【作者】 王志富；

【导师】 俞志明； 宋秀贤；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 环境科学， 2014， 博士

【摘要】 作为有害藻华的一种防治方法，改性粘土絮凝法的有效性已在中国、日本、韩国和澳大利亚等多个国家和地区被实践证明。本文中，作者在实验室内以仿刺参（Apostichopus japonicas Selenka）稚参、虾夷扇贝(Patinopecten yessoensis)稚贝和锥状斯氏藻（Scrippsiella trochoidea）为实验对象，研究了改性粘土治理藻华对三种典型底栖生物的影响，结果如下：1改性粘土治理藻华对仿刺参稚参的影响研究。96h急性毒性试验发现，改性粘土对仿刺参稚参的半致死浓度（LC50）为6.01g/L；安全浓度为0.601g/L，高于现场使用浓度（0.1g/L）；慢性毒性试验显示改性粘土对仿刺参稚参成活率和体重增长率无显著影响，不同浓度的改性粘土添加组中，发现仿刺参稚参的体壁组份（包括水分、灰分、总糖、总脂和粗蛋白）含量变化不大，改性粘土的加入对减少仿刺参稚参的种内竞争有一定积极作用。另外，针对仿刺参稚参食用包括改性粘土在内的底层颗粒物的现象，考察了不同实验组仿刺参稚参体壁中铝的含量，分析结果显示各实验组与对照组没有显著差异。在此基础上，考察了改性粘土有效去除有害藻华的过程仿刺参稚参的影响，发现与对照组相比，改性粘土不仅有效的去除了有害藻华，还明显降低了仿刺参稚参的死亡率。因此，适量的改性粘土是一种有效治理仿刺参养殖水体有害藻华、对仿刺参无负面影响的藻华治理技术。2改性粘土治理藻华对虾夷扇贝稚贝的影响研究。96h急性毒性试验发现，改性粘土对虾夷扇贝稚贝的半致死浓度（LC50）为2.3g/L，安全浓度为0.23g/L，高于其通常的现场使用浓度0.1g/L。慢性毒性试验显示，0.1到1.0g/L的改性粘土对虾夷扇贝稚贝的存活率、壳长和壳高略有影响，但是影响不显著。对稚贝滤食率的研究发现，加入改性粘土会影响其摄食，并且随着改性粘土浓度的增高影响越明显。模拟养殖水体爆发藻华后喷洒改性粘土的实验表明，改性粘土在有效去除有害藻华营养细胞，改善水体环境的同时，还能使虾夷扇贝稚贝的存活率从22%提高到38%。另外，如采用先用改性粘土治理，再向养殖水体投放稚贝的策略，稚贝的存活率会大大提高，约是对照组的3倍。本文的实验结果说明采取适当的策略和适当用量的改性粘土既可以效去除有害藻华，也可以对虾夷扇贝稚贝无害甚至有益，是一种极具应用前景的藻华治理技术方法。3改性粘土对锥状斯氏藻孢囊的影响研究。锥状斯氏藻在改良的f/2培养基中培养到对数生长期的中期（第10天）后，分别加入浓度为0,0.1,0.5和1.0g/L的改性粘土。实验发现：改性粘土对锥状斯氏藻去除效果明显，加入改性粘土24h后，随着改性粘土浓度的增加，营养细胞的去除率从69.1%增加到97.7%。而在改性粘土组中，孢囊的形成率也较高，最高的形成率出现在第四组，为24.6%。这说明改性粘土的加入，能够促使部分营养细胞停止增殖，形成孢囊沉积到水底，孢囊的形成有助于有害藻华的消亡。另外，作者发现在实验中形成的孢囊有两种主要形态：具刺孢囊和无刺孢囊。两种孢囊在对照组和粘土组中所占的比例有巨大差异，从对照组到改性粘土组，具刺孢囊所占比例分别为76.9%,46.6%,39.7%和24.1%。这种现象的原因可能是：改性粘土吸附水体中的Ca2+沉降到水底，使得孢囊形成时水体中没有足够的钙元素形成钙质刺。通过萌发实验发现，相对于具刺孢囊，无刺孢囊的萌发周期长，萌发率低。由于添加改性粘土后更易于形成无刺孢囊，这使得改性粘土组中形成的孢囊的萌发率远比对照组中的孢囊低，各组中孢囊的萌发率分别为82.1%，71.3%，47.5%和45.0%。在进行萌发实验前，为了让孢囊度过强制性休眠期，在低温黑暗中储存了3个月，发现改性粘土组孢囊数量降低，这说明在实验开始时改性粘土组形成的孢囊中，有部分是因为粘土的加入造成环境的突变而形成的暂时性孢囊，这部分孢囊只能短暂抵抗不利环境，长时间的低温保存使得孢囊细胞破裂死亡。总体来说，改性粘土对锥状斯氏藻有较好的去除效率，能够促使营养细胞转变成孢囊，从而促进藻华的消亡，粘土组中更容易形成萌发率低的无刺孢囊和暂时性孢囊，从最终结果来看，改性粘土使用不会为下次藻华的爆发留下比对照组多的种源。该研究为有害藻华的防治提供了重要的参考依据。另外，为了解释改性粘土对锥状斯氏藻孢囊的影响结果，作者研究了NO3-N和NH4-N对锥状斯氏藻生长和孢囊形成的影响。锥状斯氏藻培养在缺少氮和硅的f/2培养基中，将培养液分为两组，经过3天的饥饿培养后，第一组以硝酸盐作为氮源，第二组用铵盐作为氮源，浓度梯度均设置为0、10、30、60和90μM。最终的结果显示NO3--N能够促进藻细胞的生长，但是浓度越高越不利于孢囊的形成。而NH4-N则能同时促进藻细胞的生长和孢囊的形成。在相同浓度下，NH4-N不如NO3-N利于藻细胞的生长，但是其能明显提高孢囊的形成率。这说明NH4-N浓度是调控孢囊形成的一个重要因子。我们认为NH4-N对孢囊的诱导作用是生态系统的一种自我调节能力，具有非常重要的生态学意义。更多还原

【Abstract】 Modified clay is considered an effective protective measure against harmful algalbloom (HAB) and has been applied successfully in China, Japan, South Korea, andAustralia.We present results on the effect of modified clay on marine benthos inHABs’ controlling. We choose3marine benthos as experiment subject: Apostichopusjaponicas Selenka, Patinopecten yessoensis, Scrippsiella trochoidea.The main resultsare shown follow:1Effects of modified clay on the infant of Apostichopus japonicas.The halflethal concentration（LC50）of modified clay on the infant of A.japonicas was6.01g/Lin the96h acute test. In chronic toxicity tests, the growth rates and survival rates inmodified clays were almost as same as control groups. When less than1.0g/Lmodified clay added, the moisture, ash, sugar, fatty acid and protein contents of thetissues had no significant changes. After60days incubation experiments with theconcentration of1.0g/L modified caly, there was no significant effect on thealuminum content of the tissues. Meanwhile, the study studied the removing ofharmful algal Prorocentrum donghaiense, and the efficiency concentration ofmodified clay was0.1g/L and0.5g/L, which could simutaneously reduce the mortalityof the juvenile A.japonicas. Thus, there were no negative impacts of the modified clayflocculation on the benthic A.japonicas, and the modified clay treatment was aneffective and reliable strategy to mitigate the harmful algaes.2Effects of modified clay on the infant of Patinopecten yessoensis.The half lethalconcentration（LC50）of modified clay on the infant of P. yessoensis was2.3g/L, andthe safe concentration was0.23g/L, which was more than two times higher than thefield application concentration（0.1g/L）in96h acute toxicity test. The chronic toxicity test showed the influence of modified clay on the survival rate, shell length and heightwas insignificant when the concentration of clay increased from0.1to1.0g/L. Andthe feeding of P. yessoensis infant was effected by modified clay, with highermodified clay concentration leading to greater influence. We also studied the effectsof P. yessoensis infant when harmful algae（Prorocentrum donghaiense）bloom wascontrolled by modified clay. Compared with the control, the addition of modified clayimproved the survival rate of P. yessoensis from22%to38%. Meanwhile, it improved3times than control group, when we picked out the P. yessoensis before the additionof clay and then put P. yessoensis into the culture. To sum up, the addition of modifiedclay not only controlled HABs effectively, but also improved the survival of P.yessoensis infant. It was a meaningful technology to mitigate HABs.3Effects of modified clay on cysts of Scrippsiella trochoidea. Concentrationsof modified clay（0,0.1,0.5, and1.0g/L）were added to cultures, and observationswere made oncysts of S.trochoidea under controlled laboratory conditions. Resultsindicate that the removal rate of algal cells reached97.7%at the clay concentrationof1.0g/L. The cyst formation rate increased from4.6%to24.6%when theconcentration of clay was increased from0to1.0g/L. Two cyst metamorphs wereobserved: spinal calcareous cysts and smooth noncalcareous ones. The proportion ofthe spinal cysts decreased from76.9%to24.1%when clay concentration increasedfrom0to1.0g/L. In addition, modified clay affected cyst germination. Thegermination rate decreased with the increases in the clay concentrations.Non-calcareous cysts had a lower germination rate and a longer germination time. Weconclude that modified clay could depress algal cell multiplication and promoteformation of temporal cysts of S. trochoidea, which may help in controlling HABoutbreaks.In addition, the author also investigated the effects of nitrate and ammonium onthe growth and encystment of S. trochoidea. We incubated S. trochoidea in modifiedf/2media without nitrogen and silicate in flasks. The flasks were divided into twogroups. Nitrate was added as a nitrogen source in the first group, and ammonium was added in the second group. The concentrations of the nitrogen compounds were0,10,30,60, and90μM. The duration of the experiment was14days. The results indicatethat NO3-N favors cell growth, and cultures with a higher concentration of NO3-Nwere ineffective at forming cysts. In contrast, NH4-N promoted cell growth and cystformation. At similar concentrations as NO3-N, NH4-N had a toxic effect on cellgrowth and increased the cyst formation rate. Thus, the NH4-N concentration is animportant factor for controlling encystment. We believe that the impact of ammonia ininducing cyst formation may be a useful feedback mechanism in ecological systems.更多还原