2000 Progress Report: Genotoxicity and Occurrence Assessment of Disinfection By-Product Mixtures in Drinking Water

EPA 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, 1999 through August 31, 2000
Project Amount: $378,088
RFA: Drinking Water (1997) RFA Text |  Recipients Lists
Research Category: Water , Drinking Water

Objective:

The objectives of the research are to: (1) calibrate short-term genotoxicity assays based on mutation in Salmonella (S.) typhimurium and direct DNA damage in cultured mammalian cells using regulated disinfection byproducts (DBPs); (2) compare the relative genotoxicities of chlorinated versus brominated DBPs; (3) compare the relative genotoxicities of chlorination byproducts versus brominated byproducts; (4) compare the relative genotoxicities of DBPs derived from singular versus combination (sequential) use of ozonation and chlorination; and (5) provide a DBP occurrence database for extrapolating genotoxicity results to practice.

Progress Summary:

This project involves the merging of quantitative chemistry and biology components. We developed a new method to evaluate brominated and chlorinated DBPs based on an ion chromatograph system as the detector instead of a coulometric titration cell. Gas phase HBr and HCl that corresponded to brominated and chlorinated compounds were dissolved in water, and the aqueous sample was 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 to the chlorinated fraction of TOX during chlorine dioxide treatment was much higher than those during chlorination and chloramination. The cytotoxic and mutagenic properties of known DBPs were quantitatively compared using bacterial and mammalian cell systems. We developed and calibrated a rapid microplate cytotoxicity assay using S. typhimurium. The cytotoxicity data were incorporated into the analysis of the genotoxic potency of each DBP. Selected DBPs were assayed for mutagenicity in strains TA98, TA100, and RSJ100 under preincubation test conditions. The disinfection of drinking water generates cytotoxic and mutagenic compounds. The cytotoxic and mutagenic properties of known DBPs were quantitatively compared. Using S. typhimurium strain TA100, a rapid, semiautomated, 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. The rank order of the cytotoxicity of the DBPs and other chemicals tested is: MX>> bromoacetic acid (BA) >bromoform (BF) >dibromoacetic acid (DBA) >chloroacetic acid (CA) >tribromoacetic acid (TBA) >chloroform (CF) >ethylmethanesulfonate (+ control, EMS) >trichloroacetic acid (TCA) >ethanol (EtOH) >dichloroacetic acid (DCA) >KBrO3 >dimethylsulfoxide (DMSO). Selected DBPs were assayed for mutagenicity in S. typhimurium strains TA98, TA100, and RSJ100 under preincubation test conditions. The rank order for the mutagenic potency for each DBP and Salmonella strain are listed below. The rank order for strain TA98 -S9 was: MX>BA>DBA>CA>DCA, with TBA, BF, TCA, and CF not mutagenic; for strain TA98 +S9: MX>BF>BA>DBA, with TBA, CA, DCA, TCA, and CF not mutagenic. The rank order for strain TA100 -S9 was: MX>BA>EMS>DBA>DCA>CA, with TBA, BF, TCA, and CF not mutagenic; for strain TA100 +S9: BA>BF>CA>DCA, with TBA, MX, TCA, and CF not mutagenic. The rank order for strain RSJ100 -S9 was: BF>DCA, with BA, DBA, TBA, MX, CA, TCA, and CF not mutagenic; for strain RSJ100 +S9: none of the DBP standards expressed a mutagenic response. Mammalian cell assays can provide toxicological information that may be more relevant to human risk assessment than commonly used microbial tests. Rapid, semiautomated, 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 BA>MX>DBA>CA>KBrO3>TBA>EMS>DCA>TCA. Genotoxicity analyses of the DBPs were 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 BA>MX>CA>DBA >EMS>TBA>DCA>TCA. The relative cytotoxicity and genotoxicity of these agents were compared with S. 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.

Future Activities:

Complex DBP mixtures have been isolated from real waters after chlorination, chloramination, or ozonation. Cytotoxicity and genotoxicity analyses of these samples currently are underway and will be the focus of the work in the final project year.

Journal Articles:

No journal articles submitted with this report: View all 25 publications for this project

Supplemental Keywords:

Chinese hamster ovary cells, mammalian cells, single cell gel electrophoresis, SCGE, water disinfection byproducts., RFA, Health, Scientific Discipline, Waste, Water, Environmental Chemistry, Genetics, Health Risk Assessment, Environmental Microbiology, Risk Assessments, chemical mixtures, Drinking Water, monitoring, occurrence monitoring, Safe Drinking Water, single cell electrophoesis assay, microbial risk assessment, human health effects, chlorinated by products, exposure and effects, disinfection by-product mixtures, disinfection byproducts (DPBs), exposure, community water system, natural organic matter, genotoxicity, human exposure, treatment, bacterial genotoxicity, dietary ingestion exposures, drinking water contaminants, water treatment, drinking water treatment, cytotoxic effects, drinking water system, ozonation, DNA microarrays

Progress and Final Reports:

Original Abstract
  • 1998
  • 1999 Progress Report
  • 2001
  • Final Report