淡水环境重金属监测用“标准背角无齿蚌”群体的建立及移殖应用研究
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摘要
背角无齿蚌(Anodonta woodiana)隶属于软体动物门,瓣鳃纲,蚌目,蚌科,无齿蚌属,在全球广泛分布。基于背角无齿蚌作为指示生物的贝类监测已成功评价太湖重金属、有机锡及有机氯等持续性污染物污染状况。然而,迄今基于贝类的生物监测(包括被动监测和主动监测)均依赖于采集野生资源。这就面临着样本的规格、年龄、生长率、发育程度等生物学因子以及污染暴露史难以控制；一定程度上会破坏野生贝类资源量,在某些水域甚至采不到样本等棘手问题。创新性地开发出生物学背景相同,遗传质量稳定,污染本底值低,可向待测水域移殖和回捕,即标准化了的监测专用背角无齿蚌(本文称之为“标准背角无齿蚌”)群体,既可以解决上述问题,又能适应今后建立大规模、规范化、信息化的“淡水贝类观察”监测研究体系的需求。为实现该目的,本论文主要从以下几个方面开展了研究：
1.不同水域背角无齿蚌体内重金属积累的差异性：比较研究了中国水产科学研究院淡水渔业研究中心南泉基地(无明显外源污染)、太湖梅梁湾(历史上污染较为严重)以及云南茈碧湖(高原洁净水体)中的背角无齿蚌软组织中重金属(A1、Cr、 Mn、Fe、Co、Ni、Cu、Zn、As、Mo、Ag、Cd、Ba、T1及Pb)的积累特征。结果表明,南泉基地养殖蚌中A1、Mn、Ni、Cu、Zn、As、Cd、Ba、Fe和Cr的含量显著低于梅梁湾野生蚌(P<0.05)；茈碧湖野生蚌中A1、Mn、Ni、Cu、Zn、As、Cd、Ba和Pb的含量显著低于太湖梅梁湾(P<0.05)；虽然南泉基地养殖蚌和茈碧湖野生蚌中的重金属积累均显著低于太湖梅梁湾但前者中的Cu、As和Pb含量仍高于后者(P<0.05)。太湖梅梁湾、南泉基地和茈碧湖蚌的金属污染指数(MPI)分别为11.1、1.8和1.0。另外,南泉基地养殖蚌中A1-Cd, Cr-Mo, Fe-Zn, Fe-Cd, Fe-Pb及Zn-Pb含量间呈显著相关性(P<0.05)；茈碧湖野生蚌中Cr-Fe, Cr-As, Mn-Bs, Fe-Mo和Mo-Ba含量间呈显著相关性(P<0.05)；而梅梁湾野生蚌中Mn-Cu, Mn-Zn, Mn-Mo, Mn-Cd, Mn-Pb, Fe-Ni, Fe-Cu, Fe-Cd, Fe-Pb, Ni-Cu, Ni-Pb, Cu-Mo, Cu-Cd, Cu-Pb, Zn-Mo, Zn-Cd, Zn-Pb, Mo-Cd, Mo-Pb以及Cd-Pb含量间呈显著相关性(P<0.05)；后者的重金属积累关系远较前两者复杂。显示出背角无齿蚌中重金属的积累程度及复杂性与其生活环境的污染背景有密切关系,在较为洁净的水域繁育和养殖的背角无齿蚌应该可以控制其体内环境重金属积累在较低的水平。
2.“标准背角无齿蚌”群体的建立：首次详细研究了背角无齿蚌从受精卵至性成熟的全生活史过程的生境条件、发育机制和生长规律。结果表明：钩介幼虫指数(Gin)为0.077,属于大型；钩介幼虫寄生在黄颡鱼(Pelteobagrus fulvidraco)鳃丝上发育到刚脱落稚蚌的生物学零度是8.89℃,有效积温为86.39℃·d；寄生期间,壳长增长显著(P<0.01)。0-60d稚蚌由原钩介幼虫状逐渐向成体形态转变,斧足、鳃、心脏、肠道、出水管及入水管等器官渐渐发育完全；60d以后幼蚌的形态结构与成体相同；至750d,90%以上的个体发育至性成熟并开始进行繁殖。背角无齿蚌为异速生长,0-60d稚蚌壳长(L)与生长时间(t)呈二次函数关系,L=0.383-0.031t+0.003t2(R2=0.953,P<0.01)；60-750d幼蚌的壳长(L)、壳宽(W)、壳高(H)与生长时间(t)呈逆函数关系,分别为L=67.071-3364/t(R2=0.924,P<0.01),W=25.875-1326／t(R2=0.779,P<0.01)和H=41.441-2092/t(R2=0.907,P<0.01)。然而,壳长、壳宽和壳高之间均满足线性相关,关系式分别为L=8.168+2.208W(R2=0.921,P<0.01),L=1.557+1.571H(R2=0.973,P<0.01),W=0.641H-0.663(R2=0.920,P<0.01),L=4.868+0.875W+0.929H(R2=0.950,P<0.01),表明三者之间按照一定的比例保持等速增长。本研究人工繁育的条件下成功建立有12000余只生物学背景和遗传质量可控、标准化的背角无齿蚌养殖群体,并发现水温、水流速率和养殖密度是影响蚌存活率的关键因素。
3.不同生活史过程“标准背角无齿蚌”重金属积累的动态：应用电感耦合等离子质谱仪分析了不同生活史阶段“标准背角无齿蚌”(钩介幼虫,幼蚌软组织和壳,及母蚌软组织和壳)中重金属(Al、Cr、Mn、Fe、Co、Ni、Cu、Zn、As、Se、Mo、 Ag、Cd与T1)的积累动态,并进一步使用X射线电子探针微区分析仪分析了母蚌壳中重金属(A1、Ag、Co、Cu、Fe、Mn、Mo、Se、T1和Zn)的分层特征。结果表明：钩介幼虫中重金属总体含量较低,但Cr (0.5μg·g-1)、Ni (0.9μg·g-1)和As (5.0μg·g-1)已明显检出。幼蚌(S1)(平均壳长17.5mm)和幼蚌(S2)(平均壳长35.8mm)软组织中重金属(Cr、Ni、As、Se、Cu、Zn和Cd等)含量很低,且含量水平一致(P>0.05),但是Co、Cu、Mo、Ag和T1显著高于母蚌(P<0.05)。母蚌软组织中Ni、Zn、As、Se和Cd含量显著高于幼蚌(P<0.05),且As的含量较高,甚至超出了我国《食品中污染物限量》中的最大残留量。幼蚌和母蚌壳中重金属组成和含量总体比较稳定,基本呈Fe> Mn> A1> Ag> Mo> Cu> Co> Se> T1> Cr≈Ni≈Zn≈As≈Cd的趋势。此外,Mn的含量在珍珠层显著高于棱柱层(P<0.05),而其余重金属在壳层中分布均匀。提示除母蚌会残留原采集环境、钩介幼虫会受到母蚌重金属背景的少量影响外,全人工繁育并养殖的幼蚌中环境重金属污染程度非常轻,积累背景非常低。
4.野外水域“标准背角无齿蚌”的移殖重金属监测：移殖“标准背角无齿蚌”幼蚌至太湖五里湖和武汉东湖作为“实验组”,并保留在南泉基地的同一批蚌为对照组；每3个月定期回收样本,进行重金属监测的初步应用。结果表明：移殖3mo,南泉基地Mn、Mo、Ni、Zn、Pb、As和Ba的含量显著上升(P<0.05),而Al的含量显著下降(P<0.05)；五里湖Fe、Ni、Cr、Zn、As、Mo、Mn、Ba、T1和Pb的含量显著上升(P<0.05)；东湖Mo和As的含量显著上升(P<0.05),而Cr、A1、T1和Pb的含量显著下降(P<0.05)。其中,Mn、Fe、Zn、Cd、Ba和Pb在南泉的含量最高,而Al、Cr、Ni、Cu、As、Mo和Tl在五里湖的含量最高。南泉基地、五里湖和东湖的MPI分别为8.6、14.1和1.6。移殖6mo,南泉基地Fe的含量显著下降(P<0.05)；五里湖Fe、Mo、Cr、Ni、As、Ba、T1和Pb的含量显著下降(P<0.05)；东湖Mo的含量明显上升(P<0.05),而Fe和As的含量显著下降(P<0.05)。其中,Ni、Cu和Tl在三者中均未检出,Cr、Mn、Fe、Zn、As、Mo、Cd、Ba和Pb在南泉的含量最高,而A在五里湖的含量为最高。南泉基地、五里湖和东湖的MPI分别为1.7、0.4和0.3。有效地反映了不同水环境重金属污染的时空动态特征。
本研究客观地证实了背角无齿蚌重金属积累与不同污染程度水域的背景相关,在有效掌握了背角无齿蚌生境条件、发育机制和生长规律并建立了确保存活率方法的基础上,成功建立了生境、种质、规格、年龄、生长及发育程度等一致的“标准背角无齿蚌”群体。并通过对其不同生活史阶段的个体进行了重金属积累特征和背景的跟踪研究,为移殖对象的选择和监测结果的解释奠定了必要的基础。进而创新性地移殖“标准背角无齿蚌”至不同水域,有效地反映了不同水环境的重金属含量时空动态特征。所有这些为正在开展的淡水渔业环境重金属“背角无齿蚌观察”研究提供了移殖监测的新思路,也为今后“标准背角无齿蚌”群体的完善和应用的拓展提供了理论基础。
Anodonta woodiana (Mollusca, Lamellibranchia, Eulamellibranchia, Unionidae, Anodonta) is a worldwide-distributed freshwater mussel, which has proven useful for temporal and spatial monitoring of persistent pollutants (e.g., heavy metals, organotins, and organochlorines) in aquatic environments as bioindicators. To date, however, the utilization of mussel-based monitoring (both passive biomonitoring and active biosurveillance) is generally dependent on field-collected mussels. As a result, it is difficult to ensure that the mussels will possess the same biological factors (e.g., size, age, growth rate, sexual maturity) and exposure history of heave metals. These conditions may significantly influence the bioaccumulation pattern and fate of heavy metals. Moreover, sample collection may also be difficult or impossible as wild mussels sometimes are rare, or even absent at study locations. In addition, sampling of wild mussels possibly destroys their nature population. Herein, it must be very important to establish an innovative 'standardized' mussel population for environment monitoring (e.g.,"standardized A. woodiana" of the present study) possessing the same biological factors, stable inherited quality and low background of heavy metals. Furthermore, the mussels can subsequently be transplanted and retrieved for metal pollution monitoring. There is no doubt that that the approach of artificial propagation and culture of unionid mussels will be very helpful to set up such a population. Corresponding studies have been conducted in this study to establish a "standardized A. woodiana" population, which can contribute the large-scale, standardized, informationalized biomonitoring system of "Fershwater Mussel Watch". The main results can be summarized as follows:
1. The difference of heavy metal bioaccumulation in A. woodiana from different waters:Bioaccumulation characteristics of heavy metal (Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Mo, Ag, Cd, Ba, Tl, and Pb) in soft tissues of cultured A. woodiana from Nanquan Aquatic Base of Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences (without obvious exogenous metal pollution), and wild mussels from Meiliang Bay of Taihu Lake (suffered relatively serious pollution) and Cibihu Lake in Yunnan Province (a very clean plateau lake) were investigated by an Agilent7500ce inductively coupled plasma mass spectroscopy (ICP-MS). The results of the present study showed that concentrations of Al, Cr, Mn, Fe, Ni, Cu, Zn, As, Cd and Ba in wild mussels of Meiliang Bay were significantly higher than cultured mussel (P<0.05). Moreover, concentrations of Al, Mn, Ni, Cu, Zn, As, Cd, Ba and Pb of Meiliang Bay were significantly higher than wild mussels from Cibihu Lake (P<0.05). Additionally, concentrations of Cr, Fe, Mo and T1in cultured mussels were significantly lower than wild mussels from Cibihu Lake (P<0.05). However, concentrations of Cu, As and Pb in the former were significantly higher than the latter (P<0.05). The metal pollution index (MPI) of mussels in Nanquan Aquatic Base, Meiliang Bay, and Cibihu Lake were1.8,11.1and1.0, respectively. In addition, Al-Cd, Cr-Mo, Fe-Zn, Fe-Cd, Fe-Pb, and Zn-Pb in mussels of Nanquan Aquatic Base showed significantly positive correlation (P<0.05). Mn-Cu, Mn-Zn, Mn-Mo, Mn-Cd, Mn-Pb, Fe-Ni, Fe-Cu, Fe-Cd, Fe-Pb, Ni-Cu, Ni-Pb, Cu-Mo, Cu-Cd, Cu-Pb, Zn-Mo, Zn-Cd, Zn-Pb, Mo-Cd, Mo-Pb, and Cd-Pb in mussels of Meiliang Bay showed significantly positive correlation (P<0.05). And Cr-Fe, Cr-As, Mn-Bs, Fe-Mo, and Mo-Ba in mussels of Cibihu Lake showed significantly positive correlation (P<0.05). The study indicated that bioaccumulation of heavy metals in A. woodiana presented positive correlation with the pollution background of water-bodys. Individuals of A. woodiana with relatively low level background of heavy metals can be possibly obtained by culture of the mussels in clean water-body.
2. Establishment of a population of "standardized A. woodiana":The present study investigates firstly the habitat condition, development mechanism, and growth characteristics of A. woodiana throughout whole life cycle from fertilized egg to mature adults. The results showed that glochidia belong to large size as the glochidia index (Gin) is0.077. The biological zero and the effective temperature sum were calculated to be8.89℃and86.39°C-d respectively during the period of their parasitic metamorphosis development in the gill filaments of yellow catfish (Pelteobagrus fulvidraco). During metamorphosis from glochidia to early free-living juveniles, the shell length increased significantly (P<0.01). The shape of0-day-old juveniles is similar as glochidia but with a strong foot which could drag the body to crawl all around. Until60-day-old the morphology of juveniles begin to be as same as adults. Moreover, the organs such as visceral mass, intestine, incurrent siphon, excurrent siphon, and gills have developed. The form of the juvenile at750-day-old resembles that of a fully grown adult and more than90%females are pregnant with glochidia. It is found that the growth of A. woodiana was allometric. The relationship between shell length and growth time for0-60-day juveniles was quadratic, L=0.383-0.031t+0.003t2(R2=0.953, P<0.01). For60-750-day-old individuals, the relationships of length, width, height and time were all inverses, L-67.071-3364/t (R2=0.924, P<0.01), W=25.875-1326/t (R2=0.779, P<0.01), and H=41.441-2092/t (R2=0.907,P<0.01), respectively. In addition, the general relationships of size (length-width, length-height, and width-height as well as length-width-height) were linear, L=8.168+2.208W (R2=0.921, P<0.01), L=1.557+1.571H(R2=0.973, P<0.01), W=0.641H-0.663(R2=0.920, P<0.01), and L=4.868+0.875W+0.929H (R2=0.950, P<0.01), respectively. A population of "standardized A. woodiana" with ca.12000individuals has been successfully established in the present study, and are believed water temperature, the rate of flowing water, and carrying capacity as the key factors that influence the survival rate of A. woodiana during the process of artificial propagation and culture of this mussel.
3. The bioaccumulation dynamics of heavy metals in "standardized A. woodiana"at different stages of life cycle:The accumulation level and dynamics of heavy metals (Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, Ag, Cd, and T1) in "standardized A. woodiana" at different life stages (glochida, juveniles and gravid adults) were assessed by the Agilent7500ce ICP-MS. Furthermore, the profiles of heavy metals (Al, Ag, Co, Cu, Fe, Mn, Mo, Se, T1, and Zn) in the shell of an adult were studied by the JEOL JXA-8100electron probe microanalyzer (EPMA). The results showed most of heavy metals in glochidia were low, but Cr (0.5μg·g-1), Ni (0.9μg·g-1), and As (5.0μg·g-1) were significantly detected. Concentrations of Cr, Ni, As, Se, Cu, Zn, and Cd in juvenile (S1) with average shell length17.5mm and juvenile (S2) with average length35.8mm were much low and the ranges of concentrations were narrow (P>0.05), while the concentrations of Cr, Ni, As, Se, Cu, Zn, and Cd were significantly higher than gravid adults (P<0.05). The concentrations of Ni, Zn, As, Se, and Cd in gravid adults were significantly higher than both juvenile (S1) and juvenile (S2)(P<0.05). Noteworthily, the concentration of As was so high than exceed the limit of Maximum Levels of Contaminants in Foods (GB2762-2005) of China. As to shells, the metal concentrations of all samples were similar except the concentration of Al in juvenile (S1) was significantly higher than gravid adults (P<0.05). Moreover, the concentrations of heavy metals generally decreased in the order Fe> Mn> Al> Ag> Mo> Cu> Co> Se> T1> Cr-Ni-Zn-As-Cd. In addition, the concentration of Mn in nacreous layer were significantly higher than prismatic layer (in the umbo and ventral edge)(P<0.05), while the distribution of other metals were uniformity at different layers. The results suggested that although the accumulation levels of several heavy metals in gravid adults and glochida might keep little influence from the background of collection sites and maternal mussels, respectively, those levels in cultured juveniles were obviously low and the metal contamination in these mussels were very weak.
4. Heavy metal monitoring by transplantation of "standardized A. woodiana":The juvenile mussels of "standardized A. woodiana" from the same artificially propagated and cultured population were divided into three sister groups. Two of them were transplanted into Wulihu Bay of Taihu Lake (Wuxi city) and Donghu Lake (Wuhan City), respectively as the experimental group for assessing heavy metal pollution. Meanwhile, the remaining one was still kept in Nanquan Aquatic Base as the control group. All cages were maintained at a depth of50cm from the water surface. And the samples were recovered per3months to determine heavy metal contents. The results showed that concentrations of Mn, Mo, Ni, Zn, Pb, As, and Ba in control in Nanquan Aquatic Base for3months (CN3) were significantly higher than control for all sites from beginning (CAO)(P<0.05), while the concentration of Al in the former was significantly lower than the latter (P<0.05). The concentrations of Fe, Ni, Cr, Zn, As, Mo, Mn, Ba, T1, and Pb in mussels transplanted Wulihu Lake for3months (TW3) were significantly higher CNO (P<0.05). The concentrations of Mo and As in mussels transplanted Donghu Lake for3months (TD3) were significantly higher CNO (P<0.05), but concentrations of Cr, Al, T1, and Pb were significantly lower than the latter (P<0.05). Comparison different water-bodies, the highest concentration of Mn, Fe, Zn, Cd, Ba, and Pb presented in CN3, while the highest concentration of Al, Cr, Ni, Cu, As, Mo, and T1presented in TW3. The MPI of CN3, TW3, and TD3were8.6,14.1, and1.6, respectively. In addition, the concentrations of Fe in control in Nanquan Aquatic Base for6months (CN6) were significantly lower than CN3(P<0.05). The concentrations of Fe, Mo, Cr, Ni, As, Ba, T1, and Pb in mussels transplanted Wulihu Lake for6months (TW6) were significantly lower than TW3(P<0.05). The concentration of Mo in mussels transplanted Donghu Lake for6months (TD6) was significantly higher TD3(P<0.05), but concentrations of Fe and As were significantly lower than the latter (P<0.05). Comparison different water-bodies, the highest concentration of Cr, Mn, Fe, Zn, As, Mo, Cd, Ba, and Pb presented in CN6, while the highest concentration of Al presented in TD6. The MPI of CN6, TW6, and TD6were1.7,0.4, and0.3, respectively. The results suggested transplant "standardized A. woodiana" is useful in monitoring temporal and spatial trends of heavy metals in aquatic environments.
The present study demonstrates objectively that bioaccumulation of heavy metals in A. woodiana correlated with the pollution background of water-bodys. A "standardized A. woodiana"with the same biological factors and exposure history of heave metals can be established based on mastering the habitat necessary, development mechanism, and growth characteristics throughout the whole life cycle. By the individuals of "standardized A. woodiana" at different stages of life cycle, the temporal variations of heavy metal bioaccumulation has been understood, which provide basic information for mussel-size selection for transplantation and interpretation of the results obtained during the research period. By means of transplantation monitoring of "standardized A. woodiana" to different nature water-bodies, the temporal and spatial dynamics have been clarified. All results of the present study provide new ideas for the current project of "Freshwater Mussel Watch" monitoring, and also provide the fundamental knowledges to perfect the aforementioned "standardized A. woodiana" population and extend its future applicaion.
1.不同水域背角无齿蚌体内重金属积累的差异性：比较研究了中国水产科学研究院淡水渔业研究中心南泉基地(无明显外源污染)、太湖梅梁湾(历史上污染较为严重)以及云南茈碧湖(高原洁净水体)中的背角无齿蚌软组织中重金属(A1、Cr、 Mn、Fe、Co、Ni、Cu、Zn、As、Mo、Ag、Cd、Ba、T1及Pb)的积累特征。结果表明,南泉基地养殖蚌中A1、Mn、Ni、Cu、Zn、As、Cd、Ba、Fe和Cr的含量显著低于梅梁湾野生蚌(P<0.05)；茈碧湖野生蚌中A1、Mn、Ni、Cu、Zn、As、Cd、Ba和Pb的含量显著低于太湖梅梁湾(P<0.05)；虽然南泉基地养殖蚌和茈碧湖野生蚌中的重金属积累均显著低于太湖梅梁湾但前者中的Cu、As和Pb含量仍高于后者(P<0.05)。太湖梅梁湾、南泉基地和茈碧湖蚌的金属污染指数(MPI)分别为11.1、1.8和1.0。另外,南泉基地养殖蚌中A1-Cd, Cr-Mo, Fe-Zn, Fe-Cd, Fe-Pb及Zn-Pb含量间呈显著相关性(P<0.05)；茈碧湖野生蚌中Cr-Fe, Cr-As, Mn-Bs, Fe-Mo和Mo-Ba含量间呈显著相关性(P<0.05)；而梅梁湾野生蚌中Mn-Cu, Mn-Zn, Mn-Mo, Mn-Cd, Mn-Pb, Fe-Ni, Fe-Cu, Fe-Cd, Fe-Pb, Ni-Cu, Ni-Pb, Cu-Mo, Cu-Cd, Cu-Pb, Zn-Mo, Zn-Cd, Zn-Pb, Mo-Cd, Mo-Pb以及Cd-Pb含量间呈显著相关性(P<0.05)；后者的重金属积累关系远较前两者复杂。显示出背角无齿蚌中重金属的积累程度及复杂性与其生活环境的污染背景有密切关系,在较为洁净的水域繁育和养殖的背角无齿蚌应该可以控制其体内环境重金属积累在较低的水平。
2.“标准背角无齿蚌”群体的建立：首次详细研究了背角无齿蚌从受精卵至性成熟的全生活史过程的生境条件、发育机制和生长规律。结果表明：钩介幼虫指数(Gin)为0.077,属于大型；钩介幼虫寄生在黄颡鱼(Pelteobagrus fulvidraco)鳃丝上发育到刚脱落稚蚌的生物学零度是8.89℃,有效积温为86.39℃·d；寄生期间,壳长增长显著(P<0.01)。0-60d稚蚌由原钩介幼虫状逐渐向成体形态转变,斧足、鳃、心脏、肠道、出水管及入水管等器官渐渐发育完全；60d以后幼蚌的形态结构与成体相同；至750d,90%以上的个体发育至性成熟并开始进行繁殖。背角无齿蚌为异速生长,0-60d稚蚌壳长(L)与生长时间(t)呈二次函数关系,L=0.383-0.031t+0.003t2(R2=0.953,P<0.01)；60-750d幼蚌的壳长(L)、壳宽(W)、壳高(H)与生长时间(t)呈逆函数关系,分别为L=67.071-3364/t(R2=0.924,P<0.01),W=25.875-1326／t(R2=0.779,P<0.01)和H=41.441-2092/t(R2=0.907,P<0.01)。然而,壳长、壳宽和壳高之间均满足线性相关,关系式分别为L=8.168+2.208W(R2=0.921,P<0.01),L=1.557+1.571H(R2=0.973,P<0.01),W=0.641H-0.663(R2=0.920,P<0.01),L=4.868+0.875W+0.929H(R2=0.950,P<0.01),表明三者之间按照一定的比例保持等速增长。本研究人工繁育的条件下成功建立有12000余只生物学背景和遗传质量可控、标准化的背角无齿蚌养殖群体,并发现水温、水流速率和养殖密度是影响蚌存活率的关键因素。
3.不同生活史过程“标准背角无齿蚌”重金属积累的动态：应用电感耦合等离子质谱仪分析了不同生活史阶段“标准背角无齿蚌”(钩介幼虫,幼蚌软组织和壳,及母蚌软组织和壳)中重金属(Al、Cr、Mn、Fe、Co、Ni、Cu、Zn、As、Se、Mo、 Ag、Cd与T1)的积累动态,并进一步使用X射线电子探针微区分析仪分析了母蚌壳中重金属(A1、Ag、Co、Cu、Fe、Mn、Mo、Se、T1和Zn)的分层特征。结果表明：钩介幼虫中重金属总体含量较低,但Cr (0.5μg·g-1)、Ni (0.9μg·g-1)和As (5.0μg·g-1)已明显检出。幼蚌(S1)(平均壳长17.5mm)和幼蚌(S2)(平均壳长35.8mm)软组织中重金属(Cr、Ni、As、Se、Cu、Zn和Cd等)含量很低,且含量水平一致(P>0.05),但是Co、Cu、Mo、Ag和T1显著高于母蚌(P<0.05)。母蚌软组织中Ni、Zn、As、Se和Cd含量显著高于幼蚌(P<0.05),且As的含量较高,甚至超出了我国《食品中污染物限量》中的最大残留量。幼蚌和母蚌壳中重金属组成和含量总体比较稳定,基本呈Fe> Mn> A1> Ag> Mo> Cu> Co> Se> T1> Cr≈Ni≈Zn≈As≈Cd的趋势。此外,Mn的含量在珍珠层显著高于棱柱层(P<0.05),而其余重金属在壳层中分布均匀。提示除母蚌会残留原采集环境、钩介幼虫会受到母蚌重金属背景的少量影响外,全人工繁育并养殖的幼蚌中环境重金属污染程度非常轻,积累背景非常低。
4.野外水域“标准背角无齿蚌”的移殖重金属监测：移殖“标准背角无齿蚌”幼蚌至太湖五里湖和武汉东湖作为“实验组”,并保留在南泉基地的同一批蚌为对照组；每3个月定期回收样本,进行重金属监测的初步应用。结果表明：移殖3mo,南泉基地Mn、Mo、Ni、Zn、Pb、As和Ba的含量显著上升(P<0.05),而Al的含量显著下降(P<0.05)；五里湖Fe、Ni、Cr、Zn、As、Mo、Mn、Ba、T1和Pb的含量显著上升(P<0.05)；东湖Mo和As的含量显著上升(P<0.05),而Cr、A1、T1和Pb的含量显著下降(P<0.05)。其中,Mn、Fe、Zn、Cd、Ba和Pb在南泉的含量最高,而Al、Cr、Ni、Cu、As、Mo和Tl在五里湖的含量最高。南泉基地、五里湖和东湖的MPI分别为8.6、14.1和1.6。移殖6mo,南泉基地Fe的含量显著下降(P<0.05)；五里湖Fe、Mo、Cr、Ni、As、Ba、T1和Pb的含量显著下降(P<0.05)；东湖Mo的含量明显上升(P<0.05),而Fe和As的含量显著下降(P<0.05)。其中,Ni、Cu和Tl在三者中均未检出,Cr、Mn、Fe、Zn、As、Mo、Cd、Ba和Pb在南泉的含量最高,而A在五里湖的含量为最高。南泉基地、五里湖和东湖的MPI分别为1.7、0.4和0.3。有效地反映了不同水环境重金属污染的时空动态特征。
本研究客观地证实了背角无齿蚌重金属积累与不同污染程度水域的背景相关,在有效掌握了背角无齿蚌生境条件、发育机制和生长规律并建立了确保存活率方法的基础上,成功建立了生境、种质、规格、年龄、生长及发育程度等一致的“标准背角无齿蚌”群体。并通过对其不同生活史阶段的个体进行了重金属积累特征和背景的跟踪研究,为移殖对象的选择和监测结果的解释奠定了必要的基础。进而创新性地移殖“标准背角无齿蚌”至不同水域,有效地反映了不同水环境的重金属含量时空动态特征。所有这些为正在开展的淡水渔业环境重金属“背角无齿蚌观察”研究提供了移殖监测的新思路,也为今后“标准背角无齿蚌”群体的完善和应用的拓展提供了理论基础。
Anodonta woodiana (Mollusca, Lamellibranchia, Eulamellibranchia, Unionidae, Anodonta) is a worldwide-distributed freshwater mussel, which has proven useful for temporal and spatial monitoring of persistent pollutants (e.g., heavy metals, organotins, and organochlorines) in aquatic environments as bioindicators. To date, however, the utilization of mussel-based monitoring (both passive biomonitoring and active biosurveillance) is generally dependent on field-collected mussels. As a result, it is difficult to ensure that the mussels will possess the same biological factors (e.g., size, age, growth rate, sexual maturity) and exposure history of heave metals. These conditions may significantly influence the bioaccumulation pattern and fate of heavy metals. Moreover, sample collection may also be difficult or impossible as wild mussels sometimes are rare, or even absent at study locations. In addition, sampling of wild mussels possibly destroys their nature population. Herein, it must be very important to establish an innovative 'standardized' mussel population for environment monitoring (e.g.,"standardized A. woodiana" of the present study) possessing the same biological factors, stable inherited quality and low background of heavy metals. Furthermore, the mussels can subsequently be transplanted and retrieved for metal pollution monitoring. There is no doubt that that the approach of artificial propagation and culture of unionid mussels will be very helpful to set up such a population. Corresponding studies have been conducted in this study to establish a "standardized A. woodiana" population, which can contribute the large-scale, standardized, informationalized biomonitoring system of "Fershwater Mussel Watch". The main results can be summarized as follows:
1. The difference of heavy metal bioaccumulation in A. woodiana from different waters:Bioaccumulation characteristics of heavy metal (Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Mo, Ag, Cd, Ba, Tl, and Pb) in soft tissues of cultured A. woodiana from Nanquan Aquatic Base of Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences (without obvious exogenous metal pollution), and wild mussels from Meiliang Bay of Taihu Lake (suffered relatively serious pollution) and Cibihu Lake in Yunnan Province (a very clean plateau lake) were investigated by an Agilent7500ce inductively coupled plasma mass spectroscopy (ICP-MS). The results of the present study showed that concentrations of Al, Cr, Mn, Fe, Ni, Cu, Zn, As, Cd and Ba in wild mussels of Meiliang Bay were significantly higher than cultured mussel (P<0.05). Moreover, concentrations of Al, Mn, Ni, Cu, Zn, As, Cd, Ba and Pb of Meiliang Bay were significantly higher than wild mussels from Cibihu Lake (P<0.05). Additionally, concentrations of Cr, Fe, Mo and T1in cultured mussels were significantly lower than wild mussels from Cibihu Lake (P<0.05). However, concentrations of Cu, As and Pb in the former were significantly higher than the latter (P<0.05). The metal pollution index (MPI) of mussels in Nanquan Aquatic Base, Meiliang Bay, and Cibihu Lake were1.8,11.1and1.0, respectively. In addition, Al-Cd, Cr-Mo, Fe-Zn, Fe-Cd, Fe-Pb, and Zn-Pb in mussels of Nanquan Aquatic Base showed significantly positive correlation (P<0.05). Mn-Cu, Mn-Zn, Mn-Mo, Mn-Cd, Mn-Pb, Fe-Ni, Fe-Cu, Fe-Cd, Fe-Pb, Ni-Cu, Ni-Pb, Cu-Mo, Cu-Cd, Cu-Pb, Zn-Mo, Zn-Cd, Zn-Pb, Mo-Cd, Mo-Pb, and Cd-Pb in mussels of Meiliang Bay showed significantly positive correlation (P<0.05). And Cr-Fe, Cr-As, Mn-Bs, Fe-Mo, and Mo-Ba in mussels of Cibihu Lake showed significantly positive correlation (P<0.05). The study indicated that bioaccumulation of heavy metals in A. woodiana presented positive correlation with the pollution background of water-bodys. Individuals of A. woodiana with relatively low level background of heavy metals can be possibly obtained by culture of the mussels in clean water-body.
2. Establishment of a population of "standardized A. woodiana":The present study investigates firstly the habitat condition, development mechanism, and growth characteristics of A. woodiana throughout whole life cycle from fertilized egg to mature adults. The results showed that glochidia belong to large size as the glochidia index (Gin) is0.077. The biological zero and the effective temperature sum were calculated to be8.89℃and86.39°C-d respectively during the period of their parasitic metamorphosis development in the gill filaments of yellow catfish (Pelteobagrus fulvidraco). During metamorphosis from glochidia to early free-living juveniles, the shell length increased significantly (P<0.01). The shape of0-day-old juveniles is similar as glochidia but with a strong foot which could drag the body to crawl all around. Until60-day-old the morphology of juveniles begin to be as same as adults. Moreover, the organs such as visceral mass, intestine, incurrent siphon, excurrent siphon, and gills have developed. The form of the juvenile at750-day-old resembles that of a fully grown adult and more than90%females are pregnant with glochidia. It is found that the growth of A. woodiana was allometric. The relationship between shell length and growth time for0-60-day juveniles was quadratic, L=0.383-0.031t+0.003t2(R2=0.953, P<0.01). For60-750-day-old individuals, the relationships of length, width, height and time were all inverses, L-67.071-3364/t (R2=0.924, P<0.01), W=25.875-1326/t (R2=0.779, P<0.01), and H=41.441-2092/t (R2=0.907,P<0.01), respectively. In addition, the general relationships of size (length-width, length-height, and width-height as well as length-width-height) were linear, L=8.168+2.208W (R2=0.921, P<0.01), L=1.557+1.571H(R2=0.973, P<0.01), W=0.641H-0.663(R2=0.920, P<0.01), and L=4.868+0.875W+0.929H (R2=0.950, P<0.01), respectively. A population of "standardized A. woodiana" with ca.12000individuals has been successfully established in the present study, and are believed water temperature, the rate of flowing water, and carrying capacity as the key factors that influence the survival rate of A. woodiana during the process of artificial propagation and culture of this mussel.
3. The bioaccumulation dynamics of heavy metals in "standardized A. woodiana"at different stages of life cycle:The accumulation level and dynamics of heavy metals (Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, Ag, Cd, and T1) in "standardized A. woodiana" at different life stages (glochida, juveniles and gravid adults) were assessed by the Agilent7500ce ICP-MS. Furthermore, the profiles of heavy metals (Al, Ag, Co, Cu, Fe, Mn, Mo, Se, T1, and Zn) in the shell of an adult were studied by the JEOL JXA-8100electron probe microanalyzer (EPMA). The results showed most of heavy metals in glochidia were low, but Cr (0.5μg·g-1), Ni (0.9μg·g-1), and As (5.0μg·g-1) were significantly detected. Concentrations of Cr, Ni, As, Se, Cu, Zn, and Cd in juvenile (S1) with average shell length17.5mm and juvenile (S2) with average length35.8mm were much low and the ranges of concentrations were narrow (P>0.05), while the concentrations of Cr, Ni, As, Se, Cu, Zn, and Cd were significantly higher than gravid adults (P<0.05). The concentrations of Ni, Zn, As, Se, and Cd in gravid adults were significantly higher than both juvenile (S1) and juvenile (S2)(P<0.05). Noteworthily, the concentration of As was so high than exceed the limit of Maximum Levels of Contaminants in Foods (GB2762-2005) of China. As to shells, the metal concentrations of all samples were similar except the concentration of Al in juvenile (S1) was significantly higher than gravid adults (P<0.05). Moreover, the concentrations of heavy metals generally decreased in the order Fe> Mn> Al> Ag> Mo> Cu> Co> Se> T1> Cr-Ni-Zn-As-Cd. In addition, the concentration of Mn in nacreous layer were significantly higher than prismatic layer (in the umbo and ventral edge)(P<0.05), while the distribution of other metals were uniformity at different layers. The results suggested that although the accumulation levels of several heavy metals in gravid adults and glochida might keep little influence from the background of collection sites and maternal mussels, respectively, those levels in cultured juveniles were obviously low and the metal contamination in these mussels were very weak.
4. Heavy metal monitoring by transplantation of "standardized A. woodiana":The juvenile mussels of "standardized A. woodiana" from the same artificially propagated and cultured population were divided into three sister groups. Two of them were transplanted into Wulihu Bay of Taihu Lake (Wuxi city) and Donghu Lake (Wuhan City), respectively as the experimental group for assessing heavy metal pollution. Meanwhile, the remaining one was still kept in Nanquan Aquatic Base as the control group. All cages were maintained at a depth of50cm from the water surface. And the samples were recovered per3months to determine heavy metal contents. The results showed that concentrations of Mn, Mo, Ni, Zn, Pb, As, and Ba in control in Nanquan Aquatic Base for3months (CN3) were significantly higher than control for all sites from beginning (CAO)(P<0.05), while the concentration of Al in the former was significantly lower than the latter (P<0.05). The concentrations of Fe, Ni, Cr, Zn, As, Mo, Mn, Ba, T1, and Pb in mussels transplanted Wulihu Lake for3months (TW3) were significantly higher CNO (P<0.05). The concentrations of Mo and As in mussels transplanted Donghu Lake for3months (TD3) were significantly higher CNO (P<0.05), but concentrations of Cr, Al, T1, and Pb were significantly lower than the latter (P<0.05). Comparison different water-bodies, the highest concentration of Mn, Fe, Zn, Cd, Ba, and Pb presented in CN3, while the highest concentration of Al, Cr, Ni, Cu, As, Mo, and T1presented in TW3. The MPI of CN3, TW3, and TD3were8.6,14.1, and1.6, respectively. In addition, the concentrations of Fe in control in Nanquan Aquatic Base for6months (CN6) were significantly lower than CN3(P<0.05). The concentrations of Fe, Mo, Cr, Ni, As, Ba, T1, and Pb in mussels transplanted Wulihu Lake for6months (TW6) were significantly lower than TW3(P<0.05). The concentration of Mo in mussels transplanted Donghu Lake for6months (TD6) was significantly higher TD3(P<0.05), but concentrations of Fe and As were significantly lower than the latter (P<0.05). Comparison different water-bodies, the highest concentration of Cr, Mn, Fe, Zn, As, Mo, Cd, Ba, and Pb presented in CN6, while the highest concentration of Al presented in TD6. The MPI of CN6, TW6, and TD6were1.7,0.4, and0.3, respectively. The results suggested transplant "standardized A. woodiana" is useful in monitoring temporal and spatial trends of heavy metals in aquatic environments.
The present study demonstrates objectively that bioaccumulation of heavy metals in A. woodiana correlated with the pollution background of water-bodys. A "standardized A. woodiana"with the same biological factors and exposure history of heave metals can be established based on mastering the habitat necessary, development mechanism, and growth characteristics throughout the whole life cycle. By the individuals of "standardized A. woodiana" at different stages of life cycle, the temporal variations of heavy metal bioaccumulation has been understood, which provide basic information for mussel-size selection for transplantation and interpretation of the results obtained during the research period. By means of transplantation monitoring of "standardized A. woodiana" to different nature water-bodies, the temporal and spatial dynamics have been clarified. All results of the present study provide new ideas for the current project of "Freshwater Mussel Watch" monitoring, and also provide the fundamental knowledges to perfect the aforementioned "standardized A. woodiana" population and extend its future applicaion.
引文
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