近十年來，台灣本島近半數主要水庫及全數離島水庫都曾有優養化的現象。大量藍綠菌與藻類所產生的數百種生物活性代謝物，包含藻類毒素、臭味物質、藍綠菌胜肽等，其大量釋出在水體中不僅惡化飲用水品質，更可能引發民眾健康風險的疑慮，因此我們必須了解這些藻類代謝物在環境中的暴露水平，以保障一個健康安全的用水、飲水環境。
本研究將針對固相萃取法中數種參數進行最佳化測試，以確立其對環境樣品中藍綠菌胜肽的濃縮萃取效果，並搭配液相層析串聯式質譜儀定量分析環境水體中的數種藍綠菌胜肽(Anabaenopeptin A、Anabaenopeptin B、Microginin 527、Microginin 690及Microcystin-LR)以及傳統常見之藍綠毒素Anatoxin-a以及Cylindrospermopsin，了解其濃度範圍與流佈狀態以作為未來水質管理及處理策略之依據。
本研究成功建立以LC/MS-MS同時分析七種藍綠菌胜肽與毒素之分析方法，此方法最低定量極限為0.125 µg-L-1，且成功利用固相萃取技術濃縮純化大體積之環境樣品，此方法之濃縮倍率為1000倍，可將樣品最低定量極限降至0.04-0.08 ng-L-1。在純藻株生產藍綠菌胜肽能力調查中，大多數藻株產藍綠菌胜肽與毒素的細胞當量為10-6至10-8 ng/cell之間，而微囊藻屬產MC-LR之能力約為10-2至10-3 ng/cell之間，以微囊藻屬產藍綠菌胜肽之細胞當量最高且種類最豐富。在38個環境樣品調查結果中，藍綠菌胜肽濃度範圍0.25到94.29 ng-L-1，六種藍綠菌胜肽之中除了Anatoxin-a與Anabaenopeptin A的出現頻率小於15%外，其餘四種出現的頻率皆大於60%，其中以Anabaenopeptin B最高，Microginin 527次之。此外，本研究亦在魚體與鴨子體中發現了Microginin 527、Microginin 690、Anabaenopeptin B與Microcystin-LR的存在，最高濃度為10.61 ng-g-1，此項調查亦為首次發現MGs存在魚類與鴨類之生物體中之證據。

The secondary metabolites of cyanobacteria such as cyanopeptides and cyanotoxins can be potential risks in drinking water systems. An analytical method of cyanopeptides and applications of the developed methods in the measurement of these low concentration cyanopeptides in environmental samples was established in this study. The detection limits of these chemicals are 0.04-0.08 μg-L-1 after being concentrated by the solid-phase extraction process. Production of the studied cyanopeptides and cyanotoxins was investigated for nine strains of cultivated algae, and all strains can produce more than 3 different cyanpeptides. MG527, MG690, AP-B, and MCLR were present in >60% environmental samples, including source water, raw water, and finished water samples in Taiwan. In addition, MG527, MG690, AP-B, AP-A, and MC-LR have also been detected in fish and ducks living in algal blooming lakes, and have shown bioaccumulation and biomagnification.

Azevedo, S. M., Carmichael, W. W., Jochimsen, E. M., Rinehart, K. L., Lau, S., Shaw, G. R., & Eaglesham, G. K. (2002). Human intoxication by microcystins during renal dialysis treatment in Caruaru—Brazil. Toxicology, 181, 441-446.
Beversdorf, L. J., Rude, K., Weirich, C. A., Bartlett, S. L., Seaman, M., Kozik, C., . . . Stempa, C. (2018). Analysis of cyanobacterial metabolites in surface and raw drinking waters reveals more than microcystin. Water research, 140, 280-290.
Beversdorf, L. J., Rude, K., Weirich, C. A., Bartlett, S. L., Seaman, M., Kozik, C., . . . Miller, T. R. (2018). Analysis of cyanobacterial metabolites in surface and raw drinking waters reveals more than microcystin. Water Res, 140, 280-290. doi:10.1016/j.watres.2018.04.032
Beversdorf, L. J., Weirich, C. A., Bartlett, S. L., & Miller, T. R. (2017). Variable Cyanobacterial Toxin and Metabolite Profiles across Six Eutrophic Lakes of Differing Physiochemical Characteristics. Toxins, 9(2), 62. Retrieved from https://www.mdpi.com/2072-6651/9/2/62
https://res.mdpi.com/d_attachment/toxins/toxins-09-00062/article_deploy/toxins-09-00062-v2.pdf
Briand, E., Humbert, J.-F., Tambosco, K., Bormans, M., & Gerwick, W. H. (2016). Role of bacteria in the production and degradation of Microcystis cyanopeptides. MicrobiologyOpen, 5(3), 469-478. doi:10.1002/mbo3.343
Brookes, J. D., & Carey, C. C. (2011). Resilience to blooms. Science, 334(6052), 46-47.
Campos, A., & Vasconcelos, V. (2010). Molecular mechanisms of microcystin toxicity in animal cells. International journal of molecular sciences, 11(1), 268-287.
Cordeiro-Araújo, M. K., Chia, M. A., de Toledo Arruda-Neto, J. D., Tornisielo, V. L., Vilca, F. Z., & do Carmo Bittencourt-Oliveira, M. (2016). Microcystin-LR bioaccumulation and depuration kinetics in lettuce and arugula: Human health risk assessment. Science of The Total Environment, 566, 1379-1386.
Dinh, Q. T., Munoz, G., Simon, D. F., Duy, S. V., Husk, B., & Sauvé, S. (2021). Stability issues of microcystins, anabaenopeptins, anatoxins, and cylindrospermopsin during short-term and long-term storage of surface water and drinking water samples. Harmful Algae, 101, 101955.
Duncan, M. W. (2012). Good mass spectrometry and its place in good science. Journal of mass spectrometry, 47(6), 795-809.
Díez-Quijada, L., Guzmán-Guillén, R., Prieto Ortega, A. I., Llana-Ruíz-Cabello, M., Campos, A., Vasconcelos, V., . . . Cameán, A. M. (2018). New method for simultaneous determination of microcystins and cylindrospermopsin in vegetable matrices by SPE-UPLC-MS/MS. Toxins, 10(10), 406.
E Chlipala, G., Mo, S., & Orjala, J. (2011). Chemodiversity in freshwater and terrestrial cyanobacteria-a source for drug discovery. Current drug targets, 12(11), 1654-1673.
Egli, C. M., Natumi, R. S., Jones, M. R., & Janssen, E. M.-L. (2020). Inhibition of Extracellular Enzymes Exposed to Cyanopeptides. CHIMIA International Journal for Chemistry, 74(3), 122-128.
Gademann, K., & Portmann, C. (2008). Secondary metabolites from cyanobacteria: complex structures and powerful bioactivities. Current Organic Chemistry, 12(4), 326-341.
Gademann, K., Portmann, C., Blom, J. F., Zeder, M., & Jüttner, F. (2010). Multiple toxin production in the cyanobacterium Microcystis: Isolation of the toxic protease inhibitor cyanopeptolin 1020. Journal of natural products, 73(5), 980-984.
Gkelis, S., Lanaras, T., & Sivonen, K. (2015). Cyanobacterial Toxic and Bioactive Peptides in Freshwater Bodies of Greece: Concentrations, Occurrence Patterns, and Implications for Human Health. Mar Drugs, 13(10), 6319-6335. doi:10.3390/md13106319
Harada, K.-i. (2004). Production of secondary metabolites by freshwater cyanobacteria. Chemical and Pharmaceutical bulletin, 52(8), 889-899.
Janssen, E. M. (2019). Cyanobacterial peptides beyond microcystins - A review on co-occurrence, toxicity, and challenges for risk assessment. Water Res, 151, 488-499. doi:10.1016/j.watres.2018.12.048
Kurmayer, R., Blom, J. F., Deng, L., & Pernthaler, J. (2015). Integrating phylogeny, geographic niche partitioning and secondary metabolite synthesis in bloom-forming Planktothrix. The ISME journal, 9(4), 909-921.
Kurmayer, R., & Christiansen, G. (2009). The genetic basis of toxin production in cyanobacteria. Freshwater reviews, 2(1), 31-50.
Kurmayer, R., Deng, L., & Entfellner, E. (2016). Role of toxic and bioactive secondary metabolites in colonization and bloom formation by filamentous cyanobacteria Planktothrix. Harmful Algae, 54, 69-86.
Lenz, K. A., Miller, T. R., & Ma, H. (2019). Anabaenopeptins and cyanopeptolins induce systemic toxicity effects in a model organism the nematode Caenorhabditis elegans. Chemosphere, 214, 60-69. doi:10.1016/j.chemosphere.2018.09.076
Lopes, V. R., Ramos, V., Martins, A., Sousa, M., Welker, M., Antunes, A., & Vasconcelos, V. M. (2012). Phylogenetic, chemical and morphological diversity of cyanobacteria from Portuguese temperate estuaries. Marine environmental research, 73, 7-16.
Lyra, C., Suomalainen, S., Gugger, M., Vezie, C., Sundman, P., Paulin, L., & Sivonen, K. (2001). Molecular characterization of planktic cyanobacteria of Anabaena, Aphanizomenon, Microcystis and Planktothrix genera. International Journal of Systematic and Evolutionary Microbiology, 51(2), 513-526.
Merel, S., Walker, D., Chicana, R., Snyder, S., Baurès, E., & Thomas, O. (2013). State of knowledge and concerns on cyanobacterial blooms and cyanotoxins. Environment international, 59, 303-327.
Mumenthaler, M., & Leuenberger, H. (1991). Atmospheric spray-freeze drying: a suitable alternative in freeze-drying technology. International Journal of Pharmaceutics, 72(2), 97-110.
Neilan, B. A., Pearson, L. A., Muenchhoff, J., Moffitt, M. C., & Dittmann, E. (2013). Environmental conditions that influence toxin biosynthesis in cyanobacteria. Environmental microbiology, 15(5), 1239-1253.
Neumann, U., Forchert, A., Flury, T., & Weckesser, J. (1997). Microginin FR1, a linear peptide from a water bloom of Microcystis species. FEMS Microbiology Letters, 153(2), 475-478. doi:https://doi.org/10.1016/S0378-1097(97)00291-7
Ortiz, X., Korenkova, E., Jobst, K. J., MacPherson, K. A., & Reiner, E. J. (2017). A high throughput targeted and non-targeted method for the analysis of microcystins and anatoxin-A using on-line solid phase extraction coupled to liquid chromatography–quadrupole time-of-flight high resolution mass spectrometry. Analytical and bioanalytical chemistry, 409(21), 4959-4969.
Otten, T. G., Paerl, H. W., Dreher, T. W., Kimmerer, W. J., & Parker, A. E. (2017). The molecular ecology of Microcystis sp. blooms in the San Francisco Estuary. Environmental microbiology, 19(9), 3619-3637.
Paerl, H. W., & Huisman, J. (2008). Blooms like it hot. Science, 320(5872), 57-58.
Pichon, V. (2000). Solid-phase extraction for multiresidue analysis of organic contaminants in water. Journal of Chromatography A, 885(1-2), 195-215.
Ploutno, A., & Carmeli, S. (2002). Modified peptides from a water bloom of the cyanobacterium Nostoc sp. Tetrahedron, 58(50), 9949-9957.
Poole, C. F., Gunatilleka, A. D., & Sethuraman, R. (2000). Contributions of theory to method development in solid-phase extraction. Journal of Chromatography A, 885(1-2), 17-39.
Pouria, S., de Andrade, A., Barbosa, J., Cavalcanti, R., Barreto, V., Ward, C., . . . Codd, G. (1998). Fatal microcystin intoxication in haemodialysis unit in Caruaru, Brazil. The Lancet, 352(9121), 21-26.
Roy-Lachapelle, A., Solliec, M., Sauvé, S., & Gagnon, C. (2019). A data-independent methodology for the structural characterization of microcystins and anabaenopeptins leading to the identification of four new congeners. Toxins, 11(11), 619.
Saker, M., Fastner, J., Dittmann, E., Christiansen, G., & Vasconcelos, V. (2005). Variation between strains of the cyanobacterium Microcystis aeruginosa isolated from a Portuguese river. Journal of Applied Microbiology, 99(4), 749-757.
Sano, T., & Kaya, K. (1997). A 3-amino-10-chloro-2-hydroxydecanoic acid-containing tetrapeptide from Oscillatoria agardhii. Phytochemistry, 44(8), 1503-1505.
Santori, N., Buratti, F. M., Scardala, S., Dorne, J.-L. C., & Testai, E. (2020). In vitro detoxication of microcystins in human samples: variability among variants with different hydrophilicity and structure. Toxicology letters, 322, 131-139.
Sanz, M., Andreote, A. P. D., Fiore, M. F., Dörr, F. A., & Pinto, E. (2015). Structural characterization of new peptide variants produced by cyanobacteria from the Brazilian Atlantic coastal Forest using liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry. Marine Drugs, 13(6), 3892-3919.
Sedmak, B., Carmeli, S., & Eleršek, T. (2008). “Non-toxic” cyclic peptides induce lysis of cyanobacteria—an effective cell population density control mechanism in cyanobacterial blooms. Microbial ecology, 56(2), 201-209.
Shishido, T. K., Jokela, J., Fewer, D. P., Wahlsten, M., Fiore, M. F., & Sivonen, K. (2017). Simultaneous production of anabaenopeptins and namalides by the cyanobacterium Nostoc sp. CENA543. ACS chemical biology, 12(11), 2746-2755.
Simpson., N. J. K. (2000). Solid-Phase ExtractionPrinciples, Techniques, and Applications.
Skafi, M., Duy, S. V., Munoz, G., Dinh, Q. T., Simon, D. F., Juneau, P., & Sauvé, S. (2021). Occurrence of microcystins, anabaenopeptins and other cyanotoxins in fish from a freshwater wildlife reserve impacted by harmful cyanobacterial blooms. Toxicon, 194, 44-52.
Spoof, L., Blaszczyk, A., Meriluoto, J., Ceglowska, M., & Mazur-Marzec, H. (2015). Structures and Activity of New Anabaenopeptins Produced by Baltic Sea Cyanobacteria. Mar Drugs, 14(1), 8. doi:10.3390/md14010008
Stewart, A. K., Ravindra, R., Van Wagoner, R. M., & Wright, J. L. (2018). Metabolomics-guided discovery of microginin peptides from cultures of the cyanobacterium Microcystis aeruginosa. Journal of natural products, 81(2), 349-355.
Ujvarosi, A. Z., Hercog, K., Riba, M., Gonda, S., Filipic, M., Vasas, G., & Zegura, B. (2020). The cyanobacterial oligopeptides microginins induce DNA damage in the human hepatocellular carcinoma (HepG2) cell line. Chemosphere, 240, 124880. doi:10.1016/j.chemosphere.2019.124880
Ülker, D., BAYIRHAN, İ., & Burak, S. (2020). Assessment and comparison of commonly used eutrophication indexes. Turkish Journal of Water Science and Management, 4(1), 4-30.
Welker, M., Maršálek, B., Šejnohová, L., & Von Doehren, H. (2006). Detection and identification of oligopeptides in Microcystis (cyanobacteria) colonies: toward an understanding of metabolic diversity. Peptides, 27(9), 2090-2103.
Welker, M., & Von Döhren, H. (2006). Cyanobacterial peptides — Nature's own combinatorial biosynthesis. FEMS Microbiology Reviews, 30(4), 530-563. doi:10.1111/j.1574-6976.2006.00022.x
Zervou, S. K., Gkelis, S., Kaloudis, T., Hiskia, A., & Mazur-Marzec, H. (2020). New microginins from cyanobacteria of Greek freshwaters. Chemosphere, 248, 125961. doi:10.1016/j.chemosphere.2020.125961
Żwir-Ferenc, A., & Biziuk, M. (2006). Solid Phase Extraction Technique--Trends, Opportunities and Applications. Polish Journal of Environmental Studies, 15(5).

行政院環境保護署 (2020) 民國109年環境水質監測年報
飲用水中微囊藻毒素化學檢測方法－固相萃取與高效液相層析／串聯式質譜儀法 (NIEA W539.50B)
環境檢驗方法偵測極限測定指引(NIEA-PA107)