Use of Three Genotoxicity Tests to Evaluate the Genotoxic Activity of Organohalides, Chlorinated Fulvic Acids and Unconcentrated Water Samples Collected from a Drinking Water Treatment Plant
Since the identification of organohalides in drinking water in 1974, several investigators have detected genotoxic activity in drinking water concentrates. It is now widely admitted that the observed genotoxicity originates mainly from the reaction of chlorine on natural organic matter contained in the raw water, which leads to the formation of organohalogenated compounds. The aim of this study is to show the benefit of three short-term assays for the evaluation of the genotoxic potency of organohalogenated compounds and of complex mixtures. In a wider context, the purpose is to identify a test or a battery of tests that can contribute to the control of natural and drinking water genotoxicity. The three genotoxicity assays carried out during this work were: - the SOS chromotest, a primary DNA damage in vitro assay on Escherichia coli; - the Ames- fluctuation test, a point mutation in vitro assay on Salmonella typhimurium; - and the newt micronucleus test, a chromosomal aberration in vivo assay on the amphibian Pleurodeles waltl. These assays display a valuable advantage: the water samples under study can be analyzed without concentration prior to testing. Thus, the different concentration procedures, which may modify the original genotoxicity of the water samples, are avoided. A previous study on seven reference genotoxic chemicals had indicated that the SOS chromotest was never the most sensitive of the three tests (for a given chemical, the most sensitive assay is defined as the test which detects the lowest concentration inducing a significant genotoxic effect). On the contrary, the Ames-fluctuation test proved to be the most sensitive for compounds showing direct genotoxic activity, and the newt micronucleus test the most sensitive for chemicals with indirect genotoxic effects. None of the assays was the most sensitive for every substance analyzed. These observations suggested the need to implement a battery of tests using several cell types, different metabolization systems and detecting several genotoxicity events. This earlier study also showed, in accord with several results in the literature, that the Ames-fluctuation test (in liquid medium) demonstrated a better sensitivity than the Ames test (in agar solid medium). The first part of the present study involved testing the genotoxicity of 14 organo- halogenated compounds identified in drinking water samples or in chlorinated humic matter samples. The chemicals studied were four chlorinated and/or brominated trihalomethanes (trichloro-, bromodichloro-, chlorodibromo- and tribromomethane), five chlorinated or brominated acetonitriles with one, two or three halogens (monochloro-, dichloro-, trichloro-, monobromo- and dibromoacetonitrile) and five chlorinated propanones with one, two or three substitutions on one or two carbon atoms (monochloro-, 1,1-dichloro-, 1,3-dichloro-, 1,1,1-trichloro- and 1,1,3- trichloropropanone). Although the SOS chromotest was the most sensitive for 3 of the 14 substances analyzed, the results confirmed that this test was globally the least sensitive; the Ames-fluctuation test and the newt micronucleus test remained the most efficient assays. It is interesting to note that the Ames-fluctuation test appeared the most sensitive for all the chloropropanones tested and the newt micronucleus test, for all the haloacetonitriles analyzed. Moreover, several structure-activity relationships were demonstrated: the nature of the halogenated substituents (bromine or chlorine), the number and, above all, the position of chlorine atoms strongly influenced the genotoxicity of the organohalides studied. In the second part of the work we analyzed the effects of complex mixtures containing several organohalogenated compounds: the three tests were performed on two chlorinated fulvic acids of different origin. Pornic fulvic acid was extracted from a surface water reservoir used to produce drinking water in Vendée (France) and Pinail fulvic acid came from a forest pond near Poitiers (France). The total organic carbon was about 1 g/l in the solution subjected to chlorination and the molar chlorination ratio was 1.5 Cl2/C. The results showed the advantage of tests using bacteria: the Ames fluctuation test was the only assay able to detect the genotoxicity of both chlorinated fulvic acids; the SOS chromotest detected the genotoxic effect of only one of the chlorinated fulvic acids (Pinail). In contrast, the newt micronucleus test did not show any genotoxicity of the chlorinated fulvic acids. However, it must be pointed out that, as insufficient fulvic acid was available, the genotoxic potency of these solutions on the newt was not tested under adequate conditions (e.g., subchronic concentrations were not studied). Nevertheless, the concentrations of fulvic acid analyzed were very close to those found in the aquatic environment. The last part of the study attempted to approximate environmental and human exposure conditions: the three tests were performed on four water samples taken at several stages of a drinking water treatment plant. These samples were analyzed for genotoxicity in the three test systems without preconcentration. The plant studied is characterized by the following treatment steps: - coagulation-flocculation; - chlorination at 6 g Cl2/m3; - sand filtration; - ozonation at 1.9 g O3/m3; - final chlorination at 1.4 g Cl2/m3 before sending the treated water into the distribution system. The four samples were taken: 1. before any treatment (raw water, 10 to 13 mg total organic carbon per liter), 2. between the sand layer filter and the ozonator (chlorinated water), 3. after the ozonator (ozonated water), 4. and at the end of the treatment process (treated water). The results obtained confirmed the advantage of the Ames fluctuation test, which was the only assay able to detect a genotoxic activity in the unconcentrated water samples studied. Regarding the influence of the different chemical treatments on the mutagenicity observed, it was demonstrated that the first chlorination step led to the formation of direct-acting mutagens. The treatment with ozone, at the rate used, did not significantly modify the mutagenicity of the samples that had previously been chlorinated. Similarly, the second chlorination step did not significantly increase the direct mutagenicity detected. Practically, our study indicated that the Ames-fluctuation test is the only assay, among the three performed that is able to contribute efficiently to the control of drinking water genotoxicity. In this context, the benefit of the SOS chromotest appears only in case of accidental pollution: indeed, it is the only test able to yield results within 24 hours. The implementation of the newt micronucleus test could be useful for the control of natural or drinking water genotoxicity in case of extensive pollution.