海水重金属单一及复合污染对双壳类金属硫蛋白的影响和非生物因子的干扰
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摘要
重金属具有亲脂性、难降解性和高富集性,容易在海洋生物体内积累,通过食物链进行放大,海水中微量浓度的重金属即可引起生物机体的氧化应激和氧化损伤甚至导致生物死亡。因此,寻找能够快速、准确监测海洋环境中重金属污染水平的方法,对于及时掌握海水污染程度及生态风险性评价十分必要。
运用生物标志物法监测海水重金属污染,能够反映海水中多重金属对生物的混合效应,灵敏度高且专一性较强。海洋双壳类金属硫蛋白(MTs)即是重金属毒性监测中常用的一类生物标志物。但是,相同重金属对不同物种MTs的诱导规律存在较大差异,即使同一物种其不同组织MTs对重金属暴露的响应程度也不相同,显示在重金属生物监测方面应选择适宜的生物种类和组织类型。定量监测海水重金属污染水平则需要获得重金属浓度—MTs诱导量及暴露时间—MTs诱导量的响应关系方程。另外,由于海水温度、pH值、盐度等因子的波动会对生物产生直接或间接的干扰,影响其体内MTs的水平,造成海域现场监测结果的不准确性。
本研究以中国沿海海域常见的双壳类生物——菲律宾蛤仔(Ruditapes philippinarum)为实验对象,分别选用含单一重金属离子Cu2+、Zn2+、Cd2+、Hg2+的人工海水对蛤仔进行暴露培养,通过测定重金属在鳃和内脏中的总含量及可溶性含量变化,分析其在蛤仔组织中的富集规律；根据蛤仔鳃和内脏中MTs的水平随暴露浓度和时间的变化,选择适用于指示单一重金属污染的组织类型,并确定指示重金属污染的最佳浓度范围和暴露时间。研究结果表明：蛤仔内脏比鳃更容易蓄积海水中的Cu2+、Zn2+、Cd2+、Hg2+,50μg/LCu2+、100μg/LZn2+、50μg/L Cd2+、5μg/LHg2+分别暴露5d后蛤仔鳃对4种重金属的BCF值分别为53.6、280.8、20.6、282.8,内脏的BCF值分别为676.8、268.1、47.4、565.6。进入鳃和内脏中的Cu2+和Cd2+主要以游离态形式存在,而Zn2+、Hg2+暴露下,亚细胞组分中的Zn、Hg主要以不溶形式存在。组织内富集金属Cu、Cd、Hg与相应组织中MTs含量之间的关系呈幂函数关系或负指数关系,而Zn的蓄积与MTs之间的关系可以用直线方程表征。蛤仔内脏MTs对Cu2+暴露更为敏感,而鳃MTs则更适合指示海水Zn2+、Cd2+或Hg2+等重金属污染水平。
在研究重金属单一暴露的基础上,分别将上述重金属离子2种或4种混合,研究复合污染下蛤仔MTs的响应规律,并与单一金属暴露相比较,探讨2种或4种重金属对蛤仔MTs的联合诱导效应机制。结果表明：双重金属和四重金属联合与对照组相比均能显著诱导蛤仔鳃和内脏MTs的合成。(Cu2+-Zn2+)联合暴露5d,蛤仔鳃和内脏MTs含量随混合金属浓度升高表现出先升高后降低的趋势,随着(Cu2+-zn2+)混合浓度的增大,其对鳃MTs的联合诱导表现为协同—相加—拮抗效应,对内脏MTs则表现为协同—拮抗效应。较低浓度(Cd2+-Hg2+)联合暴露响对MTs的诱导能力远远大于高浓度组,随着(Cd2+-Hg2+)混合浓度的增大,其对鳃MTs的联合诱导表现为协同—相加—拮抗效应,对内脏MTs则表现为协同—拮抗效应。在4种重金属离子联合暴露下,重金属浓度较低时,对鳃MTs联合诱导效应表现为相加作用,随着浓度增大则转变为拮抗作用,而对内脏MTs的联合诱导均表现为拮抗作用。
研究非生物因子对蛤仔MTs的干扰程度,对复合污染下蛤仔MTs的响应值随海水温度、盐度、pH值的变化趋势进行了初步探索,辨析这些因子的干扰作用并采取适宜方法对测定结果加以校正,以便于在消除非生物因子影响的前提下进行重金属综合污染水平评价。结果表明：上述非生物因子中温度、盐度变化对MTs的响应值影响较大,pH值的影响相对较小。①在蛤仔适宜生长的海水温度范围(5～20℃)内,无论是低浓度还是高浓度重金属污染区,鳃、内脏的MTs含量均随温度升高而增加,同等污染水平在20℃时对MTs的诱导量最高。高、低浓度污染区的鳃MTs含量以及高浓度污染区的内脏MTs含量,与温度之间呈现显著正相关性,可直接采用内插法,将MTs含量实测值转换为15℃时的校正值。②蛤仔MTs对重金属暴露的响应值受盐度影响呈“阶梯”状变化。如果以盐度30时的测定值作为基准值,以下情况无需进行校正：重污染区、盐度为20-35时,鳃MTs含量；盐度在20～35范围内时,低污染、重污染区内脏MTs含量。其他盐度范围下,所测定的鳃或内脏MTs均需采用内插法进行校正。③蛤仔MTs对重金属的响应受海水pH值的影响较小。以pH8.0的测定值作为基准,如果采用鳃MTs含量指示重金属综合污染水平,在海水pH为7.5～9.0(低污染区)和pH7.5-8.0(重污染区)的测定值无需校正；如果采用内脏MTs含量指示重金属综合污染水平,在海水pH为8.0～8.5(低污染区、重污染区)时的测定值无需校正。其他pH范围下所测定的鳃或内脏MTs均以pH8.0为准,需采用内插法进行校正。
Heavy metals are highly lipophilic, hard to degrade, bioaccumulative. Amplifying through the food chain, trace metals can cause oxidative stress and oxidative damage in organisms, even result in death. Therefore, it is necessary to research suitable methods for monitoring heavy metal pollution in seawater and ecological risk assessment. Biomarkers have high sensitivity and specificity, they can monitor mixed effect of heavy metals in the seawater. Metallothioneins (MTs) of marine bivalves are common biomarkers which are used in monitoring heavy metal toxicity. However, MTs in different species, even in different tissues of the same species, respond differently to the same pollution. To quantify heavy metal pollution, the dose-or time-effect relationships between heavy metal concentrations and MTs contents need to be obtained. Furthermore, the MTs inductions by metals can be influenced by variations of the temperature, salinity, pH value and so on, which can cause inaccuracy of field monitoring.
Ruditapes philippinarum are exposed in artificial seawater containing single heavy metal ion Cu2+, Zn2+, Cd2+or Hg2+. The aims of this part is to study the rules of metal enrichment, through determining the total/soluble component of heavy metal in gills and visceral mass; finding the suitable tissue for monitoring single-metal pollution and confirming the concentration range of each metal and exposure time. The experiments indicate that visceral mass can easier accumulate metals than gills. BCF5of gills are53.6,280.8.20.6,282.8after exposed in50μg/L Cu2+,100μg/L Zn2+,50μg/L Cd2+and5μg/L Hg2+, respectively, while BCF5of gills are676.8,268.1,47.4,565.6.Cu2+and Cd2+in gills and visceral mass are mainly in free form, in clams exposed to Zn2+or Hg2+, subcellular fraction C1contained higher Zn or Hg percentage. It is confirmed by the power function or negative exponential relationship between the levels of MTs and the contents of Cu, Cd or Hg in all tissues (gills and visceral mass), while Zn and MTs is in the positive and significant relationship. The MTs of visceral mass are more sensitive to Cu2+exposure, while for monitoring metal ions Zn2+, Cd2+or Hg2+, MTs of gills are more suitable.
To better understand the effects of metals in MTs response in Ruditapes philippinarum, these clams are exposed to polymetallic mixtures (Cu2+-Zn2+, Cd2+-Hg2+or Cu2+-Zn2+-Cd2+-Hg2+). The interactions between these metals and induction mechanism of MTs by polymetallic mixtures are discussed. The results show that the MTs in all clams exposed to a mixture of2or4metals are found significantly increase, which compared to control group (p<0.01or p<0.05). During exposed to Cu2+-Zn2+for5days, MTs levels in gills and visceral mass show increase at first, then decreased. The inductive effect of Cu2+-Zn2+compound is showed synergism-addition-antagonism on MTs in gills and synergism-antagonism in visceral mass with the increase concentrations of Cu2+and Zn2+. The inducibility of MTs by Cd2+-Hg2+in lower concentrations is larger than other groups. The inductive effect of Cd2+-Hg2+compound is showed synergism-addition-antagonism on MTs in gills and synergism-antagonism in visceral mass with the increase concentrations of Cd2+and Hg2+. When exposed to polymetallic mixtures(Cu2+-Zn2+-Cd2+-Hg2+), the inductive effect of Cu2+-Zn2+-Cd2+-Hg2+compound is showed addition-antagonism on MTs in gills and antagonism in visceral mass with the increase concentrations of these four metals.
The abiotic factors have certain influences on MTs induction by heavy metals in clams. The temperature, salinity and pH value of seawater are common abiotic factors. The present study is designed to examine the interference degree of these factors on MTs induction by polymetallic mixtures and put forward the effective methods to eliminate the unfavorable effects when applying MTs to monitoring heavy metals pollution in seawater. The results indicate that temperature and salinity affect MTs contents in gills and visceral mass notably, while the influences of pH value are relatively minor.①Under the optimal growth temperature5～20℃, there are linear relationship between MTs contents in gills and visceral mass and exposure temperatures (except MTs in visceral mass of clams exposed in lower concentrations of metals). The interpolation method is used to convert actual values of MTs to standard values based on the MTs contents determined at15℃.②Stepped changes are found for MTs with salinity fluctuation. Based on the standard value determined at30ppt salinity, the values of MTs in gills at HPA with salinity range20-35ppt and MTs in visceral mass at LPA or HPA with salinity range20-35ppt don't need to be adjusted. The MTs contents determined at other salinity ranges need correction by interpolation method.③The impacts on MTs by pH value are relatively minor. Based on the standard value measured at pH8.0, the MTs contents in gills of the clams living at pH7.5-9.0(LPA) or pH7.5-8.0(HPA) don't need correction, and also for MTs contents in visceral mass at8.0-8.5(LPA, HPA).
运用生物标志物法监测海水重金属污染,能够反映海水中多重金属对生物的混合效应,灵敏度高且专一性较强。海洋双壳类金属硫蛋白(MTs)即是重金属毒性监测中常用的一类生物标志物。但是,相同重金属对不同物种MTs的诱导规律存在较大差异,即使同一物种其不同组织MTs对重金属暴露的响应程度也不相同,显示在重金属生物监测方面应选择适宜的生物种类和组织类型。定量监测海水重金属污染水平则需要获得重金属浓度—MTs诱导量及暴露时间—MTs诱导量的响应关系方程。另外,由于海水温度、pH值、盐度等因子的波动会对生物产生直接或间接的干扰,影响其体内MTs的水平,造成海域现场监测结果的不准确性。
本研究以中国沿海海域常见的双壳类生物——菲律宾蛤仔(Ruditapes philippinarum)为实验对象,分别选用含单一重金属离子Cu2+、Zn2+、Cd2+、Hg2+的人工海水对蛤仔进行暴露培养,通过测定重金属在鳃和内脏中的总含量及可溶性含量变化,分析其在蛤仔组织中的富集规律；根据蛤仔鳃和内脏中MTs的水平随暴露浓度和时间的变化,选择适用于指示单一重金属污染的组织类型,并确定指示重金属污染的最佳浓度范围和暴露时间。研究结果表明：蛤仔内脏比鳃更容易蓄积海水中的Cu2+、Zn2+、Cd2+、Hg2+,50μg/LCu2+、100μg/LZn2+、50μg/L Cd2+、5μg/LHg2+分别暴露5d后蛤仔鳃对4种重金属的BCF值分别为53.6、280.8、20.6、282.8,内脏的BCF值分别为676.8、268.1、47.4、565.6。进入鳃和内脏中的Cu2+和Cd2+主要以游离态形式存在,而Zn2+、Hg2+暴露下,亚细胞组分中的Zn、Hg主要以不溶形式存在。组织内富集金属Cu、Cd、Hg与相应组织中MTs含量之间的关系呈幂函数关系或负指数关系,而Zn的蓄积与MTs之间的关系可以用直线方程表征。蛤仔内脏MTs对Cu2+暴露更为敏感,而鳃MTs则更适合指示海水Zn2+、Cd2+或Hg2+等重金属污染水平。
在研究重金属单一暴露的基础上,分别将上述重金属离子2种或4种混合,研究复合污染下蛤仔MTs的响应规律,并与单一金属暴露相比较,探讨2种或4种重金属对蛤仔MTs的联合诱导效应机制。结果表明：双重金属和四重金属联合与对照组相比均能显著诱导蛤仔鳃和内脏MTs的合成。(Cu2+-Zn2+)联合暴露5d,蛤仔鳃和内脏MTs含量随混合金属浓度升高表现出先升高后降低的趋势,随着(Cu2+-zn2+)混合浓度的增大,其对鳃MTs的联合诱导表现为协同—相加—拮抗效应,对内脏MTs则表现为协同—拮抗效应。较低浓度(Cd2+-Hg2+)联合暴露响对MTs的诱导能力远远大于高浓度组,随着(Cd2+-Hg2+)混合浓度的增大,其对鳃MTs的联合诱导表现为协同—相加—拮抗效应,对内脏MTs则表现为协同—拮抗效应。在4种重金属离子联合暴露下,重金属浓度较低时,对鳃MTs联合诱导效应表现为相加作用,随着浓度增大则转变为拮抗作用,而对内脏MTs的联合诱导均表现为拮抗作用。
研究非生物因子对蛤仔MTs的干扰程度,对复合污染下蛤仔MTs的响应值随海水温度、盐度、pH值的变化趋势进行了初步探索,辨析这些因子的干扰作用并采取适宜方法对测定结果加以校正,以便于在消除非生物因子影响的前提下进行重金属综合污染水平评价。结果表明：上述非生物因子中温度、盐度变化对MTs的响应值影响较大,pH值的影响相对较小。①在蛤仔适宜生长的海水温度范围(5～20℃)内,无论是低浓度还是高浓度重金属污染区,鳃、内脏的MTs含量均随温度升高而增加,同等污染水平在20℃时对MTs的诱导量最高。高、低浓度污染区的鳃MTs含量以及高浓度污染区的内脏MTs含量,与温度之间呈现显著正相关性,可直接采用内插法,将MTs含量实测值转换为15℃时的校正值。②蛤仔MTs对重金属暴露的响应值受盐度影响呈“阶梯”状变化。如果以盐度30时的测定值作为基准值,以下情况无需进行校正：重污染区、盐度为20-35时,鳃MTs含量；盐度在20～35范围内时,低污染、重污染区内脏MTs含量。其他盐度范围下,所测定的鳃或内脏MTs均需采用内插法进行校正。③蛤仔MTs对重金属的响应受海水pH值的影响较小。以pH8.0的测定值作为基准,如果采用鳃MTs含量指示重金属综合污染水平,在海水pH为7.5～9.0(低污染区)和pH7.5-8.0(重污染区)的测定值无需校正；如果采用内脏MTs含量指示重金属综合污染水平,在海水pH为8.0～8.5(低污染区、重污染区)时的测定值无需校正。其他pH范围下所测定的鳃或内脏MTs均以pH8.0为准,需采用内插法进行校正。
Heavy metals are highly lipophilic, hard to degrade, bioaccumulative. Amplifying through the food chain, trace metals can cause oxidative stress and oxidative damage in organisms, even result in death. Therefore, it is necessary to research suitable methods for monitoring heavy metal pollution in seawater and ecological risk assessment. Biomarkers have high sensitivity and specificity, they can monitor mixed effect of heavy metals in the seawater. Metallothioneins (MTs) of marine bivalves are common biomarkers which are used in monitoring heavy metal toxicity. However, MTs in different species, even in different tissues of the same species, respond differently to the same pollution. To quantify heavy metal pollution, the dose-or time-effect relationships between heavy metal concentrations and MTs contents need to be obtained. Furthermore, the MTs inductions by metals can be influenced by variations of the temperature, salinity, pH value and so on, which can cause inaccuracy of field monitoring.
Ruditapes philippinarum are exposed in artificial seawater containing single heavy metal ion Cu2+, Zn2+, Cd2+or Hg2+. The aims of this part is to study the rules of metal enrichment, through determining the total/soluble component of heavy metal in gills and visceral mass; finding the suitable tissue for monitoring single-metal pollution and confirming the concentration range of each metal and exposure time. The experiments indicate that visceral mass can easier accumulate metals than gills. BCF5of gills are53.6,280.8.20.6,282.8after exposed in50μg/L Cu2+,100μg/L Zn2+,50μg/L Cd2+and5μg/L Hg2+, respectively, while BCF5of gills are676.8,268.1,47.4,565.6.Cu2+and Cd2+in gills and visceral mass are mainly in free form, in clams exposed to Zn2+or Hg2+, subcellular fraction C1contained higher Zn or Hg percentage. It is confirmed by the power function or negative exponential relationship between the levels of MTs and the contents of Cu, Cd or Hg in all tissues (gills and visceral mass), while Zn and MTs is in the positive and significant relationship. The MTs of visceral mass are more sensitive to Cu2+exposure, while for monitoring metal ions Zn2+, Cd2+or Hg2+, MTs of gills are more suitable.
To better understand the effects of metals in MTs response in Ruditapes philippinarum, these clams are exposed to polymetallic mixtures (Cu2+-Zn2+, Cd2+-Hg2+or Cu2+-Zn2+-Cd2+-Hg2+). The interactions between these metals and induction mechanism of MTs by polymetallic mixtures are discussed. The results show that the MTs in all clams exposed to a mixture of2or4metals are found significantly increase, which compared to control group (p<0.01or p<0.05). During exposed to Cu2+-Zn2+for5days, MTs levels in gills and visceral mass show increase at first, then decreased. The inductive effect of Cu2+-Zn2+compound is showed synergism-addition-antagonism on MTs in gills and synergism-antagonism in visceral mass with the increase concentrations of Cu2+and Zn2+. The inducibility of MTs by Cd2+-Hg2+in lower concentrations is larger than other groups. The inductive effect of Cd2+-Hg2+compound is showed synergism-addition-antagonism on MTs in gills and synergism-antagonism in visceral mass with the increase concentrations of Cd2+and Hg2+. When exposed to polymetallic mixtures(Cu2+-Zn2+-Cd2+-Hg2+), the inductive effect of Cu2+-Zn2+-Cd2+-Hg2+compound is showed addition-antagonism on MTs in gills and antagonism in visceral mass with the increase concentrations of these four metals.
The abiotic factors have certain influences on MTs induction by heavy metals in clams. The temperature, salinity and pH value of seawater are common abiotic factors. The present study is designed to examine the interference degree of these factors on MTs induction by polymetallic mixtures and put forward the effective methods to eliminate the unfavorable effects when applying MTs to monitoring heavy metals pollution in seawater. The results indicate that temperature and salinity affect MTs contents in gills and visceral mass notably, while the influences of pH value are relatively minor.①Under the optimal growth temperature5～20℃, there are linear relationship between MTs contents in gills and visceral mass and exposure temperatures (except MTs in visceral mass of clams exposed in lower concentrations of metals). The interpolation method is used to convert actual values of MTs to standard values based on the MTs contents determined at15℃.②Stepped changes are found for MTs with salinity fluctuation. Based on the standard value determined at30ppt salinity, the values of MTs in gills at HPA with salinity range20-35ppt and MTs in visceral mass at LPA or HPA with salinity range20-35ppt don't need to be adjusted. The MTs contents determined at other salinity ranges need correction by interpolation method.③The impacts on MTs by pH value are relatively minor. Based on the standard value measured at pH8.0, the MTs contents in gills of the clams living at pH7.5-9.0(LPA) or pH7.5-8.0(HPA) don't need correction, and also for MTs contents in visceral mass at8.0-8.5(LPA, HPA).
引文
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