Michael J. Plewa and Elizabeth D. Wagner

OBJECTIVES:

The objectives of this study were to (1) analyze USEPA priority drinking water disinfection by-products (DBPs) for their chronic cytotoxicity and acute genotoxicity in mammalian cells, (2) rank the cytotoxicity and genotoxicity of the DBPs, (3) generate comparative indices for the DBP chemical classes, and (4) employ the toxicity data to determine structure activity relationships for the analyzed DBPs.

BACKGROUND:

The drinking water community provides an exceedingly important public health service by its generation of high quality, safe palatable tap water. The disinfection of drinking water in public facilities primarily employs chemical disinfectants such as chlorine, chloramines, ozone, and chlorine dioxide. These disinfectants are oxidants that convert naturally occurring and synthetic organic material, bromide, and iodide in the raw water into chemical DBPs. DBPs are an unintended consequence and were first discovered over 30 years ago. The majority of DBPs have yet to be chemically characterized, and only a small fraction of DBPs have been evaluated for their biological and toxicological effects.

HIGHLIGHTS:

This study is a comprehensive analysis of the toxicity of priority DBPs using in vitro mammalian cell assays. The highlights of the study included the following:

This study represents the first systematic study on the mammalian cell chronic cytotoxicity and acute genotoxicity of EPA priority DBPs.

This work integrates the analytical chemistry and analytical biology of DBPs into a comprehensive structure activity relationship among major DBP chemical classes.

Emerging DBPs, especially iodinated and nitrogen-containing agents, are more cytotoxic and generally induce a greater level of genomic DNA damage in mammalian cells than currently regulated DBPs.

APPROACH:

Quantitative biological assays used cultured Chinese hamster ovary (CHO) cells. The analytical methods primarily employed two quantitative biological assays. The CHO cell chronic cytotoxicity assay measures the reduction in cell density as a function of DBP concentration over a period of approximately 3 cell divisions. Single cell gel electrophoresis (SCGE) is a molecular genetic assay that quantitatively measures the level of genomic DNA damage induced in individual nuclei of treated cells. Published statistical analyses were used to evaluate the data for each agent. Cytotoxicity and genotoxicity indices were generated to compare the responses among different chemical classes of DBPs.

RESULTS/FINDINGS:

Based on the data generated in this study, the following conclusions were reached:

A comparative mammalian cell toxicity database was prepared that encompassed 66 DBPs and related chemicals.

The CHO cell chronic cytotoxicity of the DBPs encompassed concentrations over 4 log orders of magnitude with diiodoacetamide the most cytotoxic agent and bromodichloromethane the least cytotoxic.

For CHO cell cytotoxicity, the rank order from most cytotoxic to least cytotoxic for the DBP classes was haloacetaldehydes > haloacetamides > halonitromethanes > haloacetonitriles > >2C-haloacids > haloacetic acids > halomethanes.

The CHO cell genotoxicity of the DBPs encompassed concentrations over 3 orders of magnitude. A majority (75.8 percent) induced significant levels of genomic DNA damage. In this group, iodoacetic acid was the most genotoxic. The least genotoxic was chlorodibromoacetic acid.

For induced genomic DNA damage in CHO cells, the rank order from the most genotoxic to the least genotoxic of the DBP classes was haloacetonitriles > haloacetamides > halonitromethanes > haloacetaldehydes > haloacetic acids > >2C-haloacids > halomethanes.

Iodinated DBPs were more cytotoxic and genotoxic than their brominated and chlorinated analogues.

In general, nitrogen-containing DBPs were more toxic than DBPs that did not contain nitrogen.

IMPACT:

The DBPs chosen for analysis were partially based upon data from the USEPA Nationwide Occurrence Study. Mammalian cell cytotoxicity and genotoxicity data provided a rank ordering of the relational toxicities of regulated and emerging DBPs and related agents both within an individual chemical class and among classes. Alternative disinfectants generate new DBP compounds and alter the distribution of DBP chemical classes. The water supply community will be able to consider these factors when employing alternatives to chlorine disinfection. In addition, these data will be available to prioritize DBPs for future in vivo toxicological studies and risk assessment.

PARTICIPANTS:

The research team received support from the U.S. Environmental Protection Agency, the Illinois-Indiana Sea Grant, and the Center of Advanced Materials for the Purification of Water with Systems (a National Science Foundation Science and Technology Center).