MUTAGENIC POTENTIAL OF WATERS FROM THE TUZ LAKE, TURKEY

1. MUTAGENIC POTENTIAL OF WATERS FROM THE TUZ LAKE, TURKEY Mutlu M.B., Guven K., Kutlu M. and Aydogan G. Anadolu University, Faculty of Science, Department of Biology, 26470, Eskişehir, TURKEY Summary Themutagenic potential of water samples from the Tuz Lake-Turkey was examined by applying Salmonella mutagenicity assay (Ames test) using TA 98 and TA 100 strains without metabolic activation. XAD 4 and XAD 16 column extracts of the samples were used in plate incorporation assay. The results obtained were negative in both TA 98 and TA 100.Although the number of revertants of XAD 4 extracts in one station and XAD 16 extracts in another station were higher than the revertants that of other stations for TA100 strain when compared to the solvent control, The difference was also not significant to be considered as mutagenic. The results were discussed for the future analysis. Keywords: Mutagenicity, Tuz Lake, XAD 4, XAD 16, Salmonella typhimurium Introduction Tuz Lake is a hypersaline lake with a salinity of 33% NaCl, located120 km to the south of Ankara, on the way to Cappadocia (38o 48o N-33o 36o E) and comprises 1665 km2 within a closed basin. It is the second largest lake of Turkey. Its lenght and width are respectively 90 km and 35 km. Water flows into the Tuz Lake through Melendiz Stream and drainage channels of Konya plateau. In summer, the lake dries out and a 30 cm layer of salt forms because of the evaporation. Under layer of the salt is mud. In winter, water is not more than 2 m height. It is obtained thousand tons of salt per year which is the 60% of the total salt production in Turkey (Uygun 1978, Çamur 1995). The salt mining generates industrial activity in the region, mainly related to salt processing and refining. The quality of the water in the Tuz Lake Basin, the biggest Tuz Lake in Turkey, has been deteriorated over the last years motivated by intensive agriculture, discharge of untreated wastewater and uncontrolled dumping of solid waste (both urban and sludge from the salt mining industry) (Gorostiaga, 2003). Chemical compounds of urban-industrial origin provide a significant contribution to the contamination of river and lake water, resulting in a potential ecotoxicological risk. Organic micropollutants are particularly one of the most critical mutagenic agents in the aquatic environment. Many studies of surface waters using different approaches for mutagenicity evaluation have reported that, mutagenicity on bacteria can be recommended for initial simple and effective screening in a monitoring programme (Filipic and Toman, 1996; Vargas et al.,2001). The continuing popularity of Salmonella mutagenicity assay (Ames test) depends on the ability of prescreening a large number of chemicals for the identification of genotoxic hazards and the elucidation of the biochemical mechanisms of mutagenesis (Josephy et al., 1997, Mamber et al., 1993). In addition, the test is used for its high sensitivity and recognized validity (Kaplan et al., 2004). In the present study, an initial mutagenicity screening of four different water samples collected from the Tuz Lake was performed. Salmonella mutagenicity assay was applied by using two strains of Salmonella typhimurium with frameshift mutation (TA 98) and base-pair mutation (TA 100). Plate incorporation assay was applied in the absence of metabolic activation. XAD 4 and XAD 16 columns were used for the fractionation of the water samples. RESULTS Table 1. pH and salt concentrations of the water samples from Tuz Lake. sample pH salt concentration (%) A 7.5 36 B 8.0 36 C 7.8 35 D 7.5 35 Table 2. Mutagenicity analysis of XAD 4 resin concentrated water samples from the the Tuz Lake in the absence of metabolic activation MATERIALS AND METHODS Sampling Area DISCUSSION Since the Tuz Lake is placed in a closed basin, it is the final recipient of all pollution. The natural hydrological regime has been altered severely by the construction of dams for irrigation (Cihanbeyli and Uluırmak), extraction of groundwater for irrigation and the construction of an inter-basin channel that conveys drainage water and untreated wastewater from the Konya plain to the Tuz Lake (Gorostiaga, 2003) The Ames test is based on a set of Salmonella typhimurium strains that revert to histidine independence upon exposure to mutagens. TA 98 strain is susceptible for the mutagens causing frame-shift mutations and TA 100 strain is susceptible for the mutagens causing base-pair substitution mutations. The potential for contamination by toxic substances in Tuz Lake is investigated by Salmonella mutagenicity assay. It is carried out by the preincubation method with Salmonella typhimurium TA 98 and 100 strains without metabolic activation (S9 mix). XAD 4 and XAD 16 resin columns were used to fractionate the water samples. XAD 4 resin traps a wide range of spectra of soluble organic substances in water while XAD 16 resin traps solutes of higher molecular weights (Junk et al., 1974, Guzzella et al., 2002). According to the results of the present study, waters from the four stations of Tuz Lake have not shown any mutagenic activity toward both TA 98 and TA 100 strains of Salmonella typhimurium. Number of revertants of XAD 4 extract in station D were higher than the revertants in the other stations for TA100 strain when compared to the solvent control. Number of revertants was also higher for TA 100 strain in station C. But it was not a significant difference to be considered as mutagenic. However this may be the result of contamination intensity in those stations because of the settlement and the contaminated river water draining to the lake. It should be considered that the Ames test is a prescreening method for the analysis of possible mutagenic substances in environmental samples. Further analysis of the water samples from Tuz Lake should be carried out as it is the primary salt source of Turkey. Figure 1.- Geographical locations of the Sampling sites in Tuz Lake Sample collection Samples were taken from four different locations (A, B, C and D) of the Tuz Lake in July 2005 (Figure 1). The total salt concentration of each sample was determined in situ with a hand refractometer (Eclipse) and pH values were determined by using a pH meter (TOA WQC). Extraction of Water Samples Dissolved organics were extracted by absorption of 2.5 liters of water sample separately on XAD 4 and XAD 16 resins (Aldrich) packed in a glass column (15 mm x 150 mm). After removing residual water, the organic matter in the column was eluted with consecutive application of 300 ml each of dichloromethane (CH2Cl2), acetone (CH3COCH3), diethylether ((C2H5)2O) and methanol (CH3 OH) into the column (rate of 5 ml/min) (Junk et al., 1974, Nakamuro et al., 1992). Each fraction was evaporated to dryness with a rotary evaporator in a water bath at about 40 oC and these concentrates were dissolved in 50 ml dimethylsulfoxide (DMSO) (50 times concentrated of the original sample) for the mutagenicity assay (Ohe and Haruo, 1996). Mutagenicity Testing Mutagenicity was tested by the plate incorporation assay with and without metabolic activation (Maron and Ames, 1983) using tester strains TA 98 and TA 100 kindly provided by Professor B.N. Ames (Berkeley, CA, USA). Samples are considered to be cytotoxic when the percentage of sample survival is less than 60 % of that observed for negative control (Vargas et al., 2001). In the standard plate incorporation assay, 0.1 ml of the fresh overnight culture of the tester strain and 0.1 ml of the extract to be tested were added into the capped culture tubes in 45 oC water bath containing 2 ml of soft agar and 0.2 ml of histidine/biotine solution. The test components were mixed by vortexing the tube for about 3 s at low speed and directly poured onto a minimal glucose agar plate. The plates were inverted and incubated at 37 oC for 48 h (Maron and Ames, 1983). Negative (sterile distilled water or dimethylsulfoxide, DMSO, spectrophotometric grade) and positive (1.5 g sodium azide, AZS, 20 g 4 nitro-o-phenylenediamine, NPD, per plate for strains TA 100 and TA 98 without S9 mix and 1 g 2 AF per plate for both of the two strains) controls were included in each assay. Three plates for three separate experiments were carried out for determining possible mutagens tested alongside positive and negative controls.(Mortelmans and Zeiger 2000). REFERENCES ÇAMUR, M.Z., MUTLU, H. (1995) Tuz Gölündeki mineral çökeliminin termodinamik değerlendirimi. Geological Bulletin of Turkey 38, 67-73. FILIPIC, M., TOMAN, M.J. (1996) Ecotoxicological studies using modified ames bioassay. Wat. Sci. Tech. 34, 1-7. GOROSTIAGA, A. (2003) Integrated feasibilty study for the sanitation of the Tuz Lake Basin- Turkey. Enviroment 2003 Conference. September 30-October 2 Nasr, Egypt. GUZZELLA, L., FERRETTI, D., MONARCA, S. (2002) Advanced oxidation and adsorption Technologies for organic micropollutant removal lake water used as drinking water supply. Water Research 36(17), 4307-18. JUNK, G.A., RICHARD, J.J., GREISER, M.D., WITIAK, D., WITIAK, J.L., ARGUELLO, M.D., VICK, R., SVEC, J., FRITZ, J.S., CALDER, G.V. (1974). Use of macroreticular resins in the analysis of water for trace organic contaminants. Journal of Cromatography 99, 745-762. KAPLAN, Ç., DİRİL, N., ŞAHİN, S., CEHRELİ, M.C. (2004) Mutagenic potentials of dental cements as detected by the Salmonella / microsome test. Biomaterials 25, 4019-4027. MARON, D., AMES, B.N. (1983) Revised methods for Salmonella Mutagenicity test. Mutation Research 113, 173-215. MORTELMANS, K., ZEIGER, E. (2000) The Ames Salmonella/ microsome mutagenicity assay. Mutation Research 455 29-60. NAKAMURO, K., UENO, H., SAYATO, Y. (1992) Evaluation of mutagenicity of municipal river water concentrated using XAD resin column method.. Wat.Sci.Tech. 25, 293-299. OHE, T., NUKAYA, H. (1996) Genotoxic activity of 1-nitropyrene in water from the Yodo River Japan. The science of Total Environment 181, 7-12. PARK, J.H., LEE, B.J., LEE, S.K., KIM, K., LEE, K.H., CHE, J.H., KANG, K.S., LEE.Y.S., (2000) Genotoxicity of drinking water from three Korean cities. Mutation Research 466, 173-178. UYGUN, A., ŞEN, E. (1978) Tuz Gölü havzası ve doğal kaynakları. Geological Bulletin of Turkey 21, 113-120. VARGAS, V.M.F., MIGLIAVACCA, S.B., MELO, A.C., HORN, R.C., GUIDOBONO, R.H., SÁ FERREIRA, I.C.F., PESTANA, M.H.D. (2001). Genotoxicity assesment in aquatic environments under the influence of heavy metals and organic contaminants. Mutation Research 490, 141-158. Evaluation of Results A sample is considered to be mutagenic if it gives a significant response more than the control incidence (Hrelia et al., 1998; Sarrif et al., 1997; Suter et al., 2002). Student-t test was used for the comparisons with solvent control. Results are considered to be statistically significant at P ≤ 0.05.