Contribution of Organic Bromines to the Genotoxicity of Chlorinated

Downloaded by NANYANG TECHNOLOGICAL UNIV on August 24, 2015 | http://pubs.acs.org Publication Date: August 5, 2008 | doi: 10.1021/bk-2008-0995.ch005

Chapter 5

Contribution of Organic Bromines to the Genotoxicity of Chlorinated Water: A Combination of Chromosomal Aberration Test and Total Organic Bromine Analysis 1

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Shinya Echigo , Sadahiko Itoh , and Ryo Ando

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Department of Urban Management, Graduate School of Engineering, Kyoto University, Nishikyo, Kyoto 617-8540, Japan Sumitomo Heavy Industries, Ltd., 9-11, Kita-Shinagawa 5-chome, Shinagawa-ku, Tokyo 141-8686, Japan 2

The contribution of organic bromines to the genotoxicity of a chlorinated lake water was evaluated by chromosomal aberration test at various chlorine doses and bromide concentrations. The number of chromosomal aberrations increased as a function of initial bromide ion concentration, indicating that organic bromines are major contributors to the genotoxicity. This study highlights the importance of monitoring and controlling brominated disinfection by-products.

© 2008 American Chemical Society

In Disinfection By-Products in Drinking Water; Karanfil, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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66 Bromide ion is a trace but ubiquitous anion in aquatic environment. Its origin in source waters can be both natural (salt water intrusion and dissolution from geological formation) and anthropogenic (/). Although bromide ion is quite stable in natural water, it can be involved in various chemical reactions once it enters a drinking water treatment process. Ozone (0 ) and hypochlorous acid (HOC1) used in drinking water treatment easily oxidize bromide ion to hypobromous acid (HOBr) (2). This intermediate, HOBr, rapidly reacts with natural organic matter (NOM) in source water to form organic bromines both during chlorination and ozonation (3). While the toxicity of bromide ion itself is considered negligible, some of these organic bromines are known to be toxic. Several recent studies have suggested the toxicological importance of brominated DBPs. For example, Plewa et al has shown that bromoacetic acids are more mutagenic than chloroacetic acids with an in vitro bioassay using mammalian cells (4). In particular, monobromoacetic acid is much more mutagenic than other chloro- or bromoacetic acids (4). In addition, brominated nitromethanes and brominated nitriles are more cytotoxic and genotoxic than their chlorinated analogues (5, 6) Also, Nobukawa and Sanukida (7) showed that the mutagenicity of chlorinated water increased with increasing bromide ion concentration using Ames test. Kargalioglu et al demonstrated that the rank order of the mutagenic potency in Salmonella typhimurium for the haloacetic acids was bromoacetic > dibromoacetic > chloroacetic > dichloroacetic acids (8). Further more, it is known that the products of the reaction between hypobromous acid and humic acid are several times more genotoxic than the ones between hypochlorous acid and humic acid on TOX basis (9). These results indicated that brominated compounds formed during chlorination could be major contributors to the toxicity of drinking water. Also, the importance of bromination during chlorination has been highlighted by kinetic studies on the reactions between HOBr and organic compounds. HOBr is much more reactive to phenolic compounds, one of the most reactive components in NOM, than HOC1 (10, 11). Westerhoff et al. observed the same trend for NOM isolates from a real source water: HOBr was more efficient at halogen substitution than HOC1 (12). Therefore, bromination of NOM by HOBr during chlorination should not be neglected even if bromide ion concentration is much lower than HOC1 dose. Given the toxicological and kinetic importance of bromination during drinking water treatment, it is of practical importance to quantitatively identify raw water characteristics and treatment conditions under which the contribution of brominated DBPs to the toxicity of drinking water is not negligible. However, the information on the relationship between chlorination condition (e.g., bromide ion concentration and chlorine dose) and the toxicity of chlorination by-products to mammalian cells in the presence of bromide ion is still very limited. We have reported a preliminary result on the effect of bromide ion on the toxicity of chlorinated water (13), but the chlorine dose was fixed in


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this evaluation. This is not sufficient to fully evaluate the importance of controlling brominated DBPs. The present study sheds the light on this problem. More specifically, the relationship between the activity inducing chromosomal aberrations and the formation characteristics of total organic bromines (TOBr) from the chlorination of a real source water was explored with varying bromide ion concentration and chlorine dose. For this evaluation, we employed the combination of chromosomal aberration test and the differentiation technique of between TOBr and total organic chlorine (TOC1) (14).

Materials and Methods Materials Chemicals All the chemical reagents used in this study were of reagent grade or better (mostly analytical grade), and were purchased from Wako pure chemical (Japan). All the aqueous solutions were prepared with ultra pure water treated by a Millipore Elix20 system.

Raw Water The raw water used in this study was collected from Lake Biwa, Japan on September 15,2004. This lake is the largest lake in Japan, and serves 14 million people as water source. The total sample volume was approximately 320 L. The ambient bromide concentration of the lake water was 50 μ&^. Also, DOC, pH, UV absorbance at 260 nm (E ), and the chlorine demand for 24 hours were 1.6 mg/L, 7.6, 0.03 absorbance unit (AU)/cm and 1.71 mg Cl /L, respectively. The water was filtered through 0.45 μπι membrane (ADVANTEC) to removal particulate matters right after collection, and chlorinated on the same day. 260

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Chlorination and Sample Concentration Chlorination of the Lake Biwa water was performed at five different bromide ion concentrations (50, 120,240,400, and 600 μg/L) and three different chorine doses (C1 /DOC=1.0, 1.5, and 2.0 mg Cl /L). Hence, 15 samples were prepared for chromosomal aberration test and TOBr analysis. The samples were chlorinated in the dark at 20 C for 24 hours. The chlorinated samples were 2

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68 concentrated by solid phase extraction before chromosomal aberration test in the following procedure. After pH adjustment to 2 with HC1, each sample (20 L) was passed through a pair of two Sep-Pak® Plus (Long) CSP-800 solid phase extraction cartridges (Waters) in series at a flow rate of 50 mL/min. The materials extracted onto the solid phase were eluted with dimethylsulfoxide (DMSO) at a flow rate of 0.2 mL/min. The final volume of the extract was 2 mL. That is, the concentration factor of this process was 10 . Downloaded by NANYANG TECHNOLOGICAL UNIV on August 24, 2015 | http://pubs.acs.org Publication Date: August 5, 2008 | doi: 10.1021/bk-2008-0995.ch005

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Chromosomal Aberration Test Chromosomal aberration test using Chinese hamster lung cells (CHL/IU, Dainihon Pharmaceutical) was performed to evaluate the initiating activity in the carcinogenesis process of the products produced from the chlorination of the Lake Biwa water under various chlorination conditions. The detail of the test procedure is described elsewhere (15). The dose of the extract to the cell culture media was fixed to 30 μ ί , and the sample-to-media ratio was 0.5% (v/v). The incubation time after contacting with sample solutions was 24 hours. For each sample, 50 metaphases were analyzed. The number of chromosomal aberrations was counted by visual observation under microscope.

Analytical Methods Differentiation of TOBr and TOO The detail of the procedure is described elsewhere (14). But, briefly, the off gas from a TOX furnace that contains HC1 and HBr corresponding to TOC1 and TOBr was trapped into distilled water, and the corresponding chloride and bromide ions were separately quantified by ion chromatography (see the next paragraph for detail). A TOX analyzer (ΤΟΧ-10Σ, Mitsubishi Chemical) was used as a furnace. Anion Concentrations Bromide and chloride concentrations were determined by ion chromatography (LC-VP, Shimadzu) with a Shim-pack IC-A3 analytical column (Shimadzu) protected by a Shim-pack IC-GA3 guard column (Shimadzu). The mobile phase was 50 mM of boric acid/ 8 mM of p-hydroxybenzoic acid/ 3.2 mM bistris.


69 Other Water Quality Parameters For DOC and UV absorbance measurements, a TOC analyzer (TOC-5000A, Shimadzu) and a UV/Visible spectrophotometer (Multispec-1500, Shimadzu) were used respectively. Also, solution pH was measured by a D-51 pH meter (Horiba).


Results and Discussion TOBr and TOCI Formation The effects of bromide ion concentration and chlorine dose on TOCI and TOBr are shown in Figures 1 and 2, respectively. While TOCI was slightly higher at C1 /TOC=2.0 mg/mg than other conditions, no distinct difference in TOCI was observed with respect to chlorine dose. Similarly, TOBr was almost independent of chlorine dose in the range of Cl /TOC= 1.0-2.0 mg/mg. On the other hand, bromide ion concentration had strong impact on both TOCI and TOBr. TOBr increased with increasing bromide ion, but only slightly for a bromide ion concentration higher than 200 μg/L. At the same time, TOCI decreased with increasing bromide ion concentration. TOX (=TOBr+TOCl) decreased slightly, but was not as sensitive to bromide level as TOCI and TOBr (Figure 3). The reason for these constant TOX values with different chlorine dose is not clear at this point, but one should note that the chlorine doses used in this study were approximately at or above the chlorine demand. Thus, almost all the reaction sites in NOM may have been used up even at the lowest chlorine dose, and no further halogenation occurred with additional chlorine. The reason for the constant TOBr values with different chlorine doses seems even more profound. However, this result makes sense if we assume that bromination is much faster than chlorination. That is, if we assume that bromination was so fast that bromination was completed before a competitive situation between chlorination and bromination occurs, it is reasonable to observe a constant TOBr level regardless HOC1 concentration. These assumptions are supported by the high conversion of bromide ion to organic bromines (Figure 4) and higher TOBr-to-TOX ratios than corresponding initial bromide-to-chlorine ratios (Figure 5). 2

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Chromosomal Aberration Test Figure 6 shows the result of chromosomal aberration test. The trend was similar to that of TOBr. That is, the activity inducing chromosomal aberrations


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100 200 300 400 500 600 700 Bromide concentration (pg/L)

Figure 1. Effect of bromide concentration and chlorine dose on TOOformation.

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350

~θ— CI /TOC=1.0 mg/mg

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CI /TOC=1.5 mg/mg

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- B — CI /TOC=2.0 mg/mg 2

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100 50 0 100 200 300 400 500 600 700 Bromide concentration (pg/L)

Figure 2. Effect of bromide concentration and chlorine dose on TOBr formation.



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200

Ο 150 100 50 0

-θ— CI /TOC=1.0 mg/mg 2

- a — CI /TOC=1.5 mg/mg 2

-B— Ci /TOC=2.0 mg/mg 2

100 200 300 400 500 600 700 Bromide concentration (pg/L)

Figure 3. Effect of bromide concentration and chlorine dose on TOX formation.

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Contribution of Organic Bromines to the Genotoxicity of Chlorinated

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