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环境因子影响下的聚苯乙烯微塑料对长牡蛎毒性效应研究
Alternative TitleThe influence of environmental factors on the toxicity of polystyrene microplastics to the Pacific oysters Crassostrea gigas
杜蕴超
Subtype博士
Thesis Advisor赵建民 ; 王清
2023-05-28
Training institution中国科学院烟台海岸带研究所
Degree Grantor中国科学院大学
Place of Conferral中国科学院烟台海岸带研究所
Degree Name理学博士
Degree Discipline海洋生物学
Keyword微塑料 长牡蛎 环境因子 联合胁迫效应 代谢组
Abstract微塑料(Microplastics,MPs)是海洋环境中的一类新型污染物,其对海洋生物的毒性作用和生态风险已引起人们的广泛关注。目前,对海洋贝类体内微塑料丰度的研究多为一次性采样调查,季节性采样调查研究相对缺乏,对贝类体内微塑料富集特征的研究不够系统。已有研究表明微塑料对海洋生物的毒性作用与微塑料的特性(粒径、形状和聚合物类型等)有关,但很少有研究探讨不同粒径微塑料对海洋生物联合胁迫的影响机制。沿海地区环境复杂多变,微塑料可能与环境因子对海洋生物产生联合胁迫效应。然而,目前关于微塑料和环境因子对海洋生物的联合胁迫效应研究较少,联合胁迫效应机制尚不清楚。因此,本研究首先选取山东半岛五个养殖场,探究长牡蛎(Crassostrea gigas)体内微塑料丰度的季节性特征,并测定周围表层海水中微塑料丰度和环境因子(温度、pH、盐度和营养盐)的季节性变化,以揭示实际环境条件对长牡蛎体内微塑料丰度的综合影响。然后开展环境因子(温度、pH和盐度)与微塑料对长牡蛎的联合暴露实验,从生理、生化和分子水平探讨两种粒径的聚苯乙烯微塑料(小粒径聚苯乙烯微塑料(SPS-MPs:6 μm);大粒径聚苯乙烯微塑料(LPS-MPs:50~60 μm))与环境因子(温度、pH和盐度)对长牡蛎的联合胁迫效应,结合综合生物标志物响应指数法(IBR)和主成分分析(PCA)揭示联合胁迫效应机制,并探究联合暴露条件下不同粒径微塑料对免疫、氧化应激、能量代谢的影响机制。此外,通过非靶向代谢组学技术阐释了温度、pH、盐度与SPS-MPs联合暴露对长牡蛎的胁迫效应。研究结果如下: (1)野外调查研究揭示了长牡蛎体内微塑料丰度的季节特征和影响因素。研究表明,长牡蛎体内的微塑料摄入率秋季最高,春季最低;在不同站位间,烟台站位长牡蛎摄入率最高,莱州站位最低。在对不同季节所有站位长牡蛎体内微塑料丰度的统计中发现,长牡蛎体内微塑料的季节性丰度特征为:2.40(冬季)~ 3.28(秋季)个/个体,或0.22(春季)~ 0.45(夏季)个/g (ww)。对不同站位长牡蛎体内微塑料丰度的统计中发现,以个/个体为单位时,长岛和荣成站位长牡蛎体内的微塑料丰度较低;以个/g (ww)为单位时,微塑料丰度长岛站位最高,莱州站位最低。长牡蛎体内微塑料最常见的聚合物类型是玻璃纸(CP),其次是聚对苯二甲酸乙二醇酯(PET)、聚丙烯腈(PAN)、聚酰胺(PA)和聚乙烯/聚丙烯(PE/PP);最常见的形状是纤维,其次是碎片和薄膜;大多数微塑料的粒径在0.5-1 mm之间。对于表层海水,微塑料丰度在春季显著低于夏季和秋季(p < 0.05),且长岛和荣成站位微塑料丰度相对较低。在表层海水中,占比最大的聚合物类型是PET,其次是CP、PE、PA、PP和PE/PP;占比最大的形状是纤维,其次是碎片和薄膜;占比最大的粒径范围是0.5~1 mm。冗余分析(RDA)表明,影响夏、冬两季长牡蛎和表层海水中微塑料丰度的主要环境因子分别为NH4-N和SiO3-Si。Spearman相关分析表明,当以个/个体为单位时,不同季节长牡蛎体内的微塑料丰度与环境因子(温度和NO2-N)呈显著正相关(p < 0.05)。综上可知,长牡蛎体内的微塑料特征与表层海水中的微塑料特征有关。 (2)探究了低盐与不同粒径聚苯乙烯微塑料对长牡蛎的联合胁迫效应。研究发现,低盐会降低长牡蛎体内微塑料的富集,抑制消化腺组织己糖激酶(HK)和丙酮酸羧化酶(PEPCK)基因的表达,诱导鳃组织丙酮酸激酶(PK)基因的表达,并会诱导消化腺和鳃组织超氧化物歧化酶(SOD)和谷胱甘肽S-转移酶(GST)活性;21 psu盐度会降低消化腺和鳃组织丙二醛(MDA)含量,并会诱导血淋巴细胞凋亡率。微塑料会诱导血淋巴细胞凋亡率,并会诱导消化腺组织SOD和GST酶活性;SPS-MPs相比于LPS-MPs会增加消化腺组织MDA含量。微塑料与低盐的联合作用以拮抗作用为主,其次是协同作用,且协同作用主要为SPS-MPs与低盐联合作用。联合暴露时,长牡蛎能够通过提高抗氧化酶活性和能量代谢调控,使得MDA水平有降低的趋势。微塑料与低盐对消化腺组织中PEPCK基因表达的联合作用受盐度水平的影响:26 psu低盐与微塑料联合暴露具有拮抗作用,21 psu低盐与微塑料联合暴露具有协同作用。IBR分析表明,低盐与SPS-MPs联合暴露组胁迫效应最强,表明较小粒径微塑料可造成更严重的危害。代谢组学结果表明,盐度是影响长牡蛎鳃组织代谢的主要因素,并且联合胁迫效应可能与渗透调节、能量代谢和抗氧化防御相关。PCA分析结果表明,低盐与抗氧化酶活性具有更强的相关性,而微塑料与脂质过氧化水平和应激蛋白基因具有更强的相关性。 (3)探究了海水酸化与不同粒径聚苯乙烯微塑料对长牡蛎的联合胁迫效应。研究发现,海水酸化对长牡蛎体内的微塑料富集没有影响,但会增加血细胞凋亡率,并会提高长牡蛎摄食率以增强应对胁迫的能量储备;抑制抗氧化酶活性,分别抑制和诱导消化腺和鳃组织溶酶体酶活性;诱导鳃组织p53基因表达,引起免疫应答。LPS-MPs暴露会增加血淋巴细胞ROS含量;SPS-MPs会增强鳃组织过氧化氢酶(CAT)活性,增加MDA含量。微塑料与海水酸化的联合作用以拮抗作用为主,其次是协同作用,且协同作用主要为SPS-MPs与海水酸化联合作用。联合暴露组中,长牡蛎可通过调控糖酵解基因表达量,影响能量代谢,并会抑制抗氧化酶活性,引起鳃组织氧化损伤。IBR分析表明,海水酸化与SPS-MPs联合暴露组IBR值大于SPS-MPs或海水酸化单独暴露组,表明海水酸化与SPS-MPs联合暴露对长牡蛎的胁迫效应最强。代谢组学结果表明,SPS-MPs是影响长牡蛎消化腺组织代谢的主要因素,联合胁迫效应可能与渗透调节、能量代谢和抗氧化防御相关。PCA分析结果表明,微塑料与抗氧化酶之间的相关性较强,而酸化与应激蛋白基因和溶酶体酶之间具有较强的相关性。 (4)探究了升温与不同粒径聚苯乙烯微塑料对长牡蛎的联合胁迫效应。研究发现,升温会显著降低长牡蛎消化腺中LPS-MPs含量,但对SPS-MPs的富集没有影响,会增加GST酶活性,并会启动免疫应答。微塑料暴露会增强核因子κB抑制蛋白(IκB)基因的表达,并会诱导鳃组织GST酶活性;SPS-MPs会增加长牡蛎摄食率,抑制鳃组织中ACP酶活性,增加消化腺组织中MDA含量,并会增加血淋巴细胞ROS产量;LPS-MPs会降低消化腺组织AKP酶活性。升温和微塑料对牡蛎消化腺组织中糖原含量具有显著协同作用,表明联合暴露组牡蛎具有更高的能量需求。联合胁迫组长牡蛎鳃组织中GST酶活性、MDA含量、p53基因和IκB基因表达量,以及消化腺中p53基因表达量均提示升温与微塑料之间可能存在拮抗作用。升温和微塑料对消化腺组织中hsp90和IκB基因表达量的显著交互作用受微塑料粒径的影响:SPS-MPs与升温联合暴露具有协同作用,LPS-MPs与升温联合暴露具有拮抗作用。IBR分析表明,SPS-MPs与升温联合暴露组的IBR值大于SPS-MPs或升温单独暴露组,表明SPS-MPs与升温联合暴露对长牡蛎的胁迫效应最强。代谢组学结果表明,联合暴露组的代谢谱与SPS-MPs或单独升温暴露组具有明显差异,主要涉及能量代谢、抗氧化防御和渗透调节等代谢物含量的改变。PCA分析表明,升温与氧化应激相关指标有较强相关性,微塑料与氧化应激和免疫防御指标有较强相关性。
Other AbstractMicroplastics (MPs) have become an emerging new pollutant in the marine environment. MPs have attracted remarkable attention due to their toxic effects on marine organisms and potential ecological risks. At present, most of the studies collected mollusk samples only once. Seasonal investigations of MPs pollution levels in mollusks are relatively few, and MPs uptake characteristics in mollusks have not been studied systematically. Previous studies showed that the toxicity of MPs on marine organisms is also related to MPs characteristics (e.g. particle size, shape, and polymer type). Nevertheless, few studies have investigated the stress effects of MPs size on marine organisms in combined exposure experiments. As coastal environments are complex and changeable, MPs and environmental factors may cause combined stress effects on marine organisms. However, relatively few studies have examined combined stress effects of MPs and environmental factors on marine organisms, and the mechanism of the combined stress effects is still unclear. Therefore, in this study, the seasonal MPs abundance characteristics of C. gigas from five aquaculture farms in Shandong Peninsula were investigated. In addition, we detected MPs abundance and environmental factors (temperature, pH, salinity, and nutrient salts) in surface seawater around oysters in different seasons to reveal the comprehensive influence of actual environmental conditions on the seasonal MPs abundance of C. gigas. Moreover, the combined exposure experiments of environmental factors (temperature, pH, and salinity) and MPs to C. gigas were conducted. Specifically, we investigated the combined effects of polystyrene MPs of two sizes (small polystyrene microplastics, SPS-MPs: 6 μm; large polystyrene microplastics, LPS-MPs: 50~60 μm) and environmental factors (temperature, pH, and salinity) to C. gigas at the physiological, biochemical and molecular levels. The combined stress effects mechanism was revealed based on the integrated biomarker response index (IBR) and Principal Component Analysis (PCA). The influential mechanisms of different MPs sizes on the immunity, oxidative stress, and energy metabolism of oysters under combined exposure conditions were also investigated. In addition, untargeted metabolomics techniques were used to elucidate the combined stress effects of temperature, pH, salinity, and SPS-MPs on C. gigas. The results were summarized as follows: (1)Field investigation revealed the seasonal characteristics and influencing factors of MPs abundance in C. gigas. In the present study, the ingestion rate of MPs in C. gigas was the highest in autumn and the lowest in spring, and the MPs ingestion rate in C. gigas at different sites was the highest at the Yantai site and the lowest at the Laizhou site. According to the statistics on MPs abundance of C. gigas at all sites in different seasons, the MPs abundance of C. gigas ranged from 2.40 (winter) to 3.28 (autumn) items/individual, or from 0.22 (spring) to 0.45 (summer) items/g (ww). According to the statistics on MPs abundance of C. gigas at different sites, the MPs abundances of C. gigas were relatively lower at the Changdao and Rongcheng sites when expressed as items/individual and MPs abundance of C. gigas was highest at the Changdao site and lowest at the Laizhou site when expressed as items/g (ww). In oysters, the dominant type was cellophane (CP), followed by polyethylene terephthalate (PET), polyacrylonitrile (PAN), nylon (PA), and polyethylene/polypropylene (PE/PP); the main shape of MPs was fiber, followed by fragment and film; the most abundant size class of MPs was 0.5~1 mm. In surface seawater, the abundance of MPs was significantly lower in spring than in summer and autumn (p < 0.05) and was relatively lower at the Changdao and Rongcheng sites. In surface seawater, the dominant type was PET, followed by CP, PE, PA, PP, and PE/PP; the main shape of MPs was fiber, followed by fragment and film; the most abundant size class of MPs was 0.5~1 mm. Redundancy (RDA) analysis showed that NH4-N and SiO3-Si were the main environmental factors affecting MPs abundance of oysters and surface seawater in summer and winter, respectively. Spearman correlation analysis showed that the MPs abundance of C. gigas in different seasons was significantly positively correlated with environmental factors (temperature and NO2-N) when expressed as items/individual (p < 0.05). From the above, MPs characteristics in oysters are related to MPs characteristics in surface seawater. (2)The combined stress effects of low salinity and polystyrene MPs of different sizes on C. gigas were investigated. The results showed that low salinity exposure decreased the MPs accumulation in C. gigas, inhibited the expression of Hexokinase (HK) and Phosphoenolpyruvate carboxylase (PEPCK) genes in digestive glands, induced the expression of Pyruvate kinase (PK) genes in gills, and increased Superoxide dismutase (SOD) and Glutathione S-transferase (GST) activity in digestive glands and gills; low salinity (21 psu) exposure reduced Malondialdehyde (MDA) levels in digestive glands and gills, and increased the apoptosis rate of hemolymph. MPs can induce the apoptosis rate of hemolymph, and can induce SOD and GST activity in digestive glands; SPS-MPs can increase MDA levels in digestive glands compared with LPS-MPs. The combined effects of MPs and low salinity were mostly antagonistic effects, followed by synergistic effects, and the synergistic effects were mainly induced by SPS-MPs and low salinity; in the combined exposure groups, the MDA levels of C. gigas tended to decrease by improving antioxidant enzyme activity and regulating energy metabolism. The combined effects of MPs and low salinity on PEPCK gene expression in digestive glands were influenced by salinity levels: low salinity (26 psu) combined with MPs had an antagonistic effect, and low salinity (21 psu) combined with MPs had a synergistic effect. IBR analysis showed that the combined exposure group of low salinity and SPS-MPs had the strongest stress effects, suggesting that a smaller size may cause severe damage. The metabolomics results showed that salinity was the main factor affecting metabolism in the gills of C. gigas. In addition, the results suggested that the complex stress effects might be related to osmotic regulation, energy metabolism, and antioxidant defense. The results of PCA analysis showed that low salinity was related to antioxidant enzymes, while MPs exposure was related to lipid peroxidation levels and hsp genes. (3)The combined stress effects of seawater acidification and polystyrene MPs of different sizes on C. gigas were investigated. The results showed that seawater acidification had no effect on MPs accumulation in C. gigas, but increased the apoptosis rate of hemolymph and increase the clearance rate (CR) of C. gigas to enhance energy reserves for stress adaptation; seawater acidification inhibited antioxidant enzymes and inhibited and induced lysosomal enzyme activity in digestive glands and gills, respectively; seawater acidification increased p53 gene expression in gills and induced the immune response. The exposure to LPS-MPs increased the production of ROS in hemolymph, while the exposure to SPS-MPs increased the activity of Catalase (CAT) in gills, and increased MDA levels. The combined effect of MPs and seawater acidification was mostly antagonistic, followed by a synergistic effect, and the synergistic effect was mainly the combined effect of SPS-MPs and seawater acidification. Combined exposure influenced the energy metabolism of oysters by regulating glycolysis gene expression, inhibited antioxidant enzyme activity, and induced oxidative damage in gills. The IBR analysis showed that the IBR value of the combined exposure group of seawater acidification and SPS-MPs was higher than that of the single exposure group of SPS-MPs or seawater acidification, suggesting that the stress effects of the combined exposure of seawater acidification and SPS-MPs were the strongest. The metabolomic results indicated that SPS-MPs were the main factors affecting the digestive gland tissue metabolism of C. gigas, and complex stress effects might be related to osmoregulation, energy metabolism, and antioxidant defense. PCA analysis also showed that MPs was related to antioxidant enzymes and seawater acidification was related to hsp genes and lysosomal enzymes. (4)The combined stress effects of elevated temperature and polystyrene MPs of different sizes on C. gigas were investigated. The results showed that elevated temperature significantly reduced LPS-MPs accumulation in the digestive glands of C. gigas, but it had no effect on SPS-MPs accumulation; elevated temperature increased Glutathione S-transferase (GST) activity, and induced immune response. MPs exposure increased the gene expression of Inhibitor of NF-κB (IκB), and induced GST activity in the gills; SPS-MPs exposure increased CR, inhibited ACP activity in the gills, increased the MDA content in the digestive glands, and induced ROS production of hemolymph; LPS-MPs exposure decreased AKP activity in digestive glands. There was a significant synergistic effect between elevated temperature and MPs on glycogen content in digestive glands, indicating that the combined exposure group had a higher energy demand. The results of combined exposure of elevated temperature and MPs on GST activity, MDA content, and gene expression of p53 and IκB in gills of oysters, as well as p53 gene expression in digestive glands, suggested that antagonistic interactions between MPs and elevated temperature may occurred. The combined effects of elevated temperature and MPs on hsp90 and IκB gene expression in digestive glands were influenced by MPs size: SPS-MPs combined with elevated temperature had a synergistic effect, and LPS-MPs combined with elevated temperature had an antagonistic effect. The IBR analysis showed that the IBR value of combined exposure to SPS-MPs and the elevated temperature was higher than that of single SPS-MPs and elevated temperature, suggesting that the stress effects of the combined exposure of SPS-MPs and the elevated temperature to oysters were the strongest. The metabolomics results showed that the metabolic profile of the combined exposure group was significantly different from that of the single SPS-MPs or single elevated temperature group, which mainly involved changes in metabolites such as energy metabolism, antioxidant defense, and osmotic regulation. PCA analysis showed that elevated temperature was related to oxidative stress, and MPs was related to oxidative stress and immune defense.
Pages161
Language中文
Document Type学位论文
Identifierhttp://ir.yic.ac.cn/handle/133337/32045
Collection中国科学院烟台海岸带研究所知识产出_学位论文
Recommended Citation
GB/T 7714
杜蕴超. 环境因子影响下的聚苯乙烯微塑料对长牡蛎毒性效应研究[D]. 中国科学院烟台海岸带研究所. 中国科学院大学,2023.
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