Distributing Characteristics Of Microcystins And Regression Model Of Cytotoxicity/Genotoxicity On Pollution-Spectrum With Huai River Water Organic Extract From X County

Frequent occurrence of water crisis has drawn attention on water pollution and safety of drinking water. It had been commonly recognized the safety of drinking water is the basis of ensuring public health. In recent year, with the effects of discharge the waste water of sewage, agriculture and industry, most water bodies in China have been serious eutrophic status which has increased the frequent outbreak of cyanobacteria in lakes and rivers. Pollution of cyanobacteria and cyanotoxins greatly affected to urban water supply and public health. Microcystins (MCs), as one of the risk facts to liver cancer, are existent in water bodies and sediments. How to rapid and sensitive detection of algal toxins in the environment is a prerequisite to evaluate exposure level and risk assessment. X County in Huai River Basin occurred to a high congregation of cancer, especially liver cancer gathered in part of the village, which has been concerned by the government and the scholars. These villages closed to the Shaying River, the largest tributary of Huai River with the serious pollution, the ponds existed in the village with high cancer incidence. The data from the Senate Environmental Protection Agency (SEPA) showed that many enterprises such as paper-making and printing and dyeing were in the upstream of Shaying River. The water was serious eutrophication. The cancer project I for Huai River found that cancer may be related to water pollution. Owing to lack of basic data for algae and their toxins in Shaying River, the cancer in there whether associated with the contamination of algae toxin remained unknown.As common sense, the pollutants in water bodies are consistent with the type of drinking water. The type and the amount of pollutants in drinking water were related with the water pollution. The pollutants in drinking water were complicated and could cause various health effects. The mixture exposure could cause significant threat to health with synergism even the single component was below safety level. As a result, it was important to develop methods for assessment of mixture exposure effects. The previous studies on mixture studies demonstrated that one was bottom-up—the single component and all the interactions were studied. The other was top-down—the mixture was studied as a whole. In the second study, the effects could be regressed on the spectrum derived from GC-MS and the predictive model could be used to rapid predict the effects with the spectrum.This study focused on the pollution in Shaying River of the Huai River Basin. On the basis of establishing the SPE-HPLC method for determination of MCs, the distribution of MCs in groundwater, surface water and sediment in X County of the Huai River Basin were investigated. The interaction of MX and MCs was assessed both in cytotoxicity and genotoxicity. The regression model was established using the relationship of the cytotoxicity and genotoxicity of organic extractions from water and analysis of GC-MS spectrum, the harmful effects was predicted with the Spectrum of pollution.Part One The Polluted Characteristic of Algae and MCs in X County in the Huai River BasinIn this study, MCs in water and sediments was extracted by ODS resin. After systemic optimization of extract procedure, the method of determination of MCs based on HPLC was set up and five isomeric MCs including MC-LF/LR/LW/RR/YR were simultaneous detected with one injection. The detection of limit for this method was 0.01μg/L～0.02μg/L and recovery rate was 68.78%～123.08%, which was met with the EPA standards.The surface and sediment samples were collected during median-water period and high water periods. Chemical oxygen demand (COD), total nitrogen (TN), total phosphorus (TP), chlorophyll a (Chl a), algal density and MC-LF/LR/LW/RR/YR were detected, respectively. The result show that water pollutions in the study area were serious and water quality ranged from gradeⅢto worse than gradeⅤof national standard for surface water quality. The water quality of the main river was better in high water period than in median-water period while the tributary and ditch was just the opposite. The highest density of algae was founded in ditch during high water period and reached 237.00×106/L and cyanobacteria became predominant in this time MC-RR in dissolved form and algal cells was up to 4.297μg/L and 17.731μg/L, respectively. MC-RR in sediments was 0.802μg/g.The routine items of water quality and the concentration of MCs in surface water and groundwater were surveyed every quarter from September,2008 to January,2010. The results demonstrated the routine items of water quality in groundwater was better than that of surface water (P<0.05). The density of algae and chlorophyll a in surfaced were linear correlated (r=0.94, P<0.05). The concentration of MCs in surface water ranged in 0.007μg/L-2.298μg/L while the concentration of MCs in groundwater averaged at 0.061μg/L, the maximum of MCs was 0.118μg/L. There are no significant differences on MCs concentration in different towns and in groundwater with different depths (P=0.05). The concentration of MCs in groundwater was linear correlated with river water (R2=0.95, P<0.05) and the correlation increased with the reducing of distance to river; the closer with the river is, the higher is. The river had higher level of MC-LR and lower MC-RR than groundwater while the MC-RR did not differ significantly.Part Two Cytotoxicity and Genotoxicity Induced by the Joint Effect of MX and MC-LRCytotoxicity and genotoxicity were very important aspects for recognizing the toxic characteristic of pollutants. Full factorial design was used to study the joint effects of MX and MC-LR on cytotoxicity and genotoxicity. The method of CCK-8 test was employed for determination the cytotoxicity of MX and MC-LR. Ames (Salmonella Typhimurium Reverse Mutation Assay) and cytokinesis-block micronucleus cytome assay were used to test genotoxicity induced by MX and MC-LR. The results showed 0～5μM MC-LR has no significant cytotoxicity and the interaction between MX and MC-LR was weak antagonism (R2 of interaction=0.0114,P<0.0001). IC50 of MX with 0μM,0.5μM and 5μM MC-LR was 64.61μM,67.84μM and 75.44μM respectively. With the addition of S9, MC-LR and MX showed no mutagenicity in TA100 and TA98 strains either in dependent or in combined. Without the addition of S9, just MC-LR did not have mutagenicity while MX showed strong mutagenicity in TA100 and TA98 strains. Obvious interaction, synergism, was observed when MC-LR and MX administrated jointly in TA100 and TA98 strains. 0.1nmol MC-LR could increase the number of revertants induced by MX in TA100 and TA98 strains (P<0.05). In micronucleus assay,5μM MC-LR could induce higher rate of nuclear buds and 5μM～20μM of MX could induce higher rate of micronucleus, nucleoplasmic bridges and nuclear buds. There were no interactions observed in micronucleus assay induced by MC-LR and MX.Part Three Studies on the Cytotoxicity and Genotoxicity of Organic Extracts from Water Bodies in the Huai River Basin and its Regression Model with GC-MS Pollution-SpectrumHealth effect of mixture exposure was the important issue in health risk assessment. Pollutants in water body are complicated and various, the numbers of pollutants were more than hundreds in a serious polluted water samples. It was impractical that the single pollutant was extracted and separated and assessed with its effect on health. Currently, the regular method was enriched and extracted pollutants from water samples. The enrichments were treated as a whole which was used for the evaluation of biological effects. The pollutants that contributed to biological effects were analyzed by advanced analytic technique. As the board application of GC-MS, it was possible to disclose the relationship between health effect and components, which was also one of the most important methods for health effects induced by pollutants. During this study, water samples from rivers, wells and ditches were collected from September,2008 to January,2010 in X County of the Huai River Basin. After extraction by XAD-2, cytotoxicity and mutagenicity (Salmonella Typhimurium Reverse Mutation Assay) of organic extracts were evaluated. Meanwhile, the extraction was analyzed using GC-MS. Then statistical models were used to predict the toxic effects. The results of Ames test were coded as dichotomies. Base on the amount of sample sizes, variety and multicollinearity, the principal component analysis was applied before logistic regression of Ames results on spectrum. Partial least square regression was used in modeling the cytotoxicity on spectrum.Six of the water samples including river and ground water showed mutagenicity in TA98 strains of Ames test and it is indicated that the water bodies in this area is still existed the potential of mutagenicity. The IC50 of different water bodies ranged from 0.023L/ml to 0.338L/ml. The cylotoxicity of ground water was lower than river water. In autumn and winter the cytotoxicity of river water samples and ground water samples were lower than in spring or summer. Logistic model was used in modeling the Ames result on spectrum and the area under curve derived from sensitivity-specificity analysis was 0.823, which showed this model could effectively predict the Ames test. PLS model was used in modeling the cytotoxicity on spectrum and the R-square was 0.91. The difference between practical and predicted value was not significant, indicating the model was valid. This study provided the tools for predicting effect of mixture exposure.