1999 Progress Report: Genotoxicity and Occurrence Assessment of Disinfection By-Product Mixtures in Drinking WaterEPA Grant Number: R825956
Title: Genotoxicity and Occurrence Assessment of Disinfection By-Product Mixtures in Drinking Water
Investigators: Minear, Roger A. , Plewa, Michael J.
Institution: University of Illinois at Urbana-Champaign
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 1997 through August 31, 2000 (Extended to November 16, 2001)
Project Period Covered by this Report: September 1, 1998 through August 31, 1999
Project Amount: $378,088
RFA: Drinking Water (1997) RFA Text | Recipients Lists
Research Category: Water , Drinking Water
Objective:The general hypothesis asks if the single cell gel electrophoresis (SCG COMET) assay using transgenic mammalian cells can predict human risks from drinking water disinfection by-products (DBPs) rather than bacterial genotoxicity tests, and is it sufficiently responsive to evaluate DBPs produced from different disinfection processes and under different conditions? The hypothesis is founded on the premise that mammalian cells may be more representative of direct cytotoxic and genotoxic affects from DBPs on humans than the "traditional" bacterial assays. The objectives of the proposed research are to: (1) calibrate S. typhimurium and transgenic mammalian cells genotoxicity assays using regulated DBPs, (2) compare the relative genotoxicities of chlorinated versus brominated DBPs, (3) compare the relative genotoxicities of chlorination by-products (CBPs) versus brominated ozonation by-products (OBPs), (4) compare the relative genotoxicities of DBPs derived from singular versus combination (sequential) use of ozonation and chlorination, and (5) provide a DBP occurrence data base for extrapolating genotoxicity results to current disinfection practice.
Progress Summary:Chemical Section. A new method to evaluate brominated and chlorinated DBPs was developed to compensate the inability of conventional TOX analysis to differentiate brominated and chlorinated compounds. The new method uses an ion chromatograph system as the detector instead of a coulometric titration cell. In our new method, gas phase HBr and HCl that correspond to brominated and chlorinated compounds from the furnace for TOX analysis are dissolved in water, and then the aqueous sample is applied to ion chromatography for separation and quantification. Comparison among chlorination, chloramination, and chlorine dioxide treatment of Suwannee River fulvic acid with bromide ion showed that the ratio of the brominated fraction of TOX (TOBr) to the chlorinated fraction of TOX (TOCl) during chlorine dioxide treatment was much higher than those during chlorination and chloramination, though TOX produced during chlorine dioxide treatment was the lowest among the three processes. Also, a strong correlation was found between the ratios of TOBr to TOCl and the ratios of bromine to chlorine in known DBPs such as trihalomethanes and haloacetic acids. Six raw waters were concentrated and fractionated with a RO system. Their characterization has been completed and is in evaluation.
Biological Section. The disinfection of drinking water generates cytotoxic and mutagenic compounds. The cytotoxic and mutagenic properties of known DBPs were quantitatively compared. Using Salmonella typhimurium strain TA100 a rapid, semi-automated, microplate cytotoxicity assay was developed. The assay can accommodate a concentration range of six log orders of magnitude with six replicates per concentration and requires approximately 5 hours. Data were automatically transferred from a microplate reader to a computer spreadsheet. The DBP concentration that induced 50 percent repression of growth in the cytotoxicity assay was used as the highest concentration for the S. typhimurium mutagenicity assay. Selected DBPs were assayed in S. typhimurium strains TA98, TA100, and RSJ100 under suspension test conditions. The mutagenic potency of the DBPs were calculated and compared with the cytotoxicity data. The rank order of decreasing cytotoxicity of the DBP standards and solvents was MX >> bromoacetic acid >> bromoform > dibromoacetic acid >> tribromoacetic acid > chloroform >> dimethylsulfoxide. The rank order of the mutagenic potency of the DBPs from highest to lowest was MX >>> bromoacetic acid >> bromoform > dibromoacetic acid >> tribromoacetic acid. Mammalian cell assays can provide toxicological information that may be more relevant to human risk assessment than commonly used microbial tests. Rapid, semi-automated, quantitative mammalian cell cytotoxicity and genotoxicity assays were developed to analyze drinking water DBPs. These assays employ 96-well microplates; selected DBPs were analyzed with cultured Chinese hamster ovary (CHO) cells. The concentration of the DBPs that repressed 50 percent of CHO cell growth with a 72 hour exposure was calculated as the percent C? value. Using these values, the rank order (from highest to lowest cytotoxicity) was bromonitromethane, dibromonitromethane, tribromonitromethane, bromoacetic acid, dibromoacetic acid, and tribromoacetic acid. Genotoxicity analysis of the DBPs was conducted using the single cell gel electrophoresis (SCGE) assay. This assay detects genomic DNA damage at the level of the individual nucleus. Using SCGE genotoxic potency, the rank order was bromoacetic acid > dibromonitromethane >> bromonitromethane > dibromoacetic acid > tribromoacetic acid. The relative cytotoxicity and genotoxicity of these agents were compared with Salmonella typhimurium. Studies of specific DBPs in mammalian cell systems are important to compare the toxicity of these hazardous water contaminants. Such knowledge is necessary for risk assessment and to assist in the formulation of public regulatory policies that protect the environment and the public health.