【作者】 吕鉴达；

【导师】 潘国伟；

【作者基本信息】 中国医科大学 ， 流行病与卫生统计学， 2023， 博士

【摘要】 目的:2021年世界塑料制品产量已达3.91亿吨,而世界范围内塑料制品的回收利用量仅有1020万吨。高产量低回收利用率导致大量塑料废物进入到环境中,它们在紫外线、外力磨损等作用下产生了粒径小于5 mm的微塑料。环境中微塑料的污染已引起全球范围内的关注。呼吸道和消化道是人体暴露微塑料最重要的两种途径。目前,经消化道途径暴露的研究较多,食物、饮品中的暴露评价及其毒性效应、作用机制均已被大量报道。与之相比,经呼吸道暴露的研究相对较少,尤其是健康危害评价的首要步骤暴露评价,开展的更少。分析其原因主要为:经呼吸道途径的暴露评价不仅存在外暴露具有室内外的差异且受到多种因素影响的复杂性,而且内暴露评价亦面临具有代表性的支气管肺泡灌洗液和肺组织活检样本难以获得等难题。由于新冠肺炎疫情肆虐,口罩使用量居高不下,有研究表明医用外科口罩本身为塑料材质,可成为微塑料的又一潜在污染源,在防控病原微生物侵入的同时却能够增加人体暴露微塑料的风险。但到目前为止,相关的暴露评价尚未见报道。本研究利用鼻腔灌洗液和痰液对呼吸系统内暴露负荷的良好指示作用,并且易采集、无侵害,能够在大规模人群流行病学调查中开展等优势,采用偏振光显微镜镜检和激光红外成像系统相结合的方法,于室内外典型代表性人群中开展鼻腔灌洗液和痰液中微塑料负荷评价的前探研究,阐明鼻腔灌洗液和痰液作为内暴露指示物进行检测的可行性;并于代表性污染区家养猪肺组织中进行定性、定量检测方法的验证,明确偏振光显微镜与激光红外系统结合的检测方法对不同污染水平检测的准确性。在此基础上,利用大学校园区域相对独立,建筑形式和用材较为统一,室内陈设装饰较为一致,在校学生人口学特征简单,呼吸系统疾患少,生活规律相对一致,依从性和配合度高,即开展空气中微塑料外暴露和内暴露系统评价的干扰因素少等优势,于校园内不同功能分区采集室内外空气样本,开展空气中微塑料外暴露负荷评价,阐明室内外暴露水平及特征,揭示室内外污染状况的相关性;招募在校研究生为调查对象,开展经呼吸道微塑料内暴露负荷评价,并开展不同材质口罩的干预研究,揭示人体生物样本中微塑料污染水平及特征,并阐明其影响因素,明确口罩对人体微塑料内暴露状况的影响。研究方法:1.以快递员为室外典型代表性人群,以办公室工作人员为室内典型代表性人群,以中国医科大学预防医学研究所为研究地点,共招募8名办公室工作人员和周围3 km内8名快递员为研究对象,采集研究对象的鼻腔灌洗液和痰液样本,等分后经过消化处理,一份样本经膜过滤并制作成压片,置于Leica DM4B DFC7000T偏振光显微镜下观察,记录微塑料数目、粒径及形状;另一份样本通过氮吹浓缩,放入Agilent 8700激光红外成像系统检测,明确微塑料主要成分构成情况,通过t检验分析室内和室外典型代表性人群鼻腔灌洗液和痰液中微塑料丰度差异是否具有统计学意义。以典型污染区饲养1年以上的家猪的肺组织和出生前因窒息而死亡的胎猪的肺组织为研究样本,分别进行Leica DM4B DFC7000T偏振光显微镜镜检和Agilent 8700激光红外成像系统检测,分析家猪和胎猪肺组织中微塑料丰度及特征的异同。2.采集校园内不同功能分区(生活区、学习工作区和活动区)室内外空气降尘样本,共39份,其中室内18份,室外21份。消化处理后制成压片,通过Leica DM4B DFC7000T偏振光显微镜进行观察,记录微塑料的数量、粒径及形状;随机抽取部分样本进行Agilent 8700激光红外成像系统检测,明确室内外空气降尘中微塑料成分分布情况。使用t检验和方差分析检验各场所空气降尘中微塑料丰度差异及不同室内因素对室内空气降尘中微塑料丰度的影响是否具有统计学意义,使用相关分析明确室内外空气降尘中微塑料丰度与温度和湿度的关系,采用一致性检验分析室内外空气降尘中微塑料污染的同源性。3.校园内招募学生开展呼吸道微塑料内暴露情况调查,共招募113名在校研究生。研究对象随机分组,其中54名为自然暴露组,29名为外科口罩干预组,30名为棉布口罩干预组。干预组研究对象要求佩戴所发口罩3天,且佩戴时长不低于2 h/天。于第4天,在指定实验室内,由专业人员对所有研究对象进行鼻腔灌洗液的采集及调查问卷填写,同时收回《口罩佩戴情况记录单》。鼻腔灌洗液经消化处理并制成压片,通过Leica DM4B DFC7000T偏振光显微镜明确微塑料数目、粒径及形状;每组随机抽取部分样本进行Agilent 8700激光红外成像系统检测其中微塑料成分。使用t检验或方差分析进行影响鼻腔灌洗液中微塑料丰度的单因素分析,通过广义线性模型进行多因素分析,采用卡方检验分析各组志愿者人群特征的异同以及各组鼻腔灌洗液中微塑料粒径、形状及成分分布是否存在差异,通过方差分析分析各组志愿者鼻腔灌洗液中微塑料丰度差异是否具有统计学意义,通过广义线性模型控制影响因素,进一步明确口罩使用对鼻腔灌洗液中微塑料丰度的影响。结果:1.快递员和办公人员分别作为室外和室内典型代表型人群,快递员鼻腔灌洗液中微塑料丰度(0.76±1.51个/克)显著低于办公人员(2.24±1.82个/克)(P<0.01);快递员和办公人员鼻腔灌洗液中微塑料形状均以纤维状为主,所占比例分别为83.81%和87.03%;快递员鼻腔灌洗液中微塑料以PA(25.56%)和PE(23.06%)为主,办公人员以PVC(41.40%)和PA(31.81%)为主。针对痰液中的微塑料丰度,快递员(92.09±1.76个/克)和办公人员(58.55±3.33个/克)间的差异不具有统计学意义(P=0.35);快递员(94.26%)和办公人员(83.28%)痰液中微塑料形状仍然以纤维状为主;快递员痰液中微塑料成分以PC(23.93%)和PVC(23.08%)为主,办公人员以PVC(40.21%)和PA(25.30%)为主。胎猪肺组织中微塑料丰度为6个/克,家猪肺组织中微塑料丰度为12个/克,均为纤维状;胎猪肺组织中微塑料成分以PC(32.99%)、PP(15.46%)和PVC(15.46%)为主,家猪肺组织中以PA(46.11%)、PP(21.11%)和PE(17.22%)为主。2.校园内不同功能分区中各场所室外空气降尘中微塑料丰度均值为1666.18个/(d·m~2),各功能分区间差异不具有统计学意义(P=0.78),其中,公共教学楼室外空气降尘中微塑料丰度显著高于体育场(P=0.13)。室内空气降尘中微塑料丰度均值为2244.89个/(d·m~2),其中,女生寝室高于办公楼(P=0.13)和图书馆(P=0.12),公共教学楼高于图书馆(P<0.15);湿度(P=0.03)、配置窗帘(P=0.10)和使用空调(P=0.03)等因素与室内空气降尘中微塑料丰度水平显著相关,不同清扫方式(P=0.42)和频次(P=0.55)均未对室内空气降尘中微塑料丰度产生显著影响。室内外的比较发现,室内空气降尘中微塑料丰度高于室外空气降尘中微塑料丰度,但不具有统计学意义(P=0.26)。无论是室外还是室内,空气降尘中微塑料形状均以纤维状为主,但室内纤维比例(94.06%)显著高于室外(87.52%)(P<0.01)。二者主要成分亦存在显著差异(P<0.01),室外以PA(34.45%)和PC(28.26%)为主,室内以PA(30.62%)和PE(33.33%)为主;室外(53.87%)空气降尘中微塑料粒径多集中在200μm以下,室内(62.70%)多集中在500μm以下,粒径分布差异具有统计学意义(P<0.01)。3.自然状态下,人体鼻腔灌洗液中微塑料丰度为31.90±1.77个/克,经多因素广义线性模型分析,3日内办公室空调使用情况(偶尔使用VS无/未使用:β=0.24,P=0.09;经常使用VS无/未使用:β=-0.17,P=0.22)和使用毛巾材质(不含化纤VS含有化纤:β=0.24,P=0.08)与鼻腔灌洗液中微塑料丰度显著相关;自然暴露组志愿者鼻腔灌洗液微塑料中88.06%为纤维状,主要成分以PA(43.70%)、PC(13.80%)和PE(12.83%)为主。与自然暴露组相比,经控制其它影响因素后发现,外科口罩干预组志愿者鼻腔灌洗液中微塑料丰度显著高于自然暴露组(β=0.25,P=0.01),棉布口罩干预组志愿者鼻腔灌洗液中微塑料丰度略低于自然暴露组,但差异不具有统计学意义(β=-0.11,P=0.23),外科口罩干预组(92.01%)和棉布口罩干预组(89.38%)志愿者鼻腔灌洗液中微塑料均以纤维状为主,各组志愿者鼻腔灌洗液中微塑料形状分布差异具有统计学意义(P<0.01),其主要成分分布亦不相同(P<0.01),在外科口罩干预组,微塑料成分以PC(25.30%)、PA(24.10%)和PVC(19.28%)为主,而棉布口罩干预组则以PA(28.32%)、PC(25.66%)和PE(15.04%)为主,自然暴露组(59.30%)、外科口罩干预组(64.57%)和棉布口罩干预组(58.50%)志愿者鼻腔灌洗液中微塑料粒径多集中在200μm以下,粒径分布差异具有统计学意义(P<0.01)。结论:1.室内外典型代表性人群的鼻腔灌洗液和痰液中均检测出微塑料的存在,且室内暴露水平高于室外,形状以纤维状为主;2.偏振光显微镜镜检和激光红外成像系统检测结合的方法能够有效检测生物样本中微塑料的暴露状况;3.室内外空气降尘中微塑料污染水平存在差异,微塑料粒径分布及主要成分均不同,但均以纤维状为主,室内的外暴露负荷高于室外,二者具有非同源性;4.自然暴露状态下,人体鼻腔灌洗液中微塑料丰度为31.90±1.77个/克,以纤维状为主,以PA、PC及PE为主要成分;个体行为是影响呼吸道微塑料暴露水平的主要因素;不同材质口罩显著影响人体呼吸道微塑料暴露水平,佩戴外科口罩能够显著升高鼻腔灌洗液中微塑料暴露水平。更多还原

【Abstract】 Objective:In 2021,the plastic production in the world reached 391 million tons,while only 10.2 million tons of plastic products were recycled.High production and low recovery lead to a large amount of plastic waste into the environment,which produces microplastics with particle size less than 5 mm under the action of ultraviolet light and abrasion.The pollution of microplastics in the environment has attracted worldwide attention.The two most important ways of human body being exposed to microplastics are respiratory tract and digestive tract.At present,there are many studies on digestive tract exposure,the exposure evaluation,toxic effects and mechanism of action in food and drink have been extensively reported.In contrast,there are few studies on respiratory exposure,especially the exposure evaluation of health hazard assessment which is the first step of assessment.The main reasons are as follows:The evaluation of exposure through the respiratory tract not only has difficulties as the external exposure is different between indoor and outdoor and can be influenced by many different factors,the evaluation of internal exposure also faces the difficulties of obtaining representative bronchoalveolar lavage fluid and lung tissue biopsy.Due to the epidemic of COVID-19,the use of masks remains high.There are studies showing that surgical masks are made of plastic,which can become another potential source of pollution of microplastics,and increase the risk of humans being exposed to microplastics while preventing the invasion of pathogenic microorganisms.But so far,the related exposure evaluation has not been reported.In this study,we took great advantages of the nasal lavage fluid and sputum as they can be used as good indicators of exposure load in the respiratory system.These samples are easy to collect and non-invasive,and can be carried out in large-scale population epidemiological investigations.We conducted a preliminary study of evaluating the load of microplastics in nasal lavage fluid and sputum in typical indoor and outdoor populations to clarify the feasibility of nasal lavage fluid and sputum as indicators of internal exposure by a combination of polarized light microscope and laser infrared imaging system.The qualitative and quantitative detection methods were verified in the lung tissues of domestic pigs in representative contaminated areas,and the accuracy of the detection method combined with polarized light microscope and laser infrared imaging system for different pollution levels was confirmed.On this basis,we made use of advantages as follows:the campus area of a university is relatively independent,the architectural style and materials are unified,the interior furnishings are consistent,the students’demographic characteristics are simple,the casas of respiratory system diseases are less,the life styles are relatively consistent,the compliance and cooperation degree are high,which means there are fewer interference factors in the evaluation of the external and internal exposure of microplastics in the air.We collected indoor and outdoor air samples from different functional zones on campus to evaluate the external exposure load of microplastics in the air,to clarify the level and characteristics of indoor and outdoor exposure and reveal the correlation between indoor and outdoor pollution of microplastics.Graduate students were recruited as the investigation objects to evaluate the internal exposure levels of microplastics through respiratory tract,intervention studies on different kinds of masks were conducted to reveal the levels and characteristics of microplastic pollution in human biological samples,to clarify the influencing factors,and confirm the influence of masks on human internal exposure.Methods:1.Taking couriers as the outdoor typical population,office workers as the indoor typical population,and the Institute of Preventive Medicine of China Medical University as the research site,a total of 8 office staff and 8 couriers within 3 km radius around the Institute of Preventive Medicine were recruited as the research objects.Nasal lavage fluid and sputum samples of the subjects were collected,and then divided into equal parts before digestion treatment.One of the samples was filtered through a membrane and made into a dual slide chamber,observed under Leica DM4B DFC7000T polarized light microscope and recorded its number,size and the shape of microplastics.The other one was concentrated by nitrogen blowing and put into the Agilent 8700 laser infrared imaging system to determine the main composition of microplastics.The student’s t test was used to analyze whether the difference in the abundance of microplastics in nasal lavage fluid and sputum of typical indoor and outdoor populations was statistically significant.The lung tissues of domestic pigs raised in typical contaminated areas for more than 1 year and fetal pigs that died of asphyxia before birth were taken as the research objects.Polarized light microscope and laser infrared imaging system were used to analyze the abundance and characteristics of microplastics in lung tissues of pigs and fetal pigs.2.A total of 39indoor and outdoor air dust fall samples were collected from different functional zones(living area,learning or work area and activity area)in the campus,including 18 indoor and 21 outdoor air dust fall samples.After digestion treatment,the microplastics were made into dual slide chambers,which were observed by polarizing light microscope to record the quantity,particle size and shape of microplastics.Part of samples were selected for detection by laser infrared imaging system to determine the distribution of microplastics in indoor and outdoor air dust fall.The student’s t test and ANOVA were used to analyze whether the differences in the abundance of microplastics in air dust fall in different places and whether the influences of different indoor factors on the abundance of microplastics in indoor air dust fall were statistically significant.Correlation analysis was used to clarify the relationship between the abundance of microplastics in indoor and outdoor air dust fall and temperature and humidity.Consistency test was used to analyze the homology of microplastic pollution in indoor and outdoor air dust fall.3.A total of 113graduate students were recruited to be investigated the internal exposure of respiratory microplastics on campus.Subjects were randomly divided into a natural exposure group(n=54),a surgical mask intervention group(n=29),and a cotton mask intervention group(n=30).Participants in the intervention group were asked to wear the masks for 3 days,no less than 2 hours per day.On the fourth day,they would finish the questionnaire and be collected nasal lavage fluid by professionals in the laboratory.The Mask Wearing Record sheet was retrieved at the same time.The nasal lavage fluid was digested and made into dual slide chambers,which were determined the number,size and shape of microplastics by polarizing light microscope.Part of samples were randomly selected from each group for the detection of microplastics components by Agilent 8700 laser infrared imaging system.The student’s t test or ANOVA were used to do the single factors analysis of the abundance of microplastics in nasal lavage fluid,the generalized linear model was used for multi-factor analysis and the Chi-square test was used to analyze the differences in the population characteristics of volunteers in each group and whether there were differences in the particle size,shape and composition distribution of microplastics in nasal lavage fluid in each group.ANOVA was used to analyze whether there was statistical significance in the abundance of microplastics in the nasal lavage fluid of volunteers in each group,and the influence factors were controlled by the generalized linear model to further clarify the influence of mask use on the abundance of microplastics in the nasal lavage fluid.Results:1.Couriers and office workers were regarded as typical outdoor and indoor populations,respectively.The abundance of microplastics in the nasal lavage fluid of couriers(0.76±1.51 MPs/g)was significantly lower than that of office workers(2.24±1.82MPs/g)(P<0.01).The shape of microplastics in the nasal lavage fluid of couriers and office workers was mainly fibrous,accounting for 83.81%and 87.03%respectively.PA(25.56%)and PE(23.06%)were the main microplastics in the nasal lavage fluid of couriers,while PVC(41.40%)and PA(31.81%)were the main microplastics in office workers.There was no significant difference in the abundance of microplastics in sputum between couriers(92.09±1.76 MPs/g)and office workers(58.55±3.33 MPs/g)(P=0.35).The microplastic shape in sputum of couriers(94.26%)and office workers(83.28%)was still mainly fibrous.PC(23.93%)and PVC(23.08%)were the main microplastics in the sputum of couriers,while PVC(40.21%)and PA(25.30%)were the main microplastics in the sputum of office workers.The abundance of microplastics in fetal pig lung tissue was 6 MPs/g,and that in domestic pig lung tissue was 12 MPs/g,both of which were fibrous.The main microplastics in fetal pig lung tissues were PC(32.99%),PP(15.46%)and PVC(15.46%),and PA(46.11%),PP(21.11%)and PE(17.22%)in the lung tissues of domestic pigs.2.In different functional zones on campus,the average abundance of microplastics in outdoor air dust fall was 1666.18 MPs/(d·m~2),and the difference among different functional zones was not statistically significant(P=0.78).Among them,the abundance of microplastics in outdoor air dust fall of teaching building was significantly higher than that of sports stadiums(P=0.13).The average abundance of microplastics in indoor air dust fall was2244.89 MPs/(d·m~2),of witch in the female dormitory was higher than that in office building(P=0.13)and library(P=0.12).And the abundance of microplastics of teaching building was higher than library(P<0.15).Humidity(P=0.03),curtains(P=0.10)and the use of air conditioner(P=0.03)were significantly correlated with the abundance of microplastics in indoor air dust fall.Different cleaning methods(P=0.42)and frequency(P=0.55)had no significant effect on the abundance of microplastics in indoor air dust fall.The comparison between indoor and outdoor air dust fall showed that the abundance of microplastics in indoor was higher than that in outdoor air dust fall,but it was not statistically significant(P=0.26).In both outdoor and indoor air dust fall,the main microplastic shape was fibrous,but the proportion of indoor fiber(94.06%)was significantly higher than that of outdoor(87.52%)(P<0.01).There were also significant differences between indoor and outdoor components(P<0.01).The main components were PA(34.45%)and PC(28.26%)in outdoors air dust fall,and PA(30.62%)and PE(33.33%)in indoors air dust fall.The particle size of microplastics in outdoor(53.87%)air dust fall mostly concentrated below 200μm,and in indoor(62.70%)air dust fall mostly concentrated below 500μm.The difference of particle size distribution was statistically significant(P<0.01).3.Under natural conditions,the abundance of microplastics in human nasal lavage fluid was 31.90±1.77 MPs/g.The multi-factor generalized linear model analysis showed that office air conditioning usage within 3 days(Occasionally used vs None/Not used:β=0.24,P=0.09;Frequently used vs None/Not used:β=-0.17,P=0.22)and towel material(Without chemical fiber vs Containing chemical fiber:β=0.24,P=0.08)were significantly correlated with the abundance of microplastics in nasal lavage fluid.In the natural exposure group,main shape of the microplastics was fibrous(88.06%),and the main components were PA(43.70%),PC(13.80%)and PE(12.83%).Compared with the natural exposure group,after controlling for other influencing factors,the abundance of microplastics in nasal lavage fluid in surgical mask intervention group was significantly higher than that in natural exposure group(β=0.25,P=0.01).The abundance of microplastics in nasal lavage fluid was slightly lower in the cotton mask intervention group than in the natural exposure group,but the difference was not statistically significant(β=-0.11,P=0.23).The shape of microplastics in nasal lavage fluid of volunteers in surgical mask intervention group(92.01%)and cotton mask intervention group(89.38%)was mainly fibrous.There was significant difference in the shape of microplastics in nasal lavage fluid between all groups(P<0.01).Also,the main components of microplastics in nasal lavage fluid were different(P<0.01).In the surgical mask intervention group,the main microplastics were PC(25.30%),PA(24.10%)and PVC(19.28%),while in the cotton mask intervention group were mainly PA(28.32%),PC(25.66%)and PE(15.04%).The particle size of microplastics in natural exposure group(59.30%),surgical mask intervention group(64.57%)and cotton mask intervention group(58.50%)were mostly less than 200μm,and the difference of particle size distribution was statistically significant(P<0.01).Conclusion:1.The presence of microplastics was detected in nasal lavage fluid and sputum of typical indoor and outdoor population,and the indoor exposure level was higher than that of outdoor population,mainly fibrous.2.The combination of polarized light microscope and laser infrared imaging system could effectively detect the exposure status of microplastics in biological samples.3.The pollution levels,particle size and main components of microplastics in indoor and outdoor air dust fall were different,but they were mainly fibrous,and the indoor exposure load was higher than that of outdoor air.Microplastic pollution in indoor and outdoor air has non-homology.4.Under natural exposure condition,the abundance of microplastics in human nasal lavage fluid was31.90±1.77 MPs/g,mainly fibrous,with PA,PC and PE as the main components.Individual behavior was the main factor affecting respiratory microplastic exposure.Masks of different materials could significantly affect the microplastic exposure level of human respiratory tract.Wearing surgical mask could significantly increase the level of microplastic exposure in nasal lavage fluid.更多还原