三氯杀螨醇对泥鳅的生态毒性效应研究
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摘要
随着人们对食品安全的日益重视和食品国际贸易的增加,许多国家规定了水产品中杀虫剂残留限量,同时也更加关注渔业环境质量的保护和改善。三氯杀螨醇(dicofol, DCF)作为滴滴涕杀虫剂的替代品,自问世以来就受到了许多学者的极大关注。DCF尽管对啮齿动物的毒性较低,杀螨效果好,促进了农林作物的丰收,但越来越多的研究发现其在环境中残留时间长,对水生生物的毒性很强,对动物的潜在危害很大。
利用生物化学指标预测污染物的危害或潜在影响,是当前国际环境研究的热点之一。谷胱甘肽转移酶(GST)和乙酰胆碱酯酶(AChE)等生物酶、卵黄蛋白原(Vtg)等雌性物质具有较好的生物指示作用,可在环境灾害暴发之前预警污染物的生态毒性效应。国内近年开展了DCF在一些环境介质中的含量调查,但极少像西方国家那样报道DCF对水生生物的毒性效应。
泥鳅( Misgurnus anguillicaudatus )栖息在我国大部分地区的淡水及其沉积物中,是水环境质量重要指示鱼类之一,其肉质细嫩,营养丰富,味道鲜美。渔业环境和水产品中DCF的安全隐患越来越引起人们的疑虑和关注。因此,为探索沉积物中DCF对鱼类的生物化学效应,本研究选取泥鳅作为实验生物进行初步尝试。这对于阐明DCF在渔业生态系统内的迁移和转化规律,评价和预测DCF的生态危害具有重要的意义。
本研究在实验室条件下,将泥鳅在DCF剂量分别为0(对照)、5、10、20、40 mg·kg~(-1)的人工沉积物中分别暴露24 h、48 h和96 h,然后测定其血清中Vtg浓度、GST活性和AChE活性,以及肌肉、皮、肝、肠和鳃等5种组织中DCF的残留量,此外通过实验建立了沉积物中DCF的气相色谱测定方法。主要实验结果如下:
(1)在经DCF暴露后的泥鳅血清中,GST活性、Vtg浓度均高于空白对照,AChE活性低于空白对照;GST活性随DCF剂量加大而显著增强(相关系数r = 0.998~0.999,相关显著性p<0.01),AChE活性随DCF剂量加大而较显著地下降(r = - 0.906 ~ - 0.946, p<0.10);Vtg浓度与DCF暴露剂量正相关,暴露24 h、48 h和96 h的相关性分别为r =0.825(p < 0.10)、r =0.719(p > 0.10)和r =0.885(p < 0.10)。这表明DCF可诱导泥鳅血清的GST活性,抑制其AChE活性,并程度不同地增高雄性泥鳅的雌激素水平。
(2)泥鳅组织中DCF的残留量大体随暴露时间的延长、DCF剂量的增大而增多,肝脏中DCF的残留量与作用剂量显著正相关(r = 0.976~0.994,p < 0.01),皮和肠中DCF的残留量与作用剂量较显著地正相关(r = 0.888~0.985,p < 0.05或p < 0.01);鳃和肌肉中DCF残留量与作用剂量的关系为实验24 h和48 h较显著或显著地正相关(r = 0.953 ~0.993,p < 0.05或p<0.01),实验96 h虽仍呈正相关但显著性降低(r = 0.566~0.831,p > 0.05或p >0.10)。
(3)泥鳅5种组织对DCF的吸收能力:暴露24 h的吸收能力为肠>肝>皮>鳃>肌肉,暴露48 h的吸收能力为肠>皮>肝>鳃>肌肉,5 mg·kg~(-1)剂量下吸收能力为肠>皮>鳃>肝≈肌肉,10 mg·kg~(-1)剂量下为鳃>肠>皮>肌肉>肝,20 mg·kg~(-1)剂量下为肠>皮>鳃>肝>肌肉;暴露96 h或40 mg·kg~(-1)剂量下吸收能力均为肠>鳃>皮>肝>肌肉。DCF残留总体上表现为肠中较高,肌肉中较少,皮、鳃和肝中受DCF剂量和暴露时间的影响较大。
(4)经DCF暴露后泥鳅可食用组织(皮和肌肉)中DCF残留量大多程度不同地超过欧盟、日本、加拿大、国际食品法典委员会关于畜禽肉中DCF的残留限量(0.05 mg·kg~(-1)~ 3 mg·kg~(-1)),远远超过日本对水产品中DCF的“一律限量”(0.01 mg·kg~(-1))。
(5)沉积物试样中DCF的定量检出限为3μg·kg~(-1),3~50μg·kg~(-1)加标水平的回收率在80.8%~99.5%之间,平行双样测定的相对标准偏差为5.4%~7.2%。本方法的灵敏度、准确性、再现性、可操作性均可满足沉积物中痕量DCF的测定。
With the increasing of emphasis on food security and international trade in food, maximum residual limits of pesticides in aquatic products have been enforced in many countries. More attention was paid to improve and enhance the quality of fishery environment. As an alternative pesticide of DDT, dicofol (DCF) has been concerned by a lot of scholars since its initial production. DCF has low toxic to rodents, and can efficiently control acarid, and useful in promoting the harvest of agricultural crops. However, more and more reports suggested that DCF can exist in environment for a long time, it is hypertoxic to aquatic organisms and harmful to animals.
The research on biochemical indexes which indicate the potential harm of pollutants was high lighted. Vitellogenin (Vtg), Glutathione S-transferase (GST) and Acetylcholinesterase (AChE) etc. are useful bio-indicators for predicting eco-toxicity of pollutants. Information on DCF concentrations in various environmental media was very limited. The bio-chemical effects of DCF on aqutic organisms were seldom reported in China compared with that in western countries.
Loach (Misgurnus anguillicaudatus) is widely distributed in fresh water and sediment in China. It played an important role in the monitoring of water environment. Loach’s meat is delicate, eutrophy and delicious. Potential safety hazard of DCF in fishery environment and aquatic product have been doubted and worried by more and more people. Therefore, loach is selected as an experimental organism for preliminary study of the bio-chemical effects of DCF in sediment on fish. This experiment aimed to elucidate the moving and transformation of DCF in fishery environment, and to provide useful information for evaluating and forecasting the potential harm of DCF.
This study was carried out in laboratory, loach samples were exposed to 0, 5, 10, 20 and 40 mg·kg~(-1) DCF,respectively, exposure time in each dosage last for 24 h, 48 h and 96 h respectively. Activities of GST and AChE, and Vtg concentration in the blood serum were examined, at the end of the experiment, DCF residue in the muscle, skin, liver, intestine and gill were determined. In addition, an analysis method for DCF residue in sediments by gas chromatography was developed.
The results were as following:
(1) The GST activities and Vtg concentrations in the blood serum of experimental loach were higher and AChE activities were lower than that of control, respectively. The GST activities were significantly increased with increasing DCF concentration (r=0.998~0.999, p<0.01). On the other hand, the AChE activities were significantly decreased (r= - 0.906~ - 0.946, p<0.10). A positive correlation between Vtg concentration and DCF level was observed. The correlation coefficient with significant level were 0.825 with p<0.10, 0.719 with p>0.10, 0.885 with p<0.10 for 24 h, 48 h and 96 h, respectively. These results indicated that the GST activity and estrogenic level of male loach could be increased by, but the AChE activity in blood serum could be decreased by DCF.
(2) Generally, DCF residuals in the loach tissues increased with exposure time and DCF dosage. There was significantly positive correlation between DCF residuals in liver and DCF dosage (r= 0.976~0.994, p<0.01); Also, a positive correlation between DCF residuals in skin or intestine and DCF dosage was observed (r=0.888~0.985, p<0.05 and p<0.01, respectively). There was positive correlation between DCF residual in gills or muscle and DCF dosage was existed after 24 h or 48 h exposure (r = 0.953 ~0.993, p<0.05 or p< 0.01), but indistinctively positive for 96 h exposure (r= 0.566~0.831, p> 0.05 or p>0.10).
(3) The absorption capacity for DCF was in the following order: intestine>liver>skin>gill>muscle after 24 h exposure, and intestine>skin>liver>gill> muscle after 48 h exposure, and intestine>skin>gill>liver≈muscle under the concentration of 5 mg·kg~(-1) DCF, gill > intestine > skin > muscle > liver under the concentration of 10 mg·kg~(-1) DCF, intestine>skin> gill > liver >muscle under the concentration of 20 mg·kg~(-1) DCF, and intestine> gill > skin > liver >muscle after 96 h exposure or under the concentration of 40 mg·kg~(-1) DCF. DCF levels were highest in intestine and lowest in muscle. The DCF levels in gill, skin and liver were affected by DCF concentration or exposure time.
(4) After exposure to DCF, the DCF residuals in eatable tissues (skin and muscle) were higher than the limits for meat product prescribed in European Union, Japan, Canada and CAC (Codex Alimentarius Commission) which was 0.05 mg·kg~(-1)~3 mg·kg~(-1), and higher than the limit in Japan, which is 0.01 mg·kg~(-1) .
(5) The detection limit of DCF in sediment sample is 3μg·kg~(-1).The recovery of this analysis method range from 80.8% to 99.5%, and the relative standard deviation (S.D.) of the two parallel samples is 5.4%~7.2%, when 3~50μg·kg~(-1) DCF was supplemented. The result suggested that this method is available to detect trace DCF in sediment sample, due to its relative high sensitivity, accuracy, repeatability and maneuverability.
利用生物化学指标预测污染物的危害或潜在影响,是当前国际环境研究的热点之一。谷胱甘肽转移酶(GST)和乙酰胆碱酯酶(AChE)等生物酶、卵黄蛋白原(Vtg)等雌性物质具有较好的生物指示作用,可在环境灾害暴发之前预警污染物的生态毒性效应。国内近年开展了DCF在一些环境介质中的含量调查,但极少像西方国家那样报道DCF对水生生物的毒性效应。
泥鳅( Misgurnus anguillicaudatus )栖息在我国大部分地区的淡水及其沉积物中,是水环境质量重要指示鱼类之一,其肉质细嫩,营养丰富,味道鲜美。渔业环境和水产品中DCF的安全隐患越来越引起人们的疑虑和关注。因此,为探索沉积物中DCF对鱼类的生物化学效应,本研究选取泥鳅作为实验生物进行初步尝试。这对于阐明DCF在渔业生态系统内的迁移和转化规律,评价和预测DCF的生态危害具有重要的意义。
本研究在实验室条件下,将泥鳅在DCF剂量分别为0(对照)、5、10、20、40 mg·kg~(-1)的人工沉积物中分别暴露24 h、48 h和96 h,然后测定其血清中Vtg浓度、GST活性和AChE活性,以及肌肉、皮、肝、肠和鳃等5种组织中DCF的残留量,此外通过实验建立了沉积物中DCF的气相色谱测定方法。主要实验结果如下:
(1)在经DCF暴露后的泥鳅血清中,GST活性、Vtg浓度均高于空白对照,AChE活性低于空白对照;GST活性随DCF剂量加大而显著增强(相关系数r = 0.998~0.999,相关显著性p<0.01),AChE活性随DCF剂量加大而较显著地下降(r = - 0.906 ~ - 0.946, p<0.10);Vtg浓度与DCF暴露剂量正相关,暴露24 h、48 h和96 h的相关性分别为r =0.825(p < 0.10)、r =0.719(p > 0.10)和r =0.885(p < 0.10)。这表明DCF可诱导泥鳅血清的GST活性,抑制其AChE活性,并程度不同地增高雄性泥鳅的雌激素水平。
(2)泥鳅组织中DCF的残留量大体随暴露时间的延长、DCF剂量的增大而增多,肝脏中DCF的残留量与作用剂量显著正相关(r = 0.976~0.994,p < 0.01),皮和肠中DCF的残留量与作用剂量较显著地正相关(r = 0.888~0.985,p < 0.05或p < 0.01);鳃和肌肉中DCF残留量与作用剂量的关系为实验24 h和48 h较显著或显著地正相关(r = 0.953 ~0.993,p < 0.05或p<0.01),实验96 h虽仍呈正相关但显著性降低(r = 0.566~0.831,p > 0.05或p >0.10)。
(3)泥鳅5种组织对DCF的吸收能力:暴露24 h的吸收能力为肠>肝>皮>鳃>肌肉,暴露48 h的吸收能力为肠>皮>肝>鳃>肌肉,5 mg·kg~(-1)剂量下吸收能力为肠>皮>鳃>肝≈肌肉,10 mg·kg~(-1)剂量下为鳃>肠>皮>肌肉>肝,20 mg·kg~(-1)剂量下为肠>皮>鳃>肝>肌肉;暴露96 h或40 mg·kg~(-1)剂量下吸收能力均为肠>鳃>皮>肝>肌肉。DCF残留总体上表现为肠中较高,肌肉中较少,皮、鳃和肝中受DCF剂量和暴露时间的影响较大。
(4)经DCF暴露后泥鳅可食用组织(皮和肌肉)中DCF残留量大多程度不同地超过欧盟、日本、加拿大、国际食品法典委员会关于畜禽肉中DCF的残留限量(0.05 mg·kg~(-1)~ 3 mg·kg~(-1)),远远超过日本对水产品中DCF的“一律限量”(0.01 mg·kg~(-1))。
(5)沉积物试样中DCF的定量检出限为3μg·kg~(-1),3~50μg·kg~(-1)加标水平的回收率在80.8%~99.5%之间,平行双样测定的相对标准偏差为5.4%~7.2%。本方法的灵敏度、准确性、再现性、可操作性均可满足沉积物中痕量DCF的测定。
With the increasing of emphasis on food security and international trade in food, maximum residual limits of pesticides in aquatic products have been enforced in many countries. More attention was paid to improve and enhance the quality of fishery environment. As an alternative pesticide of DDT, dicofol (DCF) has been concerned by a lot of scholars since its initial production. DCF has low toxic to rodents, and can efficiently control acarid, and useful in promoting the harvest of agricultural crops. However, more and more reports suggested that DCF can exist in environment for a long time, it is hypertoxic to aquatic organisms and harmful to animals.
The research on biochemical indexes which indicate the potential harm of pollutants was high lighted. Vitellogenin (Vtg), Glutathione S-transferase (GST) and Acetylcholinesterase (AChE) etc. are useful bio-indicators for predicting eco-toxicity of pollutants. Information on DCF concentrations in various environmental media was very limited. The bio-chemical effects of DCF on aqutic organisms were seldom reported in China compared with that in western countries.
Loach (Misgurnus anguillicaudatus) is widely distributed in fresh water and sediment in China. It played an important role in the monitoring of water environment. Loach’s meat is delicate, eutrophy and delicious. Potential safety hazard of DCF in fishery environment and aquatic product have been doubted and worried by more and more people. Therefore, loach is selected as an experimental organism for preliminary study of the bio-chemical effects of DCF in sediment on fish. This experiment aimed to elucidate the moving and transformation of DCF in fishery environment, and to provide useful information for evaluating and forecasting the potential harm of DCF.
This study was carried out in laboratory, loach samples were exposed to 0, 5, 10, 20 and 40 mg·kg~(-1) DCF,respectively, exposure time in each dosage last for 24 h, 48 h and 96 h respectively. Activities of GST and AChE, and Vtg concentration in the blood serum were examined, at the end of the experiment, DCF residue in the muscle, skin, liver, intestine and gill were determined. In addition, an analysis method for DCF residue in sediments by gas chromatography was developed.
The results were as following:
(1) The GST activities and Vtg concentrations in the blood serum of experimental loach were higher and AChE activities were lower than that of control, respectively. The GST activities were significantly increased with increasing DCF concentration (r=0.998~0.999, p<0.01). On the other hand, the AChE activities were significantly decreased (r= - 0.906~ - 0.946, p<0.10). A positive correlation between Vtg concentration and DCF level was observed. The correlation coefficient with significant level were 0.825 with p<0.10, 0.719 with p>0.10, 0.885 with p<0.10 for 24 h, 48 h and 96 h, respectively. These results indicated that the GST activity and estrogenic level of male loach could be increased by, but the AChE activity in blood serum could be decreased by DCF.
(2) Generally, DCF residuals in the loach tissues increased with exposure time and DCF dosage. There was significantly positive correlation between DCF residuals in liver and DCF dosage (r= 0.976~0.994, p<0.01); Also, a positive correlation between DCF residuals in skin or intestine and DCF dosage was observed (r=0.888~0.985, p<0.05 and p<0.01, respectively). There was positive correlation between DCF residual in gills or muscle and DCF dosage was existed after 24 h or 48 h exposure (r = 0.953 ~0.993, p<0.05 or p< 0.01), but indistinctively positive for 96 h exposure (r= 0.566~0.831, p> 0.05 or p>0.10).
(3) The absorption capacity for DCF was in the following order: intestine>liver>skin>gill>muscle after 24 h exposure, and intestine>skin>liver>gill> muscle after 48 h exposure, and intestine>skin>gill>liver≈muscle under the concentration of 5 mg·kg~(-1) DCF, gill > intestine > skin > muscle > liver under the concentration of 10 mg·kg~(-1) DCF, intestine>skin> gill > liver >muscle under the concentration of 20 mg·kg~(-1) DCF, and intestine> gill > skin > liver >muscle after 96 h exposure or under the concentration of 40 mg·kg~(-1) DCF. DCF levels were highest in intestine and lowest in muscle. The DCF levels in gill, skin and liver were affected by DCF concentration or exposure time.
(4) After exposure to DCF, the DCF residuals in eatable tissues (skin and muscle) were higher than the limits for meat product prescribed in European Union, Japan, Canada and CAC (Codex Alimentarius Commission) which was 0.05 mg·kg~(-1)~3 mg·kg~(-1), and higher than the limit in Japan, which is 0.01 mg·kg~(-1) .
(5) The detection limit of DCF in sediment sample is 3μg·kg~(-1).The recovery of this analysis method range from 80.8% to 99.5%, and the relative standard deviation (S.D.) of the two parallel samples is 5.4%~7.2%, when 3~50μg·kg~(-1) DCF was supplemented. The result suggested that this method is available to detect trace DCF in sediment sample, due to its relative high sensitivity, accuracy, repeatability and maneuverability.
引文
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