釋放到環境中的污染物有70 % 會表現出非極性麻醉的毒性作用模式，這是在大多數工業有機化學品中常觀察到的作用機轉，然而現今生態系中大多數非極性麻醉物質的濃度多介於亞致死濃度，導致環境中生物反覆且持續性的暴露於非致死濃度，因此非極性麻醉物質在環境中所引發之慢性毒性風險是需迫切探討的。傳統慢性毒性試驗花費時間長、成本高、使用的動物數量多，無法提供即時有效的環境安全評估，且伴隨著動物福祉的問題，而整合性測試策略 (Integrated Testing Strategy, ITS) 可以整合多樣化數據來源，包括電腦模式、魚類胚胎毒性試驗 (Fish Embryo Acute Toxicity Test, FET) 和亞致死效應數據，這被認為是預測環境毒物的良好預測方法。因此本研究將整合電腦模式和亞致死效應的數據來建構非極性麻醉物質的生態急毒性與慢毒性ITS。
首先，針對非極性麻醉物質的生態魚類急毒性ITS而言，電腦預測模式、FET與亞致死效應與魚類急毒性試驗表現出良好的相關性 (r > 0.7) ，但電腦模式 (42.85 %) 和FET (0 %) 的敏感度不佳，並且電腦模式 (57.14 %) 與FET (0 %) 在準確率分析上亦不佳。相較於FET與電腦模式而言，亞致死效應的EC20具有良好的非極性麻醉物質敏感性，並且亞致死效應EC20能夠呈現高的魚類急毒性的預測能力。而在慢性毒性而言，電腦預測模式因數據點不足而無法與魚類慢性毒性試驗有良好相關性，但亞致死效應的EC20相較前者可以在慢性毒性試驗中表現出良好的敏感性、相關性及模式準確率。
綜上所述，本研究證實亞致死效應指標是有效且敏感的預測非極性麻醉物質的急毒性與慢毒性，並且辨別出適於非極性麻醉物質的亞致死效應，包含心率、孵化異常率、卵黃囊水腫、心包水腫、身體彎曲、不良魚鰾狀態和體長，未來可應用於預測非極性麻醉物質的魚類急毒性和慢性毒性。

It has been demonstrated that 70% of pollutants released into the environment exhibit toxic mode of action of nonpolar narcotic, a common mechanism observed in most industrial organic chemicals. Most nonpolar narcotic substances existing in ecosystem at low concentration led to induction of sublethal effects but not acute toxicity in living organisms. The situation implies that nonpolar narcotic substances may increase the potential chronic toxicity risks in the environment. Therefore, the fish chronic toxicity testing assays urgently need new approach methodologies (NAMs) due to time-consuming, high cost, and use of abundant animals.Integrated Testing Strategy (ITS) integrate data from various NAMs including sublethal effects data, which is believed as a promising method for assessing environmental risk. Our current study established a novel ITS for determining fish acute toxicity of nonpolar narcotic substances via integrating the data from in silico, fish embryo toxicity test and sublethal effects. According to the results, the sublethal effects exhibit excellent correlation with both fish acute and chronic toxicity test, and we identified multiple sublethal effects which are appropriate for nonpolar narcotic substances, including heart rate, hatching abnormality rate, yolk sac edema, pericardial edema, body curvature, uninflated swim bladder, and body length. We concluded that the established ITS is suitable for predicting toxicity of nonpolar narcotic substances for fish acute and chronic toxicity through sublethal effects of fish embryo.

Adhikari C, Mishra BK. 2018. Quantitative structure-activity relationships of aquatic narcosis: A review. Curr Comput Aided Drug Des 14:7-28.
Agency EC. 2017. Non-animal approaches – current status of regulatory applicability under the reach, clp and biocidal products regulations:European Chemicals Agency.
Ahlers J, Nendza M, Schwartz D. 2019. Environmental hazard and risk assessment of thiochemicals. Application of integrated testing and intelligent assessment strategies (its) to fulfil the reach requirements for aquatic toxicity. Chemosphere 214:480-490.
Aluru N. 2017. Epigenetic effects of environmental chemicals: Insights from zebrafish. Curr Opin Toxicol 6:26-33.
Alves RN, Mariz CF, Paulo DVd, Carvalho PSM. 2017. Toxicity of effluents from gasoline stations oil-water separators to early life stages of zebrafish danio rerio. Chemosphere 178:224-230.
Ankley GT, Bennett RS, Erickson RJ, Hoff DJ, Hornung MW, Johnson RD, et al. 2010. Adverse outcome pathways: A conceptual framework to support ecotoxicology research and risk assessment. Environ Toxicol Chem 29:730-741.
Austin TJ, Eadsforth CV. 2014. Development of a chronic fish toxicity model for predicting sub-lethal noec values for non-polar narcotics. SAR QSAR Environ Res 25:147-160.
Baudiffier D, Audouze K, Armant O, Frelon S, Charles S, Beaudouin R, et al. 2023. Editorial trend: Adverse outcome pathway (aop) and computational strategy - towards new perspectives in ecotoxicology. Environ Sci Pollut Res Int.
Belanger SE, Rawlings JM, Carr GJ. 2013. Use of fish embryo toxicity tests for the prediction of acute fish toxicity to chemicals. Environ Toxicol Chem 32:1768-1783.
Birke A, Scholz S. 2019. Zebrafish embryo and acute fish toxicity test show similar sensitivity for narcotic compounds. Altex 36:131-135.
Blaxter JHS, Tytler P. 1978. Physiology and function of the swimbladder. In: Advances in comparative physiology and biochemistry, Vol. 7, (Lowenstein O, ed):Elsevier, 311-367.
Bohlen ML, Jeon HP, Kim YJ, Sung B. 2019. In silico modeling method for computational aquatic toxicology of endocrine disruptors: A software-based approach using qsar toolbox. J Vis Exp.
Brack W, Ait-Aissa S, Burgess RM, Busch W, Creusot N, Di Paolo C, et al. 2016. Effect-directed analysis supporting monitoring of aquatic environments--an in-depth overview. Sci Total Environ 544:1073-1118.
Bradbury SP, Russom CL, Ankley GT, Schultz TW, Walker JD. 2003. Overview of data and conceptual approaches for derivation of quantitative structure-activity relationships for ecotoxicological effects of organic chemicals. Environ Toxicol Chem 22:1789-1798.
Burden N, Benstead R, Benyon K, Clook M, Green C, Handley J, et al. 2020. Key opportunities to replace, reduce, and refine regulatory fish acute toxicity tests. Environ Toxicol Chem 39:2076-2089.
Cassar S, Adatto I, Freeman JL, Gamse JT, Iturria I, Lawrence C, et al. 2020. Use of zebrafish in drug discovery toxicology. Chem Res Toxicol 33:95-118.
Chatain B. 1994. Abnormal swimbladder development and lordosis in sea bass (dicentrarchus labrax) and sea bream (sparus auratus). Aquaculture 119:371-379.
Chen J. 2015. Cardiac toxicity by sublethal 2,3,7,8-tetrachlorodibenzo-p-dioxin correlates with its anti-proliferation effect on cardiomyocytes in zebrafish embryos. Environ Toxicol Chem 34:420-428.
Connolly M, Little S, Hartl MGJ, Fernandes TF. 2022. An integrated testing strategy for ecotoxicity (its-eco) assessment in the marine environmental compartment using mytilus spp.: A case study using pristine and coated cuo and tio(2) nanomaterials. Environ Toxicol Chem 41:1390-1406.
Cronin MT. 2017. (q)sars to predict environmental toxicities: Current status and future needs. Environ Sci Process Impacts 19:213-220.
Czesny SJ, Graeb BD, Dettmers JM. 2005. Ecological consequences of swim bladder noninflation for larval yellow perch. Transactions of the American Fisheries Society 134:1011-1020.
de Souza SML, de Vasconcelos EC, Dziedzic M, de Oliveira CMR. 2009. Environmental risk assessment of antibiotics: An intensive care unit analysis. Chemosphere 77:962-967.
Diniz MS, Salgado R, Pereira VJ, Carvalho G, Oehmen A, Reis MA, et al. 2015. Ecotoxicity of ketoprofen, diclofenac, atenolol and their photolysis byproducts in zebrafish (danio rerio). Sci Total Environ 505:282-289.
Dong G, Wang N, Xu T, Liang J, Qiao R, Yin D, et al. 2023. Deep learning-enabled morphometric analysis for toxicity screening using zebrafish larvae. Environ Sci Technol.
ECHA. 2008. Guidance on information requirements and chemical safety assessment chapter r.10: Characterisation of dose [concentration]-response for environment.
Ellison CM, Piechota P, Madden JC, Enoch SJ, Cronin MT. 2016. Adverse outcome pathway (aop) informed modeling of aquatic toxicology: Qsars, read-across, and interspecies verification of modes of action. Environ Sci Technol 50:3995-4007.
Embry MR, Belanger SE, Braunbeck TA, Galay-Burgos M, Halder M, Hinton DE, et al. 2010. The fish embryo toxicity test as an animal alternative method in hazard and risk assessment and scientific research. Aquat Toxicol 97:79-87.
Fraher D, Sanigorski A, Mellett NA, Meikle PJ, Sinclair AJ, Gibert Y. 2016. Zebrafish embryonic lipidomic analysis reveals that the yolk cell is metabolically active in processing lipid. Cell reports 14:1317-1329.
Godoy AA, Kummrow F, Pamplin PAZ. 2015. Occurrence, ecotoxicological effects and risk assessment of antihypertensive pharmaceutical residues in the aquatic environment - a review. Chemosphere 138:281-291.
Hayes AW, Li R, Hoeng J, Iskandar A, Peistch MC, Dourson ML. 2019. New approaches to risk assessment of chemical mixtures. Toxicology Research and Application 3:2397847318820768.
Hedgpeth BM, Redman AD, Alyea RA, Letinski DJ, Connelly MJ, Butler JD, et al. 2019. Analysis of sublethal toxicity in developing zebrafish embryos exposed to a range of petroleum substances. Environ Toxicol Chem 38:1302-1312.
Horie Y, Yamagishi T, Koshio M, Iguchi T, Tatarazako N. 2017. Lethal and sublethal effects of aniline and chlorinated anilines on zebrafish embryos and larvae. J Appl Toxicol 37:836-841.
Hubrecht RC, Carter E. 2019. The 3rs and humane experimental technique: Implementing change. Animals (Basel) 9.
Iwasaki T, Teruya K, Mizuta S, Hamasaki K. 2017. Swim bladder inflation as a possible cause of saddleback-like syndrome malformation in hatchery-reared red spotted grouper epinephelus akaara. Fisheries science 83:447-454.
Jeffries MK, Stultz AE, Smith AW, Stephens DA, Rawlings JM, Belanger SE, et al. 2015. The fish embryo toxicity test as a replacement for the larval growth and survival test: A comparison of test sensitivity and identification of alternative endpoints in zebrafish and fathead minnows. Environ Toxicol Chem 34:1369-1381.
Kammann U, Vobach M, Wosniok W. 2006. Toxic effects of brominated indoles and phenols on zebrafish embryos. Arch Environ Contam Toxicol 51:97-102.
Khabib MNH, Sivasanku Y, Lee HB, Kumar S, Kue CS. 2022. Alternative animal models in predictive toxicology. Toxicology 465:153053.
Krewski D, Acosta D, Jr., Andersen M, Anderson H, Bailar JC, 3rd, Boekelheide K, et al. 2010. Toxicity testing in the 21st century: A vision and a strategy. J Toxicol Environ Health B Crit Rev 13:51-138.
Krzykwa JC, Olivas A, Sellin Jeffries MK. 2018. Development of cardiovascular and neurodevelopmental metrics as sublethal endpoints for the fish embryo toxicity test. Environ Toxicol Chem 37:2530-2541.
Lammer E, Carr GJ, Wendler K, Rawlings JM, Belanger SE, Braunbeck T. 2009. Is the fish embryo toxicity test (fet) with the zebrafish (danio rerio) a potential alternative for the fish acute toxicity test? Comp Biochem Physiol C Toxicol Pharmacol 149:196-209.
Li X, Xiong D, Ding G, Fan Y, Ma X, Wang C, et al. 2019. Exposure to water-accommodated fractions of two different crude oils alters morphology, cardiac function and swim bladder development in early-life stages of zebrafish. Chemosphere 235:423-433.
Lillicrap A, Belanger S, Burden N, Pasquier DD, Embry MR, Halder M, et al. 2016. Alternative approaches to vertebrate ecotoxicity tests in the 21st century: A review of developments over the last 2 decades and current status. Environ Toxicol Chem 35:2637-2646.
Lin Z, Du J, Yin K, Wang L, Yu H. 2004. Mechanism of concentration addition toxicity: They are different for nonpolar narcotic chemicals, polar narcotic chemicals and reactive chemicals. Chemosphere 54:1691-1701.
Lungu-Mitea S, Vogs C, Carlsson G, Montag M, Frieberg K, Oskarsson A, et al. 2021. Modeling bioavailable concentrations in zebrafish cell lines and embryos increases the correlation of toxicity potencies across test systems. Environ Sci Technol 55:447-457.
Madan AK, Bajaj S, Dureja H. 2013. Classification models for safe drug molecules. Methods Mol Biol 930:99-124.
Martínez R, Navarro-Martín L, van Antro M, Fuertes I, Casado M, Barata C, et al. 2020. Changes in lipid profiles induced by bisphenol a (bpa) in zebrafish eleutheroembryos during the yolk sac absorption stage. Chemosphere 246:125704.
Massei R, Knapen D, Covaci A, Blust R, Mayer P, Vergauwen L. 2021. Sublethal effect concentrations for nonpolar narcosis in the zebrafish embryo. Environ Toxicol Chem 40:2802-2812.
Metian M, Warnau M, Chouvelon T, Pedraza F, Rodriguez y Baena AM, Bustamante P. 2013. Trace element bioaccumulation in reef fish from new caledonia: Influence of trophic groups and risk assessment for consumers. Mar Environ Res 87-88:26-36.
Mombelli E, Pandard P. 2021. Evaluation of the oecd qsar toolbox automatic workflow for the prediction of the acute toxicity of organic chemicals to fathead minnow. Regul Toxicol Pharmacol 122:104893.
Moore DRJ, Caux P-Y. 1997. Estimating low toxic effects. Environmental Toxicology and Chemistry 16:794-801.
Nendza M, Müller M, Wenzel A. 2017. Classification of baseline toxicants for qsar predictions to replace fish acute toxicity studies. Environ Sci Process Impacts 19:429-437.
Nilsen E, Smalling KL, Ahrens L, Gros M, Miglioranza KSB, Picó Y, et al. 2019. Critical review: Grand challenges in assessing the adverse effects of contaminants of emerging concern on aquatic food webs. Environ Toxicol Chem 38:46-60.
Norberg-King TJ, Embry MR, Belanger SE, Braunbeck T, Butler JD, Dorn PB, et al. 2018. An international perspective on the tools and concepts for effluent toxicity assessments in the context of animal alternatives: Reduction in vertebrate use. Environ Toxicol Chem 37:2745-2757.
OECD. 2013a. Test no. 210: Fish, early-life stage toxicity test.
OECD. 2013b. Test no. 236: Fish embryo acute toxicity (fet) test.
Pereira AC, Gomes T, Ferreira Machado MR, Rocha TL. 2019. The zebrafish embryotoxicity test (zet) for nanotoxicity assessment: From morphological to molecular approach. Environ Pollut 252:1841-1853.
Perugini M, Merola C, Amorena M, D'Angelo M, Cimini A, Benedetti E. 2020. Sublethal exposure to propylparaben leads to lipid metabolism impairment in zebrafish early-life stages. J Appl Toxicol 40:493-503.
Philibert DA, Philibert CP, Lewis C, Tierney KB. 2016. Comparison of diluted bitumen (dilbit) and conventional crude oil toxicity to developing zebrafish. Environ Sci Technol 50:6091-6098.
Ping S, Lin W, Ming R, He Y, Yin Y, Ren Y. 2022. Toxic effects of four cardiovascular drugs on the development and epigenetics of zebrafish (danio rerio). Sci Total Environ 846:157360.
PubChem. 2023.
Raies AB, Bajic VB. 2016. In silico toxicology: Computational methods for the prediction of chemical toxicity. Wiley Interdiscip Rev Comput Mol Sci 6:147-172.
Ren S. 2002. Predicting three narcosis mechanisms of aquatic toxicity. Toxicol Lett 133:127-139.
Richard AM, Judson RS, Houck KA, Grulke CM, Volarath P, Thillainadarajah I, et al. 2016. Toxcast chemical landscape: Paving the road to 21st century toxicology. Chem Res Toxicol 29:1225-1251.
Rovida C, Alépée N, Api AM, Basketter DA, Bois FY, Caloni F, et al. 2015. Integrated testing strategies (its) for safety assessment. Altex 32:25-40.
Santos D, Luzio A, Matos C, Bellas J, Monteiro SM, Félix L. 2021. Microplastics alone or co-exposed with copper induce neurotoxicity and behavioral alterations on zebrafish larvae after a subchronic exposure. Aquatic Toxicology 235:105814.
Santos SW, Cachot J, Gourves PY, Clérandeau C, Morin B, Gonzalez P. 2019. Sub-lethal effects of waterborne copper in early developmental stages of rainbow trout (oncorhynchus mykiss). Ecotoxicol Environ Saf 170:778-788.
Scholz S, Sela E, Blaha L, Braunbeck T, Galay-Burgos M, García-Franco M, et al. 2013. A european perspective on alternatives to animal testing for environmental hazard identification and risk assessment. Regul Toxicol Pharmacol 67:506-530.
Schweizer M, von der Ohe PC, Gräff T, Kühnen U, Hebel J, Heid C, et al. 2022. Heart rate as an early warning parameter and proxy for subsequent mortality in danio rerio embryos exposed to ionisable substances. Sci Total Environ 818:151744.
Settivari RS, Krieger SM, Hindle S, Gehen SC, Hollnagel HM, Boverhof DR. 2017. Current status of alternative methods for assessing immunotoxicity: A chemical industry perspective. Current Opinion in Toxicology 5:19-27.
Sobanska M, Scholz S, Nyman AM, Cesnaitis R, Gutierrez Alonso S, Kluver N, et al. 2018. Applicability of the fish embryo acute toxicity (fet) test (oecd 236) in the regulatory context of registration, evaluation, authorisation, and restriction of chemicals (reach). Environ Toxicol Chem 37:657-670.
Spence R, Gerlach G, Lawrence C, Smith C. 2008. The behaviour and ecology of the zebrafish, danio rerio. Biol Rev Camb Philos Soc 83:13-34.
Stelzer JAA, Rosin CK, Bauer LH, Hartmann M, Pulgati FH, Arenzon A. 2018. Is fish embryo test (fet) according to oecd 236 sensible enough for delivering quality data for effluent risk assessment? Environ Toxicol Chem 37:2925-2932.
Strähle U, Scholz S, Geisler R, Greiner P, Hollert H, Rastegar S, et al. 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. Reprod Toxicol 33:128-132.
Suryanto ME, Saputra F, Kurnia KA, Vasquez RD, Roldan MJM, Chen KH, et al. 2022. Using deeplabcut as a real-time and markerless tool for cardiac physiology assessment in zebrafish. Biology (Basel) 11.
Tal T, Yaghoobi B, Lein PJ. 2020. Translational toxicology in zebrafish. Curr Opin Toxicol 23-24:56-66.
Tatarazako N, Oda S. 2007. The water flea daphnia magna (crustacea, cladocera) as a test species for screening and evaluation of chemicals with endocrine disrupting effects on crustaceans. Ecotoxicology 16:197-203.
Taylor K, Gordon N, Langley G, Higgins W. 2008. Estimates for worldwide laboratory animal use in 2005. Altern Lab Anim 36:327-342.
Tian S, Teng M, Meng Z, Yan S, Jia M, Li R, et al. 2019. Toxicity effects in zebrafish embryos (danio rerio) induced by prothioconazole. Environ Pollut 255:113269.
Tollefsen KE, Scholz S, Cronin MT, Edwards SW, de Knecht J, Crofton K, et al. 2014. Applying adverse outcome pathways (aops) to support integrated approaches to testing and assessment (iata). Regul Toxicol Pharmacol 70:629-640.
Verhaar HJM, van Leeuwen CJ, Hermens JLM. 1992. Classifying environmental pollutants. Chemosphere 25:471-491.
Volz DC, Belanger S, Embry M, Padilla S, Sanderson H, Schirmer K, et al. 2011. Adverse outcome pathways during early fish development: A conceptual framework for identification of chemical screening and prioritization strategies. Toxicol Sci 123:349-358.
Werner I, Schneeweiss A, Segner H, Junghans M. 2021. Environmental risk of pesticides for fish in small- and medium-sized streams of switzerland. Toxics 9.
Yordanova D, Schultz TW, Kuseva C, Tankova K, Ivanova H, Dermen I, et al. 2019. Automated and standardized workflows in the oecd qsar toolbox. Computational Toxicology 10:89-104.
Zhang Z, Yan Q. 2014. Comparative toxicity of different crude oils on the cardiac function of marine medaka (oryzias melastigma) embryo. International Journal Bioautomation 18:389.
Zhu D, Li TT, Zheng SS, Yan LC, Wang Y, Fan LY, et al. 2018. Comparison of modes of action between fish and zebrafish embryo toxicity for baseline, less inert, reactive and specifically-acting compounds. Chemosphere 213:414-422.