Research on the Relationship between Three Isomers of Microcystins and Environmental Factors

[Objective] The relationship between three isomers of microcystins and environmental factors were studied in the fields.[Method] Three isomers of microcystins （MC-LR,RR and YR） from water of five sampling spots in a northern reservoir were observed for one year with High Performance Liquid Chromatography analytical method in order to study the relationship between three isomers and environmental factors.[Result] The three isomers of microcystins showed positive correlation with chlorophyll a;LR and YR isomers all had significant linear positive correlations with the water temperature,but the RR isomer showed no significant correlation with the water temperature;LR and YR isomers had relatively significantly correlativities with the contents of total nitrogen,nitrate nitrogen and organic nitrogen,while the RR isomer only showed a significant negative correlation with the content of nitrate nitrogen;LR and RR isomers both showed significant positive correlations with the contents of total phosphorus and organic phosphorus,while the phosphorus hardly affected the YR isomer and showed no evident correlation.[Conclusion] The relationship between three isomers of microcystins and environmental factors such as chlorophyll a,water temperature,nitrogen,phosphorus were studied and investigated the reasons,which might offered a reference for controlling the growth of blue algae in water and toxin synthesis.

Mechanism of microcystin removal from eutrophicated source water by aquatic vegetable bed

For purifying raw water for tap water treatment, the aquatic vegetable bed （AVB） experiment has been carded out in a hypertrophic waterfront of Taihu Lake, China. The average removal rates of total microcystin-RR and microcystin-LR are 63.0% and 66. 7%, respectively. Experiments indicate that lpomoea aquatica can absorb microcystin by using enzyme-linked immunosorbent assay （ELISA）, and the roots absorb more toxins than leaves and stems. Fluorescence in situ hybridization （FISH） is used to analyze the density of microcystin degrading bacteria in the AVB sediment. Two species of microcystin degrading bacteria are detected, which indicate that microcystin bio-degradation process happened in the AVB. Protozoa and metazoa are abundant in root spheres. Aspidisca sp., Vorticella sp., Philodina sp., and Lecane sp. are dominant species. The predation functions of protozoa and metazoa have a positive effect on the removal of cyanobacteria and microcystin.

Comparison on the Tolerance of Cruciferous Crops and Leguminous Crops to Microcystin

[Objective] To study the differences in the tolerance of leguminous crops and cruciferous crops to microcystin （MC）. [Methed] The cruciferous typical crops oilseed rapes, pakchois, cabbages and leguminous typical crops soybeans, peas and broad beans were selected as the materials to test the effects of MC of differ- ent concentrations on the germination, growth and development of leguminous crops and cruciferous crops. The measurement indicators included germination rate, plant height, chlorophyll, etc. [Result] The MC had great effects on the oilseed rape and pakchois of cruciferous crops, and smaller effects on cabbage, while the leguminous crops were generally not affected. [Conclusion] Leguminous crops are more tolerant to MC than cruciferous crops and more preferential in MC polluted regions.

Fast adsorption of microcystin-LR by Fe(Ⅲ)-modified powdered activated carbon

Microcystins(MCs) are cyclic hepatotoxic peptides produced by the bloom-forming cyanobacterium Microcystis and present a public health hazard to humans and livestock. The removal of MCs from contaminated water with powdered activated carbon(PAC) has been employed as a simple and economic treatment strategy. In this study, PAC-Fe(Ⅲ) was prepared and utilized for the fast and efficient removal of MCs from water. PAC-Fe(Ⅲ) exhibited superior microcystin-LR(MC-LR) removal capacity and efficiency compared to the unmodified PAC. The MC-LR removal efficiency of PAC-Fe(Ⅲ) increased with decreasing p H within the pH range of 4.3 to 9.6. PAC-Fe(Ⅲ) could be reused for 3 times by methanol elution while the MC-LR removal efficiency was still over 70 percent. The removal efficiency was positively correlated to the ionic strength of water and negatively correlated to alkalinity. Natural organic matter(NOM) such as humic acid(HA) and salicylic acid(SA) generated low interference with MC-LR adsorption by PAC-Fe(Ⅲ). The complexation reaction between Fe^(3+) in PAC-Fe(Ⅲ) and the functional groups of MCLR was suggested as the key mechanism of MC-LR removal by PAC-Fe(Ⅲ). The results suggest that Femodified PAC is a promising material for the treatment of MC-contaminated waters.

Environmental abundance and microcystin-LR production ability of toxic Microcystis in Nanquan region of Lake Taihu

The variations of environmental abundance and microcystin-LR （MC-LR） production ability of toxic Microcystis in the Nanquan region of Lake Taihu are investigated by real-time quantitative PCR （RTQ-PCR） and high performance liquid chromatography （HPLC） from May to December in 2009. Simultaneously, degrees of water pollution and eutrophication are monitored. The results indicate that the water quality in the Nanquan region of Lake Taihu is in a moderate degree of pollution and eutrophication. Algal density exceeds the threshold of bloom from May to November. The environmental abundance of toxic Microcystis is more than 40% from May to October and then significantly declines to 5.66% due to the obvious reduction in the water temperature in December. From May to December, the MC-LR production ability of toxic Microcystis ranges from 1.661 to 9.293 μg/108cells. With the significant drops in water temperature and algal density, the MC-LR production ability of toxic Microcystis is obviously increased from November to December. It is concluded that the lake presents Microcystis bloom and the toxic Microcystis becomes dominant during most of the year. The environmental abundance and the MC-LR production ability of toxic Microcystis have a close relationship with water temperature. The effective control of toxic Microcystis should be considered in both the bloom period and the non-bloom period of winter since the MC-LR production ability of toxic Microcystis obviously increases in winter.

N-TiO2/硅藻土负载型纳米材料可见光催化降解水中Microcystin-LR

以钛酸四丁酯(C16H36O4Ti)为Ti源,尿素((NH2)2CO)为N源,硅藻土(Diatomite)为载体,采用溶胶凝胶法制备硅藻土负载氮掺杂二氧化钛改性纳米材料(N-TiO2/Diatomite).利用SEM、XRD、XPS、FT-IR、UV-Vis DRS对其进行系列特性表征及可见光催化性能分析,探究Microcystin-LR初始浓度对光催化的影响,分析其降解动力学和降解途径.结果表明,硅藻土负载优化了N-TiO2分子的分散性,形成了链条型纳米孔隙球状结构;N元素掺杂、硅藻土负载不影响材料晶型,晶粒粒径减小至11.39 nm;Si、N分别与TiO2成键;光响应强度和范围红移,可见光活性显著提升.以优化制备条件(350℃煅烧、掺N的物质的量分数为8%)的改性N-TiO2/Diatomite材料可见光催化降解Microcystin-LR,当初始质量浓度为1 mg·L^-1的Microcystin-LR水溶液反应6 h后降解率可达95.0%,矿化率为83.9%;反应符合准一级动力学方程,速率常数k为0.453 3 h^-1.

微囊藻毒素Microcystin-LR体外遗传毒性

背景与目的:应用人类淋巴母细胞TK6研究微囊藻毒素(Microcystin_LR,MCLR)的体外遗传毒性。材料与方法:MCLR体外染毒TK6细胞4h或24h后检测细胞毒性、微核及tk位点突变频率。结果:4h染毒未引发明显细胞毒性,24hMCLR染毒导致TK6细胞相对存活率下降,细胞微核率及TK基因突变频率明显上升,并有剂量-反应关系。最高浓度组(80μg/ml)的细胞微核率及TK基因突变频率分别是对照组的4.8及5.1倍。MCLR诱发tk位点两种不同表型的突变细胞集落,即正常生长集落及缓慢生长集落,并以后者为主。结论:24h染毒MCLR可以诱发TK6细胞微核及基因突变,揭示MCLR可能是一种断裂剂。

Relationship Between Microcystin in Drinking Water and Colorectal Cancer

Objective To investigate the association of microcystin (MC) in drinking water with the incidence of colorectal cancer. Methods The study was designed as a retrospective cohort. Eight townships or towns were randomly selected as the study sites in Haining City of Zhejiang Province, China. 408 cases of colon and rectum carcinomas diagnosed from 1977 to 1996 in the study sites were included, and a survey on types of drinking water of these patients was conducted. Samples of different water sources (well, tap, river and pond) were collected separately and microcystin concentrations were determined by indirect competitive ELISA method. Results The incidence rate of colorectal cancer was significantly higher in population who drank river and pond water than those who drank well and tap water. Compared to well water, the relative risk (RR) for colorectal cancer was 1.88 (tap), 7.94 (river) and 7.70 (pond) respectively. The positive rate (>50 pg/mL) of microcystin in samples of well, tap, river and pond water was 0, 0, 36.23% and 17.14% respectively. The concentration of microcystin in river and pond water was significantly higher than that in well and tap water (P<0.01). Spearman rank correlation analysis showed that in the study sites, the microcystin concentration of river and pond water was positively associated with the incidence of colorectal cancer (rs= 0.881, P<0.01). Conclusions The types of drinking water are positively associated with the incidence of colorectal cancer in the study sites, and this may be related to microcystin contamination of drinking water. Further biological study is needed to support the possible causative role of mycrocystin in carcinogenesis of colon and rectum.

Biodegradation of microcystin-RR and -LR by an indigenous bacterial strain MC-LTH11 isolated from Lake Taihu

The indigenous bacterial strain MC-LTH11 with the capability of degrading microcystin-RR MC-RR and microcystin-LR MC-LR was successfully isolated from Lake Taihu.The bacterium was identified as Stenotrophomonas sp. which possessed a mlrA gene. The MC-LTH11 thoroughly degraded MC-RR and MC-LR with the initial concentration of 37.13 mg/L and 18.49 mg /L respectively in the medium containing crude microcystins extract within 6 d.The degradation rates were affected by temperature pH initial MCs concentration and the kinds of media. Additionally the bacterial strain MC-LTH11 also degraded thoroughly microcystins in the water body of Lake Taihu within 1 d.These results suggest that the Stenotrophomonas sp.MC-LTH11 has the capacity to bioremediate water bodies contaminated by microcystins and may contribute to the degradation of microcystins after the outbreak of harmful cyanobacterial blooms in Lake Taihu.

Seven microcystins from Microcystis waterbloom in Lake Dalai,China

Seven types of microcystins,isolated from Microcystis waterbloom in Lake Dalai, were characterized.The major toxins:MCYST-LR,MCYST-RR,[D-Asp^3]MCYST-LR and [Dha^7]MCYST-LR were identified by high performance liquid chromatography(HPLC),as com- pared with the authentic microcystins.The minor toxins:MCYST FR,[L-Mser^7]MCYST-LR and an unknown MCYST which was most likely to be MCYST-(H_4)YR were identified with frit- fast atom bombardment liquid chromatography/mass spectrometry(Frit-FAB LC/MS)and amino acid analysis.The toxigenic diversity in blue-green algae(cyanobacteria)was discussed.

磁性功能化纳米粒子对微囊藻毒素Microcystin-LR的吸附性能研究

通过水热合成的方法制备出磁性Fe3O4纳米粒子,并将其表面功能化,得到新型的磁性纳米粒子.对影响微囊藻毒素Microcystin-LR吸附效果的一些因素如吸附剂加入量、吸附时间、浓度、p H等进行了优化.研究结果表明,制得的磁性吸附剂具有良好的吸附性能,适用于水中痕量藻毒素的去除.

Hepatic Histopathological Characteristics and Antioxidant Response of Phytoplanktivorous Silver Carp Intraperitoneally Injected with Extracted Microcystins

Objective To investigate the hispathological characteristics and antioxidant responses in liver of silver carp after intraperitoneal administration of microcystins （MCs） for further understanding hepatic intoxication and antioxidation mechanism in fish. Methods Phytoplanktivorous silver carp was injected intraperitoneally （i.p.） with extracted hepatotoxic microcystins （mainly MC-RR and -LR） at a dose of 1000 μg MC-LReq./kg body weight, and liver histopathological changes and antioxidant responses were studied at 1, 3, 12, 24, and 48 h, respectively, after injection. Results The damage to liver structure and the activities of hepatic antioxidant enzymes including catalase （CAT）, superoxide dismutase （SOD）, and glutathione peroxide （GPX） were increased in a time-dependent manner. Conclusion In terms of clinical and histological signs of intoxication and LD50 （i.p.） dose of MC-LR, silver carp appears rather resistant to MCs exposure than other fishes. Also the significantly increased SOD activity in the liver of silver carp suggests a higher degree of response to MCs exposure than CAT and GPX.

Microcystin-LR对体外牛子宫内膜上皮细胞增殖和凋亡的影响

使用Microcystin-LR体外处理牛子宫内膜上皮细胞,探究其对细胞活率影响及分子调控机制,以期为提高牛繁殖效率提供理论基础和实践依据。试验运用CCK-8检测MC-LR处理后细胞存活率;流式细胞术检测细胞周期变化和细胞凋亡率;Annexin V-FITC试剂盒检测细胞凋亡;实时荧光定量检测Pi3k、Akt、mTOR、Cyt-c、Caspase-3、Caspase-9和p53基因mRNA表达量。Western blot检测Pi3k、Akt、Caspase-3和Caspase-9蛋白表达量。结果表明,不同浓度和时间处理下细胞存活率出现升高和降低两种相反情况,根据CCK-8检测结果,选择12 h,100μg·L^(-1)和24 h,160μg·L^(-1)两种处理方案开展后续试验。在12 h,100μg·L^(-1)条件下,Pi3k、Akt、mTOR的mRNA表达量升高(P<0.01),Cyt-c、Caspase-3和Caspase-9基因mRNA表达量下降(P<0.05),Pi3k和Akt蛋白表达量升高(P<0.05),Caspase-3和Caspase-9蛋白表达量降低(P<0.05)。处于G0/G1期细胞数量减少(P<0.01),处于G2/M和S期细胞数量增加(P<0.01);p53基因mRNA表达量升高(P<0.05)。在24 h,160μg·L^(-1)处理条件下,Caspase-3、Caspase-9、Bax/Bcl2基因mRNA表达量升高(P<0.05),Caspase-3和Caspase-9蛋白表达量升高(P<0.05)。研究表明,100μg·L^(-1)MC-LR处理细胞12 h,细胞通过增强Pi3k-Akt-mTOR信号通路,抑制线粒体凋亡通路促进细胞增殖,同时导致处于G1期细胞数量减少、G2/M期和S期细胞数量增加。160μg·L^(-1)MC-LR处理细胞24 h,细胞通过活化线粒体凋亡通路发生凋亡。

A method to extract algae toxin of microcystin-LR

A simple and low-cost method to obtain cyanobacterial toxin microcystin-LR(MC-LR) was developed. A new strain of Microcystis aeruginosa, named DC-1, producing microcystin-LR but not microcystin-RR, was separated from the field blooming algae samples of Dianchi Lake, in southwest of China. Following three times' freeze and thaw treatment, the cultivated DC-1 cells were extracted with 40% methanol in water. The extract was centrifuged and the supernatant applied to a Hydrophilic-Lipophilic Balance(HLB) SPE cartridge. Eluted impurities with a certain gradient from 30% to 50% methanol in water, MC-LR was finally eluted from the HLB cartridge with 60% methanol in water, and samples containing 3.85% to 14.8% of MC-LR were obtained. These MC-LR samples may be used in adsorption and biodegradation experiments instead of using expensive standard reagents.

Using the nematode Caenorhabditis elegans as a model animal for assessing the toxicity induced by microcystin-LR

Among more than 75 variants of microcystin （MC）, microcystin-LR （MC-LR） is one of the most common toxins. In this study, the feasibility of using Caenorhabditis elegans to evaluate MC-LR toxicity was studied. C. elegans was treated with MC-LR at different concentrations ranging from 0.1 to 80 μg/L. The results showed that MC-LR could reduce lifespan, delay development, lengthen generation time, decrease brood size, suppress locomotion behavior, and decreases hsp-16-2-gfp expression. The endpoints of generation time, brood size, and percentage of the population expressing hsp-16-2-gfp were very sensitive to 1.0μg/L of MC-LR, and would be more useful for the evaluation of MC-LR toxicity. Furthermore, the tissue-specific hsp-16-2-gfp expressions were investigated in MC-LR-exposed animals, and the nervous system and intestine were primarily affected by MC-LR. Therefore, the generation time, brood size, and hsp-16-2-gfp expression in C. elegans can be explored to serve as valuable endpoints for evaluating the potential toxicity from MC-LR exposure.

Study on the Photocatalytic Degradation of Microcystins with UV/Fenton/TiO2

[Objective] The paper aimed to study on the effects of photocatalytic degradation of microcystins MC-RR and MC-LR by UV/Fenton/TiO2 in depth lake water.[Method] With Fenton-TiO2 as photocatalyst,the influences of different reaction time,initial pH value,H2O2 concentration,Fe2+ concentration,TiO2 dosage,light intensity,initial concentration of microcystin on UV/Fenton/TiO2 heterogeneous photocatalytic degradation of microcystin were investigated,and removal effects of microcystin between heterogeneous photocatalytic degradation and UV photolysis were compared at the same time.[Result] Under the conditions that the initial concentration of H2O2 was 0.1 mmol/L,[H2O2]/[FeSO4] was 15:1,pH value was 4.0,the distance between the reaction solution and UV lamp tube was 1 cm,TiO2 dosage was 0.05 g/L,reaction temperature was (16±2) ℃,the removal rate of MC-RR with concentration of 0.35 mg/L and MC-LR with concentration of 0.29 mg/L could reach 91.5% and 90.2% after 3 minutes reacting.[Conclusion] UV/Fenton/TiO2 photocatalytic oxidation was proved to be effective in degradating microcystins.

Ecological damage of submerged macrophyte Myriophyllum spicatum by cell extracts from microcystin(MC)-and non-MC-producing cyanobacteria,Microcystis

To explore how decomposed Microcystis-dominant cyanobacterial blooms affect submerged macrophytes,the submerged plant Myriophyllum spicatum was exposed to cell extracts from microcystin(MC)-and non-MC-producing Microcystis strains in a laboratory experiment.Results showed that both Mcracystis cell extracts exerted obvious damages to plant biomass,photosynthesis,primary and secondary metabolism measures,and resistance of plant antioxidant systems,with MC-producing Microcystis having stronger effects due to the presence of MCs.Cyanotoxins other than MCs responsible for the negative effects from both strains needs further identification.The Shannon diversity and Chao1 indices of epiphytic and planktonic bacteria were decreased by the cell extracts from both Microcystis strains.However,epiphytic and planktonic bacterial communities responded differently to cell extracts at the genus level.The dominant genera of planktonic bacteria including Enterobacter,Pseudomonas,and Novosphingobium from phylum Proteobacteria,Chryseobacterium from phylum Bacteroidetes,and Microbacterium from Actinobacteriota in the treatments with cell extracts were previously reported to have strains with algicidal and MC-degrading capabilities.B acterial genes associated with energy production and conversion,amino acid transport and metabolism,and inorganic ion transport and metabolism,were more abundant in both treatments than the control for planktonic bacteria,but less abundant for epiphytic bacteria.We speculate that planktonic bacterial communities have the potential to use and degrade substances derived from Microcystis cell extracts,which may be beneficial for M.spicatum to alleviate damages from Microcystis.Further research is needed to verify the structure and function dynamics of epiphytic and planktonic bacteria in the interaction between cyanobacteria and submerged macrophytes.

Survival,recovery and microcystin release of Microcystis aeruginosa in cold or dark condition

Microcystis often dominates phytoplankton in eutrophic lakes and must survive a long period of cold or dark conditions. However, the survival strategies of Microcystis to withstand cold or dark stress are less well known. In this study, we conducted experiments on the responses of two toxic Microcystis aeruginosa strains （FACHB-905 and FACHB-915） and their microcystin release in conditions of low temperature （15℃ or 4℃, with illumination） or darkness, and subsequent recovery in standard conditions （25℃ with illumination）. On exposure to 15℃, a small decrease in cell viability was observed, but the cell number increased gradually, suggesting that M. aeruginosa FACHB-905 and FACHB-915 cells seem in general tolerant in 15℃. Interestingly, our results show that a higher carotenoid content and microcystin release potentially enhance the fitness of surviving cells at 15℃. M. aeruginosa cells exposed to lower temperature light stress （4℃） did not completely lose viability and retained the ability to reinitiate growth. In darkness, the maximum quantum yield （Fv/Fm） and the maximum electron transport rate （ETRmax） values and cell viability of M. aeruginosa cells gradually decreased with time. During the recovery period, the photosynthetic efficiency of M. aeruginosa reverted to the normal level. Additionally, M. aeruginosa FACHB-905 and FACHB-915 exposed to low temperature had increased caspase-3-1ike activity and DNA fragmentation, which suggests the occurrence of a type of cell death in M. aeruginosa cells under cold stress similar to programmed cell death. Overall, our findings could confer certain advantages on the Microcystis for surviving cold or dark conditions encountered in the annual cycle, and help explain its repeated occurrence in water blooms in large and shallow lakes.

Microbial biodegradation of microcystin-RR by bacterium Sphingopyxis sp. USTB-05

A strain, USTB-05, isolated from Lake Dianchi, China, degraded the cyanobacterial toxin microcystin-RR （MC-RR） at the rate of 16.7 mg/L per day. Analysis of 16S rDNA sequence showed that the strain was Sphingopyxis sp. Enzymatic degradation pathways for MC-RR by Sphingopyxis sp. USTB-05 were identified. Adda-Arg peptide bond of MC-RR was cleaved and then a hydrogen and a hydroxyl were combined onto the NH2 group of Adda and the carboxyl group of arginine to form a linear molecule as intermediate product within the first few hours. Then, through dehydration reaction, two hydrogen of amino group on arginine reacted with adjacent hydroxyl on carbon to form a linear MC-RR with two small peptide rings as the final product after 24 hr. These biodegradation pathways were different from those reported for other strains, implying that MC-RR may undergo different transformations and different products were formed due to various bacteria in natural lakes and reservoirs.

Identification and Determination of Microcystins in Source Water and Waterbloom Sample From Meiliang Bay,Taihu Lake,China

Objective To identify and determine the congener and level of microcystins in the source water of Taihu Lake. Methods Improved method of SPE combined with HPLC was employed to detect the concentration and varieties of microcystins in source water and bloom samples collected from Meiliang Bay, Taihu Lake. Results The contents of two predominant microcystin components, MC-RR, and MC-LR, were relatively high in samples during warm months and correlated with the phase of algae growth. The maximum concentrations of MC-RR and MC-LR in water sample reached 3.09±0.53μg/L and 2.39±0.41μg/L during the period of water bloom in September 2004, respectively. Even without waterbloom, the concentration of MC-LR in source water sample was still higher than the guideline value. Conclusion The status of microcystin pollution in this region is serious and measures to monitor and control the growth of cyanobacteria are urgently needed.