| The greatest harm of eutrophication is water blooms.60%～70%of algae in water bloom can produce microcystins (MCs). Qingcaosha Reservoir is located in the downstream of Yangtze River, the typical estuarine reservoir in China. Qingcaosha Reservoir supplies more than50%of source water of Shanghai City, and affects more than10million residents. It is of great significance for the safety of water supply in Shanghai.MCs are toxic and chemically stable. They can not be completely eliminated by conventional watertreatment technology and by boiling. Short-term exposure to high doses of MCs can cause acute poisoning in human, even death. Meanwhile, long-term exposure to low doses of MCs may pose a potential threat to human health. MCs are a strong tumor promoter and it can inhibit the activity of protein phosphatase1(PP1) and protein phosphatase2A (PP2A). The studies on the genotoxicity of MCs were mostly carried out in liver cells. Recent research suggests that microcystis extract (MCE) could induce the expression of proto-oncogene, such as c-fos and c-jun, in kidney. However, the genotoxicity study of MCs on renal cells was rarely reported.Our main aims were1) to study the pollution of water quality, algae and MCs in Qingcaosha Reservoir, to judge the degree of eutrophication of Qingcaosha Reservoir, to identify the dominant species, and to study the trend of algae.2) to explore the cytotoxocity and genotoxicity of MCE prepared from Qingcaosha Reservoir in HEK293in order to provide foundation for risk control. First Section:Study on Water Quality, Pollution of Algae and MCs in Qingcaosha ReservoirQingcaosha Reservoir, first formally used in June2011, is the largest estuarine reservoir in China. The area of the reservoir is about66km2, containing435million m3of water and having the capacity of tap water supply of7.19million m3/d. The water quality is directly affected by its upstream water quality and yield, especially by the amount of nitrogen and phosphorus. Therefore, we monitored the water quality, algae and microcystins in Qingcaosha Reservoir between June and December in2011.1. Sampling sites and monitoring indicatorsAccording to Sampling Plan for Routine Monitoring of Water Quality in Qingcaosha Reservoir,11sampling sites were set, including one in the head of reservoir (1#), one in central island of the reservoir (2#), one in the south of reclamation area (3#), one in the north of reclamation area (4#), three in the middle of the reservoir (5#,6#,7#), two in the rear of reservoir (8#,9#), one in the intake of water (10#), and one outside the reservoir sluice near the head of reservoir (11#).Monitoring indicators reflecting water quality includs total nitrogen, total phosphorus, permanganateindex, chlorophyll-a, water temperature, dissolved oxygen, water transparency, turbidity, pH value, salinity, electricalresistivity, electricalconductivity and total dissolved solids. We also carried out algal species identification, algal cell density count, and5microcystins content determination.2. Results1). Water temperature showed an obvious seasonal variation trend in Qingcaosha Reservoir. Its interquartile range was20.30～27.50℃. Dissolved oxygen mostly belonged to class I water quality standard, with its interquartile range7.899.82mg/L. Transparency was low at the sites of head of reservoir, central island of reservoir, south of reclamation area and outside the reservoirsluice. The water was alkaline, with interquartile range of pH value8.43～9.01. Salinity leveled off between July and November. Its interquartile range was0.15～0.19g/L, but in December, salinity was clearly on the rise to0.30g/L by mean. Interquartile range of resistivity, conductivity, total dissolved solids were2.48～3.20×103Ω’cm,313～402μs/cm, and156.5～201.0mg/L respectively。Chlorophyll-a was positively correlated with water temperature and CODmn.2). CTSIm of Qingcaosha Reservoir was between60and70according to the correction of Carlson index method. It means the reservoir was moderate eutrophication.3). The density of algae cell was much high in August and September, with its interquartile range of4.3×106cells/L～5.9×106cells/L. Outside the reservoir sluice the density of algae cell was mostly105cells/L, lower than reservoir water.4). The ratio of blue-green algae increased significantly between June and August, especially Microcystis, Oscillatoria, Anabaena, and Aphanizomenon. During September to December, diatom was dominant, mainly Cyclotella, Aulacoseira granulate, Navicula.5). MCs appeared in Qingcaosha Reservoir, mainly MC-RR/YR/LR. The detection rates were relatively high from June to August, particularly in the middle of reservoir (6#), in the north rear of reservoir (9#), and in the south of reclamation area (4#).Second Section:Study on Genotoxicity of Microcystis Extracts in Qingcaosha ReservoirMost studies on genotoxicity of MCs were performed in liver cells, but literatures indicated that MCs might also injure kidney. However, the studies about the effects of MCs on kidney were scarce. Due to the development of micronucleus (MN) assay in genotoxicity test, Cytokinesis-Block Micronucleus(CBMN) assay was extensively used, since it can overcome the weakness of routine MN assay which can not distinguish totally transformed cells from dividing cells for the first time. Therefore, in this section we used CBMN to assess the genotoxicity of MCE.1. Sampling and extraction of MicrocystisWater samples were collected by plankton net No.25during the period of the algae bloom in Qingcaosha Reservoir in summer of2012. Microcystiswas dominant, while Oscillatoria and Anabaenawere also observed by microscopy. All of these three species can produce MCs. Then MCE were analyzed by HPLC, and the concentrations of MCs in per gram of lyophilized algae cells were540.3μg for MC-RR,106.7μg for MC-YR,311.8μg for MC-LR,12.7μg for MC-LW, and10.3μg for MC-LF.2. The cytotoxicity of MCE in HEK293The cytotoxicity was assessed by CCK-8assay. The dosage was6.13X10"3μg lyophilized algae cells/ml,6.13×10-2μg lyophilized algae cells/ml,6.13×10-1μg lyophilized algae cells/ml,6.13μlyophilized algae cells/ml,61.3μg lyophilized algae cells/ml, and613μg lyophilized algae cells/ml. The results indicated that the average cell viability was80%when the dosage was613μg lyophilized algae cells/ml, and that the cell viability was more than90%when the dosage was lower than613μg lyophilized algae cells/ml.3. The genotoxicity of MCE in HEK293In this study CBMN was used to evaluate the genotoxicity of MCE in HEK293. The negative control was0.5%DMSO, and the positive control was1μmol/L of mitomycin C. Dosages were6.13μg lyophilized algae cells/ml,61.3μg lyophilized algae cells/ml, and613μg lyophilized algae cells/ml. The results showed that MCE could cause the formation of micronuclear at the the dosage of61.3μg lyophilized algae cells/ml, which is equivalent to19.1μg/L of MC-LR in media. The result showed that MCE could damage chromosomein HEK293dose-dependently, suggesting MCE might have genotoxicity. |
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