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研究生:黃秋菊
研究生(外文):Chiu-Chu Hwang
論文名稱:微量元素及化學藥劑對毒藻Alexandrium minutum生長及產毒之影響
論文名稱(外文):The Effects of Trace Elements and Chemicals on the Growth and Toxicity of Alexandrium minutum
指導教授:蔡永祥蔡永祥引用關係
指導教授(外文):Yung-Hsien Tsai
學位類別:碩士
校院名稱:大仁科技大學
系所名稱:環境管理研究所
學門:環境保護學門
學類:環境資源學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:93
中文關鍵詞:麻痺性貝毒毒藻殺藻劑
外文關鍵詞:toxic dinoflagellateParalytic shellfish poisoning (PSP)algicides
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台灣在1986年屏東東港和1991年嘉義東石均曾發生因食用西施舌貝中毒並造成2人死亡之案例,其中毒原因經證實為毒藻Alexandrium minutum產生麻痺性貝毒並毒化西施舌貝所導致。其次,在東港附近排水溝出海口及養殖池於每年冬季均可檢出此毒藻存在,並含有麻痺性貝毒。因此為了解水域中之微量元素是否會影響毒藻A. minutum之生長及產毒,本實驗即以不同氧化態及不同濃度之銅離子、鐵離子以及維生素B群於walne medium配置時分別加入以培養藻類,結果發現毒藻A. minutum之最適生長與產毒條件仍然以walne medium之條件最佳,即銅離子(Cu2+)濃度5 ppb、鐵離子(Fe3+)濃度270 ppb以及維生素B1 200 ppb與維生素B12 10 ppb。若利用亞銅離子(Cu+)及亞鐵離子(Fe2+)取代walne medium中之銅離子及鐵離子之含量,結果發現生長與產毒均不佳,顯示其無法取代原本培養液中銅、鐵離子之使用。以HPLC分析藻體毒素之組成種類皆為GTX1-4為主,且以GTX1和GTX4佔多數。
其次,為了解目前養殖池常用之化學殺藻劑及較熱門的殺菌劑臭氧(Ozone,O3)與幾丁聚醣(chitosan)等,是否具有抑制A. minutum生長及產毒的效果。故係添加不同濃度之臭氧(O3)、幾丁聚醣(Chitosan)與化學殺藻劑於藻液中,觀察毒藻A.minutum生長及產毒之影響。結果顯示臭氧(O3)抑制毒藻生長與產毒之效果最好,其次為BKC與(NH4)2SO4,其他化學殺藻劑與幾丁聚醣之效果較差。若以對藻類於24 hr時之影響濃度EC50(Effective concentration)來比較時,則O3、BKC、KMnO4、Malachite green與(NH4)2SO4分別為0.5、1.0、7.5、10和11.1 ppm,而96小時之EC50則分別為O3 0.5 ppm、BKC 1.0 ppm、(NH4)2SO4 1.3 ppm 、KMnO 4 5.2 ppm、Malachite green 10 ppm、Methylene blue 35 ppm 、CuSO4 100 ppm、D(+)Glucosamine (pH 6.0) 200 ppm以及Chitosan (pH 6.0) 1000 ppm。另外,各組藻細胞之總毒量會因藻體死亡而降低,但單位細胞毒量在低濃度之化學藥劑組別(除幾丁聚醣與葡萄糖胺外)有升高之趨勢,甚至高於控制組。另以HPLC分析藻體毒素之組成均為GTX1-4,而以GTX1 及GTX4之總量佔多數。
Paralytic shellfish poisoning (PSP) associated with food poisoning incidents have caused two human fatalities and many illnesses in Taiwan. The sourse of PSP in Taiwan was thought to be the toxic dinoflagellate Alexandrium minutum. To understand the trace elements in walne medium for cell growth and toxin production of A. minutum, the different concentration of copper and iron ion, and vitamin B group were added into the walne medium of A. minutum. It was found that the optimal trace elements for those of A. minutum were as follows : copper ion (Cu2+) 5.0 ppb, ferric ion (Fe3+) 270 ppb, and vitamin B1 200 ppb and vitamin B12 10 ppb. The toxic components of A. minutum were assayed by high performance liquid chromatography (HPLC), and found to contain gonyautoxin (GTX)1-4 only. Among these components, GTX1 and GTX4 were predominate components.
On the other hand, chemicals were commonly used in aquaculture ponds to controlling toxic dinoflagellate. The effects of algicides 【(NH4)2SO4、CuSO4、KMnO4、benzalkonium chloride(BKC)、methylene blue and malachite green】, chitosan and ozone on the growth and toxicity of A. minutum were elucidated. Among the tested chemicals, O3 was found to be the more effective, followed by BKC and (NH4)2SO4. At the effective concentration (EC50) of 24 hrs for A. minutum were as follows : ozone 0.5 ppm, BKC 1.0 ppm, KMnO4 7.5 ppm , malachite green 10 ppm and (NH4)2SO4 11.1 ppm. At the effective concentration (EC50) of 96 hrs for A. minutum were as follows: ozone 0.5 ppm, BKC 1.0 ppm, (NH4)2SO4 1.3 ppm, KMnO4 5.2 ppm, malachite green 10 ppm, Methylene blue 35 ppm, CuSO4 100 ppm, D(+)glucosamine (pH 6.0) 200 ppm, and chitosan (pH 6.0) 1000 ppm. The total toxicity of A. minutum in each treatment were lower than control treatment, whereas the cell toxicity in low concentration of chemical treatments (except Glucosamine and Chitosan) was higher than control treatment. The toxic components of A. minutum for each treatment were assay by HPLC, and found to contain GTX1-4 only. Among these toxin components, GTX1 and GTX4 were the predominate components.
目 錄
頁次
誌 謝......................................................................................................Ⅰ
摘 要.....................................................................................................Ⅱ
ABSTRACT............................................................................................Ⅳ
目 錄.....................................................................................................Ⅵ
一、文獻整理..............................................................................................1
1.1麻痺性貝毒.......................................................................................1
1.1.1麻痺性貝毒簡介……………………………………………...1
1.1.2麻痺性貝毒之毒理機制……………………………………...1
1.1.3麻痺性貝毒中毒之症狀……………………………………...2
1.1.4麻痺性貝毒於自然界之分布情形…………………………...2
1.2有毒渦鞭毛藻之紅潮現象………………………………………...3
1.2.1紅潮之發生…………………………………………………....3
1.2.2渦鞭毛藻簡介………………………………………………....3
1.2.3休眠孢子之萌發與增殖……………………………………....4
1.2.4環境因子之影響……………………………………………....5
1.2.5金屬離子之影響……………………………………………...6
1.2.6維生素之影響………………………………………………...7
1.3有毒渦鞭毛藻之微小亞歷山大藻.……………………………...8
1.3.1微小亞歷山大藻之型態與分布.……………………………...8
1.3.2微小亞歷山大藻之毒成分………….………………………...8
1.3.3微小亞歷山大藻在台灣分布情形…………………….……...9
1.4化學方法對藻類生長之抑制情形.….…………………………...9
1.4.1硫酸銨 ( (NH4)2SO4) ……..………………………………....10
1.4.2硫酸銅 (CuSO4) …….…….………………………………...11
1.4.3過錳酸鉀 (KMnO4) ……..……………………………..…...11
1.4.4 Benzalkonium Chloride (BKC)……………………………...12
1.4.5甲烯藍(methylene blue)與孔雀綠 (malachite green)………13
1.4.6幾丁聚醣( Chitosan)與其單體-葡萄糖胺( Glucosamine)...14
1.4.7臭氧 (Ozone) ………..……………………………………....15
1.5圖表……………………………………………………………...16
二、微量元素對毒藻Alexandrium minutum生長及產毒之影響……...19
2.1前言……………………………………………………………….19
2.2實驗材料………………………………………………………….21
2.2.1藻體………..…………………………………………………..21
2.2.2試藥…..………………………………………………………..21
2.2.3麻痺性貝毒標準品..…………………………………………..22
2.3實驗方法………………………………………………………….22
2.3.1不同微量元素對藻細胞生長影響之探討…………..………..22
2.3.1.1藻類培養…………………………………………………...22
2.3.1.2銅離子與亞銅離子………………………………………...22
2.3.1.3鐵離子與亞鐵離子………………………………………...23
2.3.1.4維生素B群………………………………………………...23
2.3.2不同微量元素對藻細胞產毒之影響..………………………..23
2.3.2.1藻類毒素之萃取及毒性試驗……………………………...23
2.3.2.2毒成分之鑑定……………………………………………...24
2.4結果……………………………………………………………….26
2.4.1不同氧化態之銅、鐵離子對藻細胞生長及產毒之影響……..26
2.4.1.1銅離子 (Cu2+) 與亞銅離子 (Cu+)………………………..26
2.4.1.2鐵離子 (Fe3+) 與亞鐵離子 (Fe2+)………………………..27
2.4.1.3維生素B群………………………………………………...28
2.5討論……………………………………………………………….29
2.6 圖表……………………………………………………………...31
三、化學藥劑對毒藻Alexandrium minutum生長及產毒之影響…….41
3.1前言………………………………………………………………41
3.2實驗材料…………………………………………………………43
3.2.1藻體…………………………………………………………...43
3.2.2試劑…………………………………………………………...43
3.2.3臭氧…………………………………………………………...43
3.2.4麻痺性貝毒標準品……………………………………….…44
3.3實驗方法…..…...………………………………………………44
3.3.1化學藥劑對藻細胞生長之影響………..………………...44
3.3.1.1藻類培養…………………..………………………….....44
3.3.1.2硫酸銨 ( (NH4)2SO4) ……………………………………45
3.3.1.3 硫酸銅 (CuSO4) ………..………………………………45
3.3.1.4 過錳酸鉀 (KMnO4) ……………………………..……...45
3.3.1.5 甲烯藍(methylene blue) …..…………………………….45
3.3.1.6 孔雀綠 (malachite green) ………..……………………..45
3.3.1.7 Benzalkonium Chloride (BKC) ..……………………...…46
3.3.1.8幾丁聚醣( Chitosan) …………………..………………...46
3.3.1.9葡萄糖胺( D(+)Glucosamine) ………………………….46
3.3.1.10臭氧 (Ozone) ..…………………..……………………46
3.3.1.11 臭氧Ozone於水中殘餘量之檢測…..…..……...47
3.3.1.11.1硫代硫酸鈉之標定…………………………..….47
3.3.1.11.2水中臭氧之測定………………………………...47
3.3.2化學藥劑對藻細胞產毒之影響……………………………48
3.3.2.1藻類毒素之萃取及毒性試驗…………………………...48
3.3.2.2毒成分鑑定..……………………………………………....48
3.4結果……………………………………………………………...50
3.4.1不同化學藥劑對藻細胞生長及產毒之影響………………50
3.4.1.1硫酸銨( (NH4)2SO4 ) ………………………………….50
3.4.1.2 硫酸銅 (CuSO4) ………………………………………51
3.4.1.3 過錳酸鉀 (KMnO4) …………………………………...51
3.4.1.4 甲烯藍(methylene blue) ……………………………….52
3.4.1.5 孔雀綠 (malachite green) ……………………………..53
3.4.1.6 Benzalkonium Chloride (BKC) ………………………...54
3.4.1.7幾丁聚醣( Chitosan) …………………………………...55
3.4.1.8葡萄糖胺( D(+)Glucosamine) ………………………….56
3.4.1.9臭氧 (Ozone) …………………………………………..57
3.5討論…………………………………………………………….......58
3.5.1化學藥劑對毒藻A. minutum生長之影響…………….......58
3.5.2化學藥劑對毒藻A. minutum產毒與毒組成之影響…....62
3.6圖表……………………………………………………………….....64
四、結論……. ………………………………………………………….80
參考文獻………………………………………………………….........82
簡 歷......................................................................................................93

















表 錄
Table 1-1 The structures of PSP components…………………...16
Table 1-2 Comparsion of oxidizing ability for five oxidants ....17
Table 1-3 Comparsion of sterilize ability for ozone and three chloride compounds………………………………………….…..18
Table 2-1. The composition of Walne medium………………………..31
Table 2-2. Various concentrations of vitamins added in walne
medium.................................................................................32
Table 2-3 Effects of cupric and ferrous ions on the toxin profile of Alexandrium minutum……………………………………..33
Table 2-4 Effects of vitamin components on the toxin profile of Alexandrium .minutum……………………………………..35
Table 3-1 Treatment concentrations of chemicals in Alexandrium minutum……………………………………………………64Table 3-2. Effective concentration (EC50) of Alexandrium minutum upon exposure to various concentrations of chemicals…....65
Table 3-3. Effects of chemicals on the toxin profile of Alexandrium minutum……………………………………………………66




圖 錄
Fig. 2-1 Variations in growth and toxicity of Alexandrium minutum under different Cu2+ concentration conditions. A:cell number , B:total toxicity , C:cell toxicity…………………………..…36
Fig. 2-2 Variations in growth and toxicity of Alexandrium minutum under different Cu+ concentration conditions. A:cell number, B:total toxicity, C:cell toxicity……………………………....37
Fig. 2-3 Variations in growth and toxicity of Alexandrium minutum
under different Fe3+ concentration conditions. A:cell number, B:total toxicity, C:cell toxicity……………………………....38
Fig. 2-4 Variations in growth and toxicity of Alexandrium minutum under different Fe2+ concentration conditions. A: cell number, B:total toxicity, C:cell toxicity……………………………....39
Fig. 2-5 Variations in growth and toxicity of Alexandrium minutum under different vitamins conditions. A:cell number , B: total toxicity, C:cell toxicity.Vitamins were added to the vitamin-omitted metals mix. Walne medium:Vitamin B1(thiamine): 200 ppb, Vitamin B12: 10 ppb………………..40
Fig. 3-1 Variations in growth and toxicity of Alexandrium minutum after (NH4)2SO4 treatment. A:cell number, B:total toxicity, C:cell toxicity……………………………………............................69
Fig. 3-2 Variations in growth and toxicity of Alexandrium minutum after CuSO4 treatment. A:cell number, B:total toxicity, C:cell toxicity………………………………………………………70
Fig. 3-3 Variations in growth and toxicity of Alexandrium minutum after KMnO4 treatment. A:cell number, B:total toxicity, C:cell toxicity…………………………………………………........71
Fig. 3-4 Variations in growth and toxicity of Alexandrium minutum after methylene blue treatment. A:ell number, B:total toxicity, C:cell toxicity. ………………………………………………72
Fig. 3-5 Variations in growth and toxicity of Alexandrium minutum after malachite green treatment. A:cell number, B:total toxicity, C:cell toxicity. …………………………………………........73
Fig. 3-6 Variations in growth and toxicity of Alexandrium minutum after BKC treatment. :cell number, B:total toxicity, C:cell toxicity....................................................................................74
Fig. 3-7 Variations in growth and toxicity of Alexandrium minutum cultured in walne medium (pH7.8) after chitosan treatment.A:cell number, B:total toxicity, C:cell toxicity….75
Fig. 3-8 Variations in growth and toxicity of Alexandrium minutum cultured in walne medium (pH6.0) after chitosan treatment. A:cell number, B:total toxicity, C:cell toxicity……………...76
Fig. 3-9 Variations in growth and toxicity of Alexandrium minutum cultured in walne medium (pH7.8) after D(+)glucosamine treatment. A:cell number, B:total toxicity, C:cell toxicity…..77
Fig. 3-10 Variations in growth and toxicity of Alexandrium minutum cultured in walne medium (pH6.0) after D(+)glucosamine treatment. A:cell number, B:total toxicity, C:cell toxicity…78
Fig. 3-11 Variations in growth and toxicity of Alexandrium minutum after ozone treatment. A:cell number, B:total toxicity, C:cell toxicity..79
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