Natural Organic Matter and Disinfection By-Products - ACS Publications

Downloaded by NANYANG TECHNOLOGICAL UNIV on August 24, 2015 | http://pubs.acs.org Publication Date: August 15, 2000 | doi: 10.1021/bk-2000-0761.ch002

Chapter 2

A New Assessment of the Cytotoxicity and Genotoxicity of Drinking Water Disinfection By-Products 1

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Yahya Kargalioglu , Brian J. McMillan1, Roger A. Minear , and Michael J. Plewa 1,3

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Departments of Crop Sciences and Civil Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 Corresponding author. 3

The disinfection of drinking water generates cytotoxic and mutagenic compounds. The cytotoxic and mutagenic properties of known disinfec tion by-products (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 6 replicates per concentration and requires approximately 5 h. Data were automatically transferred from a microplate reader to a computer spreadsheet. The DBP concentration that induced 50% 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.

Introduction The use of oxidants and disinfectants in drinking water treatment leads to the formation of mutagenic and potentially carcinogenic oxidation and disinfection by products (DBPs). Natural organic matter (NOM) and bromide ion (Br") serve as the organic and inorganic precursors, respectively, to D B P formation associated with oxidants/disinfectants such as ozone (0 ) and chlorine (Cl ) (/, 2, 3, 4, 5, 6). The use of 0 as a pre-oxidant/pre-disinfectant has become increasingly common, while C l is still 3

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© 2000 American Chemical Society

In Natural Organic Matter and Disinfection By-Products; Barrett, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.


17 widely used as a post-disinfectant. Studies conducted since the 1970s on the safety of drinking water after disinfection have primarily focused on the biological effects of chlorination of water containing N O M (/). In a review on the genotoxic activity of organic chemicals in drinking water, Meier (7) stated that the overwhelming majority of genotoxic agents in drinking water was generated during the chlorination stage of water treatment. In general, epidemiological studies suggest that individuals who consume chlorinated drinking water have a somewhat elevated risk of rectal, kidney and bladder cancer as compared to those who do not drink chlorinated water (8, 9). Using the estimated mutagenic potency of drinking water based on S. typhimurium T A 100, Koivusalo et al. (10) determined that there was a significant exposure-dependent response with the consumption of mutagenic water and the development of kidney and bladder cancer (9). Considering the assembled information on the genotoxic and carcinogenic potency of chlorination by-products it has been suggested that there should be a more judicious use of chlorine in the disinfection of drinking water (7, 7/). In the presence of bromide, brominated trihalomethanes are preferentially produced during chlorine disinfection (4). Water with elevated bromide (1 mg/L) and treated with ozone followed by secondary chlorine or chloramine induced a shift in D B P formation from the chlorinated to the brominated species (6). There is a general opinion that brominated DBPs are more genotoxic than chlorinated DBPs and the toxicity of these brominated D B P products is now attracting increased attention (12). The most prevalent DBPs in drinking water in the United States are the trihalomethanes and haloacetic acids. Although epidemiological studies demonstrated a relationship of DBPs to specific cancers, it is unclear which DBPs pose the greatest risk. The work presented here is part of a larger on-going study to quantitatively compare the cytotoxicity and genotoxicity of DBPs using bacterial and mammalian cell assays. Genetic assays based on mammalian cells are more relevant to the assessment of human risks. The goal is to quantitatively compare and rank order the cytotoxicity and genotoxicity in S. typhimurium and mammalian cells of D B P standards and D B P mixtures from disinfected waters. In this paper a method is described that allows for the quantitative comparison of the cytotoxicity and mutagenicity of DBPs in S. typhimurium. In the future this general approach will be expanded to include rapid, quantitative cytotoxicity and genotoxicity analysis of DBPs with mammalian cells.

Salmonella Microplate Cytotoxicity Assay While the S. typhimurium plate incorporation test is an excellent rapid qualitative mutagenicity assay, it cannot quantitatively determine the cytotoxicity of test agents (13). Cytotoxicity is usually ignored throughout the concentration range of a test agent in the standard Salmonella plate incorporation assay. A cytotoxicity concentration-response curve for a D B P is an indication of its biological response. It is also useful in establishing the concentration range for mutagenicity tests. However, standard methods to measure toxicity are laborious and time consuming. To address these issues a rapid, semi-automatic cytotoxicity assay to quantitatively compare selected DBPs was


18 developed. Log-phase S. typhimurium T A 100 cells previously frozen ( - 8 0 ° C ) in Luria Broth (LB) plus 10% dimethylsulfoxide (DMSO) were thawed and grown in 5 mL L B at 37°C for 2 h while shaking. The cell titer was adjusted to an optical density (OD) at 595 nm of 0.030. Treatment with each DBP standard was conducted in 2-mL glass vials sealed with Teflon in a total volume of 1 mL. Each treatment vial contained 300 μ ί of the titered T A 100 cells, a known concentration of the D B P ( ω

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Bromoacetic Acid (μΜ) Figure 7. Comparison of the linear region of the concentration-response curve for bromoacetic acid using S. typhimurium TA 100 before (O) and after (·) the data were normalizedfor cytotoxicity.


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Acknowledgments This research was funded by A W W A R F grant 554, U . S. Environmental Protection Agency grant R825956-01 and a U I U C Campus Honors Program Summer Research Grant.


References

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Natural Organic Matter and Disinfection By-Products - ACS Publications

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