| The disinfection of drinking water can effectively control the occurrence of waterborne infection disease, however at the same time generating a series of disinfection by-products(DBPs) with carcinogenic risk that pose a potential threat to water quality safety. Conventional physical and chemical tests can not fully reflect the overall pollution situation of drinking water and the health risk of human exposure, biological toxicity monitoring is a supplement for physical and chemical tests. Caenorhabditis elegans(C. elegans) has been widely used to evaluate the toxicity of chemicals and surface water due to its features of small body size, short life cycle, ease of maintenance, low costs and, high homology with human genes and so on. However to date, applications of C. elegans in studying DBPs are extremely limited, no reports have been reported for the detection of biological toxicity of drinking water. Therefore, it is of great significance to explore the potential for evaluating the toxicity of typical DBPs and drinking water samples using C. elegans as a model animal.Objective:The present study was to investigate the lethal toxicity and genotoxicity of six typical DBPs and drinking water samples(raw water, finished water and tap water) collected from four mainly water treatment plant in Wuhan, and finally to explore the feasibility of the two bioassays of C. elegans for evaluating the toxicity of DBPs and drinking water.Methods:1. The lethal toxicity and genotoxicity of six DBPs were detected using C. elegans. Six DBPs included dichloroacetic acid(DCA), trichloroacetic acid(TCA), monobromoacetic acid(MBA), dibromoacetic acid(DBA), N-nitrosodimethylamine(NDMA) and 2,6-dichloro-1,4-benzoquinone(DCBQ). For the detection of lethal toxicity, C. elegans L4 larve were exposed to six DBPs at five to seven concentrations ranged from 125 to 50000 μM for 24 h with solvent control. Then the numbers of live and dead worms were then determined and LC50 were calculated. The genotoxicity for six DBPs were detected by optimized long amplificon quantitative PCR(LA-QPCR) assay. The optimizing parameters of LA-QPCR assay mainly included the methods to break the cuticle of C. elegans, the amount of template DNA, and the cycle number. For the detection of genotoxicity, C. elegans L2 larve were exposed to six DBPs at five concentrations ranged from 100 to 25000 μM for 24 h with solvent control. Then the DNA lesions were detected by LA-QPCR.2. Raw water, finished water and tap water were collected from four mainly water treatment plant in Wuhan in August, 2015. Some important physical and chemical indexes(temperature, pH value, residual chlorine, total organic carbon, total dissolved solids, turbidity, permanganate index, ammonia nitrogen, total phosphorus) were detected. Each water sample were filtered with membrane filter to obtain filtered samples. The lethal toxicity and genotoxicity of drinking water samples and filtered samples were detected by using C. elegans lethal toxicity test and LA-QPCR assay. The toxicity results from raw water, finished water, and tap water were compared, and the water samples toxicity were compared with their filtered samples, and the correlation between the genotoxic outcomes and the physical and chemical results were analyzed.Results:1. Results indicated that MBA, DBA, DCA, TCA and DCBQ increased lethality of C. elegans in a concentration-dependent manner, and survival of C. elegans was not affected after 24 h of exposure to NDMA(50 mM, the highest concentration used). The 24 h LC50 value for six DBPs were 327.81 μM(standard deviation, 13.64 μM) for DCBQ, 770.99 μM(standard deviation, 230.19 μM) for MBA, 1217.80 μM(standard deviation, 65.41 μM) for DBA, 1430.52 μM(standard deviation, 117.29 μM) for TCA, 1716.86 μM(standard deviation, 50.31 μM) for DCA, greater than 50 mM for NDMA. The lethal toxicity order was DCBQ > MBA > DBA > TCA > DCA > NDMA. DCBQ exhibits greater lethality than others.2. The optimization of LA-QPCR assay showed that breaking the cuticle of C. elegans by grinding worms in liquied nitrogen, 15 ng DNA template and 23 cycles for long target fragment PCR and 22 cycles for small target fragment PCR were suited for our study.3. LA-QPCR assay indicated that DCBQ exposure resulted in a concentration-dependent DNA damage in C. elegans at the concentrations of 300, 400 and 500 μM. Statistically significant increases in DNA damage were observed after exposure to 25 mM NDMA. Exposure to MBA, DBA, DCA, or TCA produced no detectable DNA damage in C. elegans. DCBQ performed more genotoxic than other five tested DBPs in C. elegans.4. All raw water, finished water and tap water showed no lethal toxicity and genotoxicity in C. elegans. There was no significant difference between water samples and their filtered samples.5. The correlation had not been found between the genotoxic outcomes and the physical and chemical results.Conclusion:1. The C. elegans lethal toxicity test could be used to evaluate the general toxicity of DBPs; the C. elegans LA-QPCR assay were feasible to part of DBPs.2. Further data are needed to determine whether the C. elegans lethal toxicity test and LA-QPCR assay could be applied into the drinking water biotoxicity monitoring.3. The lethal toxicity and genotoxicity of DCBQ appled to C. elegans may be higher than other five which is worth more attention. |
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