CN106614151A - 一种降低半静态养殖水中毒死蜱诱导斑马鱼死亡率的方法 - Google Patents
一种降低半静态养殖水中毒死蜱诱导斑马鱼死亡率的方法 Download PDFInfo
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Abstract
本发明公开了一种降低半静态养殖水中毒死蜱诱导斑马鱼死亡率的方法,本发明针对半静态水中斑马鱼对毒死蜱的暴露,检测、调控水中毒死蜱的浓度,通过添加EGCG试剂,调整EGCG与水中毒死蜱的浓度比,达到降低斑马鱼的死亡率。发明方法通过原位或异位技术实施,简单易行,可提高斑马鱼暴露于毒死蜱的存活率,避免使用化学解毒剂带来的后续副作用,提高养殖的经济效益;同时避免排放废水中化学解毒剂的二次污染,提高环境经济效益。
Description
技术领域
本发明涉及水产品养殖技术领域,尤其涉及一种降低半静态养殖水中毒死蜱诱导斑马鱼死亡率的方法。
背景技术
高毒有机磷农药的替代品种毒死蜱被广泛用于农业生产和草坪杀虫剂,已造成环境水体污染。毒死蜱对人类属于中低毒农药,但是鱼类的敏感类农药。毒死蜱属于神经毒剂,其功能之一是抑制机体内神经中乙酰胆碱酯酶AChE或胆碱酯酶ChE的活性,破坏正常的神经冲动传导,引起斑马鱼中毒死亡。另外,机体在正常情况下,体内氧自由基处于动态平衡状态。 一旦氧自由基在机体内失去平衡,如超过了细胞抗氧化防护的能力,则会造成氧胁迫。研究表明,达到半致死浓度的毒死蜱会破坏机体氧自由基平衡,形成氧化胁迫,造成机体损伤、死亡。
对于斑马鱼,如果血清中毒死蜱浓度升高,酶活性将会降低,解毒功能下降。鱼类有机磷农药中毒,多用“阿托品”洒入养殖水中进行解毒,降低致死率。但是“阿托品”易于鱼体内聚集,具有明显的生理副作用。同时如果滞留在养殖废水中,扩散到其他环境介质,易造成环境危害。表没食子儿茶素没食子酸酯(EGCG)是一种来源于植物的多酚化合物,具有抗氧化、抗癌、抗突变等生理活性,这些功效归结于他们对氧自由基的清除能力。研究表明EGCG具有与肠、肝、和肺中的其他酶共同阻止某些有害物质的活力,保护机体。最新研究证明EGCG可能具有神经保护功能。到目前为止, EGCG仍然被认为是具有环境友好性的纯天然试剂。
斑马鱼是生态和环境指示性生物,在生态、环境和毒理等方面极具研究价值,同时又具有观赏性等其他用途,其养殖通常在小体积半静态水中进行。
发明内容
本发明提供了一种降低半静态养殖水中毒死蜱诱导斑马鱼死亡率的方法,采用EGCG降低水中毒死蜱诱导斑马鱼死亡率 。本发明针对半静态水中斑马鱼对毒死蜱的暴露,检测、调控水中毒死蜱的浓度,通过添加EGCG,调整半静态水中EGCG与毒死蜱浓度比 ,来降低斑马鱼的死亡率。发明方法简单易行,可提高斑马鱼暴露于毒死蜱的存活率,避免使用化学解毒剂带来的后续副作用,提高养殖的经济效益;同时避免排放废水中化学解毒剂的二次污染,提高环境经济效益。
为实现上述目的,本发明提供如下技术方案:
一种降低半静态养殖水中毒死蜱诱导斑马鱼死亡率的方法,其特征在于: 在含有毒死蜱的半静态养殖水中添加适量的EGCG ,使得养殖水中EGCG与毒死蜱的浓度比=(1-2):1,所述的毒死蜱在水中的浓度为0.48-0.70 mg/L 。
所述的一种降低半静态养殖水中毒死蜱诱导斑马鱼死亡率的方法,其特征在于,具体操作步骤如下:
(1)取适量养殖水,测量水中毒死蜱母体起始浓度、pH值、水温,调整养殖水的pH=6.8~8.5,控制水温为18~25℃;
(2)根据步骤(1)测量得到的水中毒死蜱母体起始浓度,然后按比例加水稀释至毒死蜱的浓度为0.48-0.70mg/L, 在按比例向养殖水中加入已预先配制好的高浓度EGCG水溶液,使水中EGCG与毒死蜱的浓度比=(1-2):1, 其中,最佳配比为 EGCG:毒死蜱=(0.5-1.0):0.5,(mg/L:mg/L),即可有效控制斑马鱼死亡,达到降低半静态养殖水中毒死蜱诱导斑马鱼死亡率的目的。
所述高浓度EGCG溶液是指由纯度≥90%的EGCG粉状固体配制成浓度≥5.0g/L的EGCG溶液。
本发明涉及利用纯植物提取剂EGCG的配比和使用方法,降低水中斑马鱼对毒死蜱的暴露死亡率。相关研究实验如下:
配制高浓度毒死蜱和EGCG的溶液,逐级稀释用于添加试剂。供试生物选室内驯养7d以上,自然死亡率小于2%,平均体长2~3cm的斑马鱼。试验前一天停止喂食。实验将供试鱼分为三组(Gn(1、2、3)),每组按照EGCG与毒死蜱的不同配比,分不同水平(GnL(1、2、3…7 ))。每组每个水平设3个平行样,并设置相应的对照组,用已曝气的水进行处理。实验观察到,72小时处理过程中毒死蜱与EGCG配比组,水环境pH值变化在7.6~8.1之间。观察24、48、72h后的死亡率,提取72h处理后斑马鱼头部的乙酰胆碱酯酶和肝脏的谷胱甘肽进行测定。
实验结果表明:
1斑马鱼死亡率变化:72小时驯养实验结果表明,斑马鱼的毒死蜱半致死浓度为0.5mg/L,EGCG没有诱导死亡。水中含有0.5mg/L的毒死蜱时,斑马鱼的死亡率分别是:24h为40%;48h和72h均为60%。添加不同比例的EGCG后,斑马鱼的死亡率明显降低。EGCG与毒死蜱的配比在0.25:1至4:1之间,EGCG均可以降低斑马鱼72小时内死亡率。但是不同配比,降低的程度不同。0.25:1至0.5:1之间倍比,死亡率从低配比的50%向高配比的死亡率20%逐渐降低;配比为1:1至 2:1时,72h内的死亡率均为0%;3至4倍比时死亡率为10%。实验表明存在EGCG与毒死蜱的最佳配比范围内,最大限度的降低斑马鱼死亡率; EGCG降低毒死蜱致斑马鱼死亡率有最佳配比范围,即EGCG:毒死蜱(mg/L: mg/L)= 1:1至2:1之间。同时当水中毒死蜱分别为0.6、0.7mg/L时,添加EGCG也有效的降低了斑马鱼死亡率。
2斑马鱼脑部AChE变化:斑马鱼脑部乙酰胆碱酯酶活性测定表明,比较毒死蜱存在时斑马鱼脑部AChE,EGCG的出现极大的提高了斑马鱼脑部AChE的浓度。 0.5mg/L毒死蜱浓度水平下,斑马鱼AChE浓度为3.8U/ mgprot,添加0.25:1至4.0:1之间的不同配比EGCG,AChE浓度基本按照递增规律上升,最高至14.00 U/ mgprot(EGCG:毒死蜱=2:1),之后略有下降,但仍维持在12.00 U/ mgprot。相较于没有添加EGCG,EGCG的出现提高AChE的浓度最高达368%。同时,这个AChE的水平变化与相应的斑马鱼死亡率相对应。0.6mg/L毒死蜱浓度水平下,虽然斑马鱼死亡率变化成波状不稳定的趋势,AChE的变化也基本与之相呼应。说明,EGCG对斑马鱼脑部AChE的浓度提高是影响斑马鱼死亡率的重要原因之一,并且在一定浓度的毒死蜱水平下作用有效。
3谷胱甘肽酶活性变化:0.5mg/L毒死蜱浓度降低了87.6%谷胱甘肽酶的活性浓度值。但是添加了不同配比水平的EGCG,改善了谷胱甘肽酶受毒死蜱抑制的状况,提高了谷胱甘肽酶的活性。这种提高不是随着EGCG配比的增加而增加,而是呈现不稳定的趋势。EGCG:毒死蜱=1:2(EGCG浓度为0.25mg/L)提高的比率达到最大值88.7%,另一个峰值是EGCG:毒死蜱=4:1(EGCG浓度为2.0mg/L),提高率达86.5%。
0.6mg/L,0.7mg/L毒死蜱浓度水平时,添加EGCG同样不同程度的提高了谷胱甘肽酶活性,并且和0.5mg/L水平一样,影响没有一定的规律。0.6mg/L毒死蜱,添加EGCG使EGCG:毒死蜱=1:4,提高92.0%的谷胱甘肽酶活性;另一个峰值为EGCG:毒死蜱=3:1,提高94.6%,达到最大。0.7mg/L毒死水平下,EGCG提高谷胱甘肽酶活性比率较小,峰值在EGCG:毒死蜱=1:2,和EGCG:毒死蜱=3:1处。
乙酰胆碱酯酶和谷胱甘肽酶均是有机磷农药和其他污染物的靶标物质。养殖水中注入与诱导斑马鱼死亡的毒死蜱具有一定配比的EGCG后,明显提高了被毒死蜱抑制的斑马鱼的乙酰胆碱酯酶的活性,抑制了毒死蜱诱导的斑马鱼脑部毒性;同时提高斑马鱼肝部谷胱甘肽酶活性,提高机体抗氧化力,抑制毒死蜱诱导的斑马鱼肝部毒性。这些影响可能降低了因毒死蜱诱导的水中斑马鱼的死亡率。
本发明对半静态水中斑马鱼对毒死蜱的暴露,检测、调控水中毒死蜱的浓度,通过添加EGCG试剂,调整 EGCG与水中毒死蜱的浓度比,达到降低斑马鱼的死亡率。发明方法通过原位或异位技术实施,简单易行,可提高斑马鱼暴露于毒死蜱的存活率,避免使用化学解毒剂带来的后续副作用,提高养殖的经济效益;同时避免排放废水中化学解毒剂的二次污染,提高环境经济效益。
附图说明
图 1为养殖单元处理示意图。
图2 为 0.5 mg/L 毒死蜱暴露下不同暴露时间和EGCG配比死亡率。
具体实施方式
实施例:
1 一个如图1所示的处理单元。单元体积为0.5 m3 ,EGCG配制装置。设取样口(取水样测定毒死蜱浓度),进水口,多孔进EGCG溶液口,搅拌装置等。
2 进水毒死蜱浓度调节由进水部分完成:依据测定的原水毒死蜱浓度,原水由进水口1输入,一定体积比例的稀释水由进水口2输入,混合并稀释后,使进水毒死蜱浓度为0.5mg/l , 输入处理单元。
3 EGCG注入方式1:单点重力注入,将EGCG注入稀释水中,共同由进水管输入,依赖水的流动混合后,共同注入处理单元。EGCG注入方式2:多孔重力注入,由EGCG配制设备通过多点位直接将EGCG注入处理单元,设多个溶液注入口。 两种方法均启动缓速搅拌,20min内,将EGCG与水混合均匀,达到EGCG:毒死蜱= 1:1至2:1之间的配比要求。
图2表明0.5mg/l 毒死蜱水平下,EGCG和毒死蜱的不同配比作用时, EGCG (mg/l):毒死蜱(mg/l)=0.5:0.5至1.0:0.5之间斑马鱼的死亡率最低。
Claims (3)
1.一种降低半静态养殖水中毒死蜱诱导斑马鱼死亡率的方法,其特征在于: 在含有毒死蜱的半静态养殖水中添加适量的EGCG ,使得养殖水中EGCG与毒死蜱的浓度为比=(1-2):1,所述的毒死蜱在水中的浓度为0.48-0.70mg/L 。
2.根据权利要求1所述的一种降低半静态养殖水中毒死蜱诱导斑马鱼死亡率的方法,其特征在于,具体操作步骤如下:
(1)取适量养殖水,测量水中毒死蜱母体起始浓度、pH值、水温,调整养殖水的pH=6.8~8.5,控制水温为18~25℃;
(2)根据步骤(1)测量得到的水中毒死蜱母体起始浓度,然后按比例加水稀释至毒死蜱的浓度为0.48-0.70mg/L, 再按比例向养殖水中加入已预先配制好的高浓度EGCG溶液,使水中EGCG与毒死蜱的浓度比=(1-2):1 ,即可有效控制斑马鱼死亡,达到降低半静态养殖水中毒死蜱诱导斑马鱼死亡率的目的。
3.根据权利要求1所述的降低半静态养殖水中毒死蜱诱导斑马鱼死亡率的方法,其特征在于:所述高浓度EGCG溶液是指由纯度≥90%的EGCG粉状固体配制成浓度≥5.0g/L的EGCG溶液。
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