Toxicological Analysis of New emerging Drinking Water Disinfection By-Products

Non-Technical Summary: Drinking water disinfection byproducts (DBPs) are a class of environmentally hazardous chemicals that are generated during the disinfection of drinking water. DBPs carry long-term human health risks and new federal regulation will result in the increased generation of emerging DBPs. We will conduct a quantitative and comparative toxicological analysis of new iodinated-DBPs, nitrogenous-DBPs and iodo-nitrogenous-DBPs. There are no quantitative toxicological data on these emerging DBPs. We will determine their relative mammalian cell chronic cytotoxicity and their capacity to induce genomic DNA damage. These data will prove exceedingly useful in guiding future risk assessments of DBPs and will serve as a practical resource for the water treatment community in their decisions on disinfection practice. The majority of DBPs in drinking water have not been chemically or toxicologically defined. Concerns exist about which DBPs pose the greatest health risk and, therefore, which DBPs should be the focus of heightened federal regulation. There is less total organic halide (TOX) formed in drinking water treated with alternative disinfectants (e.g., chloramines, chlorine dioxide, ozone), as compared to chlorine, but the distribution of new emerging DBPs increases. Recently the U.S. EPA mandated new DBP rules that increased the stringency of previously regulated DBPs. As water utilities move from chlorine to alternative disinfectants to meet the new DBP regulation, they may generate greater amounts of highly toxic emerging DBPs for which we have little data. <P> Approach: All of the chemical characterization and identification of the emerging DBPs will be conducted at the National Exposure Research Laboratory, U.S. EPA, Athens GA. EPA will commission and pay for the synthesis of analytical grade DBPs that are not available from commercial sources. The analytical chemistry and the analytical biology will be integrated together in this project. The microplate cytotoxicity assay measures the reduction in cell density as a function of the DBP concentration over a 72 hour period. For each DBP concentration 8 replicate wells will be analyzed per experiment and the experiments will be repeated twice. For each cytotoxicity concentration-response curve we will conduct regression analysis and calculate the concentration that induces a cell density that is 50% of the negative control. We will use a powerful molecular genetic assay, single cell gel electrophoresis (SCGE), to determine if a DBP is a genotoxin. Each DBP will be dissolved in DMSO and diluted in F12 medium. We will treat the CHO cells with a concentration range of each DBP, for 4 hours. The cells will be embedded in layers of agarose prepared with an electrolyte and the cell membranes will be lysed. The microgels will be electrophoresed, stained and the amount of DNA that migrates from each nucleus will be analyzed. A computerized image analysis system will be used to measure the tail moment (integrated value of migrated DNA density multiplied by the migration distance) as the primary measure of DNA damage. The digitalized data will be transferred from the CCD camera to a computer-based spreadsheet for statistical analysis. We shall integrate the data generated from the emerging DBPs into our CHO cell chronic cytotoxicity DBP database and into our CHO cell genomic DNA damage DBP database. These databases are currently the largest available and are being used for structure function activity analyses as well as for the development of algorithms to determine the toxic action of mixtures of DBPs. The addition of the emerging DBPs into this evolving database will provide a wealth of information for scientists, risk assessors, regulators and water utility operators.