Optimization Of C.Elegans-Based OCR Assay And Its Feasibility Study In Toxicity Assessment Of Drinking Water Disinfection By-Products

The process of disinfecting drinking water inadvertently leads to the formation of numerous disinfection byproducts?DBPs?. More than 600 DBPs have been reported and new DBPs were continuously identified in drinking water, some known DBPs have a present or potential hazard to human health. So evaluation of toxicity of DBPs is of great significance for managing health risks from drinking water. In basic toxicity studies of DBPs, median lethal concentration(LC₅₀) is a main toxicity parameters indicator while indexes of sublethal toxicity are rarely used. However, human exposure to DBPs presents the characteristics of long-term and low-dose, and thus repeated exposure to sublethal stress is the main risks for human health. Oxygen consumption rate?OCR? is indicative of an organism's metabolic state. Changes in OCR indicated the metabolic response of the aerobic organism to the toxicant, thereby is a more sensitive toxicity indicator than lethality. Respirometric toxicity assays that are based on monitoring the OCR of organisms have high potential for toxicity screening. Scientists have successfully developed a sensitive and high-throughput OCR assay based on quenched fluorescence oxygen sensing and plate reader for toxicity assessment of chemicals and toxicity screening of water samples by using V.fischeri, D.magna, Danio rerio and Caenorhabditis elegans?C. elegans? as model organisms. Because the instruments and reagents used in OCR assay are constantlyoptimized, accordingly assay parameters also need to be optimized to ensure the good performance. Due to its features of small size, short life cycle, low costs and easy to cultivate, C. elegans has been used as a classical model animal to evaluate the toxicity of almost every type of known toxicant. Schouest has employed C. elegans-based OCR assay to evaluate the toxicity of several classical kinds of toxicants including heavy metal ions, aromatic compounds, pesticides, and microcystin by using EC₅₀?median concentration causing 50% reduction in respiration compared with untreated C. elegans? as the toxic endpoint. C. elegans-based OCR assay was demonstrated as a more sensitive, specific, and high-throughput method for toxicity assessment than lethality assay of rodents, and showed great potential on rapid toxicity assessment of chemicals. However, it is still not clear whether C. elegans-based OCR assay can be used to evaluate the toxicity of drinking water DBPs.Objective:?i?To optimize the parameters of C. elegans-based OCR assay used in our laboratory.?ii?By comparing reported toxicity data and results of C. elegans-based OCR assay of five representative regulated DBPs, namely, dichloroacetic acid?DCA?,trichloroacetic acid?TCA?, monobromoacetic acid?MBA?, dibromoacetic acid?DBA?,N-nitrosodimethylamine?NDMA? and a newly identified 2,6-dichloro-1,4-benzoquinone?DCBQ?, the study aimed to evaluate the feasibility of employing C. elegans-based OCR assay for toxicity evaluation of DBPs and to explore the toxicity of DCBQ. The results of this study will lay a foundation for applying this assay to toxicity assessment of newly identified DBPs in drinking water in future.Methods:?i?To optimize the parameters of C. elegans-based OCR assay, by evaluating the performance of fluorescence dynamic curve measured by plate reader under different parameter including developmental stages, number of individual nematodes and final concentrations of DMSO, the optimum parameter could be determined; besides, the method of counting nematodes also was explored.?ii?The lethal effect of five regulated DBPs, and a newly identified DCBQ in C.elegans was investigated by 24 h LC₅₀ assay. C. elegans L4 larvae were exposed to each DBP at 5 to 7 concentrations ranged from 0 to 3000 ?M for 24 h with 0 ?M as positive control, the survival rate of C. elegans for each concentration were measured under a dissecting microscope and then LC₅₀ were determined.?iii?The sublethal respiratory toxicity of aforementioned six DBPs in C.elegans was investigated by C. elegans-based OCR assay. C. elegans young adult were exposed to each DBP at 5 to 7 concentrations ranged from 0 to 400 ?M for 24 h.Fluorescence dynamic curve were measured by plate reader to calculate standard OCR of each C. elegans and then EC₅₀ were determined. Positive control?0 ?M?,blank control and negative control were also tested. OCR is the indicator of sublethal toxicity; as a measure of respiratory toxicity of DBPs.?iiii?Correlation analysis was carried out on the LC₅₀ and EC₅₀ values and the results between lethal assay and C. elegans-based OCR assay were compared; in addition, results of C. elegans-based assay also were compared with reported toxicity data of five representative regulated DBPs and DCBQ; Based on above analysis, the feasibility of employing C. elegans-based OCR assay for toxicity assessment of DBPs was evaluated.Results:?i?Under our current laboratory conditions, final concentration of DMSO?0.1%?v/v?? and 50 to 75 young adults were optimal parameters well suited for C.elegans-based OCR assay.?ii?Both regulated DBPs and DCBQ caused concentration-dependent increases in mortality of C. elegans, the 24 h LC₅₀ values presented in the form of means±standard deviation of 6 DBPs are DCBQ, 327.81±13.64 ?M; MBA,770.99±230.19 ?M; DBA, 1217.80±65.41 ?M; DCA, 1430.52±117.29 ?M; TCA,1716.86±50.31 ?M; NDMA: LC₅₀>50000 ?M. The lethal toxicity order was DCBQ?MBA>DBA>TCA>DCA>NDMA. DCBQ exhibits greater lethality than MBA, DBA, TCA, DCA, and NDMA.?iii?Both regulated DBPs and DCBQ caused concentration-dependent reductions in OCR of C. elegans, the 24 h EC₅₀ values presented in a form of means±standard deviation of 6 DBPs are DCBQ, 6.37±0.40 ?M; MBA, 11.82±1.58 ?M; DBA,23.93±1.53 ?M; DCA, 28.00±4.74 ?M; TCA, 52.00±37.18 ?M, NDMA, 83.13±35.71?M. The order of the respiratory toxicity of the six DBPs was DCBQ?MBA>DBA>TCA>DCA>NDMA. DCBQ demonstrates the highest respiratory toxicity potency among the tested DBPs.?iiii?A statistically significant correlation was found between EC₅₀ values and LC₅₀ values of the six DBPs in C. elegans?R=0.929, P=0.022?. Except for toxicity order of MBA and DCBQ, identical orders of toxicity for other tested DBPs was observed in in vitro assay, in our 24 h lethal assay and in C. elegans-based OCR assay.In our OCR assay, the EC₅₀ value was at least 30-fold lower than the LC₅₀ value for each tested DBP.Conclusions:?i?A C. elegans-based OCR assay well suited to present laboratory situation has been established.?ii?C. elegans-based OCR assay can provide a rapid, sensitive, and high-throughput tool for toxicity assessment of DBPs in drinking water.?iii?DCBQ is more potent in inducing lethal toxicity and sublethal respiratory toxicity than some regulated DBPs; the potential health effect of DCBQ cannot be ignored.