烟台近岸不同老化状态聚丙烯微塑料表面生物膜形成过程与特征分析

由于塑料制品的耐用性，大多数合成聚合物在海洋环境中的降解性较差，但当其结构变得脆弱后能够在物理或化学作用下分解成小颗粒，即产生微塑料。微塑料通常是指最大尺寸小于5mm的塑料颗粒。由于其粒径小、易漂浮、降解缓慢，因此微塑料在各种海洋环境介质中无处不在，并对海洋生物产生危害。目前，微塑料污染已成为海洋环境面临的主要问题之一。微塑料在环境中易受到化学、物理甚至生物过程的影响而发生老化，其中太阳辐射是导致微塑料老化最主要的因素。此外，微塑料作为海洋生态系统中一类相对较新的污染物，微生物能够附着于其表面形成生物膜。微塑料生物膜能够为附着微生物提供重要的生态优势，如提供营养物质、形成保护屏障等。由于微塑料的漂浮性和持续性强于大多数天然基质，因此可能成为某些污染物和有毒有害生物运输的载体。本论文以碎片状、颗粒状以及纤维状聚丙烯（Polypropylene，PP）微塑料作为微生物附着对象，并将部分微塑料进行紫外辐照处理，获得老化和非老化PP微塑料。分别于春夏两季将微塑料样品投放于烟台近岸海洋、河口以及河流生境中进行围隔实验，各站位分别孵育样品6周。通过对生物膜形貌特征、微生物群落特征分析、碳源利用能力以及抗生素抗性基因分析等，探讨影响生物膜生长速率和菌群组成的关键环境因素。我们试图将研究结果应用于筛选降解塑料菌，从而应用于塑料治理的相关内容。研究结果可为微塑料在海洋环境中的生态风险评估提供数据支撑，并为海洋微塑料污染管控提供科学依据。本论文的主要研究结果如下： （1）微塑料样品经紫外辐照15天后（UVC，4 × 60 W），使用电子能谱仪表征材料表面元素含量变化。结果表明，微塑料老化后其表面氧元素含量显著增加，表明紫外辐照能够引起微塑料表面含氧基团的产生。显微红外光谱结果显示，老化微塑料表面出现羰基附加振动峰，说明微塑料表面发生了氧化效应。扫描电子显微镜结果显示，紫外线加速照射会使微塑料表面产生细小裂纹和褶皱，粗糙度增加。此外，野外环境下孵育形成的生物膜对微塑料老化的影响结果显示，附着生物膜的微塑料表面氧碳比随着培养时间的延长而增加，因此生物膜的形成也会影响微塑料的老化过程。 （2）微塑料生物膜含量随孵育时间的增加而增加，且老化微塑料上生物膜含量更高。因此孵育时间、老化处理均与微塑料生物膜生长呈正相关性。与海洋和河口站位相比，烟台近岸淡水站位在春夏两季为微生物最适生长环境，推测可能与该季节下淡水环境温度、营养物质含量高，而离子强度低有关。与春季相比，夏季温度高、光照时间长、溶解氧含量低、细胞代谢速度快。因此，微塑料表面生物膜含量在夏季（1.004 ± 0.54 Abs）要显著高于春季（0.103 ± 0.08 Abs）。 （3）微塑料表面生物膜含量与孵育地点的环境因子相关性分析结果表明，盐度和温度为影响春季微塑料生物膜生长的主要环境因素。温度主要区分了微塑料生物膜发育早期和晚期阶段的区别，盐度主要区分了不同生境间生物膜含量的差异，河流生境离子强度显著低于河口和海洋，因而更适合微生物定植。营养盐含量为限制夏季微塑料生物膜生长的主要环境因素，表现为生物膜发育早期与营养盐含量成正比，但在发育后期生物膜含量已不再随营养盐含量增加而增加。 （4）孵育地点对微塑料生物膜微生物群落的α多样性具有显著影响，其中河流生境微塑料生物膜微生物群落α多样性指数显著高于海洋和河口生境，但微塑料老化状态对其影响较弱。微生物群落组成分析表明，变形菌门为春季和夏季微塑料生物膜微生物群落中的主要优势菌门。此外，春季条件下相对丰度较高的细菌还包括蓝细菌门和拟杆菌门，而夏季条件下相对丰度较高的细菌还包括拟杆菌门、厚壁菌门、酸杆菌门和浮霉菌门。微塑料生物膜中还检测到一定丰度的潜在致病菌，春季样品中主要的潜在致病菌为黄杆菌与莫拉氏菌，而夏季样品中的潜在致病菌为伯克氏菌、假单胞菌、气单胞菌以及黄杆菌。 （5）孵育地点以及微塑料老化状态能够影响春季微塑料生物膜微生物群落分布，但对夏季微塑料生物膜的微生物群落分布影响较小。功能预测结果显示，与人类病原体相关、氢氧化物氧化、硫酸盐还原、光合自养等基因在生物膜群落中表达较为丰富，其中某些代谢功能与生物地球化学循环密切相关。值得注意的是，人类病原体相关基因在老化微塑料中表达量显著高于原始微塑料。这一结果为老化微塑料生物膜可能携带更多人类病原体提供了依据，说明老化微塑料或许能对人类健康构成更大潜在风险。 （6）夏季微塑料生物膜微生物群落碳利用能力分析结果表明，不同孵育地点微塑料表面生物膜微生物碳利用能力存在差异，其中河流中微塑料生物膜附着的微生物群落具有较高的碳源代谢能力。微塑料老化状态对其表面生物膜的微生物碳源利用无显著影响，说明附着基质的表面性质对生物膜碳源利用的影响并不明显。微塑料生物膜上的微生物群落对31种碳源的利用效率存在差异，表明微塑料能够显著影响水生生态系统中微生物介导的碳循环过程，对碳的地球生物化学循环具有重要的生态学意义。 （7）分析了夏季微塑料生物膜中抗性基因的相对丰度，其中河口站位抗性基因相对丰度最高，推测可能是由于河口区域人类渔业、生产活动更为频繁所致。老化微塑料上抗性基因含量高于原始微塑料，可能是老化处理改变了微塑料的表面电荷和疏水性，增强了其对污染物的吸附能力。另一方面，老化微塑料可能与抗性基因载体形成更稳定的氢键。在所有检测到的抗性基因类型中，氨基糖苷类基因、氟喹诺酮类基因等为主要抗生素抗性基因类型。因此，老化微塑料可能增加抗生素抗性基因在海洋环境中远距离传播的风险

Effects of nanoplastics and nanosized titanium dioxide on hemocytes function and antioxidant enzyme activities of Litopenaeus vannamei via dietary exposure

The increasing use of nanoplastics and nanometal oxide particles in different fields have aroused concern about the environmental risks associated with these nanomaterials entering the aquatic system. In the present study, through dietary exposure route, the interference of hemocytes function and oxidative balance of the white shrimp Litopenaeus vannamei after exposure to 100 nm nano-PS and nano-TiO_2 was analyzed. Shrimps of treatment groups were fed with diets spiked with nano- PS and nano-TiO_2 at doses of 200 mg/kg and 2 000 mg/kg respectively for 28 days. The ROS production, phagocytosis and apoptosis rate of hemocytes as well as the enzymatic activities of total antioxidant capacity (T-AOC), superoxidase dismutase (SOD), glutathione-S-transferase (GST), malondialdehyde level (MDA) and Na+/K+-ATPase in hepatopancreas were measured at Day-14 and Day-28. The results showed that both nano-PS and nano-TiO_2 caused a significant increase in the apoptosis rate of hemocytes. Besides, the activities of T-AOC, SOD and GST in hepatopancreas were elevated to varying degrees, while the significant decrease of Na~+/K~+-ATPase activity was observed in the treatment groups. In addition, compared with nano-PS, nano-TiO_2 caused more serious oxidative damage to shrimps after 28 days of exposure

Toxic effects of polystyrene microplastics and pyrene on Ruditapes philippinarum

The pollution of microplastics and polycyclic aromatic hydrocarbons (PAHs) in marine environment is increasingly serious. The toxic effects of polystyrene microplastics and pyrene on the clam Ruditapes philippinarum were investigated in this study. R. philippinarum was exposed to two polystyrene microplastics sizes (0.3 mum and 6 mum, 20 mug/L) and two pyrene concentrations (10 mug/L and 100 mug/L) for 21 days, respectively. For individuals in each treatment, physiological responses (condition index and clearance rate), immune defense and oxidative stress parameters were measured. It was found that except for the pyrene single exposure group, the clearance rate of R. philippinarum in the other exposure groups was significantly lower than that of the control group, but the two pollutants had no significant effect on the condition index of R. philippinarum. Exposure to microplastics and pyrene led to impaired immune function of R. philippinarum, manifested by increased hemocyte apoptosis and inhibited phagocytosis. The apoptosis rate of hemocytes in the exposure group with small size microplastics and high concentration pyrene was significantly higher than that in the control group,and the apoptosis rate of hemocytes in the combined exposure group was the highest. Except for the large size microplastic exposure group,the phagocytic activity of hemocytes in each exposure group was significantly lower than that of the control group. The antioxidant system of R. philippinarum could not remove the free radicals produced in the body in time,leading to oxidative damage in the body,which was manifested as the change of antioxidant enzyme activity and the increase of malonic dialdehyde (MDA) content. The combined exposure of microplastics and pyrene significantly increased the activities of glutathione S-transferase (GST) in gill tissues, and significantly decreased the activities of catalase (CAT) and superoxide dismutase (SOD) in digestive gland tissues. Integrated biomarker response (IBR) analysis showed that the stress caused by combined exposure on R. philippinarum was higher than that of the single exposure group