臺灣博碩士論文加值系統

藍綠細菌(cyanobacteria)及其所產生之毒素(cyanotoxins)已被發現存在於多數優養化的池塘、湖泊，甚至在以飲用為目的之水庫當中，台灣地區亦不例外。為了解台灣水庫或凈水廠當中是否有藻類毒素的存在種類及濃度，本研究首先開發改良式固相萃取法(SPE)結合液相層析質譜儀(LC/MS)之分析方法，並應用於偵測水樣當中的微量藻類毒素。研究中開發之方法總共可以同步偵測九種不同型式之藻類毒素，包括：六種微囊藻毒素(MC-LR, -RR, -YR, -LW, -LF, -LA)，一種節球藻毒素(nodularin, NOD)，一種魚腥藻毒素(anatoxin-a, ATX)及一種柱孢藻毒素(cylindrospermopsin, CYN)。方法中並應用擬似及內標準分析法，以作為分析之品管措施。其中擬似內標物(1,9-diaminononane)協助品管固相萃取法之回收率，內標物(2,3,5-trimethylphenyl methyl carbamate)則協助品管液相層析質譜儀之穩定度。方法偵測極限(MDL)分別為：微囊藻毒素與節球藻毒素2到10 ng/L，魚腥藻毒素46 ng/L，柱孢藻毒素100 ng/L。
當分析方法確立後，研究中對台灣地區9個主要水庫及7個淨水廠展開藻類毒素流佈之調查。調查的結果顯示，所有被調查的水庫當中，皆可偵測出微囊藻毒素的存在，毒素濃度在2到30 ng/L之間；而魚腥藻毒素亦在金門四個水庫當中被偵測出。在自然的環境當中，藍綠細菌不僅會產生毒素，也會產生其他的代謝物質如：臭味物質(Geosmin和2-MIB)等，為了解藻類毒素與其他代謝物及環境因子與水質參數之間的關係，研究於牡丹水庫與曾文水庫同步收集22個因子或參數的相關數據，並進行逐步迴歸分析(stepwise regression)與相關性(correlation)的分析，結果顯示2-MIB的濃度、水溫、氣溫皆與微囊藻毒素濃度有很好的相關性，而據此建立的逐步迴歸方程式將可協助預測藻類毒素的濃度，達到預警的目的。
由於金門外島地區的飲用水水源，經常地被偵測出藍綠細菌之代謝物質，如：藻類毒素與臭味物質等，為進一步了解其毒素型式、濃度分佈等資訊，本研究展開大規模的採樣與調查，其中包含：三座主要的蓄水湖泊(太湖、榮湖、陽明湖)，及兩座淨水廠(太湖淨水廠和榮湖淨水廠)。在採樣的規劃方面，除於各湖泊表水規劃多點採樣點外，亦包括於淨水廠進水口位置的不同深度採樣、淨水廠各處理單元的採樣、清水的採樣及家戶使用端的採樣等，共約40個樣品。在湖水的分析結果顯示，三個湖泊皆可被發現藻類毒素的存在，包括：微囊藻毒素、節球藻毒素與柱孢藻毒素，濃度從80 ng/L到36,000 ng/L不等，相關淨水廠對藻類毒素的去除效率平均約為60%左右，這也說明了微量藻類毒素為何可以於清水與用戶端被偵測出來。清水與用戶端部分的水樣當中，僅微量的微囊藻毒素及節球藻毒素可被發現，但多數的水樣中，卻可以發現柱孢藻毒素的存在，且出廠清水之濃度最高可達8,600 ng/L，用戶端最高濃度達2,200 ng/L，值得注意。
在生物技術發展與應用，分為兩方面，第一為傳統的生物技術，主要用於藍綠細菌的純化與放大培養，第二則為分子生物技術，主要運用於鑑定水庫中，藻類毒素的主要產生者。在傳統生物技術方面，共4株微囊藻於牡丹水庫及1株浮絲藻於鳳山水庫被分離純化出來，其中4株微囊藻皆為產毒性之微囊藻，藉由基因的定序與NCBI的比對，皆不同於資料庫中之菌株(strain)，故命名為TWNCKU strains (TWNCKU01-TWNCKU04)。在分子生物技術方面，除相關的基礎技術，如：DNA萃取、複製、放大與克隆複製外，主要應用的分子生物技術為：濃度梯度電泳(DGGE)與即時基因定量(real-time PCR)等；前者主要用於監測樣品當中，藍綠細菌組成之種類社群與產毒菌株的存在與否，而後者則利用所設計的高專一性引子(primer)配合UPL探針進行目標基因的定量，而定量的目標基因主要可分為兩大類，第一類為合成藻類毒素的官能性基因，如：mcy基因，第二類為藍綠細菌所獨有的藻藍素合成基因，如：PC-IGS (cpcB)基因。在分析的結果方面，DGGE的藍綠細菌藻類族群的監測與目標基因的定量，皆可再次地證實水樣當中的確存在具產毒性的藍綠細菌。配合以上分子生物工具，及培養與觀測單株純化藍綠細菌之生長情形與產生毒素型式、濃度等，幾乎可以確定牡丹水庫微囊藻毒素的主要產生者，也第一次成功地應用UPL探針於藻類基因的定量運用。

Cyanobacteria are present in many drinking water reservoirs in Taiwan and the world, and some of them may produce cyanotoxins and release them to natural water bodies. However, the information relevant to the presence of toxic cyanobacteria and cyanotoxins in Taiwan’s drinking water reservoirs are very limited. Therefore, a systematic investigation of their occurrence is urgently needed. The objectives of this dissertation is to develop and apply different analytical approaches, including chemical and bio-molecular methods, for the determination of toxigenic cyanobacteria and cyanotoxins in Taiwan’s drinking water reservoirs and waterworks. In this dissertation, a solid phase extraction (SPE) coupled with liquid chromatography (LC)-mass spectrometry (MS) method was first developed to concentrate and detect nine commonly observed cyanobacterial toxins simultaneously, including six microcystins (MCs) congeners, nodularin (NOD), anatoxin-a (ATX) and cylindrospermopsin (CYN), in water samples. A surrogate standard (SS) and internal standard (IS) were applied in the analytical method for better quality control. The method detection limit (MDL) was 2-10 ng/L for MCs and NOD in pure water, and was 46 ng/L for ATX and 100 ng/L for CYN, respectively. In more complicated water matrix, reservoir water with high concentration of Microcystis spp., the MDL for the cyanotoxins increased by a factor of 3 to 10, with CYN = 500 ng/L as the highest.
The analytical method developed was then applied to monitor two groups of cyanotoxins (MCs and ATX) in nine major drinking water reservoirs and seven associated waterworks. Monitoring results suggested that microcystins were present in all the drinking water reservoirs studied, and some of them had concentration higher than the WHO guideline of MC-LR (1 μg/L). In addition, ATX was also found in four reservoirs, in Kinmen Island. In order to correlate the two groups of cyanobacterial metabolites (cyanotoxins and off-flavour compounds) and other environmental parameters, 22 water quality and meteorological parameters were monitored for two source waters (Moo Tan Reservoir, MTR, and Tseng Wen Reservoir, TWR) in south Taiwan from August 2003 to April 2005. Monitoring results showed that the cyanotoxins and off-flavour compounds (2-MIB and Geosmin) were present in the source waters. Concentrations of 2–30 ng/L of 2-MIB was observed for the two reservoirs, while that of the summation of five microcystin congeners measured were between 30 and 340 ng/L. The concentration of both 2-MIB and microcystins showed higher concentrations in warmer seasons. A stepwise regression technique was employed to correlate 2-MIB and MCs concentrations with all the corresponding water quality and meteorological parameters. Good correlations among 2-MIB concentration, MC concentration, water temperature and air temperature were found in the water samples collected from both reservoirs. The correlations may provide a simple means for the water utility to anticipate the two groups of cyanobacterial metabolites in the two source waters.
In addition to the two reservoirs monitored, the cyanobacterial metabolites were also commonly observed in reservoirs and their associated waterworks in Kinmen Island. To have a better water treatment efficiency in Kinmen’s waterworks, a more precise understanding of the algal metabolites at different time and depths in the water sources as well as the change of metabolites in the treatment processes are needed. Therefore, the diurnal concentration change of the two major cyanobacterial metabolites were monitored in a major source water (Tai Lake Reservoir, TLR and major waterworks (Tai Lake Waterworks, TLW) of the island. The samples for the reservoir water were collected at/near the water intake, and one of them was sampled at 4 different depths. Most of the parameters measured varied significantly at different depths and different time, and only 2-MIB concentration remained almost constant through out the 24 hour period and at different depths. This may imply that 2-MIB was likely to uniformly distribute in the reservoir water. For most of the cyanobacteria and cyanobacterial metabolites measured, no strong correlations were observed. However, a good correlation between Microcystis spp. and MCs concentrations was found, indicating that the probable relationship between the toxins and their producers. This simple correlation may also be used in the estimation of the cell-bound and dissolved concentration of MCs in the reservoir water. For the samples collected for the waterworks, more than 98% of cyanobacteria were removed in the treatment processes, and most were removed at the dissolved air floatation (DAF) unit. Although the overall removal efficiency of microcystins and 2-MIB in TLW is >75%, unlike that for the cyanobacertia cells, only 20-30% were removed before DAF. This may be attributed to that DAF cannot effectively remove dissolved microcystins that was already present in the raw water or was released into water from the breakage of mcirocystis cells by pre-chlorination. Compared with other conventional waterworks, the slow sand filters may provide an extra 20-30% of 2-MIB removal for TLW.
Finally, in order to identify the potential MC producers in MTR and its associated waterworks, two molecular methods were developed and employed to determine the DNA sequences and characteristics of cyanobacteria community, and to quantify the functional gene concentrations in water samples. Four toxigenic Microcystis spp. strains (TWNCKU01 - TWNCKU04) were first isolated from different locations in MTR. After laboratory cultivation, two of the strains, TWNCKU01 and TWNCKU02, were found to mainly produce MC-RR, and another two may produce MC-LR, -RR and -YR at different ratios. The bio-molecular results based on mcyA and mcyB sequencing showed that all the strains are toxic Microcystis spp. and may produce MCs. The two higher diversified regions, PC-IGS (cpcB) and 16S-23S rDNA (ITS), are used to further identify the four strains. In addition, the ITS region was also used in DGGE for the construction of a clone library and bio-makers for 11 strains observed in MTR. These ITS-DGGE biomarkers were successfully applied in monitoring the community changes of potential microcystin producers over a period of 5 years. To develop a rapid method for quantifying microcystin-producing genes, two highly specific primers were designed based on UPL probes to measure mcyB and cpcB concentrations in water samples, where the former one represents gene concentration for MC producers and the latter one is for gene concentrations of cyanobacteria. In the long term monitoring results of MTR, 39 of the 41 DGGE samples contained Microcystis spp., with 36 samples being TWNCKU01 or -02, the MC-RR producers. In addition, 3 of the samples contain Planktothrix spp. After analyzing the data from UPL-based real time PCR and other reservoir water quality parameters, including Microcystis cell counts, MC concentrations, and others, the gene concentrations based on UPL-mcyB correlates well with MC-RR concentrations, the major toxin type in the reservoir, and water temperature. In addition, the gene concentrations based on UPL-cpcB correlate with cyanobacteria as well as Microcystis cell concentrations in the water samples. Both DGGE and UPL-probe methods were further successfully applied in the water samples from MTW. Although toxin concentrations were very low, the DGGE bands clearly demonstrated the presence of MC-RR producers in both process water and finished water samples. The results of UPL-real time PCR also showed that mcyB concentrations were detected to be around 200 copies/mL in the finished samples, proving that Microcystis cells may penetrate through the treatment processes and pose a potential risk in drinking water systems.

Abstract (In Chinese)………………………………………………. i
Abstract………………………….. iv
Acknowledgements…………………………… ix
Table of Contents…………………………. x
List of Figures……………………………… xiii
List of Tables………………………….. xv
Chapter I: Introduction……………………... 1
1.1 Background……………………… 1
1.2 Literature Review………………... 2
1.2.1 Cyanobacteria............................. 2
1.2.2 Cyanotoxins classifications………................... 2
1.2.3 Properties of major cyanotoxins…………………. 3
1.2.4 Environmental parameters relevant to cyanobacterial blooms…………… 4
1.2.5 Episodes relevant to cyanobacterial toxins……………... 5
1.2.6 Drinking water standard………………..………. 6
1.2.7 Analysis methods for cyanotoxins………………………. 7
1.2.8 Molecular tools for cyanobacteria……………….......... 8
1.3 Scope and Objectives………………. 9
1.4 Dissertation overview…………… 11
1.5 Reference………………........... 16
Chapter II: Analysis of cyanotoxins in drinking water using dual solid-phase extraction and liquid chromatography-mass spectrometry…… 35
2.1 Introduction……………….. 36
2.2 Experimental………………….. 38
2.2.1 Reagents…...…………………. 38
2.2.2 Liberation of cyanotoxins………………………………. 39
2.2.3 SPE concentration…………… 39
2.2.4 Liquid chromatography coupled with electrospray ionization mass spectrometry…… 40
2.2.5 Field sites and sampling……………... 41
2.3 Results and Discussion………………….. 43
2.3.1 Qualification and quantification of cyanotoxins… 43
2.3.2 Solid phase extraction (SPE) of MC-LR and CYN…………… 44
2.3.3 Recovery in SPE…………....... 46
2.3.4 Method application in natural waters… 47
2.4 Conclusions…………………… 50
2.5 References……………………….. 52
Chapter III: Cyanobacteria toxins and toxin producers in nine drinking water reservoirs in Taiwan…… 69
3.1 Introduction………………………….... 70
3.2 Materials and Methods……………………... 71
3.2.1 Sampling locations………….......... 71
3.2.2 Chemicals………………………….......... 71
3.2.3 Extraction procedure…………......... 71
3.2.4 Analysis…………………………......... 72
3.2.5 Strain isolation and culture conditions…….. 73
3.2.6 DNA isolation, PCR amplification, DGGE screening and sequencing….... 73
3.3 Results and Discussion………………….. 73
3.3.1 Toxin concentrations in source waters……………………73
3.2 Microcystins removals in the water purification plants……… 74
3.3.3 Identification of microcystin producers…………… 75
3.4 Conclusions………………. 76
3.5 Reference…………………………... 78
Chapter IV: Correlating 2-MIB and microcystins concentrations with environmental parameters in two reservoirs in south Taiwan……… 83
4.1 Introduction………………….... 84
4.2 Materials and Methods…………... 85
4.2.1 Site description……………………. 85
4.2.2 Chemicals…………………………….. 86
4.2.3 Extraction of microcystins……………. 87
4.2.4 Analysis of microcystins………….... 87
4.2.5 Analysis of 2-MIB and geosmin……. 88
4.2.6 Analysis of water quality and collection of meteorological parameters…... 88
4.3 Results and Discussion………………….. 89
4.3.1 Long-term monitoring results………… 89
4.3.2 Water temperature and air temperature… 91
4.3.3 Correlation among 2-MIB, microcystins, and mean air temperature……... 91
4.4 Conclusions……………………….... 93
4.5 References………………………………….. 95
Chapter V: Occurrence and removal of microcystins and 2-MIB in Tai-Lake Reservoir and waterworks……………….. 102
5.1 Introduction……………………........ 103
5.2 Site Description……………….......... 104
5.3 Experimental Procedures…………………... 105
5.3.1 Water sampling…………………….. 105
5.3.2 Chemicals……………………….. 106
5.3.3 Extraction of microcystins…………… 106
5.3.4 Analysis of microcystins...106
5.3.5 Analysis of 2-MIB and geosmin………… 107
5.3.6 Cell counts of cyanobacteria and Microcystis…………………………….. 108
5.4 Results and Discussion…………………….. 108
5.4.1 Microcystins and 2-MIB concentrations in the raw water………………... 108
5.4.2 Concentrations of algae and algal metabolites………………………… 109
5.4.3 Correlation of cyanobacteria and cyanobacterial metabolites………….... 110
5.4.4 Removal of cyanobacteria and their metabolites in TLW…………………. 111
5.5 Conclusions…………………………… 114
5.6 References…………………………………….. 115
Chapter VI: Development and application of DGGE and real time PCR methods for the characterization and quantification of major microcystin producers in Moo-Tan Reservoir and Waterworks………………. 124
6.1 Introduction……………………………… 126
6.2 Methods and Materials…..………………… 129
6.2.1 Field sites and sampling………………………… 129
6.2.2 Cell culture................................ 130
6.2.3 Cell counts……………………… 130
6.2.4 Extraction and amplification of DNA……130
6.2.5 Clone…………………………. 131
6.2.6 Traditional PCR conditions……………………………………………….. 131
6.2.7 DGGE profiling………………………. 132
6.2.8 Real-Time PCR conditions……………… 132
6.2.9 Sequence analysis and phylogenetic construction………………………… 133
6.2.10 Water quality and meteorological parameters…………………………… 133
6.2.11 Nucleotide sequence accession numbers…………………………………. 134
6.3 Results and Discussion………………….. 134
6.3.1 Growth curve of isolated cyanobacteria…………………………………... 134
6.3.2 Analysis of sequence and phylogenesis for TWNCKU strains…………….. 136
6.3.3 Design and validation for UPL primers and probes…………………..… 138
6.3.4 Reservoir monitoring results….. 140
6.3.5 Monitoring of process water…………… 141
6.4 Conclusions………………………………… 142
6.5 References………….. 144
Chapter VII: Conclusions…………………... 166
7.1 Summary………………………… 165
7.2 Implications and future research directions...169
7.3 Closing remark……………………… 171
Appendix…………………… 173
VITA…………………….. 177

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37.Tillett, D., Dittmann, E., Erhard, M., Dohren, H.V., Borner, T., and Neilan, B.A., (2000), Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system, Chemistry and Biology, 7, 753-764.
38.Nishizawa, T., Ueba, A., Asayama, M., Fujii, K., Harada, K.I., Ochi, K., and Shirai, M., (2000), Polyketide synthase gene coupled to the peptide synthetase module involved in the biosynthesis of the cyclic heptapeptide microcystin, Journal of Biochemistry, 127, 779-789.
39.Kurmayer, R. and Kutzenberger, T., (2003), Application of real-time PCR for quantification of microcystin genotypes in a population of the toxic cyanobacterium Microcystis sp., Applied and Environmental Microbiology, 69(11), 6723-6730.
40.Welker, M., (2007), New challenges in cyanotoxin research, 7th International Conference on Toxic Cyanobacetria, 05-10 August, Rio das Pedras, Brazil.
41.New Zealand Ministry of Health, (2005), Drinking Water Quality Standards of New Zealand.
42.Chorus, I., Editorial Summary, in: I. Chorus (ed.) Current Approaches to Cyanotoxin Risk assessment, Risk Management and Regulations in Different Countries, Federal Environmental Agency, Berlin, Germany. (http://www.umweltbundesamt.de)
43.World Health Organization, (2006), WHO Guidelines for Drinking-Water Quality: Vol. 1. Recommendations, First addendum to 3rd edn., World Health Organization, Geneva.
44.National Health and Medical Research Council (NHMRC) of Australian Government, (2004), Australian Drinking water Guidelines.
45.Health Canada, (2007), Guidelines for Canadian Drinking Water Quality.
46.Ministry of Health of China, (2006), Drinking Water Sanitation Standard-GB5749-2006.
47.Wakayama, H., (2005), Revision of drinking water quality standards and QA/QC for drinking water quality analysis in Japan, Technical note of national institute for land and infrastructure management, 264, 74-88.
48.James, H., and Fawell, J., (1991), Detection and removal of cyanobacterial toxins from freshwaters FR 0211, Murlow, Buckinghamshire Foundation for Water Research, UK.
49.Villareal, T.A., and Carpenter, E.J., (2003), Bouyancy regulation and the potential for vertical migration in the oceanic cyanobacterium Trichodesmium, Microbial Ecology, 45, 1-10.
50.Newcombe, G., and Burch, M., (2003), Toxic blue-green algae: coming to a neighbourhood near you?, Opflow, 29(5), 1-7.
51.Zurawell, R.W., Chen, H., Burke, J.M., Prepas, E.E., (2005), Hepatotxic cyanobacteria: a review of the biological importance of microcystins in freshwater environments, Journal of Toxicology and Environmental Health: Part B, 8, 1-37.
52.Mikalsen, B., Boison, G., Skulberg, O.M., Fastner, J., Davies, W., Gabrielsen, T.M., Rudi, K., and Jakobsen, K.S., (2003), Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains, Journal of Bacteriology, 185, 2774-2785.
Chapter II
1.Sivonen, K. and Jones, G., (1999), Cyanobacterial toxins. In: Chorus, I. and Bartram, J. (Eds.) Toxic Cyanobacteria in Water: A Guide to their Publish Health Consequences, Monitoring and Management, pp. 41-111. London: E.& F.N. Spon.
2.National Health and Medical Research Council (NHMRC) of Australian Government, (2004), Australian Drinking Water Guidelines.
3.Poon, K.F., Lam, M.H.W., Lam, P.K.S., and Wong, B.S.F., (2001), Determination of microcystins in cyanobacterial blooms by solid-phase microextraction -high-performance liquid chromatography, Environmental Toxicology and Chemistry, 20, 1648-1655.
4.Herry, S.E., Fathalli, A., Rejeb, A.J.B., Bouaïcha, N., (2008), Seasonal occurrence and toxicity of Microcystis spp. and Oscillatoria tenuis in the Lebna Dam, Tunisia, Water Research, 42, 1263-1273.
5.Krishnamurthy, T., Carmichael, W.W., and Sarver, E.W., (1986), Toxic peptides from freshwater cyanobacteria (blue-green algae): isolation, purification and characterization of peptides from Microcystis aeruginosa and Anabaena flos-aquae, Toxicon, 26, 865-873.
6.Msagati, T.A.M., Siame, B.A., and Shushu, D.D., (2006), Evaluation of methods for the isolation, detection and quantification of cyanobacterial hepatotoxins, Aquatic Toxicology, 78, 382-397.
7.Norris, R.L.G., Eaglesham, G.K., Shaw, G..R., Senogles, P., Chiswell, R.K., Smith, M.J., Davis, B.C., Seawright, A.A., and Moore, M.R., (2001), Extraction and purification of the zwitterions cylindrospermopsin and deoxycylin- drospermopsin from Cylindrospermopsis raciborskii, Environmental Toxicology, 16, 391-396.
8.Harada, K.I., Ohtani, I., Iwamoto, K., Suzuki, M., Watambe, M.F., Watanabe, M., and Terao, K., (1994), Isolation of cylindrospermopsin from a cyanobacterium Umezakia natans and its screening method, Toxicon, 32. 73-84.
9.Barco, M., Rivera, J., and Caixach, J., (2002), Analysis of cyanobacterial hepatotoxins in water samples by microbore reversed-phase liquid chromatography–electrospray ionisation mass spectrometry, Journal of Chromatography A, 959, 103-111.
10.Namikoshi, M., Murakami, T., Watanabe, M.F., Oda, T., Yamada, J., Tsujimura, S., Nagai, H., and Oishi, S., (2003), Simultaneous production of homoanatoxin-a, anatoxin-a, and a new non-toxic 4-hydroxyhomoanatoxin-a by the cyanobacterium Raphidiopsis mediterranea Skuja, Toxicon, 42, 533-538.
11.Bogialli, S., Bruno, M., Curini, R., Corcia, A.D., Fanali, C., and Lagana, A., (2006), Monitoring algal toxins in lake water by liquid chromatography tandem mass spectrometry, Environmental Science and Technology, 40, 2917-2923.
12.Cong, L., Huang, B., Chen, Q., Lu, B., Zhang, J., and Ren, Y., (2006), Determination of trace amount of microcystins in water samples using liquid chromatography coupled with triple quadrupole mass spectrometry, Analytica Chimica Acta, 569, 157-168.
13.Ott, J.L. and Carmichael, W.W., (2006), LC/ESI-MS method development for the analysis of hepatotoxic cyclic peptide microcystins in animal tissues, Toxicon, 47, 734-741.
14.Kikuchi, S., Kubo, T., and Kaya, K., (2007), Cylindrospermopsin determination using 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) as the internal standard, Analytica Chimica Acta, 583, 124-127.
15.Dahlmann, J., Budakowski, W.R., and Luckas, B., (2003), Liquid chromatography-electrospray ionization-mass spectrometry based method for the simultaneous determination of algal and cyanobacterial toxins in phytoplankton from marine waters and lakes followed by tentative structural elucidation of microcystins, Journal of Chromatography A, 994, 45-57.
16.USEPA, (1996), Guide to Method Flexibility and Approval of EPA Water Methods, 27.
17.Chorus, I., Editorial Summary, in: I. Chorus (ed.) Current Approaches to Cyanotoxin Risk assessment, Risk Management and Regulations in Different Countries, Federal Environmental Agency, Berlin, Germany. (http://www.umweltbundesamt.de)
18.Richard, J.W. and Pleter, G.T., (1990), Environmental factors affecting the production of peptide toxins in floating scums of the cyanobacterium Microcystis aeruginosa in a hyperytophic African reservoir, Environmental Science and Technology, 24, 1413-1418.
19.Diehnelt, C.W., Dugan, N.R., Peterman, S.M., and Budde, W.L., (2006), Identification of Microcystin Toxins from a Strain of Microcystis aeruginosa by Liquid Chromatography Introduction into a Hybrid Linear Ion Trap-Fourier Transform Ion Cyclotrin Resonance Mass Spectrometer, Analytical Chemistry, 78, 501-512.
20.Maizels, M. and Budde, W.L., (2004), A LC/MS Method for the Determination of Cyanobacteria Toxins in Water, Analytical Chemistry, 76, 1342-1351.
21.Lawton, L.A., Edwards, C., Beattie, K.A., Pleasance, S., Dear, G.J., and Codd, G.A., (1995), Isolation and characterization of microcystins from laboratory cultures and environmental samples of Microcystis aeruginosa and from an associated animal toxicosis, Natural Toxins, 3, 50-57.
22.Jang, M.H., Ha, K., and Takamura, N., (2008), Microcystin production by Microcystis aeruginosa exposed to different stages of herbivorous zooplankton, Toxicon, 51, 882-889.
23.Spoof, L., Berg, K.A., Rapala, J., Lahti, K., Lepistö, L., Metcalf, J.S., Codd, G.A., Meriluoto, J., (2006), First observation of cylindrospermopsin in Anabaena lapponica isolated from the boreal environment (Finland), Environmental Toxicology, 21, 552-560.
24.Dyble, J., Tester, P., and Litaker, R., (2006), Effects of light intensity on cylindrospermopsin production in the cyanobacterial HAB species Cylindrospermopsis raciborskii, African Journal of Marine Science, 28(2), 309-312.
25.Aráoz, R., Nghiêm, H.O., Rippka, R., Palibroda, N., Marsac, N.T.D., and Herdman, M., (2005), Neurotoxins in axenic oscillatorian cyanobacteria: coexistence of anatoxin-a and homoanatoxin-a determined by ligand-binding assay and GC/MS, Microbiology, 151, 1263-1273.
26.Laamanen, M.J., Gugger, M.F., LehtimÄki, J.M., Haukka, K., and Sivonen, K., (2001), Diversity of toxic and nontoxic Nodularia isolates (cyanobacteria) and filaments from the Baltic Sea, Applied and Environmental Microbiology, 67, 4638-4647.
27.The Drinking Water Quality Report in Kinmen County, (2007), http://water.kinmen.gov.tw/html/09607.doc.
28.Newcombe, G. and Nicholson B., (2004), Water treatment options for dissolved cyanotoxins, Water Science and Technology: Water Supply, 227-239.
29.New Zealand Ministry of Health, (2005), Drinking Water Quality Standards of New Zealand.
30.World Health Organization, (2006), WHO Guidelines for Drinking-Water Quality: Vol. 1. Recommendations, First addendum to 3rd Edn., World Health Organization, Geneva.
31.Carlson R.E., (1977), A trophic state index for lakes, Limnology and Oceanography, 2, 361-369.
Chapter III
1.TWEPA (2003), Taiwan Environmental Protection Statistical Data Book, Taiwan Environmental Protection Administration.
2.Hummert, C., Reichelt, M., Weiβ, J., Liebert, H., and Luckas, B., (2001), Identification of microcystins in cyanobacteria from the Bleiloch former drinking-water reservoir (Thuringia Germany), Chemosphere, 44, 1581-1588.
3.Oberholster P.J., Botha, A.M., and Grobbelaar, J.U., (2004), Microcystis aeruginosa: source of toxic microcystins in drinking water, African Journal of Biotechnology, 3, 159-168.
4.Barco, M., Rivera, J. and Caixach, J., (2002), Analysis of cyanobacterial hepatotoxins in water samples by microbore reversed-phase liquid chromatography-electrospray ionization mass spectrometry, Journal of Chromatography A, 959, 103-111.
5.Pyo D. and Shin H., (2002), Extraction and analysis of microcystins RR and LR in cyanobacteria using a cyano cartridge, Journal of Biochemical and Biophysical Methods, 51, 103-109.
6.Ruangyuttikarn W., Miksik I., Pekkoh J., Peerapornpisal Y. and Deyl Z., (2004), Reversed-phase liquid chromatographic-mass spectrometric determination of microcystin-LR in cyanobacteria blooms under alkaline conditions, Journal of Chromayography B, 800, 315-319.
7.Stirling D. J. and Quilliam M. A., (2001), First report of the cyanobacterial toxin cylindrospermopsin in New Zealand. Toxicon, 39, 1219-1222.
8.Rapala J., Sivonen K., Lyra C. and Niemela S.I., (1997), Variation of Microcystins, Cyanobacterial Hepatotoxins, in Anabaena spp. as a Function of Growth Stimuli, Applied and Environmental Microbiology, 63(6), 2206-2212.
9.Tillett D., Parker D. L. and Neilan B. A., (2001), Dtection of toxigenicity by a probe for the microcystin synthetase a gene (mcyA) of the cyanobacterial genus Microcystis: comparison of toxicities with 16S rRNA and phycocyanin operon (phycocyanin intergenic spacer) phylogenies, Applied and Environmental Microbiology, 67(6), 2810-2818.
10.Mikalsen B., Boison G., Skulberg O. M., Fastner J., Davies W., Gabrielsen T. M., Rudi K. and Jakobsen K. S., (2003), Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains. Journal of Bacteriology, 185(9), 2774-2785.
11.Ishii H., Nishijima M. and Abe T., (2004), Characterization of degradation process of cyanobacterial hepatotoxins by a gram-negative aerobic bacterium. Water Research, 38, 2667-2676.
Chapter IV
1.Haider, S., Naithani, V., Viswanathan, P.N. and Kakkar, P., (2003), Cyanobacterial toxins: a growing environmental concern, Chemosphere, 52, 1–21.
2.Carlson, R.E., (1977), A trophic state index for lakes, Limnology and Oceanography, 22, 361–369.
3.TWEPA (2005), Environmental Protection Statistical Data Book, Taiwan Environmental Protection Administration, Taipei, Taiwan.
4.Lin, T.F. and Tseng, I.C., (2005), Investigation of Cyanobacteria and Cyanotoxins in Drinking Water Systems, Taiwan Environmental Protection Administration, Project Report EPA-94-U1U1-02-101 (in Chinese).
5.Falconer, I.R., (2005), Cyanobacterial Toxins of Drinking Water Supplies: Cylindrospermopsins and Microcystins, CRC Press, Boca Raton, FL, USA.
6.Jones, G.J. and Korth, W., (1995), In-situ production of volatile odour compounds by river and reservoir phytoplankton population in Australia, Water Science and Technology, 31(11), 145–151.
7.Carmichael, W.W., (2001), Assessment of Blue-Green Algal Toxins in Raw and Finished Drinking Water, American Water Works Association Research Foundation, Denver, Colorado, USA.
8.Lin, T.F., Wong, J.Y. and Kao, H.P., (2002), Correlation of musty odor and 2-MIB in two drinking water treatment plants in Southern Taiwan, Science of Total Environment, 289, 225–235.
9.Lin, T.F., Liu, C.L., Yang, F.C. and Hoang, S.W., (2003), Effect of residual chlorine on the analysis of geosmin, 2-MIB and MTBE in drinking water using the SPME technique, Water Research, 37, 21–26.
10.APHA (1995), Standard Methods for the Examination of Water and Wastewater, 19th ed, American Public Health Association, Washington, DC, USA.
11.Garcia-Villavova, R.J., Garcia, C., Gomez, J.A., Garcia, M.P. and Ardanuy, R., (1997), Formation, evolution and modeling of trihalomethanes in the drinking water of a town: I. at the municipal treatment utilities, Water Research, 31, 1299–1308.
Chapter V
1.Chorus, I., (2005), (ed.) Current Approaches to Cyanotoxin Risk Assessment, Risk Management and Regulations in Different Countries, Federal Environmental Agency, Berlin, Germany.
2.TWEPA (2005), Taiwan Environmental Protection Statistical Data Book, Taiwan Environmental Protection Administration.
3.Suffet, I.H., and Mallevialle, J., (1995), Taste-and-odor problems observed during drinking water treatment, In Advances in taste-and-odor treatment and control. Ed. by Suffet, I.H., Mallevialle, J., and Kawczynski, E., American Water Works Association Research Foundation, Denver, Coloroda, USA.
4.Lin, T.F., and Tseng, I.C., (2005), Investigation of Cyanobacteria and Cyanotoxins in Drinking Water Systems, Taiwan Environmental Protection Administration, Project Report EPA-94-U1U1-02-101. (In Chinese).
5.Lin, T.F., Wong J.Y., and Kao, H.P., (2002), Correlation of Musty Odor and 2-MIB in Two Drinking Water Treatment Plants in Southern Taiwan, Sci. Total Env., 289, 225-235.
6.Wu, J.T., (2004), The Relation between Water Quality and Algae in the Reservoirs of Eastern King-Men Island, Taiwan. (In Chinese)
7.Yen, H.K., Lin, T.F., Tseng, I.C., and Su, Y.T., (2005), Cyanobacteria toxins and toxin producers in nine drinking water reservoirs in Taiwan, Proceeding of the First IWA-ASPAC Conference, Singapore, July 10-15.
8.APHA, AWWA, and WPCF., (2000), Standard Methods for the Examination of Water and Wastewater, 20th ed., Washington, D.C., USA.
9.Lin, T.F. and Chang, E.E., (2002), Investigation and Mitigation of Taste and Odor Compounds in Drinking Water Distribution Systems, Environmental Protection Administration, Taiwan, ROC. (In Chinese)
10.Drikas, M., Newcombe, G., and Nicholson, B., (2002), Water Treatment Options for Cyanobacteria and Their Toxins, In Blue-Green Algae: Their significance and management within water supplies, The Cooperative Research Centre for Water Quality and treatment, Occasional Paper 4.
11.Sigee, D.C., (2005), Freshwater Microbiology, Wiley, West Sussex, England, UK.
12.National Health and Medical Research Council (NHMRC) of Australian Government (2004), Australian Drinking water Guidelines.
13.Hrudey, S., Burch, M, Drikas, M., and Gregory, R., (1999), Remedial Measures, In Toxic Cyanobacteria in Water: A guide to their public health consequences,monitoring and management, Ed. by IChorus, I. and Bartram, J., World Health Organization, Geneva, Switzerland.
14.NZMOH (New Zealand Ministry of Health), (2000), Drinking Water Standards for New Zealand.
Chapter VI
1.Verspagen, J.M.H., Passarge, J., Johnk, K.D., Visser, P.M., Peperzak, L., Boers, P., Laanbroek, H.J., and Huisman, J., (2006), Water management strategies against toxic Microcystis blooms in the Dutch delta, Journal of Applied Ecology, 16, 313-327.
2.Tillett, D., Dittmann, E., Erhard, M., Dohren, H.V., Borner, T., and Neilan, B.A., (2000), Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system, Chemistry and Biology, 7, 753-764.
3.Falconer, I.R. and Jackson, A.R.B., (1981), Liver pathology in mice in poisoning by the blue-green alga in Microcystis aeruginosa, Australian Journal of Biological Science, 34, 179-187.
4.Chu, F.S. and Hung, X., (1990), Enzyme-linked immunosorbent assay for microcystins in blue-green algal blooms, Journal of the Association of Official Analytical Chemists, 73, 451-456.
5.Yoshizawa, S., Matsushima, R., Watanabe, M.F., Harada, K.I., Ichihara, A., Carmichael, W.W., and Fujiiki, H., (1990), Inhibition of protein phosphatases by microcystis and nodularin associated with hepatotoxicity, Journal of Cancer Research and Clinical Oncology, 116, 609-614.
6.Metcalf, J.S., Beattie, K.A., Saker, M.L., and Codd, G.A., (2002), Effects of organic solvents on the high performance liquid chromatographic analysis of the cyanobacterial toxin cylindrospermopsin and its recovery from environmental eutrophic waters by solid phase extraction, FEMS Microbiology Letters, 216, 159-164.
7.McElhiney, J. and Lawton, L.A., (2005), Detection of the cyanobacterial hepatotoxins microcystins, Toxicology and Applied Pharmacology, 203, 219-230.
8.Bogialli, S., Bruno, M., Curini, R., Corcia, A.D., Fanali, C., and Lagana, A., (2006), Monitoring algal toxins in lake water by liquid chromatography tandem mass spectrometry, Environmental Science and Technology, 40, 2917-2923.
9.Metcalf, J.S., Bell, S.G., and Codd, G.A., (2000), Production of novel polyclonal antibodies against the cyanobacterial toxin microcystin-LR and their application for the detection and quantification of microcystins and nodularin, Water Research, 34, 2761-2769.
10.Briand, E., Gugger, M., François, J.-C., Bernard, C., Humbert, J.-F., and Quiblier, C., (2008), Temporal variations in the dynamics of potentially microcystin-producing strains in a bloom-forming Planktothrix agardhii (cyanobacterium) population, Applied and Environmental Microbiology, 74, 3839-3848.
11.Neilan, B.A., Jacobs, D., Dot, T.D., Blackall, L.L., Hawkin, P.R., Cox, P.T., and Goodman, A.E., (1997), rRNA sequences and evolutionary relationships among toxic and nontoxic cyanobacteria of the genus Microcystis, International Journal of Systematic Bacteriology, 47, 693-697.
12.Neilan, B.A., (1995), Identification and phylogenetic analysis of toxigenic cyanobacteria using a multiplex randomly amplified polymorphic DNA PCR, Applied and Environmental Microbiology, 61, 2286-2291.
13.Fahrenkrug, P.M., Bett, M.B., and Parker, D.L., (1992), Base composition of DNA from selected strains of the cyanobacterial genus Microcystis, International Journal of Systematic Bacteriology, 42, 182-184.
14.Neilan, B.A., Jacobs, D., and Goodman, A.E., (1995), Genetic diversity and phylogeny of toxic cyanobacteria determined by DNA polymorphisms within the phycocyanin locus, Applied and Environmental Microbiology, 61, 3875-3883.
15.Janse, I., Meima, M., Kardinall, W.E.A., and Zwart, G., (2003), High-resolution differentiation of cyanobacteria by using rRNA-internal transcribed spacer denaturing gradient gel electrophoresis, Applied and Environmental Microbiology, 69, 6634-6643.
16.Rudi, K., Skulberg, O.M., Larsen, F., and Jakobsen, K.S., (1997), Strain characterization and classification of oxyphotobacteria in clone cultures on the basis of 16S rRNA sequences from the variable regions V6, V7, and V8, Applied and Environmental Microbiology, 63, 2593-2599.
17.Asayama, M., Kabasawa, M., Takahashi, I., Aida, T., and Shirai, M., (1996), Highly repetitive sequences and characteristics of genomic DNA in unicellular cyanobacterial strains, FEMS Microbiology Letter, 137, 175-181.
18.Muyzer, G., Waal, E.C., and Uitterlinden, A.G., (1993), Profiling of complex microbial populations by denaturing gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA, Applied and Environmental Microbiology, 59, 695-700.
19.Temmerman, R., Masco, L., Vanhoutte, T., Huys, G., and Swings, J., (2003), Development and validation of a nested-PCR-denaturing gradient gel electrophoresis method for taxonomic characterization of bifidobacterial communities, Applied and Environmental Microbiology, 69, 6380-6385.
20.Nubel, U., Garcia-Pichel, F., and Muyzer, G., (1997), PCR primer to amplify 16S rRNA genes from cyanobacteria, Applied and Environmental Microbiology, 63, 3327-3332.
21.Tillett, D., Parker, D.L., and Neilan, B.A., (2001), Detection of toxigenicity by a probe for the microcystin synthetase A gene (mcyA) of the cyanobacterial genus Microcystis: comparison of toxicities with 16S rRNA and phycocyanin operon (phycocyanin intergenic spacer) phylogenies, Applied and Environmental Microbiology, 67, 2810-2818.
22.Nishizawa, T., Ueba, A., Asayama, M., Fujii, K., Harada, K.I., Ochi, K., and Shirai, M., (2000), Polyketide synthase gene coupled to the peptide synthetase module involved in the biosynthesis of the cyclic heptapeptide microcystin, Journal of Biochemistry, 127, 779-789.
23.Higuchi, R., Dollinger G., Walsh, P.S., and Griffith, R., (1992), Simultaneous amplification and detection of specific DNA sequences, Biotechnology, 10 413-417.
24.Mouritzen, P., Noerholm, M., Nielsen, P.S., Jacobsen, N., Lomholt, C., Pfundheller, H.M., Tolstrup, N, (2005), ProbeLibrary: a new method for faster design and execution of quantitative real-time PCR, Nature Methods, 2, 313–316.
25.Rippka, R., Deruelles, J.,Waterbury, J.B., Herdman, M., Stanier, R.Y., (1979), Genetic assignments, strain histories and properties of pure cultures of cyanobacteria, Journal of General Microbiology, 111, 1-61.
26.Gorham, P.R., McLachlan, J., Hammer, U.T., and Kim, W.K., (1964), Isolation and culture of toxic strains of Anabaena flos-aquae (Lyngb.) de Breb, Internationale Vereinigungfür theoretische und angewandte Limnologie, Verhandlungen, 15, 796-804.
27.APHA, AWWA, and WPCF, (2000), Supplement to standard methods for the examination of water and wastewater, 20th ed., Washington, D.C., USA.
28.Mikalsen, B., Boison, G., Skulberg, O.M., Fastner, J., Davies, W., Gabrielsen, T.M., Rudi, K., and Jakobsen, K.S., (2003), Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains, Journal of Bacteriology, 185, 2774-2785.
29.Hoeger, S.J., Shaw, G., Hitzfeld, B.C., and Dietrich, D.R., (2004), Occurrence and elimination of cyanobacterial toxins in two Australian drinking water treatment plants, Toxicon, 43, 639-649.
30.Cadel-Six, S., Dauga, C., Castets, A.M., Rippka, R., Bouchier, C., Marsac, N.T., and Welker, M., (2008), Halogenase genes in nonribosomal peptide synthetase gene clusters of Microcystis (cyanobacteria): sporadic distribution and evolution, Molecular Biology and Evolution, 25, 2031-2041.