| Butachlor is one of the most commonly used herbicides in China. The large use of it in field has resulted in the ubiquitous leftover of it in aquatic environment. Therefore, the potential toxicity of butachlor to aquatic organisms has attracted great attentions of the society. In this study the acute in vivo and in vitro toxicity of butachlor on flounder, Paralichihys olivaceus, and flounder gill (FG) cells, as well as the possible mechanisms were investigated for the risk assessment of butachlor to marine fish species.Firstly, the cytotoxic effects of butachlor on the cellular morphology, proliferation, activities of antioxidant enzymes and ATP level of FG cells were examined. Results indicated that: (1) the 24h-IC50 values (median inhibition concentration) of butachlor in FG cells were 43.32, 43.86 and 44.91μmol/L for neutral red (NR) uptake, MTT assay and cell protein assay, respectively. No acute cytotoxicity was observed in FG cells exposed to butachlor, which was lower than 10μM. Above 20μmol/L, the cytotoxicity of butachlor to FG cells was obvious and dose-dependent; (2) Obviously morphological alterations like shrinkage and vacuolation began to appear in FG cells exposed to 30μmol/L butachlor for 24h; (3) Butachlor can markedly affect the activities of antioxidant enzymes of Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in FG cells exposed to 30μmol/L butachlor. And a quick increase of the enzyme activity was observed in the initial 2h exposure period, then drop to the level of control cells after 6 or 8h treatment, and stop at a constant level after 12 or 24 h treatment with a level lower than that of the control; (4) ATP contents in all the treated FG cells, the doses of butachlor ranging from 0.1 to 40μmol/L, were inhibited with a dose-dependent responsive. All the above in vitro results indicated that butachlor is high toxic to FG cells. It can result in the changes of the cellular morphology, inhibition of the cell proliferation and antioxidant enzyme activity, and drop of the ATP level in treated FG cells.Secondly, acute in vivo effects of butachlor on flounder were investigated. Results indicated that: (1) butachlor caused much higher acute lethality on live flounder and the 96h-LC50 value (median lethal concentration) for live fish was 6.55 nmol/L, about 1/7000 of 24h-IC50 value in FG cells; (2) In live fish, histopathological examination showed that gill was the primary target organ of butachlor. butachlor, badly impaired the respiration of gills, by formation of lesions, breakdown of pillar cells, edema, lifting and detachment of lamellar epithelium, and blood congestion. The disfunction of gills caused suffocation to fish with extremely high acute lethality to flounder by butachlor; (3) ATP content in the gill tissues of flounder exposed to 3.84 nmol/L butachlor was also decreased for all the treatment intervals. All the in vivo results indicated that butachlor posed much higher lethality on flounder than in vitro cultured FG cells. This might be related to the extreme toxic of butachlor to the structure and function of flounder gill.In the end, genotoxicity of butachlor on flounder and FG cells were studied. Results indicated that: (1) the fragmentation of genomic DNA of FG cells exposed to 30μmol/L butachlor was first observed after 8h exposure, and as increase of exposure time, the DNA damage became more severe; (2) the ratios of micronucleus in the erythrocytes of flounder exposed to 1.28~3.84 nmol/L butachlor for 96h were significantly increased in contrast to control. The above genetoxicity results indicated that butachlor was genotoxic to flounder and FG cells.Taken together, butachlor is high toxic to marine fish flounder. Therefore, great caution should be taken in the field use of butachlor, such as keep away from raining and sea shore, to avoid any disaster to marine organism. And also, the results obtained can provide us a good guidance for the forecast of in vivo toxicity from the in vitro cytotoxic data.
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