TLC Fractionation and Characterization of Ames Mutagenic Substances in Chlorine-treated 4-methylphenol Solution in the Presence of Bromide Ion

Abstract

The diethyl ether extracts from aqueous 4-methylphenol solutions in the presence of bromide ion after treatment with chlorine were mutagenic to the Ames salmonella test strain TA100 in the absence of rat liver homogenate. Gas chromatography/mass spectrometry (GC/MS) showed the occurrence of halogenated products in the extracts: bromo-4methylphenols, bromo-4-methylquinones and brominated 4-methylphenol dimers. The diethyl ether extracts were fractionated into several fractions by polyamide thin-layer chromatography (TLC). The fractionated components were examined for mutagenicity by means of Ames assays, and were identified by GC/MS. TLC fraction of the extracts revealed that two components present in the extracts are mutagenic. GC/MS analysis indicated the presence of 2,6-dibromo-4-hydroxy-4-methylcyclohexa-2,5-lien-1-one (2,6-dibromotolquinol) as the major mutagen and brominated 4-methylphenol dimers as minor mutagenic compounds in the chlorinated 4-methylphenol solutions containing bromide ion. Production of 2,6-dibromotolquinol in water was found to be dependent on the equivalents of chlorine per mole of compound and the reaction pH.

Characterization of Polyhalogenated 4-Methylphenol Dimers (Br/Cl-Predioxins) Formed during Aqueous Chlorination of 4-Methylphenol Solution in the Presence of Bromide Ion

“…The GC/MS analysis also suggests that MMPPs are present in the chlorine-treated 4-methylphenol solution. Furthermore, these results suggest that the chlorine treatment of water contaminated with 4-methylphenol and bromide ion leads to the formation of Brpredioxins, which have been shown to be mutagenic, 8) and are also precursor of the highly toxic PBDDs. 10) Figure 4 shows a typical mass chromatogram (total ion current) of the unknown compounds occurring in the polyamide TLC fraction of a diethyl ether extract from a chlorinated alkaline 4-methylphenol solution in the presence of bromide ion, and the mass spectra of several peaks.…”

“…3 + ) in the mass spectrum, this compound is considered to be dimethylated 4-phenoxyphenol (DM-4-PP). [4][5][6][7][8] The compounds corresponding to the peaks 2 and 3 in Fig. 3 yielded the same molecular ion at m/z 292 (C 14 H 13 BrO 2 + ) and several fragment ion.…”

“…18,19) Our previous papers [4][5][6][7][8] have also demonstrated that these PCPPs are formed in the reactions of phenolic compounds with chlorine in aqueous solutions. In addition, several researchers have demonstrated that chlorination or ozonation of surface and ground water containing bromide ion produces high concentrations of brominated trihalomethanes (THMs), 12,13) according to the following reactions:…”

“…4 were interpreted to be Cl-DMPP for the peak 1, Cl 2 -DMPP isomers for the peaks 2 and 3, Cl 3 -DMPP isomers for the peaks 8 and 9, and Cl 4 -DMPP for the peak 14, respectively, as compared with those reported in the previous papers. [4][5][6][7][8] The compound corresponding to the peak 9 in Fig. 4 yielded a characteristic fragment ion at m/z 160 (C 7 H 6 Cl 2 + ), suggesting this compound to be a 2-phenoxyphenol.…”

“…2,3) This hypothesis has been directly supported by our previous findings that demonstrated that phenolic compounds react with chlorine to form the condensation products of their chlorinated compounds. [4][5][6][7][8] Recently, polybrominated diphenyl ethers (PBDEs) and dibenzo-p-dioxins (PBDDs) have attracted attention because the presence of these compounds in human milk has been shown. 9) Further, they exhibit high toxicity in the early life stages of fish.…”