足類短角異劍水蚤族群成長及脂肪酸組成之影響，國立臺灣大學漁業科學研究所碩士論文。
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63.鄭新鴻，2002，添加人工培養基醱酵液對培養模糊許水蚤的增殖效果，水產試驗所年報。

第四章 毒性鑑定（TIE）評估方法建立研究
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3.Aguayo S, Muñoz MJ, de la Torre A, Roset J, de la Peña E, Carballo M. Identification of organic compounds and ecotoxicological assessment of sewage treatment plants (STP) effluents. Sci Total Environ.2004; 328: 69-81.
4.Júnior HM, Silva Jd, Arenzon A, Portela CS, Ferreira ICFdS, Henriques JAP. Evaluation of genotoxicity and toxicity of water and sediment samples from a Brazilian stream influenced by tannery industries. Chemosphere .2007; 67: 1211-1217.
5.Teodorović I, Bečelić M, Planojević I, Ivančev-Tumbas I, Dalmacija B. The relationship between whole effluent toxicity (WET) and chemical-based effluent quality assessment in Vojvodina (Serbia). Environ Monit Assess .2008; 158: 381.
6.USEPA. Methods for Aquatic Toxicity Identification Evaluations: Phase I Toxicity Characterization Procedures. Second Edition. EPA/600/6-91/003(1991), 1991.
7.USEPA. Methods for Aquatic Toxicity Identification Evaluations: Phase II Toxicity Identification Procedures for Samples Exhibiting Acute and Chronic Toxicity. EPA/600/R-92/080(1993), 1993a.
8.USEPA. Methods for Aquatic Toxicity Identification Evaluations: Phase III Toxicity Confirmation Procedures for Samples Exhibiting Acute and Chronic Toxicity. EPA/600/R-92/081(1993), 1993b.
9.行政院環境保護署，生物急毒性檢測方法－水蚤靜水式法(NIEA B901)
10.行政院環境保護署，生物急毒性檢測方法－羅漢魚靜水式法(NIEA B902)
11.行政院環境保護署，生物急毒性檢測方法－鯉魚靜水式法(NIEA B904)
12.行政院環境保護署，生物急毒性檢測方法－細菌冷光法(NIEA B301)
13.行政院環境保護署，監測井地下水採樣方法（NIEA W103）
14.行政院環境保護署，河川、湖泊及水庫水質採樣通則（NIEA W104）
15.行政院環境保護署，事業放流水採樣方法（NIEA 109）
16.行政院環境保護署，環境樣品採集及保存作業指引（NIEA-PA102）
17.行政院環境保護署，水中揮發性有機化合物檢測方法吹氣捕捉／氣相層析質譜儀法（NIEA W785）。
18.行政院環境保護署，水中半揮發性有機化合物檢測方法－氣相層析質譜儀法（NIEA W801）。
19.行政院環境保護署，水中總有機碳檢測方法－過氧焦硫酸鹽／紫外光氧化法（NIEA W532）
20.行政院環境保護署，水中總溶解固體及懸浮固體檢測方法－103～105℃乾燥(NIEA W210)
21.行政院環境保護署，水中餘氯檢測方法－分光光度計法(NIEA W408.51A)
22.行政院環境保護署，水中氨氮檢測方法－分立分析系統比色法(NIEA W457)
23.行政院環境保護署，水中陰離子檢測方法－離子層析法（NIEA W415）

第五章 污水處理廠放流水（潛在性毒物）的環境宿命
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11.Brack, Werner,2011. Effect-Directed Analysis of Complex Environmental Contamination. The Handbook of Environmental Chemistry.
12.Qi, H., Li, H., Wei, Y., Mehler, W.T., Zeng, E.Y., You, J., 2017. Effect-Directed Analysis of Toxicants in Sediment with Combined Passive Dosing and in Vivo Toxicity Testing. Environmental Science & Technology 51, 6414-6421.
13.Muschket, M., Di Paolo, C., Tindall, A.J., Touak, G., Phan, A., Krauss, M., Kirchner, K., Seiler, T.-B., Hollert, H., Brack, W., 2018. Identification of Unknown Antiandrogenic Compounds in Surface Waters by Effect-Directed Analysis (EDA) Using a Parallel Fractionation Approach. Environmental Science & Technology 52, 288-297.
14.US EPA, 1991. Methods for Aquatic Toxicity Identification Evaluations: Phase I Toxicity Characterization Procedures. Second Edition. EPA-600-R-91-003.
15.US EPA, 1993.Methods for Aquatic Toxicity Identification Evaluations: Phase II Toxicity Identification Procedures for Samples Exhibiting Acute and Chronic Toxicity. EPA-600-R-92-080.
16.US EPA, 1993.Methods for Aquatic Toxicity Identification Evaluations: Phase III Toxicity Confirmation Procedures for Samples Exhibiting Acute and Chronic Toxicity. EPA-600-R-92-081.
17.M., B.R., T., H.K., Werner, B., Marja, L., 2013. Effects-directed analysis (EDA) and toxicity identification evaluation (TIE): Complementary but different approaches for diagnosing causes of environmental toxicity. Environmental Toxicology and Chemistry 32, 1935-1945.
18.Schmitt, S., Reifferscheid, G., Claus, E., Schlüsener, M., Buchinger, S., 2012. Effect directed analysis and mixture effects of estrogenic compounds in a sediment of the river Elbe. Environmental Science and Pollution Research 19, 3350-3361.
19.Eric, H., Stefanie, G., D., J.P., Tobias, S., Thomas-B., S., Lübcke-von, V.U., Werner, B., Jan, W., Hanno, Z., P., G.J., Henner, H., Markus, H., 2012. Endocrine disrupting, mutagenic, and teratogenic effects of upper Danube River sediments using effect-directed analysis. Environmental Toxicology and Chemistry 31, 1053-1062.
20.Mathur, P., Guo, S., 2010. Use of zebrafish as a model to understand mechanisms of addiction and complex neurobehavioral phenotypes. Neurobiology of Disease 40, 66-72.
21.G, K., U, R., P, D.A., 2007. Zebrafish: An Emerging Model System for Human Disease and Drug Discovery. Clinical Pharmacology & Therapeutics 82, 70-80.
22.Louhimies, S., 2003. Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes.
23.Hollert, H., Keiter, S., König, N., Rudolf, M., Ulrich, M., Braunbeck, T., 2003. A new sediment contact assay to assess particle-bound pollutants using zebrafish (Danio rerio) embryos. Journal of Soils and Sediments 3, 197-207.
24.Thomas, K., V., H.A., Georg, R., Werner, M., Thomas, B., Henner, H., 2006. A novel contact assay for testing genotoxicity of chemicals and whole sediments in zebrafish embryos. Environmental Toxicology and Chemistry 25, 2097-2106.
25.M., S., A., O., T., B., 2002. Effects of sediment eluates and extracts from differently polluted small rivers on zebrafish embryos and larvae. Journal of Fish Biology 61, 24-38.
26.Kosmehl, T., Hallare, A.V., Braunbeck, T., Hollert, H., 2008. DNA damage induced by genotoxicants in zebrafish (Danio rerio) embryos after contact exposure to freeze-dried sediment and sediment extracts from Laguna Lake (The Philippines) as measured by the comet assay. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 650, 1-14.
27.Hicken, C.E., Linbo, T.L., Baldwin, D.H., Willis, M.L., Myers, M.S., Holland, L., Larsen, M., Stekoll, M.S., Rice, S.D., Collier, T.K., Scholz, N.L., Incardona, J.P., 2011. Sublethal exposure to crude oil during embryonic development alters cardiac morphology and reduces aerobic capacity in adult fish. Proceedings of the National Academy of Sciences 108, 7086-7090.
28.Legler, J., van Velzen, M., Cenijn, P.H., Houtman, C.J., Lamoree, M.H., Wegener, J.W., 2011. Effect-Directed Analysis of Municipal Landfill Soil Reveals Novel Developmental Toxicants in the Zebrafish Danio rerio. Environmental Science & Technology 45, 8552-8558.
29.Chiranjib, C., Chi Hsin, H., Zhi Hong, W., Chang Shing, L., Govindasamy, A., 2009. Zebrafish: A Complete Animal Model for In Vivo Drug Discovery and Development. Current Drug Metabolism 10, 116-124.
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31.Zon, L.I., Peterson, R.T., 2005. In vivo drug discovery in the zebrafish. Nature Reviews Drug Discovery 4, 35.