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研究生: 陳逸廷
Chen, Yi-Ting
論文名稱: 水中新型藻毒素β-甲氨基-L-丙氨酸的氧化處理研究
Oxidation treatment of a novel cyanotoxin β-methylamino-L-alanine in Water
指導教授: 林財富
Lin, Tsair-Fu
學位類別: 博士
Doctor
系所名稱: 工學院 - 環境工程學系
Department of Environmental Engineering
論文出版年: 2018
畢業學年度: 106
語文別: 英文
論文頁數: 108
中文關鍵詞: 藻毒素氧化β-甲氨基-L-丙氨酸飲用水系統
外文關鍵詞: algal toxins, drinking water, β-N-methylamino-L-alanine, oxidation, kinetic
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  • 水庫優養化及其影響是當前水領域研究的重點之一,其中藻類毒素一直是研究者關注的重點。近年研究指出大多數藍綠菌(藻)具備合成神經性毒素β-甲氨基-L-丙氨酸(β-N-methylamino-L-alanine, BMAA)的能力,這種毒素可能引發肌萎縮性側索硬化症/帕金森氏症或者阿茲海默症。這意味著以優養化水庫水為飲用水源時,可能會因BMAA 的存在而影響民眾的身體健康。
    由於氯是淨水廠中最常使用的氧化劑,因此本研究首先探討以氯氧化BMAA時的反應途徑及機制。BMAA在加氯反應過程中共會生成由一或兩個氯原子與BMAA結合的四種含氯中間產物,反應速率k1介於 2.1  103 ~1.21 105M-1s-1,並會隨著pH值增加而變大;含氯中間產物的自體降解反應速率常數於pH=5.8時k2 = 0.012 min-1,pH =7-9時變化並不大,約為0.0027 min-1;自由餘氯與含氯中間產物之反應速率常數則不受pH質及水體天然有機物的含量所影響,反應速率k3=17.8M-1s-1。當系統中有還原性物質存在時,其含氯中間產物會被還原成BMAA。若於飲用水系統中,不僅會有檢測上的疑慮,更可能造成民眾健康上的風險。
    此外,本研究亦探討過錳酸鉀、過氧化氫/UV系統及臭氧等常見氧化劑,在不同pH值和天然水體中對於BMAA 之去除效能及氧化反應動力參數。氧化實驗結果顯示在四種氧化劑中,以氫氧自由基反應速率常數為最高,其反應速率常數在pH 6.5時為1.11 × 108 M-1s-1 ,而當pH > 6.5時為5.51 × 109- 1.35 × 1010 M-1s-1 ,其反應速率與pH值間的關係與氯十分相似。臭氧的反應速率常為1.88 ×106 – 3.72 × 1010 M-1s-1,並且與pH值有相當良好的線性正相關係;過錳酸鉀及過氧化氫的反應速率常數最小,三小時內幾乎無法降解BMAA。此外,天然水體中的天然有機物會降低BMAA被氧化的速率。研究中所獲得之反應速率常數,目前國際上尚無文獻報導,極具有參考價值。
    除BMAA外,本研究亦建立同為神經毒素的BMAA同份異構物2,4-diaminobutyric acid (DAB) and N-(2-aminoethy)glycine (AEG)的衍生分析方法,選用6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)做為衍生劑,並搭配Hypersil Gold管柱,以SRM模式進行定量分析衍生後的BMAA、DAB及AEG。並調整樣品量與衍生劑之比例,可得最低定量濃度降低至BMAA、AEG為2μg/L,DAB為50μg/L。並以固相萃取法濃縮環境水體,金門的太湖及榮湖水庫的湖庫水體、原水及太湖水庫的清水中均檢測到BMAA的存在,AEG也出現在榮湖原水及太湖水庫清水中。實驗室所培養之銅綠微囊藻Microcystis aeruginosa中亦檢測到游離態之BMAA和AEG,以及蛋白質鍵結態之BMAA。

    Eutrophication of lakes and reservoirs has become a worldwide issue in recent years due to increased nutrient loading from human activities. One issue associated with eutrophication is the overgrowth of cyanobacteria in lakes and reservoirs. This is especially important for the lakes and reservoirs served as drinking water sources, as many cyanobacteria may produce some second metabolites such as cyanotoxins and odorants, posing additional risk to public health and affecting the quality of drinking water.
    A novel neurotoxin, β-N-Methylamino-L-alanine (BMAA), has been reported to be produced by more than 20 genera of cyanobacteria. The chemical may be biomagnified in the food chain and may cause amyotrophic lateral sclerosis/parkinsonism–dementia complex (ALS/PDC) or Alzheimer's disease. This implies that there is a possibility for public exposure to BMAA if the drinking water sources are with high concentrations of cyanobacteria. However, to date, the removal and fate of BMAA in drinking water systems has never been reported before. As chlorine, ozone, permanganate and OH radical are the most commonly used oxidants in water treatment plants, the reactions and kinetics between BMAA and these oxidants were investigated in this study.
    The reaction pathway of BMAA, the formation of intermediates and their reaction kinetics during chlorination process were elucidated in the first part of this study. A method based on liquid chromatograph equipped with triple quadrupole mass spectrometry (LC/MS-MS) was developed for the analysis of BMAA and its chlorinated intermediates. Upon chlorination, four chlorinated intermediates, each with 1 or 2 chlorines, were identified. The reaction of BMAA with free chlorine follows a second-order reaction and was pH-dependent. The rate constants k1 increased dramatically from 2  103 M-1s-1 at pH 5.8 to 4.93  104 M-1s-1 at pH 7, and kept in a relatively stable level at pH 7-9.5. The chlorinated intermediates were found to further react with free chlorine, exhibiting a second-order rate constant k3 = 17.75 M-1s-1 under different pH conditions. After all free chlorine was consumed, the chlorinated intermediates auto-decomposed slowly with a first order rate constant k2 = 0.0121 min-1 at pH=5.8, and about 0.0023 - 0.0029 min-1 between pH=7-9.5; when a reductant was added, these chlorinated intermediates were then reduced back to BMAA. BMAA and its chlorinated intermediates can be degraded by ~90% if the CT value = 150 mg/L•min in both deionized and natural water.
    The oxidation of BMAA with ozone and OH radical also followed the second order reaction rate law. The rate constants of OH radicals were 1.11 × 108 M-1s-1 at pH 6.5 and 5.51 × 109- 1.35 × 1010 M-1s-1 at pH > 6.5, with similar pH dependency to that with chlorine. The pH dependency of chlorine and the OH radical may be attributed to the neutral form of BMAA with free lone pair electrons readily to be attacked by oxidants. However, for ozonation of BMAA, the rate constants were 1.88 ×106 – 3.72 × 1010 M-1s-1, with a linear dependency on pH. Higher hydroxide concentration may accelerate the reaction of ozone and form more reactive oxidants for BMAA. For both permanganate and H2O2 only, the removal of BMAA was negligible. The reaction rate was in the order of OH radicals > ozone >> chlorine >>permanganate ~ H2O2 ~ direct photolysis. In addition, the natural organic matters was found to slow down the degradation of BMAA if compared with that in deionized water. The results as described shed a useful light to the reactivity, appearance, and removal of BMAA in the oxidation process of a drinking-water system.
    A method to detect BMAA and its isomers, 2,4-diaminobutyric acid (DAB) and N-(2-aminoethy)glycine (AEG) was also evaluated. A liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) was employed for the analysis of BMAA, AEG, and DAB, using derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The results show that the detection limits of BMAA, AEG, DAB were 2 μg L-1, 2 μg L-1, and 50 μg L-1, respectively. Both AEG and BMAA were detected to be present in the raw water of Rong-Hu Water Treatment Plant (WTP) and the finished water of Tai-Hu WTP. In addition, BMAA and AEG were also found in the laboratory culture samples of Microcystis aeruginosa.

    摘要 I Abstract III Acknowledgments VI Table of contents VIII List of Tables X List of Figures XI ChapterⅠ- Introduction 1 1.1 Background 1 1.1.1 Cyanobacteria and cyanotoxins 1 1.1.2 What is BMAA? 4 1.1.3 BMAA and neurodegenerative diseases 9 1.1.4 Oxidation 10 1.2 Scope and objectives 12 1.3 Dissertation overview 12 Chapter Ⅱ- Reaction pathways and kinetics of a cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) during chlorination1 16 2.1 Introduction 17 2.2 Materials and Methods 19 2.2.1 Chemicals and Preparations 19 2.2.2 Chlorination Experiments 20 2.2.3 Analyses of BMAA and Reaction Intermediates 20 2.3 Results and Discussion 22 2.3.1 Chlorine residual and BMAA disappearance 22 2.3.2 Identification of chlorination intermediates 26 2.3.3 BMAA under dechlorinated condition 34 2.3.4 Reaction Pathway 36 2.3.5 BMAA Reaction Kinetics with Chlorine 39 Chapter Ⅲ- Oxidation of cyanobacterial neurotoxin beta-N-methylamino-L-alanine (BMAA) with chlorine, permanganate, ozone, hydrogen peroxide and hydroxyl radical2 50 3.1 Introduction 51 3.2 Materials and Methods 53 3.2.1 BMAA analyses 53 3.2.2 Chlorination Experiments 54 3.2.3 Competition Experiments 55 3.2.4 Oxidation with permanganate 56 3.2.5 Oxidation with hydrogen peroxide and hydroxyl radical 57 3.3. Results and Discussion 58 3.3.1 Reaction kinetics of BMAA with chlorine under alkaline conditions and in natural water 58 3.3.2 BMAA oxidation by KMnO4 64 3.3.3 Ozonation of BMAA 65 3.3.4 Reaction with hydrogen peroxide and OH radicals 68 3.3.5 pH dependency of BMAA oxidation 71 3.3.6 The effects of natural water matrix 74 3.4 Conclusions 76 Chapter Ⅳ- The optimization of analytical method for BMAA, DAB and AEG and a preliminary investigation of environmental samples 78 Abstract 78 4.1 Introduction 79 4.2 Material and methods 83 4.2.1 Chemicals and reagents 83 4.2.2 Analysis of BMAA, DAB and AEG 83 4.2.4 AQC derivatization 83 4.2.4 Sample Extraction 84 4.2.5 Solid Phase Extraction (SPE) 84 4.3 Results and discussion 86 4.3.1 Analysis of BMAA, DAB and AEG with LC/MS/MS 86 4.3.2 Optimization of derivatization 86 4.3.3 Extraction of BMAA, DAB and AEG in environmental and cyanobacterial samples 88 Chapter Ⅴ – Conclusions 94 5.1 Summary 94 5.2 Implications and future research directions 97 References 99

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