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Interaction of polycyclic aromatic hydrocarbons and heavy metals on soil enzyme
Chemosphere 61 (2005) 1175–1182 www.elsevier.com/locate/chemosphere
Interaction of polycyclic aromatic hydrocarbons and heavy metals on soil enzyme Guoqing Shen a, Yitong Lu a
b
a,*
, Qixing Zhou b, Jingbo Hong
a
Department of Environment and Resource, College of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 201101, PR China Key Laboratory of Terrestrial Ecological Process, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, PR China Received 12 August 2004; received in revised form 23 January 2005; accepted 22 February 2005 Available online 13 April 2005
Abstract Actions and interactions of heavy metals (cadmium, zinc and plumbum) and polycyclic aromatic hydrocarbons (PAHs) [phenanthrene, fluoranthene, benzo(a)pyrene] on the soil urease and dehydrogenase activity were studied after 49 days exposure. The experimental approach was based on the uniform design which can cut the experiment time and improve the efficiency of experiments. Data treatment was essentially based on the multiple regression technique. The results showed that the action and interaction between heavy metals and PAHs were strongly dependent on the time of pollution. The dehydrogenase exhibits more sensitive to the combined pollution than urease. The negative interaction between Zn and Cd to hydrogenase activity and the combined stimulatory activity of Phenanthrene and Benzo(a)pyrene (or fluoranthene) to soil enzyme were observed. The interactions between Zn (Cd) and phenanthrene towards urease (dehydrogenase) were positive, and the interaction between Zn and benzo(a)pyrene to urease activity was negative. This study corresponds to exploratory phase in order to reveal interaction effects of heavy metals and PAHs on the soil enzyme and then to set up more in-depth analysis to increase progressively the understanding of the ecotoxicological mechanisms involved. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Heavy metals; PAH; Urease; Dehydrogenase; Uniform design
1. Introduction Pollution of soil environment has become a serious problem in many countries, including China. Heavy metals (HM) and polycyclic aromatic hydrocarbons (PAHs) are two of the most abundant and potentially
* Corresponding author. Tel.: +86 21 6478 5941; fax: +86 21 6478 7938. E-mail address: [email protected] (Y. Lu).
harmful pollutants found in most polluted soil (Jones et al., 1989; Baran et al., 2004). Heavy metal pollution in soil is of major environmental concern on a world scale and in China in particular with the rapid development of industry. Beside their natural occurrence, heavy metals may enter the ecological environment through anthropogenic activities, such as mining, smelting, sewage sludge disposal, application of pesticides and inorganic fertilizers, and atmospheric deposition. PAHs are a ubiquitous group of hazardous organic pollutants which exhibit strong carcinogenic and toxic properties.
0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.02.074
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They are formed by incomplete burning of fossil fuels and can enter the soil via atmospheric deposition. It is estimated that more than 90% of the total burden of PAH resides in the surface soils (Wild and Jones, 1995). Both of HM and PAH introduced into the soil exert an influence on the microbiota, which manifests itself in changes in enzyme activity (Baran et al., 2004). Soil enzymes are the catalysts of important metabolic processes including the decomposition of organic pollutants and the detoxification of xenobiotics (Margesin et al., 2000b). Enzyme activities are considered to be sensitive to pollution and have the further advantage of being easy to determine without expensive, sophisticated instruments. So, soil enzyme activities have been proposed as indicators for measuring the degree of soil pollution. Dehydrogenase and urease are most important soil enzyme and frequently used test for determining the influence of the various pollutants (heavy metals, pesticide, crude oil, etc.) on the microbiological quality of soil (Margesin et al., 2000a,b). Several researchers investigated the effect of heavy metals and PAHs on urease and dehydrogenase activity. Margesin et al. (2000a) proved a positive influence of naphthalene and phenanthrene on urease activities and gave the correlation for urease activity and phenanthrene content. A positive correlation between dehydrogenase activity and the content of phenanthrene and also fluoranthene, chrysene and dibenzo[ah]anthracene was also observed (Baran et al., 2004). The inhibition of heavy metal pollution on urease and dehydrogenase activity was reported by many scientists (Zheng et al., 1999). The sequence of inhibition of urease activity was generally in the decreasing order of Cr > Cd > Cu > Zn > Mn > Pb. However, the stimulating effects of heavy metals were also documented (Ba˚a˚th, 1989). The aforementioned studies are mostly concentrated on the effect of a single pollutant. In the real environment, however, contaminated soils usually contain mixtures of chemicals rather than a pure substance. As a consequence, soil organisms are exposed to multiple toxicants. The actual risk to which these organisms are exposed is determined by the composition and availability of the mixture. Still, most ecotoxicologically based risk assessment methods evaluate contaminated soils by single-substance criteria, derived from single-substance toxicity data, by that, neglecting mixture effects (Weltje, 1998). Recently, several investigations were devoted to the combined effect of PAH and HM on the soil enzyme (Gogolev and Wilke, 1997; Gong et al., 1997; Irha et al., 2003; Maliszewska-Kordybach and Smreczak, 2003). The presence of metals in PAHs contaminated soils is not rare, especially at coking plants or gas works (Saison et al., 2004). However, it is an area of research that has not been covered to any extent. The contradictory and somewhat confusing results are often reported about this topic. So, the firm conclusions
are still difficult to draw from these little literatures. The difficulties inherent in studying this interaction may be linked with the methodological choices for devising protocols and the methods used for the data treatment (Ribryre et al., 1995). The laboratory situation where usually large doses of the heavy metal and PAH added is far from the real-world exposure although this process may provide some information. In addition, many studies considered the interactions only at specific endpoint, thus make the controvertible conclusion. In this respect, experimental uniform design that uniformly distributes the experimental points in the factor space and multiple regressions can provide an important contribution to research the interaction between pollutants. This design can cut the experiment time and improve the efficiency of experiments. The first aim of this research was to evaluate the action and interaction of heavy metals (Cd, Zn, Pb) and PAHs [phenanthrene, fluoranthene, benzo(a)pyrene] on the soil urease and dehydrogenase activity over 49 days. These pollutants were chosen for the present study because of their abundance and the lack of toxicity data in soil environment. The structure and solubility of three PAHs were different: 3, 4 and 5 aromatic rings, and water solubility of 1.300, 0.260 and 0.003 mg l1. The concentrations tested in this study simulated the real soil environment. The second objective is to recommend a relative new method to research the interaction of combined pollutants in soil. The protocol was based on a uniform design with 6 pollutants and 10 contamination levels for each pollutant.
2. Materials and methods 2.1. Experimental soils The experimental soils used for this study was collected from an agricultural land (0–20 cm in depth) in Shanghai city, China (31.14 0 N, 121.29 0 E). The soil type was a paddy soil. After transportation to the lab, the soil was air-dried, ground, passed through a 3 mm mesh and stored as the stock sample for this study. The basic soil properties were determined using standard methods recommended by the Chinese Society of Soil Science (Lu, 1999). The soils had the following basic properties: pH 8.18, organic matter 16.7%, cation exchange capacity (CEC) 15.6 cmol kg1, total N 1.14 g kg1 and total P 1.36 g kg1, sand 51.49%, silt 28.37%, clay 20.14%, Fe2O3 5.53%, Al2O3 14.38%, and SiO2 58.7%. 2.2. Experimental design A uniform design was employed to study the combined effect of heavy metals and PAHs. Fang (1980),
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professor of the Mathematical Research Institute of Chinese Academy of Sciences, put forward a uniform design method adopting number theory to distribute the experimental points uniformly in the factor space. This design is similar to the orthogonal one. It has two characteristics: (1) The occurrence of each level of each factor in the experiments is once only; (2) The number of experimental trials equals the level number of factors. For a study of multi-factors with multiple levels, the uniform design needs much fewer experiments than the Orthogonal design does (Fang, 1980; Liang et al., 2001; Cheng et al., 2002). It can cut the experiment time and improve the efficiency of experiments. For experiments with 6-factors and 10-levels, the experimental number of orthogonal design is 100 and the complete design is 106, while that of Uniform design is only 10, which is shown in Table 1. Uniform design tables can be expressed as Un(qs), where U stands for Uniform design, n for number of experimental trials, q for number of levels and s for maximum number of factors. In Table 1, each column corresponds to a factor and each row corresponds to an experimental trial to be carried out. As shown in this table, the Uniform design has more
levels so as to establish regression equations of higher precision and estimate the main and interaction effects of the experimental factors (Fang, 1980). Now, uniform design has been gradually popularized in China, particularly in agriculture, industry, science researches, military sciences and chemistry, and chemical engineering. Three PAH compounds with different properties [phenanthrene, fluoranthene, benzo(a)pyrene] and three elements (cadmium, zinc, plumbum) that often emitted from the same as PAHs were considered as the main pollutants in soil, and were selected as the tested pollutants for this study. The choice of concentrations was based on the environmental quality standards for soils of China and Canada. In China, the soil environmental quality standards for Cd, Zn and Pb are less than 0.2, 100 and 35 mg kg1 soil, respectively (Chinese National Environmental Protection Agency, 1995). The standard for PAHs is less than 0.1 mg kg1 in Canada (Cerniglia, 1984; Zhang and Xia, 2000). The concentrations of heavy metals and PAHs added to soil samples are listed in Table 2. The uniform designs and concentrations of heavy metals and PAHs added to soil samples are listed in Table 3.
Table 1 Uniform design table U10(106) for 6-factor and 10-level
2.3. Preparation of samples
Sample no.
The studies were performed as pot experiments. The dimension of containers was 1000 ml. Stock soil sample (800 g) was first thoroughly mixed with a water solution of HM salts [Zn(CH3COO)2, Pb(CH3COO)2, and Cd(CH3COO)2] and CH2Cl2 solution of PAHs according to the design and concentrations given in Table 3, and then incubated at room temperature for 7 weeks. Control without any artificial contaminated received the same amount of distilled water and CH2Cl2. There were three replications. Moisture in the incubated soil samples was kept at 60% of the full soil water holding capacity by weighting daily. This level was kept during the time of the experiment.
Factors
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11
X1
X2
X3
X4
X5
X6
1 2 3 4 5 6 7 8 9 10 0
2 4 6 8 10 1 3 5 7 9 0
3 6 9 1 4 7 10 2 5 8 0
5 10 4 9 3 8 2 7 1 6 0
7 3 10 6 2 9 5 1 8 4 0
10 9 8 7 6 5 4 3 2 1 0
Table 2 Experimental factors and levels (mg kg1 soil) Factor
Level 1
2
3
4
5
6
7
8
9
10
Cd Zn Pb Phea Flab Bapc
0 0 0 0 0 0
0.003125 12.5 4.3625 0.0125 0.0125 0.0125
0.0125 25 8.725 0.025 0.025 0.025
0.05 50 17.5 0.05 0.05 0.05
0.2 100 35 0.1 0.1 0.1
0.8 200 70 0.2 0.2 0.2
3.2 400 140 0.4 0.4 0.4
12.8 800 280 0.8 0.8 0.8
51.2 1600 560 1.6 1.6 1.6
204.8 3200 1120 3.2 3.2 3.2
a b c
Phe denotes phenanthrene. Fla denotes fluoranthene. BaP denotes benzo(a)pyrene.
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Table 3 Experimental treatments and concentration of HM and PAH added to soil (mg kg1 soil) Treatments
Cd
Zn
Pb
Phea
Flab
BaPc
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11
0 0.03125 0.0125 0.05 0.2 0.8 3.2 12.8 51.2 204.8 0
12.5 50 200 800 3200 0 25 100 400 1600 0
8.725 70 560 0 17.5 140 1120 4.3625 35 280 0
0.1 3.2 0.005 1.6 0.025 0.8 0.0125 0.4 0 0.2 0
0.4 0.025 3.2 0.2 0.0125 1.6 0.1 0 0.8 0.005 0
3.2 1.6 0.8 0.4 0.2 0.1 0.005 0.025 0.0125 0 0
a b c
Phe denotes phenanthrene. Fla denotes fluoranthene. BaP denotes benzo(a)pyrene.
2.4. Determination of urease and dehydrogenase activity The activities of urease and dehydrogenase were determined after 1, 3 and 7 weeks. Soil urease activity was determined by the method of Tabatabai and Bremner, expressed as mg NH4-N kg1 h1 (Tabatabai and Bremner, 1972). Dehydrogenase activity was tested by reduction of 2, 3, 5-triphenyltetrazolium chloride (TTC). After 24 h at 37 °C, the triphenyl formazan (TPF) released was extracted with methanol and assayed at 485 nm in an Perkin–Elmer UV spectrophotometer (Casida et al., 1964). The unit of dehydrogenase activity was TPF mg g1 24 h1. The results were expressed as an average of three parallel determinations of the mixture of there duplicates soil samples. The enzyme activities in Fig. 1 are expressed as % of control. 2.5. Data treatment Generally, to account for combined effect of different important independent variables, second-order polynomial models are preferred (Poorna and Kulkarni, 1995). Therefore, it was thought of interest to develop a second-order polynomial model describing the effect of six selected independent variables, namely, the concentration of cadmium, zinc, plumbum, phenanthrene, fluoranthene, benzo(a)pyrene on the urease and dehydrogenase activity in soil. The model was expressed as Y ¼ b0 þ
m X i¼1
bi X i þ
X
bij X i X j þ e;
i
where Y is the predicted value of soil enzyme activity; X is the independent variables corresponding to the concentration of heavy metals and PAHs; and b is the regression coefficients estimated by the stepwise regression method with a confidence level of 95%.
The results obtained were analyzed using SPSS (version 12.0) software.
3. Results and discussion 3.1. Comparison of the enzyme inhibition effect Fig. 1 shows the effect of combined pollution of PAHs and heavy metals on urease and dehydrogenase activities during the whole course of the experiment (49 days). The results were expressed as % of control (activity of control = 100). Although the enzyme activities in all soil samples were inhibited exception of urease on 49th day, the extent of enzyme inhibition varied with incubation proceeded. The dehydrogenase exhibits more sensitive than urease. In the case of dehydrogenase, the enzyme activity was dropped below 50% of the control soil on 7th day, 22.22–100% on 21st day and 23.53–88.24% on 49th day in all soil samples. The maximal rate of inhibition was 97.82% in sample of S4, S6, S7 and S9 on 7th day. However, the activity of urease was different from dehydrogenase. The activity was inhibited before 21st day. The strongest inhibition occurred in S5 on 7th day—the enzyme activity dropped to 46.57% of the control soil. On day 49, the activities of urease were activated in all soils with the exception of S5 and S10, whose activity decreased by 71.61% and 67.5%, respectively. The extent of activation of urease ranged from 115.6% (in S9) to 150.4% (in S6). Several studies have demonstrated that dehydrogenase enzyme activity of microorganisms is among most sensitive parameters for evaluation of toxicity. Maliszewska-Kordybach and Smreczak (2003) demonstrated that dehydrogenase activity is the most sensitive to the combine toxic effect of heavy metals and PAHs in soils.
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Enzyme Activity (% of control)
160 140
Urease
7 days
Dehydrogenase
120 100 80 60 40 20 0 S1
S2
S3
S4
S5 S6 S7 Soil Samples
S8
S9
S10
Enzyme Activity (% of control)
160 Dehydrogenase
Urease
140
21 days
120 100 80 60 40
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(Ba˚a˚th, 1989) or the degradation of PAHs and sorption of heavy metals and PAHs, which change the bioavailability of pollutants (Gong et al., 1997). It is now widely accepted that ageing phenomenon of some PAHs and heavy metals exists in soil. The behavior of aged contaminants is different from that of freshly added contaminants in the environment. Due to long-time processes (ageing), the bioavailability of contaminants decreases with time (Trivedi and Axe, 2000; Nam and Kim, 2002; Lock and Janssen, 2003). Among the possible mechanisms are the association of contaminants with natural organic matter (Carroll et al., 1994) and the penetration of contaminants into small pores in soil (Wu and Gschwend, 1986). In addition, it can be also seen that the change of enzyme activity was from 2.08% to 270% among the soil samples S2, S3, S5 and S7 with the same component of pollutants and different concentration in Fig. 1. It can be assumed that the combined effect of heavy metal and PAHs on soil enzyme is strongly dependent on concentration of pollutants. In comparison between two enzymes, dehydrogenase appears to be more suitable to be the indicator for the combined pollution of heavy metals and PAHs as urease, especially at the early stage of pollution. This result supports the previous reports (Ba˚a˚th, 1989; Yang and Liu, 2000).
20
3.2. Interactions between heavy metals and PAHs 0 S1
S2
S3
S4 S5 S6 Soil Samples
S7
S8
S9
S10
160 Enzyme Activity (% of control)
Urease
49 days
Dehydrogenase
140 120 100 80 60 40 20 0 S1
S2
S3
S4 S5 S6 Soil Samples
S7
S8
S9
S10
Fig. 1. Soil enzyme activity in different soil samples at different incubation times.
Our work supported these reports. As for the increasing activity with the incubation proceeded, a proper mechanism explaining this situation may be the ageing phenomenon of heavy metals and PAHs, the tolerance and adaptation of soil microorganism to pollutants
The type and magnitude of interaction may be dependent not only on the component in the mixture but also on the incubation time. In this work, the interaction between heavy metals and PAHs towards to soil enzyme (urease and dehydrogenase) were investigated through stepwise regression analysis. The dependent variable in all case was soil enzyme activity (% control). The independent variables used comprised the content of selected heavy metals and PAHs. Table 4 gives the summary of stepwise regression equations. 3.2.1. Interaction between heavy metals In Table 4, Eq. (4) showed that interaction between Zn and Cd to dehydrogenase activity on 7th day (the coefficient is 2.77) were the negative. The toxicity interaction between zinc and other metals has been investigated in diatoms, aquatic macrophytes and fish (Moreau et al., 1999). Results from those experiments ranged from antagonistic to synergistic responses, though the overall trend was for Zn to reduce toxicities of other metals. The negative interaction between Zn and Cd was probably the result of the competition between Zn and Cd for sorption sites. Zn is a major competitor for Cd sorption sites. This may be due to the general chemistry of Zn is somewhat comparable to the chemistry of Cd and that Zn concentrations in the environment usually are much higher than Cd
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Table 4 Relationship between soil enzyme activity (Y) and content of PAH and HM (X) No.
Multiple regression model
R2
1 2 3 4 5 6
Yu1 = 0.969XZn Yu3 = 1.06XZnXBaP + 0.387XZnXPhe 2.06XCd + 0.148XPb Yu7 = 0.743XZn 0.399XCd + 0.152XPheXFla YD1 = 0.65XBaP + 3.3XCdXPhe 2.77XCdXZn YD3 = 0.615XZn + 0.587XPheXBaP YD7 = 0.638XCd
0.969 0.996 0.985 0.933 0.937 0.956
Note: Yui (i = 1, 3, 7) denotes urease activity at ith week. YDi (i = 1, 3, 7) denotes dehydrogenase activity at ith week. Phe—phenanthrene, Fla—fluoranthene, BaP—benzo(a)pyrene.
concentrations (100–1000 times; Christensen, 1987). In soil of Cd contamination, Cd can make the Zn release to soil solution and enhance the bioavailability of zinc (Christensen, 1987). 3.2.2. The interaction between PAHs The influence of PAH on soil enzyme depends mainly on the amount and type of pollutants introduced into the environment (Boopathy, 2000). Wyszkowska et al. (2002) reported that diesel oil contamination of soil strongly inhibited the activity of dehydrogenases and soil urease. However, in some cases, the stimulatory activity also was observed (Boopathy, 2000; Margesin et al., 2000b). In our work, the combined stimulatory activity of phenanthrene and benzo(a)pyrene (or fluoranthene) was founded (Table 4, Eqs. (4) and (3)). It may be attributable to the gradual adaptation of microorganisms to the pollutants and the utilization of xenobiotics as a source of carbon and energy (Baran et al., 2004). There were known cases where bacterial, fungal or actinomycete populations are formed in polluted with substances originating from crude oil or organic compounds, capable of their decomposition. After the period of ‘‘stress’’, there is an increase in respiration intensity, enzyme activity, development of microorganisms and a gradual decomposition of pollutants (Boopathy, 2000; Baran et al., 2004). The mechanism of interaction between PAHs is not clear. 3.2.3. Interaction between heavy metals and PAHs In natural soils, heavy metals exhibit toxic activity towards soil biota which may lead to the decrease of the number and the activity of soil microorganisms and reduce the rate of PAH microbial transformations playing an important role in dissipation of these compounds in the soil environment (Wild and Jones, 1995). There is also a possibility of positive interaction of heavy metals and PAHs (Maliszewska-Kordybach and Smreczak, 2003). In this study, the positive interaction between Zn (Cd) and phenanthrene towards urease (dehydrogenase) were observed (Table 4, Eqs. (2) and (4)). One of the possible explanations of this phenomenon is that
the adsorption of phenanthrene was significantly higher after the addition of the metals (Saison et al., 2004). However, the interaction between Zn and benzo(a)pyrene to urease (Table 4, Eq. (2)) was negative. The interaction between zinc and phenanthrene enhances the urease activity on the 21st days (Table 4, Eq. (2)). This finding was in agreement with the previous reports. Moreau et al. (1999) investigated the interaction between phenanthrene and zinc in their toxicity to the sheepshead minnow; Cyprinodon variegates and suggested that the presence of zinc may result in an increased detoxification of phenanthrene. While there is no empirical evidence to support this, experiments with another metal (cadmium) have shown that Cd may increase levels or activities of xenobiotic detoxification enzymes in fish (Fair, 1986; Lemaire-Gony et al., 1995) and that Cd and benzo[a]pyrene may act synergistically in this respect (Lemaire-Gony and Lemaire, 1992). In Eq. (2), it can also be seen that the interaction between Zn and benzo(a)pyrene weaken the urease activity at 21st day (Table 4). One of the possible explanations of this phenomenon can be based on the recent assumptions (Sikkema et al., 1994) that lipophilic compounds such as PAH have a narcotic mode of toxic action and may interact with lipophilic components of cytoplasmitic membranes of bacteria, thus affecting their permeability and structure. Hence, in PAH contaminated soil, heavy metals may penetrate easier into microbial cells and affect their functions. The similar combination of toxic effects of heavy metals (Zn, Cd and Cu) and PAH (phenanthrene) towards soil bacteria was reported by Gogolev and Wilke (1997), who used agar plate technique to eliminate interaction of pollutants with soil matrix and received results that are more pronounced. In summary, the combined effect of PAH and HM on the soil enzyme activity depends largely on the incubation time. This may be related to bioavailability. The link between bioavailability and mixture toxicity is the competition among pollutants for sorption sites, which results in one pollutant displacing the weaker competing pollutant from soil particles into the soil solution. The ratio of pollutant concentration in the soil solution is
G. Shen et al. / Chemosphere 61 (2005) 1175–1182
thus different from the ratio of total pollutant concentration in soil at different incubation time. The distribution of pollutants over the soil phases is of crucial importance to the interaction between PAHs and heavy metals towards soil enzyme, because only the metal concentration in the liquid phase is considered available to them (Weltje, 1998).
4. Conclusions The results obtained from this study of combined effect of PAHs and heavy metals on the soil urease and dehydrogenase activities reveal a set of action and interaction between these pollutants. The experiment setup corresponds to an exploratory phase and suggests several orientations for further research. Contamination levels should remain ‘‘realistic’’ in relation to concentrations found in the environment. Uniform design is a powerful tool in studying the interaction of mixtures. More experimentation is necessary to better understand the mechanisms underlying the interactions between PAHs and heavy metals, but it is evident that these interactions may considerably affect their toxicities. Moreover, the magnitude and type of interaction may be strongly dependent on the level of response and the relative proportion of the components in the mixture. These factors should therefore be considered in risk assessments and in the setting of soil quality criteria.
Acknowledgements This work was supported by National Key Basic Research Program of China (No. 2004CB418503), National Natural Science Foundation of China (No. 20337010) and Key Program of Basic Research of Shanghai City (No. 04JC14051).
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Weltje, L., 1998. Mixture toxicity and tissue interactions of Cd, Cu, Pb and Zn in earthworms (Oligochaeta) in laboratory and field soils: a critical evaluation of data. Chemosphere 36 (12), 2641–2660. Wild, S.R., Jones, K.C., 1995. Polynuclear aromatic hydrocarbons in the United Kingdom environment: a preliminary source inventory and budget. Environ. Pollut. 88, 91–108. Wu, S., Gschwend, P.M., 1986. Sorption kinetics of hydrophobic organic compounds to natural sediments and soils. Environ. Sci. Technol. 20, 717–725. Wyszkowska, J., Kucharski, J., Waldowska, E., 2002. The influence of diesel oil contamination on soil enzymes activity. Rostlinna´ Vyroba 48 (2), 58–62. Yang, Z.X., Liu, S.G., 2000. Effect of single element and compound pollution of Cd, Zn and Pb on soil enzyme activities. Soil Environ. Sci. 9 (1), 15–18. Zhang, C., Xia, L.J., 2000. Bioremediation of Contaminated Soils. China Environmental Science Press, p. 164. Zheng, C.R., Tu, C., Chen, H.M., 1999. Effect of combined heavy metal pollution on nitrogen mineralization potential, urease and phosphatase activities in a Typic Udic Ferrisol. Pedosphere 9 (3), 251–258.
Interaction of polycyclic aromatic hydrocarbons and heavy metals on soil enzyme Guoqing Shen a, Yitong Lu a
b
a,*
, Qixing Zhou b, Jingbo Hong
a
Department of Environment and Resource, College of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 201101, PR China Key Laboratory of Terrestrial Ecological Process, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, PR China Received 12 August 2004; received in revised form 23 January 2005; accepted 22 February 2005 Available online 13 April 2005
Abstract Actions and interactions of heavy metals (cadmium, zinc and plumbum) and polycyclic aromatic hydrocarbons (PAHs) [phenanthrene, fluoranthene, benzo(a)pyrene] on the soil urease and dehydrogenase activity were studied after 49 days exposure. The experimental approach was based on the uniform design which can cut the experiment time and improve the efficiency of experiments. Data treatment was essentially based on the multiple regression technique. The results showed that the action and interaction between heavy metals and PAHs were strongly dependent on the time of pollution. The dehydrogenase exhibits more sensitive to the combined pollution than urease. The negative interaction between Zn and Cd to hydrogenase activity and the combined stimulatory activity of Phenanthrene and Benzo(a)pyrene (or fluoranthene) to soil enzyme were observed. The interactions between Zn (Cd) and phenanthrene towards urease (dehydrogenase) were positive, and the interaction between Zn and benzo(a)pyrene to urease activity was negative. This study corresponds to exploratory phase in order to reveal interaction effects of heavy metals and PAHs on the soil enzyme and then to set up more in-depth analysis to increase progressively the understanding of the ecotoxicological mechanisms involved. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Heavy metals; PAH; Urease; Dehydrogenase; Uniform design
1. Introduction Pollution of soil environment has become a serious problem in many countries, including China. Heavy metals (HM) and polycyclic aromatic hydrocarbons (PAHs) are two of the most abundant and potentially
* Corresponding author. Tel.: +86 21 6478 5941; fax: +86 21 6478 7938. E-mail address: [email protected] (Y. Lu).
harmful pollutants found in most polluted soil (Jones et al., 1989; Baran et al., 2004). Heavy metal pollution in soil is of major environmental concern on a world scale and in China in particular with the rapid development of industry. Beside their natural occurrence, heavy metals may enter the ecological environment through anthropogenic activities, such as mining, smelting, sewage sludge disposal, application of pesticides and inorganic fertilizers, and atmospheric deposition. PAHs are a ubiquitous group of hazardous organic pollutants which exhibit strong carcinogenic and toxic properties.
0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.02.074
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They are formed by incomplete burning of fossil fuels and can enter the soil via atmospheric deposition. It is estimated that more than 90% of the total burden of PAH resides in the surface soils (Wild and Jones, 1995). Both of HM and PAH introduced into the soil exert an influence on the microbiota, which manifests itself in changes in enzyme activity (Baran et al., 2004). Soil enzymes are the catalysts of important metabolic processes including the decomposition of organic pollutants and the detoxification of xenobiotics (Margesin et al., 2000b). Enzyme activities are considered to be sensitive to pollution and have the further advantage of being easy to determine without expensive, sophisticated instruments. So, soil enzyme activities have been proposed as indicators for measuring the degree of soil pollution. Dehydrogenase and urease are most important soil enzyme and frequently used test for determining the influence of the various pollutants (heavy metals, pesticide, crude oil, etc.) on the microbiological quality of soil (Margesin et al., 2000a,b). Several researchers investigated the effect of heavy metals and PAHs on urease and dehydrogenase activity. Margesin et al. (2000a) proved a positive influence of naphthalene and phenanthrene on urease activities and gave the correlation for urease activity and phenanthrene content. A positive correlation between dehydrogenase activity and the content of phenanthrene and also fluoranthene, chrysene and dibenzo[ah]anthracene was also observed (Baran et al., 2004). The inhibition of heavy metal pollution on urease and dehydrogenase activity was reported by many scientists (Zheng et al., 1999). The sequence of inhibition of urease activity was generally in the decreasing order of Cr > Cd > Cu > Zn > Mn > Pb. However, the stimulating effects of heavy metals were also documented (Ba˚a˚th, 1989). The aforementioned studies are mostly concentrated on the effect of a single pollutant. In the real environment, however, contaminated soils usually contain mixtures of chemicals rather than a pure substance. As a consequence, soil organisms are exposed to multiple toxicants. The actual risk to which these organisms are exposed is determined by the composition and availability of the mixture. Still, most ecotoxicologically based risk assessment methods evaluate contaminated soils by single-substance criteria, derived from single-substance toxicity data, by that, neglecting mixture effects (Weltje, 1998). Recently, several investigations were devoted to the combined effect of PAH and HM on the soil enzyme (Gogolev and Wilke, 1997; Gong et al., 1997; Irha et al., 2003; Maliszewska-Kordybach and Smreczak, 2003). The presence of metals in PAHs contaminated soils is not rare, especially at coking plants or gas works (Saison et al., 2004). However, it is an area of research that has not been covered to any extent. The contradictory and somewhat confusing results are often reported about this topic. So, the firm conclusions
are still difficult to draw from these little literatures. The difficulties inherent in studying this interaction may be linked with the methodological choices for devising protocols and the methods used for the data treatment (Ribryre et al., 1995). The laboratory situation where usually large doses of the heavy metal and PAH added is far from the real-world exposure although this process may provide some information. In addition, many studies considered the interactions only at specific endpoint, thus make the controvertible conclusion. In this respect, experimental uniform design that uniformly distributes the experimental points in the factor space and multiple regressions can provide an important contribution to research the interaction between pollutants. This design can cut the experiment time and improve the efficiency of experiments. The first aim of this research was to evaluate the action and interaction of heavy metals (Cd, Zn, Pb) and PAHs [phenanthrene, fluoranthene, benzo(a)pyrene] on the soil urease and dehydrogenase activity over 49 days. These pollutants were chosen for the present study because of their abundance and the lack of toxicity data in soil environment. The structure and solubility of three PAHs were different: 3, 4 and 5 aromatic rings, and water solubility of 1.300, 0.260 and 0.003 mg l1. The concentrations tested in this study simulated the real soil environment. The second objective is to recommend a relative new method to research the interaction of combined pollutants in soil. The protocol was based on a uniform design with 6 pollutants and 10 contamination levels for each pollutant.
2. Materials and methods 2.1. Experimental soils The experimental soils used for this study was collected from an agricultural land (0–20 cm in depth) in Shanghai city, China (31.14 0 N, 121.29 0 E). The soil type was a paddy soil. After transportation to the lab, the soil was air-dried, ground, passed through a 3 mm mesh and stored as the stock sample for this study. The basic soil properties were determined using standard methods recommended by the Chinese Society of Soil Science (Lu, 1999). The soils had the following basic properties: pH 8.18, organic matter 16.7%, cation exchange capacity (CEC) 15.6 cmol kg1, total N 1.14 g kg1 and total P 1.36 g kg1, sand 51.49%, silt 28.37%, clay 20.14%, Fe2O3 5.53%, Al2O3 14.38%, and SiO2 58.7%. 2.2. Experimental design A uniform design was employed to study the combined effect of heavy metals and PAHs. Fang (1980),
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professor of the Mathematical Research Institute of Chinese Academy of Sciences, put forward a uniform design method adopting number theory to distribute the experimental points uniformly in the factor space. This design is similar to the orthogonal one. It has two characteristics: (1) The occurrence of each level of each factor in the experiments is once only; (2) The number of experimental trials equals the level number of factors. For a study of multi-factors with multiple levels, the uniform design needs much fewer experiments than the Orthogonal design does (Fang, 1980; Liang et al., 2001; Cheng et al., 2002). It can cut the experiment time and improve the efficiency of experiments. For experiments with 6-factors and 10-levels, the experimental number of orthogonal design is 100 and the complete design is 106, while that of Uniform design is only 10, which is shown in Table 1. Uniform design tables can be expressed as Un(qs), where U stands for Uniform design, n for number of experimental trials, q for number of levels and s for maximum number of factors. In Table 1, each column corresponds to a factor and each row corresponds to an experimental trial to be carried out. As shown in this table, the Uniform design has more
levels so as to establish regression equations of higher precision and estimate the main and interaction effects of the experimental factors (Fang, 1980). Now, uniform design has been gradually popularized in China, particularly in agriculture, industry, science researches, military sciences and chemistry, and chemical engineering. Three PAH compounds with different properties [phenanthrene, fluoranthene, benzo(a)pyrene] and three elements (cadmium, zinc, plumbum) that often emitted from the same as PAHs were considered as the main pollutants in soil, and were selected as the tested pollutants for this study. The choice of concentrations was based on the environmental quality standards for soils of China and Canada. In China, the soil environmental quality standards for Cd, Zn and Pb are less than 0.2, 100 and 35 mg kg1 soil, respectively (Chinese National Environmental Protection Agency, 1995). The standard for PAHs is less than 0.1 mg kg1 in Canada (Cerniglia, 1984; Zhang and Xia, 2000). The concentrations of heavy metals and PAHs added to soil samples are listed in Table 2. The uniform designs and concentrations of heavy metals and PAHs added to soil samples are listed in Table 3.
Table 1 Uniform design table U10(106) for 6-factor and 10-level
2.3. Preparation of samples
Sample no.
The studies were performed as pot experiments. The dimension of containers was 1000 ml. Stock soil sample (800 g) was first thoroughly mixed with a water solution of HM salts [Zn(CH3COO)2, Pb(CH3COO)2, and Cd(CH3COO)2] and CH2Cl2 solution of PAHs according to the design and concentrations given in Table 3, and then incubated at room temperature for 7 weeks. Control without any artificial contaminated received the same amount of distilled water and CH2Cl2. There were three replications. Moisture in the incubated soil samples was kept at 60% of the full soil water holding capacity by weighting daily. This level was kept during the time of the experiment.
Factors
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11
X1
X2
X3
X4
X5
X6
1 2 3 4 5 6 7 8 9 10 0
2 4 6 8 10 1 3 5 7 9 0
3 6 9 1 4 7 10 2 5 8 0
5 10 4 9 3 8 2 7 1 6 0
7 3 10 6 2 9 5 1 8 4 0
10 9 8 7 6 5 4 3 2 1 0
Table 2 Experimental factors and levels (mg kg1 soil) Factor
Level 1
2
3
4
5
6
7
8
9
10
Cd Zn Pb Phea Flab Bapc
0 0 0 0 0 0
0.003125 12.5 4.3625 0.0125 0.0125 0.0125
0.0125 25 8.725 0.025 0.025 0.025
0.05 50 17.5 0.05 0.05 0.05
0.2 100 35 0.1 0.1 0.1
0.8 200 70 0.2 0.2 0.2
3.2 400 140 0.4 0.4 0.4
12.8 800 280 0.8 0.8 0.8
51.2 1600 560 1.6 1.6 1.6
204.8 3200 1120 3.2 3.2 3.2
a b c
Phe denotes phenanthrene. Fla denotes fluoranthene. BaP denotes benzo(a)pyrene.
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Table 3 Experimental treatments and concentration of HM and PAH added to soil (mg kg1 soil) Treatments
Cd
Zn
Pb
Phea
Flab
BaPc
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11
0 0.03125 0.0125 0.05 0.2 0.8 3.2 12.8 51.2 204.8 0
12.5 50 200 800 3200 0 25 100 400 1600 0
8.725 70 560 0 17.5 140 1120 4.3625 35 280 0
0.1 3.2 0.005 1.6 0.025 0.8 0.0125 0.4 0 0.2 0
0.4 0.025 3.2 0.2 0.0125 1.6 0.1 0 0.8 0.005 0
3.2 1.6 0.8 0.4 0.2 0.1 0.005 0.025 0.0125 0 0
a b c
Phe denotes phenanthrene. Fla denotes fluoranthene. BaP denotes benzo(a)pyrene.
2.4. Determination of urease and dehydrogenase activity The activities of urease and dehydrogenase were determined after 1, 3 and 7 weeks. Soil urease activity was determined by the method of Tabatabai and Bremner, expressed as mg NH4-N kg1 h1 (Tabatabai and Bremner, 1972). Dehydrogenase activity was tested by reduction of 2, 3, 5-triphenyltetrazolium chloride (TTC). After 24 h at 37 °C, the triphenyl formazan (TPF) released was extracted with methanol and assayed at 485 nm in an Perkin–Elmer UV spectrophotometer (Casida et al., 1964). The unit of dehydrogenase activity was TPF mg g1 24 h1. The results were expressed as an average of three parallel determinations of the mixture of there duplicates soil samples. The enzyme activities in Fig. 1 are expressed as % of control. 2.5. Data treatment Generally, to account for combined effect of different important independent variables, second-order polynomial models are preferred (Poorna and Kulkarni, 1995). Therefore, it was thought of interest to develop a second-order polynomial model describing the effect of six selected independent variables, namely, the concentration of cadmium, zinc, plumbum, phenanthrene, fluoranthene, benzo(a)pyrene on the urease and dehydrogenase activity in soil. The model was expressed as Y ¼ b0 þ
m X i¼1
bi X i þ
X
bij X i X j þ e;
i
where Y is the predicted value of soil enzyme activity; X is the independent variables corresponding to the concentration of heavy metals and PAHs; and b is the regression coefficients estimated by the stepwise regression method with a confidence level of 95%.
The results obtained were analyzed using SPSS (version 12.0) software.
3. Results and discussion 3.1. Comparison of the enzyme inhibition effect Fig. 1 shows the effect of combined pollution of PAHs and heavy metals on urease and dehydrogenase activities during the whole course of the experiment (49 days). The results were expressed as % of control (activity of control = 100). Although the enzyme activities in all soil samples were inhibited exception of urease on 49th day, the extent of enzyme inhibition varied with incubation proceeded. The dehydrogenase exhibits more sensitive than urease. In the case of dehydrogenase, the enzyme activity was dropped below 50% of the control soil on 7th day, 22.22–100% on 21st day and 23.53–88.24% on 49th day in all soil samples. The maximal rate of inhibition was 97.82% in sample of S4, S6, S7 and S9 on 7th day. However, the activity of urease was different from dehydrogenase. The activity was inhibited before 21st day. The strongest inhibition occurred in S5 on 7th day—the enzyme activity dropped to 46.57% of the control soil. On day 49, the activities of urease were activated in all soils with the exception of S5 and S10, whose activity decreased by 71.61% and 67.5%, respectively. The extent of activation of urease ranged from 115.6% (in S9) to 150.4% (in S6). Several studies have demonstrated that dehydrogenase enzyme activity of microorganisms is among most sensitive parameters for evaluation of toxicity. Maliszewska-Kordybach and Smreczak (2003) demonstrated that dehydrogenase activity is the most sensitive to the combine toxic effect of heavy metals and PAHs in soils.
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Enzyme Activity (% of control)
160 140
Urease
7 days
Dehydrogenase
120 100 80 60 40 20 0 S1
S2
S3
S4
S5 S6 S7 Soil Samples
S8
S9
S10
Enzyme Activity (% of control)
160 Dehydrogenase
Urease
140
21 days
120 100 80 60 40
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(Ba˚a˚th, 1989) or the degradation of PAHs and sorption of heavy metals and PAHs, which change the bioavailability of pollutants (Gong et al., 1997). It is now widely accepted that ageing phenomenon of some PAHs and heavy metals exists in soil. The behavior of aged contaminants is different from that of freshly added contaminants in the environment. Due to long-time processes (ageing), the bioavailability of contaminants decreases with time (Trivedi and Axe, 2000; Nam and Kim, 2002; Lock and Janssen, 2003). Among the possible mechanisms are the association of contaminants with natural organic matter (Carroll et al., 1994) and the penetration of contaminants into small pores in soil (Wu and Gschwend, 1986). In addition, it can be also seen that the change of enzyme activity was from 2.08% to 270% among the soil samples S2, S3, S5 and S7 with the same component of pollutants and different concentration in Fig. 1. It can be assumed that the combined effect of heavy metal and PAHs on soil enzyme is strongly dependent on concentration of pollutants. In comparison between two enzymes, dehydrogenase appears to be more suitable to be the indicator for the combined pollution of heavy metals and PAHs as urease, especially at the early stage of pollution. This result supports the previous reports (Ba˚a˚th, 1989; Yang and Liu, 2000).
20
3.2. Interactions between heavy metals and PAHs 0 S1
S2
S3
S4 S5 S6 Soil Samples
S7
S8
S9
S10
160 Enzyme Activity (% of control)
Urease
49 days
Dehydrogenase
140 120 100 80 60 40 20 0 S1
S2
S3
S4 S5 S6 Soil Samples
S7
S8
S9
S10
Fig. 1. Soil enzyme activity in different soil samples at different incubation times.
Our work supported these reports. As for the increasing activity with the incubation proceeded, a proper mechanism explaining this situation may be the ageing phenomenon of heavy metals and PAHs, the tolerance and adaptation of soil microorganism to pollutants
The type and magnitude of interaction may be dependent not only on the component in the mixture but also on the incubation time. In this work, the interaction between heavy metals and PAHs towards to soil enzyme (urease and dehydrogenase) were investigated through stepwise regression analysis. The dependent variable in all case was soil enzyme activity (% control). The independent variables used comprised the content of selected heavy metals and PAHs. Table 4 gives the summary of stepwise regression equations. 3.2.1. Interaction between heavy metals In Table 4, Eq. (4) showed that interaction between Zn and Cd to dehydrogenase activity on 7th day (the coefficient is 2.77) were the negative. The toxicity interaction between zinc and other metals has been investigated in diatoms, aquatic macrophytes and fish (Moreau et al., 1999). Results from those experiments ranged from antagonistic to synergistic responses, though the overall trend was for Zn to reduce toxicities of other metals. The negative interaction between Zn and Cd was probably the result of the competition between Zn and Cd for sorption sites. Zn is a major competitor for Cd sorption sites. This may be due to the general chemistry of Zn is somewhat comparable to the chemistry of Cd and that Zn concentrations in the environment usually are much higher than Cd
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Table 4 Relationship between soil enzyme activity (Y) and content of PAH and HM (X) No.
Multiple regression model
R2
1 2 3 4 5 6
Yu1 = 0.969XZn Yu3 = 1.06XZnXBaP + 0.387XZnXPhe 2.06XCd + 0.148XPb Yu7 = 0.743XZn 0.399XCd + 0.152XPheXFla YD1 = 0.65XBaP + 3.3XCdXPhe 2.77XCdXZn YD3 = 0.615XZn + 0.587XPheXBaP YD7 = 0.638XCd
0.969 0.996 0.985 0.933 0.937 0.956
Note: Yui (i = 1, 3, 7) denotes urease activity at ith week. YDi (i = 1, 3, 7) denotes dehydrogenase activity at ith week. Phe—phenanthrene, Fla—fluoranthene, BaP—benzo(a)pyrene.
concentrations (100–1000 times; Christensen, 1987). In soil of Cd contamination, Cd can make the Zn release to soil solution and enhance the bioavailability of zinc (Christensen, 1987). 3.2.2. The interaction between PAHs The influence of PAH on soil enzyme depends mainly on the amount and type of pollutants introduced into the environment (Boopathy, 2000). Wyszkowska et al. (2002) reported that diesel oil contamination of soil strongly inhibited the activity of dehydrogenases and soil urease. However, in some cases, the stimulatory activity also was observed (Boopathy, 2000; Margesin et al., 2000b). In our work, the combined stimulatory activity of phenanthrene and benzo(a)pyrene (or fluoranthene) was founded (Table 4, Eqs. (4) and (3)). It may be attributable to the gradual adaptation of microorganisms to the pollutants and the utilization of xenobiotics as a source of carbon and energy (Baran et al., 2004). There were known cases where bacterial, fungal or actinomycete populations are formed in polluted with substances originating from crude oil or organic compounds, capable of their decomposition. After the period of ‘‘stress’’, there is an increase in respiration intensity, enzyme activity, development of microorganisms and a gradual decomposition of pollutants (Boopathy, 2000; Baran et al., 2004). The mechanism of interaction between PAHs is not clear. 3.2.3. Interaction between heavy metals and PAHs In natural soils, heavy metals exhibit toxic activity towards soil biota which may lead to the decrease of the number and the activity of soil microorganisms and reduce the rate of PAH microbial transformations playing an important role in dissipation of these compounds in the soil environment (Wild and Jones, 1995). There is also a possibility of positive interaction of heavy metals and PAHs (Maliszewska-Kordybach and Smreczak, 2003). In this study, the positive interaction between Zn (Cd) and phenanthrene towards urease (dehydrogenase) were observed (Table 4, Eqs. (2) and (4)). One of the possible explanations of this phenomenon is that
the adsorption of phenanthrene was significantly higher after the addition of the metals (Saison et al., 2004). However, the interaction between Zn and benzo(a)pyrene to urease (Table 4, Eq. (2)) was negative. The interaction between zinc and phenanthrene enhances the urease activity on the 21st days (Table 4, Eq. (2)). This finding was in agreement with the previous reports. Moreau et al. (1999) investigated the interaction between phenanthrene and zinc in their toxicity to the sheepshead minnow; Cyprinodon variegates and suggested that the presence of zinc may result in an increased detoxification of phenanthrene. While there is no empirical evidence to support this, experiments with another metal (cadmium) have shown that Cd may increase levels or activities of xenobiotic detoxification enzymes in fish (Fair, 1986; Lemaire-Gony et al., 1995) and that Cd and benzo[a]pyrene may act synergistically in this respect (Lemaire-Gony and Lemaire, 1992). In Eq. (2), it can also be seen that the interaction between Zn and benzo(a)pyrene weaken the urease activity at 21st day (Table 4). One of the possible explanations of this phenomenon can be based on the recent assumptions (Sikkema et al., 1994) that lipophilic compounds such as PAH have a narcotic mode of toxic action and may interact with lipophilic components of cytoplasmitic membranes of bacteria, thus affecting their permeability and structure. Hence, in PAH contaminated soil, heavy metals may penetrate easier into microbial cells and affect their functions. The similar combination of toxic effects of heavy metals (Zn, Cd and Cu) and PAH (phenanthrene) towards soil bacteria was reported by Gogolev and Wilke (1997), who used agar plate technique to eliminate interaction of pollutants with soil matrix and received results that are more pronounced. In summary, the combined effect of PAH and HM on the soil enzyme activity depends largely on the incubation time. This may be related to bioavailability. The link between bioavailability and mixture toxicity is the competition among pollutants for sorption sites, which results in one pollutant displacing the weaker competing pollutant from soil particles into the soil solution. The ratio of pollutant concentration in the soil solution is
G. Shen et al. / Chemosphere 61 (2005) 1175–1182
thus different from the ratio of total pollutant concentration in soil at different incubation time. The distribution of pollutants over the soil phases is of crucial importance to the interaction between PAHs and heavy metals towards soil enzyme, because only the metal concentration in the liquid phase is considered available to them (Weltje, 1998).
4. Conclusions The results obtained from this study of combined effect of PAHs and heavy metals on the soil urease and dehydrogenase activities reveal a set of action and interaction between these pollutants. The experiment setup corresponds to an exploratory phase and suggests several orientations for further research. Contamination levels should remain ‘‘realistic’’ in relation to concentrations found in the environment. Uniform design is a powerful tool in studying the interaction of mixtures. More experimentation is necessary to better understand the mechanisms underlying the interactions between PAHs and heavy metals, but it is evident that these interactions may considerably affect their toxicities. Moreover, the magnitude and type of interaction may be strongly dependent on the level of response and the relative proportion of the components in the mixture. These factors should therefore be considered in risk assessments and in the setting of soil quality criteria.
Acknowledgements This work was supported by National Key Basic Research Program of China (No. 2004CB418503), National Natural Science Foundation of China (No. 20337010) and Key Program of Basic Research of Shanghai City (No. 04JC14051).
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