Identification, Genotoxicity, And Potential Carcinogenicity Of Iodoacetic Acid And Iodoform In Drinking Water

Iodo-disinfection byproducts are a kind of new unregulated disinfection byproducts in drinking water treatment process in recent years. Iodo-disinfection byproducts are easy to form when high iodide in raw water and using chloramination. Salt water intrusion strongly influences drinking water source because Shanghai located in the intersection zone of river and sea. Most of works used conventional treatment process (preoxidation, coagulation, sedimentation, sand filtration, and post chloramination.) to produce drinking water. Special environmental conditions and conventional treatment process are in favor of iodo-disinfection byproducts formation. Otherwise, the concentrations of iodo-disinfection byproducts are very low in drinking water but they are toxic strongly. Previous studies indicated that iodo-disinfection byproducts were more cytotoxic and genotoxic than regulated disinfection byproducts. Therefore, it is necessary to detect the levels and influencing factors of iodoacetic acid and iodoform in drinking water in Shanghai in order to evaluate population exposure. MTT assay, CCK-8 assay, Ames test, cellular y-H2AX, and cytokinesis-block micronucleus assay were used to evaluate their cytotoxicity and genotoxicity and their mechanism. Then the potential carcinogenicity and its mechanism of iodoacetic acid and iodoform were assessed by cell transformation assay in vitro. The study would provide the important scientific evidences for disinfection byproducts control and treatment process improvement. Meanwhile, it is helpful to the health risk assessment of iodoacetic acid and iodoform and the establishment of standard for drinking water quality.1 Determination of iodoacetic acid and iodoform in drinking water in Shanghai1.1 Establishment of methods for determining chloride, bromide, and iodide in drinking waterThe method for determining chloride and bromide in drinking water by ion chromatography was established on the base of U.S. Environmental Protection Agency 300.1 method, Iodobutanone derivative was identified by gas chromatography/mass spectrometry, and then gas chromatography coupled to electron capture detector was made to analyze iodide in water. The results of ion chromatography showed that chloride and bromide were separated completely. Their peaks were symmetrical, no tail and not disturbed by peaks of solvent or impurity. The total time of chromatogram separation was 12 minutes. External standard calibration curve was selected to quantitative analysis. The linear ranges of chloride and bromide were 1-1000 mg/L and 1-1000μg/L respectively, and their coefficients of determination were both 0.999. The mean recoveries were between 89.7% and 112.3%, and the relative standard deviations were less than 2.9%. Method detections limits of chloride and bromide were 55.6μg/L and 0.37μg/L respectively. Their continuing calibration checks were between 87.9% and 113.2%. Surrogate response was between 93.2% and 103.4%. Their relative percent difference for duplicates were between 0.0% and 8.0%. The results of mass spectrometry showed that iodide would form iodobutanone which could generate 1-iodo-2-butanone and 3-iodo-2-butanone isomers by derivatization. The data of qualitative analysis by two capillary columns revealed that iodobutanone were separated completely and the total time of chromatogram separation was 19.33 minutes.3-iodo-2-butanone with the high response value was selected to quantitative analysis. The linear range was 1-100μg/L, and the coefficient of determination (r2) was 0.999. The limit of detection was 13 ng/L. The mean recovery was 102.0%, and relative standard deviation was 5.1%. The continuing calibration checks were between 97.2% and 108.8%. Internal standard responses were between 92.00% and 110.05%. The relative percent difference for duplicates were between 4.4% and 11.6%. The improved method possesses higher degree of sensitivity and accuracy of qualitative and quantitative analysis. Its quality control measured up to methods of United States Environmental Protection Agency. And it is fit for trace analysis of chloride, bromide, and iodide in water.1.2 Determination of iodoacetic acid, iodoform, four trihalomethanes, and nine haloacetic acids in drinking water by gas chromatography with electron capture detectionBase on U.S. Environmental Protection Agency 551.1 and 552.3 methods, the pretreatment and detection conditions of methods for identifying iodoacetic acid, iodoform, four trihalomethanes(chloroform,bromoform, bromodichloromethane, and dibromochloromethane) and nine haloacetic acids (chloroacetic acid, dichloroacetic acid, trichloroacetic acid, bromoaceic acid, dibromoacetic acid, tribromoacetic acid, bromodichloroacetic acid, chlorodibromoacetic acid, and bromochloroacetic acid) with two capillary columns coupled to gas chromatography/electron capture detector were improved and optimized. The results of gas chromatography showed that iodoacetic acid, iodoform, four trihalomethanes, and nine haloacetic acids were separated completely. Their peaks were symmetrical, no tail and not disturbed by peaks of solvent or impurity. The total time of iodoform and four trihalomethanes separation was 40.67 minutes. The total time of iodoacetic acid and nine haloacetic acids separation was 23.50 minutes. Internal standard calibration curve was selected to quantitative analysis. The linear ranges of iodoacetic acid and iodoform were 0.01-2.5μg/L and 0.05-5μg/L respectively. The linear ranges of four trihalomethanes and nine haloacetic acids were both 1-100μg/L. Their coefficients of determination were more than 0.994. The mean recoveries were between 93.5% and 111.1%, and the relative standard deviations were less than 7.0%. Method detections limits of iodoacetic acid and iodoform were 6.2 ng/L and 17.7 ng/L respectively. Method detections limits of four trihalomethanes and nine haloacetic acids were between 191.4 ng/L and 502.2 ng/L. The continuing calibration checks were between 87.1% and 114.0%. Internal standard responses were between 85.2% and 120.0%. Surrogate response was between 85.8% and 116.5%. Their relative percent difference for duplicates were between 0.1% and 10.3%. The modified methods possesses good separation, fast, high sensitivity, high accuracy, and good consistency. It is fit for analysis in normal laboratory because of its economical and automatic characteristic.1.3 Pollution of iodoacetic acid and iodoform in drinking water in ShanghaiThe object of this study is to detect the levels of iodoacetic acid and iodoform in water of each treatment processes in works that their source waters come from Yangtze River and Huangpu River in Shanghai and evaluate the relationship of iodoacetic acid and iodoform formation to water qulity, treatment processes, and disinfectants. Thirteen water treatment works that are at central regions of Shanghai were chosen for investigation in low water period and high water period. Eleven works used conventional water treatment process. Two works used advanced water treatment process. Water samples of different treatment processes were selected to analyze for pH value, ammonia nitrogen, dissoluble organic carbon, UV absorbance, specific UV absorbance, chloride, bromide, iodide, iodoacetic acid, iodoform, four trihalomethanes, and nine haloacetic acids.The results showed that pH values of Yangtze River and Huangpu River were alkalescent (pH=7.34). The concentrations of ammonia nitrogen were less than 0.5 mg/L. The levels of organic matter were not high. Medians of dissoluble organic carbon, UV absorbance, and specific UV absorbance were 6.00 mg/L、0.23 l/cm、3.90 L/(mg·m) respectively. The pH value of Huangpu River was lower than that of Yangtze River. The pH values of two rivers in high water period were higher than low water period. The ammonia nitrogen level of Huangpu River was closed to Yangtze River in high water period. In low water period, the ammonia nitrogen level of Huangpu River was higher than Yangtze River. The dissoluble organic carbon and UV absorbance of Huangpu River was higher than that of Yangtze River. The dissoluble organic carbon levels of two rivers in high water period were higher than low water period, but the UV absorbance levels reversely. In high water period, specific UV absorbance level of Huangpu River was higher than Yangtze River, but reversely in high water period. Chloride, bromide, and iodide levels of Huangpu River were higher than that of Yangtze River. But chloride, bromide, and iodide levels of two rivers in high water period were lower than low water period. The concentrations of iodoacetic acid and iodoform were between 0.03μg/L and 1.66μg/L in finished water in works. The level of four trihalomethanes and nine haloacetic acids were between 0.28μg/L and 63.74μg/L in finished water in works.The concentrations of iodoacetic acid and iodoform in finished water in works which based on Huangpu River as water source were higher than that of works which based on Yangtze River as water source. The concentrations of iodoacetic acid and iodoform in finished water in low water period were higher than high water period. The results of multiple liner regression analysis showed that the relationship of pH value and iodo-disinfecton byproducts formation were negative correlation. Otherwise, the relationship of UV254 and iodide levels and iodoform formation were positive correlation.More iodo-disinfecton byproducts were formed when using chloramination compared with ozone. The level of iodo-disinfecton byproducts were between 0.2μg/L and 1.7μg/L when using chloramination. There were not sighnificant differences between chloramination and chlorination. The treatment processes of each work had no effect on the level of pH value, ammonia nitrogen, dissoluble organic carbon, chloride, and bromide. But the levels of UV254 and specific UV absorbance decreased. The level of iodide decreased in finished water in conventional treatment process works. Iodoacetic acid and iodoform were not detected in raw water. But iodoacetic acid and iodoform were detected in finished water in every work. Iodoacetic acid, iodoform, four trihalomethanes, and nine haloacetic acids were formed in conventional treatment process. Iodoacetic acid and iodoform were not detected before activated carbon in advanced treatment process. Only few four trihalomethanes and nine haloacetic acid were detected in finished water in advanced treatment process works. The results indicated that trace iodoacetic acid and iodoform were detected in finished water in every work at the central regions of Shanghai. Low pH value, high natural organic matter, high iodide, and chloramination were easy to form iodoacetic acid and iodoform. The conventional and advanced treatment processes had no effect on pH value, ammonia nitrogen, natural organic matter, chloride, bromide, iodoacetic acid and iodoform.2 Cytotoxicity of iodoacetic acid and iodoformThe object of this research is to study the cytotoxicity and its mechanism of iodoacetic acid and iodoform by a group of different cytotoxic end points assay. NIH/3T3 cells were treated by different doses iodoacetic acid and iodoform for 72 hours respectively. Then the proliferation and mitochondria damage of NIH/3T3 cell induced by iodoacetic acid and iodoform were evaluated by methylthiazoletrazolium assay and CCK-8 assay. The morphology of cells was observed in the microscope. The level of lactate dehydrogenase, ATP, and reduced glutathione were measured respectively. Cell cycle and apoptosis of cells were analyzed by flow cytometer. The results showed that NIH/3T3 cells viability decreased after they were exposed to iodoacetic acid and iodoform for 72 hours. There were also dose-response relationships. Iodoacetic acid was 2×more cytotoxic than bromoacetic acid and 218×more cytotoxic than chloroacetic acid in NIH/3T3 cells. Iodoform was 81×more cytotoxic than bromoform. The prohibition of cellular proliferation, apoptosis, and necrosis also could be observed in the microscope. Iodoacetic acid and iodoform could cause extracellular lactate dehydrogenase increase and intracelluar ATP decrease after 72 hours incubation (P<0.05). But they had no effect on reducing glutathione (P>0.05). Iodoacetic acid and iodoform could induced an increase in the proportion of apoptosis and necrosis. Cells cycle were arrested in S and G2/M phase respectively after incubation with iodoacetic acid and iodoform. Mitochondria damage, ATP depletion, and membrane damage are possibly involved in the induction of cytotoxicity by iodoacetic acid and iodoform. Mitochondria pathway would be possible one of mechanisms of cell death caused by iodoacetic acid and iodoform. Cells cycle arrest indicated that iodoacetic acid and iodoform can damage cellular DNA.3 Genotoxicity of iodoacetic acid and iodoformThe genotoxicity of iodoacetic acid and iodoform were evaluated by Ames test, cellularγ-H2AX, and cytokinesis-block micronucleus assay. Salmonella typhimurium TA100 and TA98 were treated by different doses iodoacetic acid and iodoform. Un-preincubation and preincubation methods were used in iodoacetic acid treatment process. Un-preincubation was just used in iodoform treatment process. Numbers of Salmonella typhimurium were counted after 48 hours. In cytokinesis-block micronucleus assay, the treatment time of iodoacetic acid and iodoform were detected by NIH/3T3 cell growth curve. Then doses of iodoacetic acid and iodoform were selected by CCK-8 assay. Micronuclei were counted after cells were treated by iodoacetic acid and iodoform for 40 hours using cytokinesis-block micronucleus assay. Doses of iodoacetic acid and iodoform were determined by CCK-8 assay. Then levels ofγ-H2AX were analyzed by flow cytometer after treatment for 24 hours. The results showed that iodoacetic acid could not induce Salmonella typhimurium TA100 and TA98 increase significantly with or without S9. Iodoform was mutagenic in Salmonella typhimurium TA100 and TA98 with or without S9. Iodoacetic acid could cause an increase in cellularγ-H2AX. And the results also showed a dose-response relationship. Iodoform could induceγ-H2AX formation in the cell, but not a dose-response relationship. Iodoacetic acid and iodoform both have no effect on micronucleus. The results of Ames test, concentration ofγ-H2AX in the cell, and cytokinesis-block micronucleus assay showed that iodoacetic acid and iodoform were possible genotoxicants.4 Potential carcinogenicity of iodoacetic acid and iodoformThe potential carcinogenicity and its mechanism of iodoacetic acid and iodoform were assessed by cell transformation assay in vitro. Doses of iodoacetic acid and iodoform were determined by cell colony formation assay. Then NIH/3T3 cells were treated by different levels of iodoacetic acid and iodoform for 72 hours. Transformation colonies were counted after 10 days culture. Then transformed cells were identified by Concanavalin A agglutination assay, soft agar assay, and assay of tumorigenicity in nude mice. Cell cycle of transformed cells was analyzed by flow cytometer. The expression of p53 protein in transformed cells was detected by immunocytochemistry assay. The results showed that iodoacetic acid could induce NIH/3T3 cells transformation. Transformed cells could be agglutinated by Concanavalin A. Meanwhile, they could form colony in soft agar and tumor in nude mice. The results of histological examination showed that the tumor was low pathologic differentiated fibrosarcoma. Cell cycle of transformed cells was arrested in G0/G1 phase. The proportions of cells in S phase and G2/M phase decreased. The expression of p53 protein in transformed cells was not high. Iodoform could not cause NIH/3T3 cells transformation in vitro. Cell transformation assay in vitro indicated that iodoacetic acid has potential carcinogenicity. But iodoform has not yet.