湖库藻华监测与微囊藻毒素细胞毒性风险评价方法研究
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摘要
随着社会经济的发展,水体富营养化问题日益严重,由此引起的藻华及其毒素直接危及饮用水安全,藻华及毒素的监控技术与水生态健康风险评价方法已成为当今科学研究的热点之一
本文围绕湖库藻华及其毒素的污染问题,从湖库藻华及其毒素的水生态健康风险、微囊藻毒素(Microcystins, MCs)产生机制和MCs与水质相关性阐述湖库藻华与水质安全研究进展；以杭州市临安青山湖水库为例,研究了湖库微囊藻毒素时空分布规律,探索了湖库藻华主要诱因(NH4+, NO3等营养盐)及环境因子对有害蓝藻和毒素的调控规律,建立了MCs污染的细胞毒性评价方法；结合实地测定的藻类生物量参数(Chl-a),构建了水质反演的多元线性回归遥感定量模型,探索了不同形态氮对硅藻指示生物的影响及其敏感性差异。主要研究结论如下：
(1)明确了青山湖主要优势藻类及其季节性演变规律,揭示了调控青山湖藻类演变及藻毒素分布规律的主要环境因子。青山湖全年主要优势藻类为硅藻、蓝藻和绿藻,季节性演变规律为硅藻(冬季)——绿藻(冬春季)——蓝藻(夏秋以微囊藻Microcystis为主、念珠藻Nostoc、鱼腥藻Anabaena和颤藻Oscillatoria为辅)；应用高效液相色谱-质谱联用分析技术(HPLC-MS)和酶联免疫吸附法(ELISA),检测到青山湖表层水MCs浓度范围为0.00±0.00～1.28±0.05μg/LMCLR,主成分分析的结果表明,MCs的产生主要受TP、水温和蓝藻生物量(Cyanobacteria Biovolume, CBV)调控,而蓝藻的相关丰度与TP呈极显著正相关(r=0.53,p<0.001),表明夏季TP负荷的削减可能是青山湖藻华及藻毒素控制和饮用水安全保障的关键。
(2)提出了青山湖夏季典型月的Chl-a浓度遥感反演模型。选用Landsat7ETM+为遥感数据源,采用波段比值法,结合夏季藻华易暴发的2个典型月份实地监测数据,通过多元线性回归建立模型,5月份Chl-a浓度遥感反演模型为：Log(Chl-a)=1.65+0.87*(ETM+1/ETM+3)-3.39*(ETM+2/ETM+3)+0.89*(ETM+3/ETM+4)(R2=0.72, p<0.01),8月份Chl-a浓度反演模型为:Log(Chl-a)=2.94-1.37*(ETM+1/ETM+3)+0.40*(ETM+2/ETM+3)-0.20*(ETM+3/ETM+4)(R2=0.92, p<0.001),均可以有效反演青山湖中Chl-a浓度分布特征和富营养化状态污染特征,有助于实现藻华识别和早期预警。
(3)建立了基于鱼体淋巴细胞凋亡EC50值的MCs细胞毒性评价方法。选取草鱼淋巴细胞为研究对象,以细胞凋亡半数有效浓度EC50为毒性指标,经1nmol·L-1(约1μg/L)的微囊藻毒素MCLR、MCRR暴露2h,即能检测到典型的细胞凋亡,并且与MCs暴露浓度呈显著的剂量-效应关系；草鱼淋巴细胞经浓度为1nmol·L-1的MCLR.MCRR分别与浓度为1、5、10、50、100nmol-L-’的CHCl2Br、 CHClBr2复合暴露2h,亦能检测到细胞凋亡,且呈显著的剂量-效应关系。应用联合作用系数法,分析4个复合暴露实验组(MCLR-CHCl2Br、MCLR-CHClBr2、 MCRR-CHCl2Br和MCRR-CHClBr2)的联合作用结果均为相加作用。
(4)明确了3种常见淡水硅藻对不同形态氮胁迫的敏感性差异。为了探索淡水硅藻在富营养化条件下作为蓝藻竞争藻种的可行性,以太湖流域三种典型淡水硅藻(Nitzschia sp., Cyclotella meneghiniana, Gomphonema parvulum)为研究对象,透射电镜(TEM)为表征手段,通过观察叶绿体超微结构的变化,结合硅藻光合放氧速率和抗氧化酶系统的响应,表明Cyclotella meneghiniana是氨氮胁迫的敏感指示种,Gomphonema parvulum和Nitzschia sp在生长上对的NH4+耐受度高于C. meneghiniana;NH4+对C. meneghiniana的光合系统存在着直接的胁迫,并且N03-的共存能缓解NH4+直接的光合胁迫作用,可能是由于不同的N03-转运系统引起。透射电镜(TEM)的结果表明,Nitzschia sp叶绿体中出现大量的电子密集型的黑色颗粒,硅藻细胞膜完整性的保持可能是对NH4+胁迫的一个过渡适应机制。
Eutrophication problems in aquatic ecosystems have deteriorated as the economy advances leading to algal blooms and microcystins (MCs) production that pose significant risks to drinking water security worldwide, then monitoring techniques of algal blooms and associated microcystin, and methods for aquatic health risk assessment have recently gained increasing attention, especially in China.
This dissertation focuses on the pollution of algal blooms and associated MCs in reservoirs, recent advances on health risks caused by algal blooms and associated toxins, production mechanism of microcystins, and the relationship between environmental factors and microcystins were discussed in this dissertation. We selected Qingshan Lake Reservoir in Lin'an of western Hangzhou as the study region, to study the spatial and temporal distribution of microcystin, to explore major nutrients (NH4+and NO3-) and environmental factors regulating harmful algal growth and microcystin production. Furthermore, an original method of assessing cytotoxicity induced by microcystin pollution was established. We combined Landsat ETM+remote sensing images with field measurements of algal biomass (Chloropyll-a, Chl-a) to establish a quantitative multiple linear regression model with water quality inversion techniques. Moreover, three common freshwater diatoms (Nitzschia sp., Cyclotella meneghiniana, Gomphonema parvulum) in Taihu Lake watershed were selected, to determine the influence of different nitrogen species on diatom indicators, and its sensitivity difference of common freshwater diatoms under stress of different nitrogen source. The main conclusions are listed as follows:
(1) The dominant algae and seasonal distribution in Qingshan Lake was identified and major environmental factors regulating algal dominance and microcystin distribution were determined in QingshanLake. Algal succession in Qingshan Lake was Diatom (winter)--green algae (winter-spring)--Cyanobacteria (summer, dominated by Microcystis, followed by Nostoc, Anabaena, and Oscillaloria);HPLC-MS and ELISA were used to measure MCs concentrations in surface water samples collected from Qingshan Lake, MCs concentrations ranged from0.00±0.00-1.28±0.05μg/L (MCLR); principal component analysis indicated that MCs production was mainly regulated by total phosphorus (TP), water temperature and cyanobacteria biovolume (CBV). The relative abundance of cyanobacteria positively related to TP (r=0.53, p<0.001) implies that the reduction of TP load is the key measure to control algal blooms, microcystin production and ensure drinking water safety.
(2) A Chl-a concentrations prediction model based on typical month in the summer for Qingshan Lake was proposed. Landsat7ETM+images were combined with field monitoring data to establish multiple linear regressions, linear regressions model in May was Log(Chl-a)=1.65+0.87*(ETM+l/ETM+3)-3.39*(ETM+2/ETM+3)+0.89*(ETM+3/ETM+4)(R2=0.72, p<0.01), and linear regressions model in August was Log(Chl-a)=2.94-1.37*(ETM+l/ETM+3)+0.40*(ETM+2/ETM+3)-0.20*(ETM+3/ETM+4)(R2=0.92, p<0.001), and it turned out that the models effectively predicted Chl-a concentrations distribution and eutrophication pollution characteristics in Qingshan Lake. The regression models can help identifying and early warning of algal blooms.
(3) An original method assessing cytotoxicity induced by MCs was established based on EC50values of fish lymphocyte apoptosis. Typical apoptosis in Grass carp lymphocyte was detected after exposure under lnmol/L MCLR or MCRR for2h, significant dose-effect was determined with increasing MCs concentrations. Similar typical apoptosis in Grass carp lymphocyte and significant dose-effect was also determined after exposure under lnmol/L MCLR or MCRR with1,5,10,50100nmol/L CHCl2Br or CHClBr2for2h. Joint effect coefficient analysis indicated that the joint effect between MCLR-CHCl2Br, MCRR-CHCl2Br, MCLR-CHClBr2and MCRR-CHClBr2were all additive, implying the joint pollution between microcystins and disinfection by-products will enhance the threat to drinking water safety caused by microcystins joint pollution.
(4) The sensitivity difference of common freshwater diatoms under stress of different nitrogen source was confirmed. To explore the possibility of diatoms acting as competitive species against cyanobacteria, different sensitivity of three common freshwater diatoms(Cyclotella meneghiniana, Nitzschia sp. and Gomphonema parvulum) in Taihu watershed to different nitrogen species was studied, transmission electron microscopy (TEM) was used to characterize the ultrastructural changes of chloroplast. Response of photosynthetic and antioxidant enzyme systems indicated that Cyclotella meneghiniana was the most sensitive indicator under NH4+stress, Gomphonema parvulum and Nitzschia sp. has more tolerance under NH4+stress on the growth than C.meneghiniana. NH4+has direct stress on photosynthetic system of C.meneghiniana, and the stress can be alleviated by the coexistance of NO3, which may be caused by different transport system of NO3-. Transmission electron microscopy (TEM) results showed that a large number of electronic dense dark particles were identified in the chloroplast, but the integrity of the membraneof Nitzschia sp. may be due to a transitional adaptation mechanism under NH4+stress.
本文围绕湖库藻华及其毒素的污染问题,从湖库藻华及其毒素的水生态健康风险、微囊藻毒素(Microcystins, MCs)产生机制和MCs与水质相关性阐述湖库藻华与水质安全研究进展；以杭州市临安青山湖水库为例,研究了湖库微囊藻毒素时空分布规律,探索了湖库藻华主要诱因(NH4+, NO3等营养盐)及环境因子对有害蓝藻和毒素的调控规律,建立了MCs污染的细胞毒性评价方法；结合实地测定的藻类生物量参数(Chl-a),构建了水质反演的多元线性回归遥感定量模型,探索了不同形态氮对硅藻指示生物的影响及其敏感性差异。主要研究结论如下：
(1)明确了青山湖主要优势藻类及其季节性演变规律,揭示了调控青山湖藻类演变及藻毒素分布规律的主要环境因子。青山湖全年主要优势藻类为硅藻、蓝藻和绿藻,季节性演变规律为硅藻(冬季)——绿藻(冬春季)——蓝藻(夏秋以微囊藻Microcystis为主、念珠藻Nostoc、鱼腥藻Anabaena和颤藻Oscillatoria为辅)；应用高效液相色谱-质谱联用分析技术(HPLC-MS)和酶联免疫吸附法(ELISA),检测到青山湖表层水MCs浓度范围为0.00±0.00～1.28±0.05μg/LMCLR,主成分分析的结果表明,MCs的产生主要受TP、水温和蓝藻生物量(Cyanobacteria Biovolume, CBV)调控,而蓝藻的相关丰度与TP呈极显著正相关(r=0.53,p<0.001),表明夏季TP负荷的削减可能是青山湖藻华及藻毒素控制和饮用水安全保障的关键。
(2)提出了青山湖夏季典型月的Chl-a浓度遥感反演模型。选用Landsat7ETM+为遥感数据源,采用波段比值法,结合夏季藻华易暴发的2个典型月份实地监测数据,通过多元线性回归建立模型,5月份Chl-a浓度遥感反演模型为：Log(Chl-a)=1.65+0.87*(ETM+1/ETM+3)-3.39*(ETM+2/ETM+3)+0.89*(ETM+3/ETM+4)(R2=0.72, p<0.01),8月份Chl-a浓度反演模型为:Log(Chl-a)=2.94-1.37*(ETM+1/ETM+3)+0.40*(ETM+2/ETM+3)-0.20*(ETM+3/ETM+4)(R2=0.92, p<0.001),均可以有效反演青山湖中Chl-a浓度分布特征和富营养化状态污染特征,有助于实现藻华识别和早期预警。
(3)建立了基于鱼体淋巴细胞凋亡EC50值的MCs细胞毒性评价方法。选取草鱼淋巴细胞为研究对象,以细胞凋亡半数有效浓度EC50为毒性指标,经1nmol·L-1(约1μg/L)的微囊藻毒素MCLR、MCRR暴露2h,即能检测到典型的细胞凋亡,并且与MCs暴露浓度呈显著的剂量-效应关系；草鱼淋巴细胞经浓度为1nmol·L-1的MCLR.MCRR分别与浓度为1、5、10、50、100nmol-L-’的CHCl2Br、 CHClBr2复合暴露2h,亦能检测到细胞凋亡,且呈显著的剂量-效应关系。应用联合作用系数法,分析4个复合暴露实验组(MCLR-CHCl2Br、MCLR-CHClBr2、 MCRR-CHCl2Br和MCRR-CHClBr2)的联合作用结果均为相加作用。
(4)明确了3种常见淡水硅藻对不同形态氮胁迫的敏感性差异。为了探索淡水硅藻在富营养化条件下作为蓝藻竞争藻种的可行性,以太湖流域三种典型淡水硅藻(Nitzschia sp., Cyclotella meneghiniana, Gomphonema parvulum)为研究对象,透射电镜(TEM)为表征手段,通过观察叶绿体超微结构的变化,结合硅藻光合放氧速率和抗氧化酶系统的响应,表明Cyclotella meneghiniana是氨氮胁迫的敏感指示种,Gomphonema parvulum和Nitzschia sp在生长上对的NH4+耐受度高于C. meneghiniana;NH4+对C. meneghiniana的光合系统存在着直接的胁迫,并且N03-的共存能缓解NH4+直接的光合胁迫作用,可能是由于不同的N03-转运系统引起。透射电镜(TEM)的结果表明,Nitzschia sp叶绿体中出现大量的电子密集型的黑色颗粒,硅藻细胞膜完整性的保持可能是对NH4+胁迫的一个过渡适应机制。
Eutrophication problems in aquatic ecosystems have deteriorated as the economy advances leading to algal blooms and microcystins (MCs) production that pose significant risks to drinking water security worldwide, then monitoring techniques of algal blooms and associated microcystin, and methods for aquatic health risk assessment have recently gained increasing attention, especially in China.
This dissertation focuses on the pollution of algal blooms and associated MCs in reservoirs, recent advances on health risks caused by algal blooms and associated toxins, production mechanism of microcystins, and the relationship between environmental factors and microcystins were discussed in this dissertation. We selected Qingshan Lake Reservoir in Lin'an of western Hangzhou as the study region, to study the spatial and temporal distribution of microcystin, to explore major nutrients (NH4+and NO3-) and environmental factors regulating harmful algal growth and microcystin production. Furthermore, an original method of assessing cytotoxicity induced by microcystin pollution was established. We combined Landsat ETM+remote sensing images with field measurements of algal biomass (Chloropyll-a, Chl-a) to establish a quantitative multiple linear regression model with water quality inversion techniques. Moreover, three common freshwater diatoms (Nitzschia sp., Cyclotella meneghiniana, Gomphonema parvulum) in Taihu Lake watershed were selected, to determine the influence of different nitrogen species on diatom indicators, and its sensitivity difference of common freshwater diatoms under stress of different nitrogen source. The main conclusions are listed as follows:
(1) The dominant algae and seasonal distribution in Qingshan Lake was identified and major environmental factors regulating algal dominance and microcystin distribution were determined in QingshanLake. Algal succession in Qingshan Lake was Diatom (winter)--green algae (winter-spring)--Cyanobacteria (summer, dominated by Microcystis, followed by Nostoc, Anabaena, and Oscillaloria);HPLC-MS and ELISA were used to measure MCs concentrations in surface water samples collected from Qingshan Lake, MCs concentrations ranged from0.00±0.00-1.28±0.05μg/L (MCLR); principal component analysis indicated that MCs production was mainly regulated by total phosphorus (TP), water temperature and cyanobacteria biovolume (CBV). The relative abundance of cyanobacteria positively related to TP (r=0.53, p<0.001) implies that the reduction of TP load is the key measure to control algal blooms, microcystin production and ensure drinking water safety.
(2) A Chl-a concentrations prediction model based on typical month in the summer for Qingshan Lake was proposed. Landsat7ETM+images were combined with field monitoring data to establish multiple linear regressions, linear regressions model in May was Log(Chl-a)=1.65+0.87*(ETM+l/ETM+3)-3.39*(ETM+2/ETM+3)+0.89*(ETM+3/ETM+4)(R2=0.72, p<0.01), and linear regressions model in August was Log(Chl-a)=2.94-1.37*(ETM+l/ETM+3)+0.40*(ETM+2/ETM+3)-0.20*(ETM+3/ETM+4)(R2=0.92, p<0.001), and it turned out that the models effectively predicted Chl-a concentrations distribution and eutrophication pollution characteristics in Qingshan Lake. The regression models can help identifying and early warning of algal blooms.
(3) An original method assessing cytotoxicity induced by MCs was established based on EC50values of fish lymphocyte apoptosis. Typical apoptosis in Grass carp lymphocyte was detected after exposure under lnmol/L MCLR or MCRR for2h, significant dose-effect was determined with increasing MCs concentrations. Similar typical apoptosis in Grass carp lymphocyte and significant dose-effect was also determined after exposure under lnmol/L MCLR or MCRR with1,5,10,50100nmol/L CHCl2Br or CHClBr2for2h. Joint effect coefficient analysis indicated that the joint effect between MCLR-CHCl2Br, MCRR-CHCl2Br, MCLR-CHClBr2and MCRR-CHClBr2were all additive, implying the joint pollution between microcystins and disinfection by-products will enhance the threat to drinking water safety caused by microcystins joint pollution.
(4) The sensitivity difference of common freshwater diatoms under stress of different nitrogen source was confirmed. To explore the possibility of diatoms acting as competitive species against cyanobacteria, different sensitivity of three common freshwater diatoms(Cyclotella meneghiniana, Nitzschia sp. and Gomphonema parvulum) in Taihu watershed to different nitrogen species was studied, transmission electron microscopy (TEM) was used to characterize the ultrastructural changes of chloroplast. Response of photosynthetic and antioxidant enzyme systems indicated that Cyclotella meneghiniana was the most sensitive indicator under NH4+stress, Gomphonema parvulum and Nitzschia sp. has more tolerance under NH4+stress on the growth than C.meneghiniana. NH4+has direct stress on photosynthetic system of C.meneghiniana, and the stress can be alleviated by the coexistance of NO3, which may be caused by different transport system of NO3-. Transmission electron microscopy (TEM) results showed that a large number of electronic dense dark particles were identified in the chloroplast, but the integrity of the membraneof Nitzschia sp. may be due to a transitional adaptation mechanism under NH4+stress.
引文
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