磺胺二甲嘧啶对斑马鱼胚胎的急性毒性作用
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摘要
通过研究磺胺二甲嘧啶(sulfamethazine,SMZ)对斑马鱼胚胎的急性毒性作用,确定关键毒理学参数,分析SMZ暴露浓度、暴露时间对斑马鱼胚胎死亡率、孵化率的影响及致畸效应,评估SMZ对斑马鱼胚胎的发育毒性作用,为预测磺胺类污染物的环境风险和生态毒性提供基础数据。以斑马鱼胚胎为受试生物,进行梯度浓度SMZ溶液96 h急性毒性暴露实验,暴露方法为半静态法,每隔24 h更换一次溶液,溶液更换前后进行高效液相色谱分析,确定暴露溶液浓度在理论浓度±20%以内。体视显微镜观察统计12、24、48、72、96 hpf胚胎死亡率和48、72、96 hpf胚胎孵化率,Origin Pro 8.0进行数据分析;显微观察并记录胚胎发育过程中出现的畸形现象。以死亡率为参数,低浓度SMZ(0.001~10 mg/L)暴露对斑马鱼胚胎死亡率无显著性影响(P>0.05);中等浓度SMZ(10~300 mg/L)暴露下斑马鱼胚胎死亡率显著降低(P<0.05);高浓度SMZ(500~1 000 mg/L)导致斑马鱼胚胎死亡率显著性上升(P<0.05),致死效应主要作用于胚胎发育早期(48 hpf内);在1 000 mg/L SMZ浓度暴露下,96 hpf胚胎致死率约为28%。以孵化率为参数,低于1 mg/L的SMZ对胚胎孵化率无显著性影响(P>0.05);1~700 mg/L SMZ对斑马鱼48 hpf胚胎孵化率显著高于对照组(P<0.05);高浓度1 000 mg/L SMZ下48 hpf胚胎孵化率显著低于对照组(P<0.05)。以致畸率为参数,0.001mg/L以上SMZ诱导48和72 hpf斑马鱼胚胎出现致畸效应,表现为胚胎脊柱弯曲、心包囊肿、色素生成增加、尾部缺失、躯干发育不全等现象。研究表明,低浓度SMZ(0.001 mg/L)对斑马鱼胚胎即表现出较强的致畸性,但其致死效应并不明显;中等浓度SMZ对斑马鱼胚胎孵化具有促进作用,同时降低96 hpf胚胎死亡率,对96 hpf胚胎的最低效应浓度为10 mg/L;高浓度SMZ暴露下,96 hpf斑马鱼胚胎死亡率显著上升,且孵化率受到抑制。
To evaluate the developmental toxicity of sulfamethazine( SMZ) in zebrafish embryos and provide fundamental data for the environmental risk forecast of sulfanilamide pollutions,the study was designed to determine the acute toxicity and main toxic parameters of SMZ to zebrafish embryos. The effects of SMZ exposure concentration and time on mortality rate,hatching rate and malformation were analyzed. The 96 h acute toxicity of SMZ was conducted with zebrafish embryos under semi-static condition. Culture solutions were renewed per 24 h to maintain the constant concentration( mean ± 20%) and monitored under high performance liquid chromatography. The number of death and hatch of zebrafish embryos in different developmental stages was observed with stereomicroscope and analyzed by Origin Pro 8 software. In addition,the developmental deformities were also observed and recorded. With mortality rate as parameter,the mortality rates in low concentrations( 0. 001-10 mg/L) of SMZ were not significantly changed compared to control( P > 0. 05),while the values in moderate concentrations groups decreased significantly( P < 0. 05)and high concentration groups increased significantly( P < 0. 05). With hatching rate as parameter,the embryo hatching rates were not significantly affected by SMZ under 1 mg/L( P > 0. 05). However,the 48 hpf hatching rates in1-700 mg/L SMZ groups increased significantly( P < 0. 05),and the hatching rate in 48 hpf decreased significantly when SMZ were above 1 000 mg/L( P < 0. 05). With respect to teratogenicity,SMZ above 0. 001 mg/L led to zebrafish embryo malformation at 48 and 72 hpf,including spinal curvature,pericardium,abnormal pigmentogenesis,tail defect and immature trunk. Our results suggested that low concentrations of SMZ induced severe teratogenesis with 0. 001 mg/L as teratogenic effective concentration whereas unconspicuous lethal effect; moderate concentrations of SMZ promoted the embryo hatch and reduced the mortality rate with the minimal effective concentration as 10 mg/L at 96 hpf;while the embryo mortality rate at 96 hpf rose up and thatching rate decreased under high concentrations of SMZ exposure.
To evaluate the developmental toxicity of sulfamethazine( SMZ) in zebrafish embryos and provide fundamental data for the environmental risk forecast of sulfanilamide pollutions,the study was designed to determine the acute toxicity and main toxic parameters of SMZ to zebrafish embryos. The effects of SMZ exposure concentration and time on mortality rate,hatching rate and malformation were analyzed. The 96 h acute toxicity of SMZ was conducted with zebrafish embryos under semi-static condition. Culture solutions were renewed per 24 h to maintain the constant concentration( mean ± 20%) and monitored under high performance liquid chromatography. The number of death and hatch of zebrafish embryos in different developmental stages was observed with stereomicroscope and analyzed by Origin Pro 8 software. In addition,the developmental deformities were also observed and recorded. With mortality rate as parameter,the mortality rates in low concentrations( 0. 001-10 mg/L) of SMZ were not significantly changed compared to control( P > 0. 05),while the values in moderate concentrations groups decreased significantly( P < 0. 05)and high concentration groups increased significantly( P < 0. 05). With hatching rate as parameter,the embryo hatching rates were not significantly affected by SMZ under 1 mg/L( P > 0. 05). However,the 48 hpf hatching rates in1-700 mg/L SMZ groups increased significantly( P < 0. 05),and the hatching rate in 48 hpf decreased significantly when SMZ were above 1 000 mg/L( P < 0. 05). With respect to teratogenicity,SMZ above 0. 001 mg/L led to zebrafish embryo malformation at 48 and 72 hpf,including spinal curvature,pericardium,abnormal pigmentogenesis,tail defect and immature trunk. Our results suggested that low concentrations of SMZ induced severe teratogenesis with 0. 001 mg/L as teratogenic effective concentration whereas unconspicuous lethal effect; moderate concentrations of SMZ promoted the embryo hatch and reduced the mortality rate with the minimal effective concentration as 10 mg/L at 96 hpf;while the embryo mortality rate at 96 hpf rose up and thatching rate decreased under high concentrations of SMZ exposure.
引文
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[20]Cao F,Liu X,Wang C,et al.Acute and short-term developmental toxicity of cyhalofop-butyl to zebrafish(Danio rerio)[J].Environ Sci Pollut R,2016,23(10):10080-10089.
[2]Hirsch R,Ternes T,Haberer K,et al.Occurrence of antibiotics in the aquatic environment[J].Sci Total Environ,1999,225(1/2):109-118.
[3]Kolpin D W,Furlong E T,Meyer M T,et al.Pharmaceuticals,hormones,and other organic wastewater contaminants in U.S.streams,1999—2000:a national reconnaissance[J].Environ Sci Technol,2002,36(6):1202-1211.
[4]Managaki S,Murata A,Takada H,et al.Distribution of macrolides,sulfonamides,and trimethoprim in tropical waters:ubiquitous occurrence of veterinary antibiotics in the Mekong Delta[J].Environ Sci Technol,2007,41(23):8004-8010.
[5]Shimizu A,Takada H,Koike T,et al.Ubiquitous occurrence of sulfonamides in tropical Asian waters[J].Sci Total Environ,2013,452:108-115.
[6]Huang D,Hou J,Kuo T,et al.Toxicity of the veterinary sulfonamide antibiotic sulfamonomethoxine to five aquatic organisms[J].Environ Toxicol Phar,2014,38(3):874-880.
[7]Bartlett A J,Balakrishnan V K,Toito J,et al.Toxicity of four sulfonamide antibiotics to the freshwater amphipod Hyalella azteca[J].Environ Toxicol Chem,2013,32(4):866-775.
[8]Strhle U,Scholz S,Geisler R,et al.Zebrafish embryos as an alternative to animal experiments—a commentary on the definition of the onset of protected life stages in animal welfare regulations[J].Reprod Toxicol,2012,33:128-132.
[9]杨颖,杜碧莹.HPLC法测定磺胺二甲嘧啶钠有关物质[J].中国医药指南,2015(13):14-15.
[10]OECD.Test No.236:Fish embryo acute toxicity(FET)test[S].Paris:OECD,2013.
[11]Whitehouse P,Brown B,Wilkinson H,et al.Guidance Document No.27.Technical guidance for deriving environmental quality standards[R].Brussels:European Commission,2011.
[12]谢美萍,颜勇.水体中3种磺胺类药物对斑马鱼的生态毒性效应[J].环境科技,2015(2):30-34.
[13]Palacio-Cortes A M,Signorini-Souza I D L,Yoshio-Hara E L,et al.Polybrominated diphenyl ethers(PBDEs)effects on Chironomus sancticaroli larvae after short-term exposure[J].Ecotox Environ Safe,2017,139:308-315.
[14]Martinez-Sales M,García-Ximénez F,Espinós F.Zebrafish(Danio rerio)as a possible bioindicator of epigenetic factors present in drinking water that may affect reproductive function:is chorion an issue?[J].Zygote,2015,23(3):447-452.
[15]Qin L,Liu F,Liu H,et al.Evaluation of HODE-15,FDE-15,CDE-15,and BDE-15 toxicity on adult and embryonic zebrafish(Danio rerio)[J].Environ Sci Pollut R,2014,21(24):14047-14057.
[16]Manjunatha B,Peng W,Liu K,et al.The effects of henna(hair dye)on the embryonic development of zebrafish(Danio rerio)[J].Environ Sci Pollut R,2014,21(17):10361-10367.
[17]Ducharme N A,Reif D M,Gustafsson J,et al.Comparison of toxicity values across zebrafish early life stages and mammalian studies:implications for chemical testing[J].Reprod Toxicol,2015,55:3-10.
[18]David A,Pancharatna K.Developmental anomalies induced by a non-selective COX inhibitor(ibuprofen)in zebrafish(Danio rerio)[J].Environ Toxicol Pharm,2009(27):390-395.
[19]Zhu L,Mu X,Wang K,et al.Cyhalofop-butyl has the potential to induce developmental toxicity,oxidative stress and apoptosis in early life stage of zebrafish(Danio rerio)[J].Environ Pollut,2015,203:40-49.
[20]Cao F,Liu X,Wang C,et al.Acute and short-term developmental toxicity of cyhalofop-butyl to zebrafish(Danio rerio)[J].Environ Sci Pollut R,2016,23(10):10080-10089.