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The Temporal and Spatial Distribution of Heavy Metals in Helice tridens in the Tidal Flat of YU Na, SUN Xinjin, LI Xiaobo, WANG Qun School of Life Science, East Normal University, Shanghai (200062) E-mail: [email protected] Abstract Between November 2006 and July 2007, we determined the heavy metal content in Helice tridens crabs inhabiting the tidal flat of Yancheng. The results showed that the accumulation of the essential elements Fe, Zn, Cu, Mn, and Ni was higher than that of the toxic element Pb; the specific relationship between elemental concentrations was Fe > Zn > Cu > Mn > Ni > Pb. We analyzed the seasonal distribution of the heavy metal content in the crabs, and found that there were seasonal differences in the contents of the six heavy metals. The seasonal distributions of the elements were in the following order: Fe content in spring > summer > autumn; Cu and Mn content in autumn > summer > spring; Zn, Ni, and Pb content in summer > spring > autumn. Thus, it was considered that temperature is not the principal factor influencing the accumulative rate of the heavy metals in H. tridens individuals. For example, there were certain differences in the heavy metal content in the crabs collected from different sampling points in autumn. In the cluster analysis, ten sampling points were approximately classified into three groups, which represented the coastal salt-industry culture area, the nature-reserve central area, and the estuarine industrial water area. The results of the cluster analysis suggested that the pollutants in the crabs’ habitats influenced the accumulation of heavy metals in their bodies. Moreover, we compared related data and found that Cu accumulation in the bodies of the crabs inhabiting the tidal flat of Yancheng was relatively low while the accumulation of Zn, Mn, Ni, and Pb was relatively high. Keywords: Tidal flat of Yancheng, Helice tridens, heavy metal distribution

Yancheng, which is located in eastern China, has a 582-km-long eastern coastline bordered by the . The tidal flat of Yancheng, with its northern end in the Guanhe estuary in junction with and its southern end in the new port gate bordering , has an area of 457,000 hm2, which accounts for 3/4th of the area of province and 1/7th of the area of the entire country. It is the most extensive tidal flat in China, and it is one of the 17 key biodiversity areas that have been chosen as priority conservation areas in China (State Environmental Protection Administration, 2003). The tidal flat has an exceptionally large resource potential for Jiangsu province and for the whole country. However, in recent years, continuous discharge of large amounts of wastewater from the towns and industries, particularly the discharge of water containing heavy metal pollutants that can accumulate continuously in tidal flat sediments and animal bodies, has resulted in direct and potential damage to the tidal-flat ecosystem(Chen et al., 2000). The zoobenthos, which inhabit the tidal flats for their entire lifetime, are one of the members of the food chain of the tidal-flat ecosystem, and the pattern of heavy metal accumulation in their bodies can partly reflect the status of the habitat (Li et al., 2007). In this study, we aimed to provide basic information for facilitating ecological protection of the Yancheng tidal flat. Therefore, between November 2006 and July 2007, we collected H. tridens crabs inhabiting the Yancheng tidal flat and analyzed the temporal and spatial distribution of heavy metals in the bodies of the crabs. 1 Sampling and analysis

1.1 Sampling-point arrangement and sample collection We selected 10 representative sampling points between the northern and southern ends of the tidal flat. The sediment samples and biotic samples were collected in three quarters, i.e., early November (autumn) in 2006, early April (spring) in 2007, and early July (summer) in 2007.

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Sampling was carried out once in a quarter. The sampling points were Zhongshan estuary, Binhai port, Guhuang estuary, Biandan port, the north of the Sheyang estuary, the core protected district, Simaoyou port, Zhugang gate, Chuanshui port, and the Chinese sturgeon reserve, as shown in Figure 1.

No. Sampling points Latitude Longitude 1 Zhongshan estuary 34°21′20.4″ 120°09′00.7″ 2 Binhai port 34°16′56.9″ 120°16′43.7″ 3 Guhuang estuary 34°15′29.1″ 120°16′66.3″ 4 Biandan port 34°06′45.6″ 120°19′13.9″ 5 North of the Sheyang estuary 33°49′13.3″ 120°28′33.3″ 6 Core protected district 33°34′30.3″ 120°32′49.0″ 7 Simaoyou port 33°20′13.2″ 120°41′05.4″ 8 Zhugang gate 33°03′03.8″ 120°48′57.3″ 9 Chuanshui port 32°57′13.0″ 120°57′34.1″ 10 Dongtai Chinese sturgeon 32°44′35.4″ 120°51′51.6″

Figure 1 Sampling points on the tidal flat of Yancheng

1.2 Sample preparation and analysis The crabs were cleaned with water from the sample points, sealed in plastic bags, and preserved in a portable freezer, which was taken back to the laboratory within two days. Then, the body of the animal was wiped dry and weighed. We randomly selected 10 adults with similar body sizes. The entire soft bodies, except for the legs, were properly dried, fully ground in an agate mortar, and passed through a 100-mesh sieve. Each sample was accurately weighed using an electronic balance, placed in a 25-ml porcelain crucible, and cinerated in a muffle furnace at 500℃ for 24 hours to remove the organic matter. All the measurements of weight performed during the cineration process were exact to 0.01 mg. The acid-dissolution method

(HNO3-HF-HClO4) was used to digest and dilute the samples. We used an M6-type atomic absorption-spectrometry apparatus (ThermoElemental Ltd., UK) to determine the Fe, Zn, Cu, Pb, Mn, and Ni contents and analyze the blank solutions. 1.3 Analysis Method The heavy metal accumulation in the organism was calculated on the basis of dry weight (DW). Statistica 6.0 was used for cluster analysis of the accumulation of the six heavy metals in the bodies of the animals. 2 Results

2.1 Seasonal distribution of heavy metals in H. tridens H. tridens is a common species of crab found in the Yancheng tidal flat. In this study, we analyzed the accumulation of six heavy metals in the bodies of these crabs in autumn, spring, and summer. The results show that there are differences between the average accumulation of different

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heavy metals in the bodies of the crabs; the relative distribution of the six heavy metals in the three seasons was Fe > Zn > Cu > Mn > Ni > Pb (see Figure 2).

200 DW 150

(mg/K

100 g ) 50

0 Autumn Fe Zn Spring Cu Summer Mn Ni Pb

Figure 2 The temporal distribution of the six heavy metals in Helice tridens individuals

There were differences in the accumulation of the six heavy metals, but the differences were insignificant. The seasonal differences in the accumulation of heavy metals in the bodies of the crabs can be observed in Figure 2. The seasonal variation in Fe accumulation was in the following order: spring (153.09 mg/kg) > summer (148.43 mg/kg) > autumn (143.87 mg/kg), and the Fe accumulation in spring was higher than the average value (148.46 mg/kg) of the three seasons. The seasonal changes in the accumulation of Cu and Mn were in the order autumn > summer > spring; the Cu concentrations were 52.37 mg/kg, 47.87 mg/kg, and 46.80 mg/kg, with an average value of 49.01 mg/kg. The Mn concentrations were 14.39 mg/kg, 13.47 mg/kg, and 11.51 mg/kg, with an average value of 13.12 mg/kg. The seasonal changes in the accumulation of Zn, Ni, and Pb were in the order summer > spring > autumn. The accumulation of Zn in summer, spring, and autumn was 144.00 mg/kg, 143.91 mg/kg, and 143.39 mg/kg, respectively; the Zn content in summer was higher than the average value (143.77 mg/kg) of the three seasons. The Ni concentrations in the three seasons were 8.00 mg/kg, 7.34 mg/kg, and 6.68 mg/kg; the Ni content in summer and spring was higher than the average value (7.34 mg/kg) of the three seasons. The Pb concentrations in the three seasons were 5.11 mg/kg, 4.54 mg/kg, and 3.97 mg/kg; the Pb content in autumn was lower than the average value (4.54 mg/kg) of the three seasons. 2.2 Horizontal distribution of heavy metal in H. tridens The horizontal distribution of the heavy metal accumulation in the bodies of H. tridens individuals is slightly different in the three seasons (no details in this paper). In this study, the sediment structure and the biological community structure of the sample plot were influenced by the Sea Embankment Target Project, which was started in early 2007 in the northern region of the Yancheng tidal flat, and the First Phase Regulation Project of the Channel under the Sheyang River Gate. Therefore, we analyzed only the accumulation of heavy metals in the crabs collected from the 10 sampling points during the autumn of 2006. The results show that there are certain differences in the accumulation of heavy metals in the crabs collected from different sampling points, as seen in Figure 3. There were significant differences in the accumulation of Cu in the crabs collected from the 10 sampling points (P < 0.01); the Cu accumulation in crabs collected from Zhugang gate, Biandan port, and Zhongshan estuary were 80.89 mg/kg, 78.60 mg/kg, and 78.29 mg/kg, respectively, and these values were significantly higher than those from the other

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sampling points. Moreover, the Cu accumulation value in the crabs obtained from the core protected district was 52.63 mg/kg. The Cu-accumulation values of these four sampling points were higher than the average accumulation value (52.37 mg/kg) of the 10 sampling points. We observed significant differences in Fe accumulation in the crabs obtained from different sampling points (P < 0.01). The Fe-accumulation value in crabs obtained from Binhai port was 197.68 mg/kg, which was significantly higher than that from other sampling points. The Fe-accumulation values in crabs obtained from Guhuang estuary, Biandan port, and Zhongshan estuary were 170.15 mg/kg, 162.40 mg/kg, and 151.09 mg/kg, respectively, and these values exceeded the average value (171.43 mg/kg). There were significant differences in the Mn-accumulation values obtained from the 10 sampling points (P < 0.01), and the average value was 14.39 mg/kg. The maximum value (24.73 mg/kg) was observed in the Dongtai Chinese sturgeon reserve while the minimum value (3.90 mg/kg) was observed in Zhongshan estuary. In addition, the Mn-accumulation values in the animals obtained from 6 sampling points, namely, Chuanshui port, Zhugang gate, north of the Sheyang estuary, Biandan port, the core protected district, and Simaoyou port, were higher than the average value of all points. The Pb-accumulation values in the animals collected from different sampling points were significantly different (P < 0.01). The Pb-accumulation values in the animals from Dongtai Chinese sturgeon reserve, Biandan port, Simaoyou port, and Guhuang estuary were 8.40 mg/kg, 6.06 mg/kg, 4.40 mg/kg, and 4.40 mg/kg, respectively; these values exceeded the average (3.97 mg/kg); the minimum value (2.05 mg/kg) was observed in the animals from Zhugang gate. The Zn accumulation in the muscles of the animal bodies was relatively higher. The highest Zn-accumulation value (179.75 mg/kg) was obtained in animals sampled from the Zhongshan estuary; the second-highest value (172.60 mg/kg) was obtained in animals sampled from the north of the Sheyang estuary. The Zn-accumulation value in animals sampled from the core protected district, Guhuang estuary, and Binhai port were 168.68 mg/kg, 167.38 mg/kg, and 161.28 mg/kg, respectively, and all these values were higher than the average value (143.39 mg/kg). The average value of Ni accumulation in the animals from the 10 sampling points was 6.68 mg/kg, with a maximum of 10.81 mg/kg in Biandan port and a minimum of 3.50 mg/kg in Guhuang estuary.

200 Zhongshan estuary 180 Binhai port 160 Guhuang estuary 140 Biandan port (mg/Kg )

120 North of the Sheyang DW estuary 100 Core protected district 80 Simaoyou port 60 Zhugang gate 40 Chuanshui port

20 Dongtai Chinese sturgeon reserve 0 Cu Fe Mn Pb Zn Ni

Figure 3 Horizontal distribution of heavy metal content in Helice tridens in autumn

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2.3 Cluster analysis of the habitat of the Yancheng tidal flat on the basis of the accumulation of heavy metals in the bodies of H. tridens individuals The results of the study on the horizontal distribution of heavy metal accumulation in the bodies of the crabs in autumn reveal that there were differences between the values obtained from different sampling points, suggesting that the external environment influences heavy metal accumulation in animal bodies. In this study, we analyzed the accumulation of six heavy metals in the bodies of the crabs collected in the autumn of 2006 and used Statistica 6.0 to perform cluster analysis of the heavy metal distribution in the sediments of the Yancheng tidal flat (Figure 4). We were eventually able to establish three groups: type I includes three sampling points to the north of the Yancheng tidal flat, i.e., Zhongshan estuary, Binhai port, and Guhuang estuary; type II includes the sampling points in the early-formed tidal flat channels that are located at a certain distance from the estuary (port), including the north of the Sheyang estuary, the core district of the national Yancheng rare birds nature reserve, and the Dongtai Chinese sturgeon reserve; and type III mainly includes the sampling points in the estuaries (port), including Biandan port on the boundary of and Sheyang county, Simaoyou port and Zhugang gate in Dafeng county, and Chuanshui port in Dongtai county. On the basis of the functional area division of offshore area environmental quality in Jiangsu province, type I includes the Coastal Salt Industry Culture Area; type II includes the Core Protected District; and type III includes the Estuarine Industrial Water Area.

Zhongshan中山河口 estuary Binhai滨海港口 port Guhuang故黄河口 estuary North of the Sheyang射阳河口北 estuary Core protected保护区核心区 district Dongtai Chinese sturgeon中华鲟保护区 reserve Biandan扁担港口 port Simaoyou四卯酉港口 port Zhugang竹港口 gate Chuanshui川水港口 port

25 30 35 40 45 50 Distance

Figure 4 Cluster analysis of the 10 sampling points according to the accumulation of heavy metals in the bodies of the animals

3. Discussion We analyzed the seasonal distribution of heavy metals in the bodies of H. tridens crabs in the Yancheng tidal flat, and the results show that the accumulation of the essential elements Fe, Zn, Cu, Mn, and Ni was higher than that of the toxic element Pb. Although some recent studies have suggested that essential elements are easily absorbed by organisms through active absorption processes (He et al., 2001), the accumulation of the toxic elements Pb and Hg in the bodies of Harpodon nehereus (a kind of fish) and Metopograpsus maculates (a kind of crab) inhabiting the Gulf of Cambay, India are higher than that of the essential microelement Co (Reddy et al., 2007). Moreover, the accumulation of the toxic element Cd in M. maculates was higher than that of Co.

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Because of the limited number of the heavy metals tested in this study, we were not able to determine the differences between the accumulation of essential and non-essential metal elements. The accumulation and distribution of heavy metals in the bodies of zoobenthos are influenced by biological factors such as the size of the individual and abiotic factors such as external environment (Bass, 1977). Li et al.(2005) considered that higher temperature facilitated the absorption of Cu, Zn, and Ni in Sesarma dehaani; however, in this study, the maximum accumulation of the six heavy metals in H. tridens individuals inhabiting the Yancheng tidal flat was observed in three different seasons (see Figure 2). The crabs selected in this study were of similar sizes and of the same gender, which indicates that temperature is not the principal factor influencing the accumulation rate of heavy metals in H. tridens individuals. The studies on Corbicula fluminea (Sun et al., 2003) also proved that seasonal change had little relation to the accumulation of heavy metals in animal bodies. Our study conjectures that the heavy metal content of the habitat may be the key factor influencing the accumulation of heavy metals in animal bodies. In this study, the horizontal distribution of heavy metal accumulation in H. tridens was analyzed, and the results suggest that there were differences in the heavy metal accumulation values among the sampling points. Another report on the distribution of heavy metal accumulation in S. dehaani in the tidal flat of Changjiang Estuary (Li et al., 2006) also indicated that the accumulation of heavy metals in the bodies of crabs is influenced by the heavy metal content of the habitat. In a cluster analysis, we classified the ten sampling points into three groups that coincided with the functional area division of offshore area environmental quality in the Jiangsu province. Because of the different social functions in each functional area, the status of environmental pollution was different. Therefore, the result of the cluster analysis proved that the pollutants in the habitat influenced the accumulation of heavy metals in the bodies of the animals. In this study, the order of accumulation of the six heavy metals in H. tridens individuals in the Yancheng tidal flat was Fe > Zn > Cu > Mn > Ni > Pb. However, the order of accumulation of these heavy metals in the bodies of M. maculates individuals inhabiting the Gulf of Cambay in India was Cu > Fe > Zn > Mn > Ni > Pb. The Fe-accumulation value in H. tridens in the Yancheng tidal flat (171.43 mg/kg) was slightly higher than that in M. maculates in the Gulf of Cambay (155.85 mg/kg). The Cu-accumulation value in H. tridens in the Yancheng tidal flat (52.37 mg/kg) was only 1/3rd of that in M. maculates in the Gulf of Cambay in India (175.45 mg/kg), lower than that of S. dehaani in the tidal flat of Changjiang Estuary (103.23 mg/kg), and slightly lower than that of Paralithodes camtschaticus in the northeastern Bering Sea in Arctic Alaska (75–78 mg/kg) (Jewett et al., 2000). The Zn-accumulation value in H. tridens in the Yancheng tidal flat (143.39 mg/kg) was more than three times higher than that in M. maculates in the Gulf of Cambay (44.22 mg/kg), similar to that of P. camtschaticus in northeastern Bering Sea in Arctic Alaska (147–159 mg/kg), and higher than that of S. dehaani in the tidal flat of Changjiang Estuary (93.41 mg/kg). The Mn, Ni, and Pb accumulation in H. tridens in the Yancheng tidal flat were higher than of that of P. camtschaticus in the northeastern Bering Sea in Arctic Alaska (Jewett et al., 2000) and S. dehaani in the tidal flat of Changjiang estuary (Li et al., 2006). The ability to accumulate heavy metals differs among different biological groups (Reddy et al., 2007); however, from a physiological standpoint, the same biological groups employ the same pathways for absorption and detoxification of heavy metals. Therefore, the heavy metal accumulation ability is similar among various crabs. On the basis of the comparative analysis, the Cu accumulation in the sediment of the Yancheng tidal flat is lower than that in the other areas mentioned above; however, the Zn-, Mn-, and Pb-accumulation in the Yancheng tidal flat have

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already exceeded the accumulation values in the other areas, a phenomenon which warrants immediate attention. There is a dearth of detailed data on heavy metal pollution in the Yancheng tidal flat; therefore, the reasons causing these results are still undetermined, and further research on the organisms inhabiting these areas is necessary. Acknowledgements: The authors acknowledge Zhanghuabin in Yancheng Normal College for the help in the sampling process. This work has been directly or indirectly supported by funds from the National Science Foundation of China (30700064, 30671607), the Fund of research for selecting young excellent teachers in University of Shanghai (79001351) and the Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresource and Environmental Protection.

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