Establishment Of A "Standardized Population Of Anodonta Woodiana" And Its Transplant Biomonitoring Of Heavy Metal In Freshwater Environment

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.