| Due to the rapid development of industry and agriculture, the surge in volume of sewage discharge has increased water pollution, many major water bodies in China were in increasing eutrophication. Water blooms resulted from algae outbreak could induce water pollution and affected the safety of water supply, which posed health threat to population. Microcystins (MCs), the metabolites of cyanobacteria, played a role in tumor promoting. Exposure in low dose for long term could increase the risk of liver cancer. Shaying River, the largest tributary of the Huai River, had occurred serious water pollutions, and many water quality indicators had been detected exceeding the national standard limit. X County near Shaying River sufferred a high incidence of cancers, especially the high congregation of digestive cancers in part of the village, which had been concerned by the government and scholars. The previous studies found that the cancer might have some relationship with water pollution. Due to the lack of continuous data about the pollution of algaes and microcystins in water bodies of Shaying River, whether the cancers there were associated with the contamination of algal toxins remained unclear.There were studies showing that the co-existence of various pollutants could induced a synergistic effect even if the concentration of single component was below safety limit, which resulted in more significant threat to the health of population, and the combination effects between multiple pollutants could be studied through the method of factorial design. Due to the mild and wet climate in Huai River Basin, the crops contaminated with aflatoxin(AFT) had been regularly reported. And the previous studies also demostrated that, the high incidence of liver cancer in our country was closely related with the contamination of AFB1. MCs were known as tumor promotor, and AFT were definite genotoxic carcinogen, the occurrence of combination toxicity of MCs and AFT was possible. Therefore, the study of the combination biological effects of two toxins was of great value, providing the basis for the explaination of high incidence of cancers. This study focused on the pollution in Shaying River of Huai River Basin, the pollution status of eutrophication, algaes and microcystins in major water bodies of X County during 2012-2013 were analysed, and the interaction of MCs and AFB1 was assessed both in cytotoxicity and genotoxicity.Part One Pollutions of eutrophication, algae and microcystins of water bodies in X County of the Huai River BasinIn this study, surface water samples were collected from S River, Y River and a representative pond from April 2012 to March 2013. Total nitrogen (TN), total phosphorus (TP), chlorophyll-a (Chi a), transparency (SD), chemical oxygen demand (CODMn) algal density were determined and analyzed. The concentrations of microcystins (MCs), including dissolved MCs in water and cell bound MCs in algae, were determined by high performance liquid chromatography with solid phase extraction. The water quality of the surface water in X County was evaluated according to the National Standard (GB3838-2002), and the TSIM was employed to assess surface water eutrophic state. The results showed that, different water bodies of ShenQiu County were serious, most items of water quality exceeded the national standard for surface water quality Class V. Total nitrogen in the mainstream of S river was serious, the maximum concentration was 13.15 mg/L, and highest concentrations of TP and CODMn were up to 0.61 mg/L,9.3 mg/L,respectively; The CODMn of tributary Y river was up to 15.39 mg/L, and its SD value was as high as 2.10 m; Total phosphorus and CODMn were serious in the pond where the maximum concentrations were 2.01 mg/L and 43.32 mg/L, respectively, and the minimum Transparency of the pond was 0.14 m. The eutrophication level of different water bodies were all beyond eutrophic level. The peak of total algal cell density was 3.54x107/L in Pond water of July. Algae species in water bodies showed seasonal changes, the Chlorophyta and Bacillariophyta were dominant in spring and winter, respectively. Cyanobacteria was dominant in summer and significantly increased. Meanwhile, cyanobacteria in Y River reached 68%in July. The maximum concentrations of dissolved and cell bound MCs were observed in pond water of July,3.69 μg/L and 6.21 μg/L, respectively. The correlation analysis indicated that the Chlorophyll-a in different water bodies had a significant positive correlation with the cyanobacteria density (P<0.0\) and cellular microcystins (P<0.05). And the density of cyanobacteria was also positive correlated with cellular microcystins (P<0.05). In sum, Algae and microcystins of surface water in ShenQiu County were heavily polluted. The cyanobacteria in different water bodies was dominant during summer.Part Two Cytotoxicity and Genotoxicity Induced by the Combination Effect of MC-LR and AFB1Cytotoxicity and genotoxicity were very valuable aspects in evaluating the safety of exposure to environmental pollutants, this study investigated the combination cytotoxicity of MC-LR and AFB1, and analysis for interactions by factorial design was used to study the combination effects of MC-LR and AFB1 on genotoxicity. The method of CCK-8 test kit was used for detecting the cell viability as the endpoint to determinate the cytotoxicity of MCs and AFB1. The genotoxicity induced by MCs and AFB1 was evaluated by Salmonella Typhimurium Reverse Mutation Assay (Ames assay) and cytokinesis-block micronucleus cytome assay (CBMNT). The results showed that, MC-LR had no significant cytotoxicity (P>0.05). From regression analysis, the IC50 of AFB1 with 0.0μM,0.5μM and 5.0μM MC-LR were 0.99μM, 0.92μM and 0.69μM, respectively. With the addition of S9, MC-LR in dependent showed no mutagenicity, while the AFB1 in dependent in this experimental conditions showed mutagenicity in TA98 and TA100, and the combination effect of these two toxins exhibited significant interaction of mutagenic synergisim in Salmonella typhimurium TA98, TA100 strains(P<0.05), 0.1μg MC-LR could significantly increase the number of revertants induced by AFB1 in TA98 and TA100 (P<0.05). Without the addition of S9, MC-LR did not have mutagenicity while MC-LR and AFB1 in combined just showed weak mutagenicity in both strains. In micronucleus assay,0.2～0.6μM of AFB 1 in dependent could induced significantly higher rate of micronucleus(P<0.05), and there was a dose-response relationship for this effect, but MC-LR in dependent had no this effect. There was no interactions observed in micronucleus assay induced by MC-LR and AFB 1 in combined (P>0.05)... |
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