导　　师： 霍霞

学科专业： J0102

授予学位： 博士

作　　者： ;

机构地区： 汕头大学

摘　　要： 在孕妇分娩后立即采集新生儿脐带血并离心成血清备用。通过液-液萃取的方式提取血清脂肪,并干燥器恒重。脂肪经正已烷溶解、硫酸净化、酸性硅胶管柱净化后,用配有自动进样器和化学源/(chemical ionization, CI/)的Agilent 7890A//5975C气质联用仪/(gas chromatograph-mass spectrometer, GC//MS/)进行脐带血PBDEs含量检测。检测在NCI/(Negative Chemical Ionization/)模式下以选择性离子监测/(Selective ion monitoring, SIM/)的方式进行。对于低溴同系物,使用的是DB-5 MS弱极性毛细管色谱柱/(30 m×0.25 mm i.d., 0.25μm film thickness, J/&W Scientific/),以氦气为载气、甲烷为反应气,离子源、四极杆、接口温度分别为200、150与280℃;对于BDE-209使用的是DB-5 HT高温柱/(15 m×0.25 mm i.d., 0.1μm film thickness/),其它条件相同。按照标准溶液建立了各同系物的保留时间。低溴同系物的监测离子为m//z 79、81;BDE-209的监测离子为m//z 79、81、486.7、488.7。低溴同系物的升温程度为:80℃/(1min/)→20℃//min→310℃/(10min/);BDE-209升温程序为:110℃/(1min/)→20℃//min→310℃/(5min/)。在仪器检测过程中,每进8个样品运行一次溶剂空白和程序空白以排除干扰和污染。 PBDEs检测数据按脂肪重量进行了校正,并以中位数和上下限范围表示。检测了8种PBDE同系物BDE-28, 47, 99, 100, 153, 154, 183及209,并以∑PBDE作为体内这8种PBDE同系物的总含量。在摩尔浓度的基础上计算了每种同系物对∑PBDE水平的贡献率。由于各样本PBDEs含量的非正态分布特征,采用了Mann-Whitney U检验来比较两组的差别,采用Kruskal Wallis H检验来比较多组之间的差别。Spearman相关及多元线性逐步回归被用来探索PBDEs水平与各因素的相关性及最终新生儿脐带血PBDEs暴露水平的影响因素。采用二分类的Logistic回归分析探索影响新生儿不良出生结局的危险因素。所有数据在Microsoft Exel 2003中进行了整理并以SPSS 13.0统计学软件/(SPSS Inc., Chicago, IL, USA/)进行统计分析。显著性水平设为双侧0.05。 结果: 对其中102名来自贵屿及51名来自潮南的孕妇在分娩后采集了新生儿脐带血。来自贵屿的脐带血样本中∑PBDE浓度/(中位数:13.84;范围1.14 ~504.97 ng g-1 lipid/)要高于对照组/(中位数: 5.23;范围0.29~363.70 ng g-1 lipid/) /(P<0.05/)。另外,除了BDE-209之外,贵屿组脐带血样本中每种PBDE同系物的水平均要高于对照地区脐带血样本/(P<0.05/)。在贵屿与潮南的所有脐带血样本中均检测出了BDE-28、47及183的存在,其它同系物如BDE-99、100、153及154的检出率,同样除了BDE-209/(检出率分别为57.84/%与56.86/%, P=0.908/)之外,贵屿组/(99.02/%, 88.24/%, 95.10/%, 72.55/%/)均要高于潮南组/(70.59/%, 72.55/%, 82.35/%, 35.29/%/),P值均小于0.05。虽然BDE-209对∑PBDE浓度贡献较大,BDE-47仍为主要的同系物,接下来 在孕妇分娩后立即采集新生儿脐带血并离心成血清备用。通过液-液萃取的方式提取血清脂肪,并干燥器恒重。脂肪经正已烷溶解、硫酸净化、酸性硅胶管柱净化后,用配有自动进样器和化学源/(chemical ionization, CI/)的Agilent 7890A//5975C气质联用仪/(gas chromatograph-mass spectrometer, GC//MS/)进行脐带血PBDEs含量检测。检测在NCI/(Negative Chemical Ionization/)模式下以选择性离子监测/(Selective ion monitoring, SIM/)的方式进行。对于低溴同系物,使用的是DB-5 MS弱极性毛细管色谱柱/(30 m×0.25 mm i.d., 0.25μm film thickness, J/&W Scientific/),以氦气为载气、甲烷为反应气,离子源、四极杆、接口温度分别为200、150与280℃;对于BDE-209使用的是DB-5 HT高温柱/(15 m×0.25 mm i.d., 0.1μm film thickness/),其它条件相同。按照标准溶液建立了各同系物的保留时间。低溴同系物的监测离子为m//z 79、81;BDE-209的监测离子为m//z 79、81、486.7、488.7。低溴同系物的升温程度为:80℃/(1min/)→20℃//min→310℃/(10min/);BDE-209升温程序为:110℃/(1min/)→20℃//min→310℃/(5min/)。在仪器检测过程中,每进8个样品运行一次溶剂空白和程序空白以排除干扰和污染。 PBDEs检测数据按脂肪重量进行了校正,并以中位数和上下限范围表示。检测了8种PBDE同系物BDE-28, 47, 99, 100, 153, 154, 183及209,并以∑PBDE作为体内这8种PBDE同系物的总含量。在摩尔浓度的基础上计算了每种同系物对∑PBDE水平的贡献率。由于各样本PBDEs含量的非正态分布特征,采用了Mann-Whitney U检验来比较两组的差别,采用Kruskal Wallis H检验来比较多组之间的差别。Spearman相关及多元线性逐步回归被用来探索PBDEs水平与各因素的相关性及最终新生儿脐带血PBDEs暴露水平的影响因素。采用二分类的Logistic回归分析探索影响新生儿不良出生结局的危险因素。所有数据在Microsoft Exel 2003中进行了整理并以SPSS 13.0统计学软件/(SPSS Inc., Chicago, IL, USA/)进行统计分析。显著性水平设为双侧0.05。 结果: 对其中102名来自贵屿及51名来自潮南的孕妇在分娩后采集了新生儿脐带血。来自贵屿的脐带血样本中∑PBDE浓度/(中位数:13.84;范围1.14 ~504.97 ng g-1 lipid/)要高于对照组/(中位数: 5.23;范围0.29~363.70 ng g-1 lipid/) /(P<0.05/)。另外,除了BDE-209之外,贵屿组脐带血样本中每种PBDE同系物的水平均要高于对照地区脐带血样本/(P<0.05/)。在贵屿与潮南的所有脐带血样本中均检测出了BDE-28、47及183的存在,其它同系物如BDE-99、100、153及154的检出率,同样除了BDE-209/(检出率分别为57.84/%与56.86/%, P=0.908/)之外,贵屿组/(99.02/%, 88.24/%, 95.10/%, 72.55/%/)均要高于潮南组/(70.59/%, 72.55/%, 82.35/%, 35.29/%/),P值均小于0.05。虽然BDE-209对∑PBDE浓度贡献较大,BDE-47仍为主要的同系物,接下来依次为BDE-28, BDE-153, BDE-183与BDE-99。 母亲从事电子垃圾拆解工作的新生儿脐带血PBDE各同系物水平明显高于对照组。我们还发现对于居所同时为电子垃圾拆解作坊的孕妇,其新生儿脐带血某些PBDE同系物如BDE-28、47、99与100的负荷也明显增加。多元线性回归显示孕妇在贵屿镇长时间居住、孕妇及其丈夫从事电子垃圾拆解工作,以及居所同时为电子垃圾拆解作坊是新生儿脐带血PBDEs含量增高的主要危险因素。 脐带血PBDEs水平对新生儿性别及分娩方式并无影响/(P>0.05/)。研究组及对照组新生儿在身长、体重、胎龄、分娩方式、性别或早产的发生率方面并无差别/(P值均>0.05/),但研究组新生儿的Apgar评分要低于对照组/(P<0.05/)。在贵屿108例新生儿中,有8例死胎与1例无脑畸形。正常出生的新生儿与不良出生结局新生儿/(含早产、低体重儿及死胎/)进行对比后发现,后者体内有着更高的PBDEs暴露,其脐带血∑PBDE及BDE-28、47、99、153及183水平均显著增高/(P<0.05/)。Logistic回归分析发现,BDE-99/(OR=3.33; 95/%CI: 1.22~9.08/)及BDE-153/(OR=1.42; 95/%CI: 1.09~1.85/)是发生不良出生结局的危险因素。 结论: /(1/)本研究采用了人群流行病学调查、人体标本PBDEs水平实验室检测、以及孕妇与新生儿的健康检查等手段,首次开展了对电子垃圾拆解区新生儿脐带血PBDEs暴露及其健康效应关系的研究。来自电子垃圾拆解区贵屿的新生儿人群处于PBDEs的暴露之下。在所检测的贵屿新生儿脐带血样本中,∑PBDE浓度要高于对照组;除了BDE-209之外,7种不同PBDE同系物单体的暴露水平及检出率均要高于对照组。 /(2/)电子垃圾拆解区新生儿脐带血样本PBDEs各同系物的分布特征:BDE-209在部分样品中含量较高,但检出率较低;除BDE-209之外,BDE-47是主要的同系物,接下依次为BDE-28、BDE-153及BDE-183。 /(3/)在我们所研究的样本中新生儿脐带血PBDEs含量增高的主要危险因素,与孕妇在贵屿居住时间、居住环境、是否从事电子垃圾拆解工作以及配偶也从事相关工作密切相关。 /(4/)在这项研究中检测到的新生儿脐带血PBDEs暴露水平可能已经影响到新生儿健康及其生长发育,并产生不良出生结局。通过对电子垃圾拆解区新生儿出生前PBDEs暴露、不良出生结局及相关影响因素分析,为PBDEs对人群健康潜在的危害提供了一些研究线索,并为降低新生儿出生缺陷率和提高人口素质提供了科学依据。但PBDEs的具体生物学作用及人群潜在健康效应仍需更大样本量的随机流行病学研究来证实和评估。 Background: Expansion of the global market for electrical and electronic products continues to accelerate, and the rapid technologic development, decrease in prices, has decreased the lifespan of the products, which has resulted in a corresponding explosion in electronic scrap. This situation creates a large volume of obsolete electrical and electronic devices termed e-waste which if not properly handled can be a source of pollutants posing a risk to the environment and humans. Guiyu, China is famous for its involvement in processing primitive electronic-waste /(e-waste/). Persistence toxic substances such as polybrominated diphenyl ethers /(PBDEs/) are continually released to the environment. These extremely hazardous and dangerous e-waste recycling operations cause heavy pollution of air, water and soil in Guiyu, which also poses a threat to the health of the local people. PBDEs are a group of brominated compounds that have been widely used as brominated flame retardants for several decades because they provide long escape times in case of fire, which saves lives and reduces damage from fire. Despite their societal benefits, PBDEs seem to pose a threat to the environment and people. Still, insufficient knowledge exists on the potential adverse effects of PBDEs on public health. Human samples such as serum//plasma, milk, and adipose tissue have been used to evaluate the extent of human exposure to PBDEs. To date, most of these reports are from Europe and North America. However, no studies have reported on PBDE levels in umbilical cord blood /(UCB/) in e-waste recycling areas and their health effects on neonates in China. Objective: We aimed to evaluate the PBDE exposure of neonates from Guiyu, China, and a control area, Chaonan, China, through umbilical cord blood /(UCB/), the health effects, and relevant factors contributing to PBDE concentrations. Methods: We enrolled 167 healthy pregnant women as participants /(108 from Guiyu and 59 from Chaonan/), who voluntarily donated UCB after birth. The study protocol was approved by the Human Ethical Committee of Shantou University Medical College, China. All participants gave their written informed consent after receiving detailed explanations of the study and potential consequences prior to enrollment. Questionnaires were addressed to collect information on potential routes of exposure to PBDEs, as well as general demographic and health parameters, and UCB was collected shortly after birth from the pregnant women from Guiyu and Chaonan between May and July 2007. Serum samples were prepared by liquid-liquid extracting methods. The lipid content of the serum was determined by gravimetric methods after solvent evaporation. Lipid was dissolved with n-hexane and washed with H2SO4,and then cleaned up with a silica//sulfuric acid column.PBDE congeners /(including BDE-28, -47, -99, -100, -153, -154, -183, and -209/) were determined by Agilent 7890A gas chromatography coupled with 5975C mass spectrometry /(Agilent Technologies, USA/) and negative chemical ion analysis. Auto splitless injection onto a J/&W Scientific DB-5 MS capillary column /(30 m×0.25 mm i.d., 0.25μm film thickness/) was used for the determination of less-brominated PBDE congeners, with helium /(1 mL min-1/) used as the carrier gas. Methane was used as a buffer gas in the NCI mode. The ion source, quadrupole and interface temperatures were set to 200, 150 and 280℃, respectively. The same conditions were used with a 15 m DB-5 HT capillary column /(0.25 mm i.d., 0.1μm film thickness/) for the analysis of BDE-209. Retention times for PBDE congeners were established with standard solutions. In addition, ion fragments at 79 and 81 m//z/(/[Br/]?/) were monitored for less-brominated PBDE congeners, and 79, 81, 486.7, and 488.7 m//z for BDE-209. Oven temperature was ramped from 80℃/(1 min/) to 310℃/(8 min/) at 20℃//min for less-brominated PBDE congeners, and from 110℃/(1 min/) to 310℃/(5 min/) at 20℃//min for BDE-209. For every sequence of 8 samples, a solvent blank and a procedural blank were added to ensure that the samples and the analysis process were free of contamination. Data are expressed as median and ranges. We defined∑PBDE as the serum sum of congeners: BDE-28, -47, -99, -100, -153, -154, -183, and -209. The mean congener contribution to the∑PBDE was calculated as the average of congener contribution to the∑PBDE of each sample based on molar concentration. The Mann-Whitney U and Kruskal Wallis H tests were used because of the nonnormal distribution of the PBDE data. Potential associations between PBDE concentrations and related factors were explored by Spearman correlation analysis. Multiple stepwise regression analysis was used to evaluate factors related to PBDE concentrations. P values were calculated by the chi-square test for categorical data. Statistical analyses involved use of SPSS v13.0 /(SPSS Inc., Chicago, IL, USA/) and Microsoft Excel. A p<0.05 was considered statistically significant. Results: A total of 108 women from Guiyu and 59 women from Chaonan were enrolled and completed the questionnaires. A total of 102 women from Guiyu and 51 women from Chaonan provided UCB samples after giving birth. The pregnant women from Guiyu had a lower educational level, higher tea consumption and longer time on the road every day than did women from Chaonan. The total PBDE concentration was higher in UCB from Guiyu than in that from Chaonan /(median 13.84, range 1.14 to 504.97 ng g-1 lipid; vs. 5.23, range 0.29 to 363.70 ng g-1 lipid/) /(P<0.05/). Also, median individual congener concentrations, with the exception of BDE-209, were higher in UCB samples from Guiyu than from Chaonan samples /(P<0.05/). BDE-28, -47 and -183 were detected in all samples from Guiyu and Chaonan, and the detection frequencies of other congeners were all higher in UCB samples from Guiyu than from Chaonan samples /(99.02/%, 88.24/%, 95.10/%, 72.55/%; vs. 70.59/%, 72.55/%, 82.35/%, 35.29/% for BDE-99, -100, -153, and -154, respectively, all P<0.05/), except for BDE-209 /(57.84/% vs. 56.86/%, P=0.908/). BDE-209 was the dominant PBDE congener, followed by BDE-47, -153, and -28. We found some highest PBDE concentrations, largely contributed by BDE-209, in some samples in our study. When we compared our data with that from UCB concentrations around the world, the median concentrations of PBDEs in Guiyu samples were higher than those reported in Europe and other areas in China, but still lower than some data from the United States. Residing in Guiyu, which is involved in e-waste recycling and residence also used as a family workshop were significant factors contributing to PBDE concentrations in UCB. UCB samples from mothers involved in e-waste recycling showed higher concentrations of PBDE congeners than did UCB from women in the control region, especially for low-brominated congeners such as BDE-28, -47 and -99. We found high concentrations of some PBDE congeners /(BDE-28, -47, -99, and -100/) in UCB from the pregnant women living in houses also served as workshops in Guiyu. PBDE concentrations did not differ by sex or delivery mode /(all P>0.05/). Neonates from Guiyu and Chaonan did not differ in height, weight, gestational age, delivery mode, sex, or premature delivery rate /(all P>0.05/), but mean Apgar score in Guiyu was lower than that in Chaonan /(P=0.001/). Among the 108 births from Guiyu there were 8 stillbirths and one case of anencephalus. PBDE levels significantly differed in neonates by normal birth and still birth, low birth weight and premature delivery /(P<0.05/). Conclusions: /(1/) This is the first study concerning PBDE levels in UCB samples and their effect on neonate health, involving investigation by questionnaire, birth health checks and detection of blood PBDE levels. We studied a vulnerable population of neonates with exposure to multiple hazardous chemicals in an e-waste recycling area. The total PBDE concentration was higher in UCB from Guiyu than in that from Chaonan. Except BDE-209, both concentrations and detection frequencies of other 7 PBDE congeners in Guiyu samples were higher than in Chaonan samples. /(2/) Except for BDE-209, BDE-47 was the dominant congener and was detected in all blood samples, following by BDE-28, BDE-153 and BDE-183. /(3/) In this study, the mothers’residence in Guiyu for years and being involved in e-waste recycling largely contributed to PBDE concentrations in UCB samples; other significant factors were the house also being the family workshop and the husband being involved in e-waste recycling. /(4/) PBDEs at levels determined in this study may have affected the neonates’health and development. This finding provides some clues for the potential health risk of PBDEs to humans. Prenatal exposure to PBDEs may have affected neonates’health. The biological effects and the potential health risks of PBDEs still need to be evaluated in random studies with larger samples.

关 键 词： 多溴联苯醚 脐带血 电子垃圾 不良出生结局 气质联用

分 类 号： [X13 X78]

领　　域： [环境科学与工程] [环境科学与工程]