养殖水体中敌百虫对鲤鲫鱼安全浓度及检测方法研究
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摘要
本论文内容包括水体中敌百虫安全浓度的研究和水体中敌百虫检测方法的研究。通过大量的实验工作,建立了水体中敌百虫残留的检测方法—气相色谱法,并通过了实验室验证工作,形成了养殖水体中敌百虫残留的检测草案。
在水体中敌百虫安全浓度的研究中,选取了耐受性较强的鲤鱼和鲫鱼进行了安全浓度的验证,同时对鲫鱼进行了敌百虫的急性毒理学实验。根据水体中有毒有害物质的最高限量确定的方法,采用Turabell公式:0.1×96hLC50进行确定。通过96h急性毒性实验敌百虫对鲫鱼的LC50为63.97mg/L, 95%可信限为1.81±0.048 mg/L,95%可信区间为57.81~72.11 mg/L。分别以1/2、1/4、1/8及1/16LC50为染毒浓度,于实验开始后24、48、72及96h采样分析,结果表明:与对照组相比,随着暴露时间的延长,各浓度组实验鱼血浆胆碱酯酶的活性均有一定的升高,但各实验组间没有表现出明显的区别。实验剂量下的敌百虫对鲫鱼血浆GST活性有一定的影响,呈现出较为明显的先扬后抑的结果;1/2LC50实验组血浆CAT在实验进行至96h活性明显高于对照组,而其他剂量组在实验期间CAT活性没有明显的改变。本实验中敌百虫对鲫鱼的遗传性在微核率上未表现出差异,但高浓度核异常率在实验24、48h明显高于对照组。鲤鱼及鲫鱼种分别养殖于在敌百虫浓度为0.2、0.3、0.4、0.5、1.0mg/L的水体中,并于实验开始后的5、10、15、20、30、40及60d取实验鱼肌肉进行高效气相色谱分析,检测鱼肉中敌百虫含量,结果表明:此检测方法下线性范围为0.02~5.00μg/mL,测定限量为0.02mg/kg,且回收率和精密度均符合检测要求,从实验结果中可看出,养殖水体中敌百虫低于0.5mg/L时,鱼体中的敌百虫残留量均符合卫生标准,可安全食用。
在养殖水体中敌百虫检测方法的研究中,通过采样及保存方法的选择(敌百虫易转化)、提取及净化方法的选择及优化、色谱条件的优化(进样口温度及检测器的选择为重点)、水体环境中敌百虫(亚磷酸二甲酯)背景值的测定及评价、水体及鱼体中中敌百虫含量测定方法的确定,建立了水体中敌百虫残留的检测方法—气相色谱法。在下列色谱条件下,DB-225毛细管柱,规格为30m×0.25μm×0.32mm。柱温在70℃(保持1min),10℃/min的速度升至180℃(保持2min),再30℃/min的速度升至230℃(保持2min)。进样口温度为280℃,不分流进样,检测器温度为250℃。载气为高纯氮,纯度≥99.999%,1.8mL/min。氢气纯度99.9%,不分流进样,进样量为1μL。通过二个实验室间的验证工作,建立养殖水体中敌百虫残留的检测标准。本方法标准添加浓度为0.004 mg/L~0.250 mg/L时,回收率≥75%、方法检出限为0.004 mg/L、方法精密度≤10%、方法线性范围为0.4~25.0μg/mL。
This thesis is on the safety level determination of trichlorfon in breeding waters and related monitoring methods were determinned. In addition, Monitoring methods by gas chromatograph of trichlorfon in breeding waters and testing standard of trichlorfon residue in breeding waters were established based on a great of laboratory investigations.
In the experiment of determination of safety level of trichlorfon in breeding waters, the common carp and the crucian carp, which had strong tolerance ability to trichlorfon, were used. The acute toxicity of trichlorfon on crucian carp was carried out. The Turabell Formula: 0.1×96hLC50 was use to determined the top limit of toxic and harmful substances in waters.Toxicity test of trichlorfon on crucian of 96 hour was conducted. the result showed that: LC50 is 63.97mg/L, 95% confidence limit is 63.97mg/L, 95% confidence interval is 57.81~72.11 mg/L. The exposure concentration is respectively is 1/2、1/4、1/8 and 1/16LC50, sample was analysis at 24、48、72and 96 hour after begin of test. The result showed that the activities of cholinesterase in plasma of crucian of test group all were higher than control group; but difference among test group was not found. The trichlorfon influnced the activities of GST in liver of crucian, the activities were first raised and then decreased. The activitiy of CAT in liver of crucian of 1/2LC50 is higher than control group at 96hour after test. The micronuclear rates of crucian almost did not changed, but the dyskaryosis of test group was higher than control group. The common carp and crucian carp were put in the water in the concentration of 0.2, 0.3, 0.4, 0.5, 1.0mg/L of trichlorfon respectively. After 5, 10, 15, 20, 30, 40 and 60days, the concentration of the trichlorfon in the meat were detected with high performance gas chromatography. The result showed that trichlorfon the linear range was 0.02～5.00μg/mL, and the determination limit was 0.02mg/kg, the recover rate and the degree of precision were consistent with the detection requirement. The result also indicated that the residual level of trichlorfon in the fish body conformed to the sanitary standard and the fish meat was safely edible.In the experiment of determination of monitoring methods of trichlorfon in breeding waters, monitoring methods by gas chromatograph was established based on Selection Optimizations including the following parameters: collection and preservation of samples (trichlorfon was easily transtomed), extraction and purification methods, Chromatographic conditions (mainly on the parameters of injection port temperature and detector type), and the methods to determine the background values of trichlorfon (dimethyl phosphite) in the waters and animal bodies.
The gas chromatography methods: The column was DB-225 capillary column (30 m×0.25μm×0.32mm), with an oven temperature of 70℃(1min)→180℃(10℃/min,2min)→23℃(30℃/min, 2min), injection port temperature was at 280℃, detector temperature was at 250℃, nitrogen with high purity of >99.999% was used as carrier gas with flow rate of 1.8ml/min, the purity of hydrogen was 99.9%, 1μl samples were injected to GC splitlessly. Accoding to the results of two labs confomatory tests, the standand method of monitoring trichlorfon residue in beeeding waters was estalsihed: the samples are spiked at 0.004~0.250mg/L, the average recovery efficiency of the method was≥75%, The minimum detectable concentration 0.004 mg/L, the variation coefficient≤10%, the linear range was at 0.4~25.0μg/mL.
在水体中敌百虫安全浓度的研究中,选取了耐受性较强的鲤鱼和鲫鱼进行了安全浓度的验证,同时对鲫鱼进行了敌百虫的急性毒理学实验。根据水体中有毒有害物质的最高限量确定的方法,采用Turabell公式:0.1×96hLC50进行确定。通过96h急性毒性实验敌百虫对鲫鱼的LC50为63.97mg/L, 95%可信限为1.81±0.048 mg/L,95%可信区间为57.81~72.11 mg/L。分别以1/2、1/4、1/8及1/16LC50为染毒浓度,于实验开始后24、48、72及96h采样分析,结果表明:与对照组相比,随着暴露时间的延长,各浓度组实验鱼血浆胆碱酯酶的活性均有一定的升高,但各实验组间没有表现出明显的区别。实验剂量下的敌百虫对鲫鱼血浆GST活性有一定的影响,呈现出较为明显的先扬后抑的结果;1/2LC50实验组血浆CAT在实验进行至96h活性明显高于对照组,而其他剂量组在实验期间CAT活性没有明显的改变。本实验中敌百虫对鲫鱼的遗传性在微核率上未表现出差异,但高浓度核异常率在实验24、48h明显高于对照组。鲤鱼及鲫鱼种分别养殖于在敌百虫浓度为0.2、0.3、0.4、0.5、1.0mg/L的水体中,并于实验开始后的5、10、15、20、30、40及60d取实验鱼肌肉进行高效气相色谱分析,检测鱼肉中敌百虫含量,结果表明:此检测方法下线性范围为0.02~5.00μg/mL,测定限量为0.02mg/kg,且回收率和精密度均符合检测要求,从实验结果中可看出,养殖水体中敌百虫低于0.5mg/L时,鱼体中的敌百虫残留量均符合卫生标准,可安全食用。
在养殖水体中敌百虫检测方法的研究中,通过采样及保存方法的选择(敌百虫易转化)、提取及净化方法的选择及优化、色谱条件的优化(进样口温度及检测器的选择为重点)、水体环境中敌百虫(亚磷酸二甲酯)背景值的测定及评价、水体及鱼体中中敌百虫含量测定方法的确定,建立了水体中敌百虫残留的检测方法—气相色谱法。在下列色谱条件下,DB-225毛细管柱,规格为30m×0.25μm×0.32mm。柱温在70℃(保持1min),10℃/min的速度升至180℃(保持2min),再30℃/min的速度升至230℃(保持2min)。进样口温度为280℃,不分流进样,检测器温度为250℃。载气为高纯氮,纯度≥99.999%,1.8mL/min。氢气纯度99.9%,不分流进样,进样量为1μL。通过二个实验室间的验证工作,建立养殖水体中敌百虫残留的检测标准。本方法标准添加浓度为0.004 mg/L~0.250 mg/L时,回收率≥75%、方法检出限为0.004 mg/L、方法精密度≤10%、方法线性范围为0.4~25.0μg/mL。
This thesis is on the safety level determination of trichlorfon in breeding waters and related monitoring methods were determinned. In addition, Monitoring methods by gas chromatograph of trichlorfon in breeding waters and testing standard of trichlorfon residue in breeding waters were established based on a great of laboratory investigations.
In the experiment of determination of safety level of trichlorfon in breeding waters, the common carp and the crucian carp, which had strong tolerance ability to trichlorfon, were used. The acute toxicity of trichlorfon on crucian carp was carried out. The Turabell Formula: 0.1×96hLC50 was use to determined the top limit of toxic and harmful substances in waters.Toxicity test of trichlorfon on crucian of 96 hour was conducted. the result showed that: LC50 is 63.97mg/L, 95% confidence limit is 63.97mg/L, 95% confidence interval is 57.81~72.11 mg/L. The exposure concentration is respectively is 1/2、1/4、1/8 and 1/16LC50, sample was analysis at 24、48、72and 96 hour after begin of test. The result showed that the activities of cholinesterase in plasma of crucian of test group all were higher than control group; but difference among test group was not found. The trichlorfon influnced the activities of GST in liver of crucian, the activities were first raised and then decreased. The activitiy of CAT in liver of crucian of 1/2LC50 is higher than control group at 96hour after test. The micronuclear rates of crucian almost did not changed, but the dyskaryosis of test group was higher than control group. The common carp and crucian carp were put in the water in the concentration of 0.2, 0.3, 0.4, 0.5, 1.0mg/L of trichlorfon respectively. After 5, 10, 15, 20, 30, 40 and 60days, the concentration of the trichlorfon in the meat were detected with high performance gas chromatography. The result showed that trichlorfon the linear range was 0.02～5.00μg/mL, and the determination limit was 0.02mg/kg, the recover rate and the degree of precision were consistent with the detection requirement. The result also indicated that the residual level of trichlorfon in the fish body conformed to the sanitary standard and the fish meat was safely edible.In the experiment of determination of monitoring methods of trichlorfon in breeding waters, monitoring methods by gas chromatograph was established based on Selection Optimizations including the following parameters: collection and preservation of samples (trichlorfon was easily transtomed), extraction and purification methods, Chromatographic conditions (mainly on the parameters of injection port temperature and detector type), and the methods to determine the background values of trichlorfon (dimethyl phosphite) in the waters and animal bodies.
The gas chromatography methods: The column was DB-225 capillary column (30 m×0.25μm×0.32mm), with an oven temperature of 70℃(1min)→180℃(10℃/min,2min)→23℃(30℃/min, 2min), injection port temperature was at 280℃, detector temperature was at 250℃, nitrogen with high purity of >99.999% was used as carrier gas with flow rate of 1.8ml/min, the purity of hydrogen was 99.9%, 1μl samples were injected to GC splitlessly. Accoding to the results of two labs confomatory tests, the standand method of monitoring trichlorfon residue in beeeding waters was estalsihed: the samples are spiked at 0.004~0.250mg/L, the average recovery efficiency of the method was≥75%, The minimum detectable concentration 0.004 mg/L, the variation coefficient≤10%, the linear range was at 0.4~25.0μg/mL.
引文
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