【作者】 戴伟；

【导师】 许梓荣；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2008， 博士

【摘要】 饲料铅作为水产养殖生产中铅污染的重要来源,不仅会导致水产动物发生铅中毒,而且还能够通过食物链损害人类健康。本试验以罗非鱼为试验对象,探讨不同水平饲料铅在罗非鱼体内的残留及其对罗非鱼的毒性作用,并对应用以上残留量指标及毒性效应指标作为监测饲料铅污染的早期预警指示物的可行性进行分析；与此同时,应用硅酸盐纳米级微球(Layer silicate nanoparticle, LSN)吸附饲料中的铅,并从罗非鱼组织中铅残留及多项毒性效应指标的变化探讨LSN的驱铅效果,为LSN作为驱铅产品在实际生产中的广泛应用提供科学依据。主要研究内容和结果如下：1饲料铅在罗非鱼体内的残留及其对罗非鱼的毒性作用选择个体初重为32.17±0.34g罗非鱼480尾,随机分为4组,每组6个重复,每个重复20尾鱼。试验组分别饲喂：基础日粮(对照组),基础日粮添加100μg／g铅(100μg Pb／g暴露组),基础日粮添加400 gg／g铅(400μg Pb／g暴露组),基础日粮添加800μg/g铅(800μg Pb／g暴露组)。试验预试期为21d,正试期为60d,分别于第15d、30d、45d和60d取样,并进行相关指标测定。通过本试验发现：随着铅暴露剂量的增加及暴露时间的延长,罗非鱼体内肠、肾脏(中肾)、骨、胃、肝脏、鳃、脾脏、精巢、肉(背肌)和脑中铅残留量均呈上升趋势;铅在罗非鱼体内的蓄积分布具有不同的组织亲和性,在不同组织中的残留量由高到低依次为：肠>肾脏(中肾)>骨>胃>肝脏>鳃>脾脏>精巢>肉(背肌)>脑。以上研究结果揭示：肾脏和骨等铅残留量高的组织能够敏感地反映环境中铅胁迫情况,而胃、肠组织中铅残留量不仅可以作为饲料铅污染还可作为天然食料铅污染的监测指标。随着饲料铅暴露剂量的增加,罗非鱼肾脏、骨、肝脏、脾脏和精巢中锌、铁、铜含量均呈下降趋势,在不同的被测组织中,锌、铁、铜含量下降幅度不同；罗非鱼肝脏、肾脏中MDA含量呈上升趋势,而T-AOC及GSH水平呈下降趋势。另外,罗非鱼肝脏、肾脏中GSH-Px及SOD活性呈相反的变化趋势：肝脏中GSH-Px和SOD活性被铅诱导升高,而肾脏中GSH-Px和SOD活性呈抑制性下降；罗非鱼肝脏、肾脏中AP、Na, K-ATPase和Ca, Mg-ATPase活性均呈下降趋势。罗非鱼肝脏中ALT、AST和LDH活性呈上升趋势,而肾脏中ALT、AST和LDH活性呈下降趋势；罗非鱼肝脏、肠和胃中淀粉酶、胰蛋白酶和脂肪酶活性均呈下降趋势；罗非鱼血清中T3和T4水平呈下降趋势。与对照组罗非鱼相比,铅暴露组罗非鱼血细胞彗尾DNA含量、彗尾长、尾距、Olive尾距显著升高,且各指标均呈剂量-效应关系；与对照组相比,铅暴露组罗非鱼肝脏、肾脏和脾脏显微结构呈现病理变化,并且随着铅暴露剂量的增加,病理损伤逐渐加剧。以上研究结果表明：饲料铅对罗非鱼具有多种毒性效应,并且以上毒性效应指标的变化与铅污染水平间具有剂量-效应关系,协同使用以上多个指标非特异性地指示铅污染情况具有一定的可行性。2硅酸盐纳米级微球减轻饲料铅毒害影响的研究选择个体初重为32.244±0.38g罗非鱼240尾,随机分为4组,每组3个重复,每个重复20尾鱼。试验组分别饲喂：基础日粮(对照组),基础日粮添加0.5%LSN(LSN组),基础日粮添加100μg／g铅和0.5%LSN (Pb+LSN组),基础日粮添加100μg/g铅(Pb组)。饲养试验结束后,处死试验鱼并进行取样分析,试验结果表明：与对照组罗非鱼相比,基础日粮中添加LSN后罗非鱼肠、肾脏、骨、胃和脾脏中铅残留量显著下降(P<0.05)。与Pb组罗非鱼相比,Pb+LSN组罗非鱼肠、肾脏、骨、胃、肝脏、鳃、脾脏、背肌、精巢和脑中铅的残留量均显著下降(P<0.05)；LSN组罗非鱼肾脏、骨、肝脏、脾脏和精巢中锌、铁和铜的含量与对照组罗非鱼相比差异不显著(P>0.05)。与Pb组罗非鱼相比,LSN+Pb组罗非鱼被测组织中锌、铁和铜的含量升高(P>0.05)；LSN组罗非鱼肾脏中MDA含量、T-AOC水平、GSH含量、GSH-Px和SOD活性与对照组罗非鱼相比差异不显著(P>0.05)。与Pb组罗非鱼相比,LSN+Pb组罗非鱼肾脏中MDA含量下降(P>0.05),GSH含量、GSH-Px和SOD活性升高(P>0.05),T-AOC水平显著升高(P<0.05)；LSN组罗非鱼血细胞彗尾DNA含量、彗尾长、尾距、Olive尾距与对照组罗非鱼相比差异不显著(P>0.05)。与Pb组罗非鱼相比,LSN+Pb组罗非鱼血细胞彗尾DNA含量、彗尾长、尾距、Olive尾距均显著降低(P<0.05)；LSN组罗非鱼肾脏中ALT、AST、AP、LDH、Na, K-ATPase、Ca, Mg-ATPase活性与对照组罗非鱼相比差异不显著(P>0.05)。与Pb组罗非鱼相比,LSN+Pb组罗非鱼肾脏中ALT、AST、AP、LDH、Na, K-ATPase、Ca, Mg-ATPase活性升高(P>0.05);LSN组罗非鱼肝脏中淀粉酶、胰蛋白酶与脂肪酶活性与对照组罗非鱼相比差异不显著(P>0.05)。与Pb组罗非鱼相比,LSN+Pb组罗非鱼肝脏中淀粉酶、胰蛋白酶与脂肪酶活性升高(P>0.05)。以上试验结果揭示：LSN能够有效吸附饲料中的铅,减少罗非鱼体内铅的残留量,降低铅致罗非鱼的毒害影响。更多还原

【Abstract】 Dietary Pb, being one of the important Pb pollutants in aquaculture, could lead to toxic effects on aquatic animal and endangered the human health through food chains. In this study, the toxic effects of dietary Pb on tilapia (Oreochromis niloticus) were investigated, and the potential of using exposure effect index and toxicicty effect indexes to monitor dietary Pb contamination was evaluated. In addition, the experiment was carried out to investigate the effectiveness of layer silicate nanopartilce (LSN) on dietary Pb adsorption, and the effects of LSN on reducing dietary Pb toxicity was conducted on Pb residues in tissues and toxicity effect indexes. The main results were presented as follows:1 Effects of dietary Pb on Pb residues in tissues of tilapia and toxic effects of dietary Pb on tilapia480 fish (mean weight,32.17±0.34g) were selected and randomly divided into four treatments with six replicates (20 fish per replicate). After three weeks of acclimation, fish were fed with diet (0,100,400 and 800μg Pb/g dw diet) at 3-3.5% fresh body weight twice daily. At 15d,30d,45d and 60d of exposure period, fish were killed and weighted. Blood and tissue samples were collected and determined for corresponding indexes. The main results were shown as follows:It was indicated that Pb residues in tissues of tilapia increased with dietary Pb exposure concentrations and period. Moreover, Pb accumulated in sampled tissues in the following order:intestine> kidney> bone> stomach> liver> gill> spleen> testis> muscle> brain. The results showed that Pb residues in kidney and bone exhibited high Pb burden could be used as surrogate biomarkers to monitor Pb pollution, and Pb residues in intestine and stomach could indicate Pb contamination in diet specially.Pb concentration-dependent decreases in trace elements (Zn, Fe and Cu) content were observed in kidney, bone, liver, spleen and testis of tilapia. In different sampled tissues, trace elements (Zn, Fe and Cu) metabolisms were disturbed at various levers. It was showed that malonyldialdehyde (MDA) levels in liver and kidney increased with dietary Pb concentrations, total antioxidant capacity (T-AOC) and glutathione (GSH) levers in liver and kidney decreased with dietary Pb exposure concentrations. It was also demonstrated that glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities in liver and kidney were affected by dietary Pb in a dissimilar way:Pb concentration-related decreases in GSH-Px and SOD activities were observed in kidney, while these two enzyme activities in liver were stimulated in a Pb concentration-dependent manner. It was observed that tail length (TL), TDNA%, tail moment (TM) and olive tail moment (OTM) of peripheral blood cells in tilapia increased with the increasing dietary Pb concentrations. Alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) activities in liver and kidney were affected by dietary Pb in a contrary way:Pb concentration-related decreases in ALT, AST and LDH activities were observed in kidney, while Pb concentration-dependent stimulative effects on above enzyme activities were observed in liver. It was also demonstrated that the inhibitory effects of dietary Pb on alkaline phosphatase (AP), Na, K-ATPase, Ca, Mg-ATPase activities in both liver and kidney were Pb concentration-dependent. The inhibitory effects of dietary Pb on amylase, trypsin and lipase enzyme activities were dietary Pb concentration-dependent. It was found that T3 and T4 levels in serum were inhibited in a Pb concentration-dependent manner. By observation of histological sections of liver, kidney and spleen in optical microscope, Pb concentration-related lesions could be detected in these tissues of Pb-exposed treatments. The results revealed that dietary Pb could lead to Pb concentration-dependent toxicity on tilapia, and cooperative above toxic effect indexes could indicate dietary Pb contamination.2 Effects of LSN on reducing dietary Pb toxicity240 fish (mean weight,32.24±0.38g) were selected and randomly divided into four treatments with three replicates (20 fish per replicate). The feeding experiment lasted for 60d after three weeks of acclimation. The treatments were denominated as follows: control treatment (basic dietary), LSN treatment (basic dietary added with 0.5% LSN), Pb treatment (basic dietary added with 100μg Pb/g dw), Pb+LSN treatment (basic dietary added with 100μg Pb/g dw and 0.5% LSN). After 60d of exposure period, fish were killed and weighted. Tissue samples were collected and determined for corresponding indexes. The main results were presented as follows:As compared with control treatment, supplementation of 0.5% LSN significantly reduced Pb residues in intestine, kidney, bone, stomach and spleen (P< 0.05). Compared to Pb treatment, Pb residues in intestine, kidney, bone, stomach, liver, gill, spleen, muscle, testis and brain of Pb+LSN treatment significantly decreased (P< 0.05), respectively. No significant differences on trace elements (Zn, Fe and Cu) content in kidney, bone, liver, spleen and testis were observed between control treatment and LSN treatment (P> 0.05). Compared to Pb treatment, trace elements (Zn, Fe and Cu) content in sampled tissues of Pb+LSN treatment increased insignificantly (P> 0.05). No significant differences on levels of MDA, GSH, T-AOC and activities of GSH-Px, SOD in kidney were observed between control treatment and LSN treatment (P> 0.05). Compared to Pb treatment, MDA level in Pb+LSN treatment decreased insignificantly (P> 0.05), while GSH level, GSH-Px activity and SOD activity were enhanced insignificantly (P> 0.05) and T-AOC level increased significantly(P< 0.05). No significant differences on TL, TDNA%, TM and OTM of peripheral blood cells were observed between control treatment and LSN treatment (P > 0.05). Significant decreases in TL, TDNA%, TM and OTM of peripheral blood cells of Pb+LSN treatment were observed when compared with that of Pb treatment. No significant differences on ALT, AST, AP, LDH, Na, K-ATPase, Ca, Mg-ATPase activities in kidney were observed between control treatment and LSN treatment (P>0.05). As compared with Pb treatment, ALT, AST, AP, LDH, Na, K-ATPase, Ca, Mg-ATPase activities in kidney of Pb+LSN treatment were insignificantly stimulated (P>0.05). No significant differences on amylase, trypsin and lipase activities in liver were observed between control treatment and LSN treatment (P>0.05). Compared to Pb treatment, amylase, trypsin and lipase activities in liver of Pb+LSN treatment increased insignificantly (P>0.05).In conclusion, LSN could reduce Pb residues in tissues and Pb toxicity through its effectiveness on Pb adsorption.更多还原