城市生活垃圾填埋场中抗生素残留浓度征及抗性基因相关性研究

发布时间：2017-12-27 05:16

本文关键词：城市生活垃圾填埋场中抗生素残留浓度征及抗性基因相关性研究　出处：《华东师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：抗生素(Antibiotics,ABs)滥用是目前许多国家和地区面临的重要问题,其在环境中的残留加速诱导了环境中耐药性细菌以及抗生素抗性基因(Antibiotic Resistance Genes,ARGs)的产生、传播,对周边环境带来潜在生态和健康风险。我国是抗生素生产和使用大国,药物因回收体制不健全,大多混入日常生活垃圾进入城市生活垃圾填埋场,但目前对于城市生活垃圾填埋场中抗生素残留及其浓度特征的研究却鲜有报道。关注城市生活垃圾填埋场环境介质中的抗生素残留及其抗性基因之间相关性研究,对提高关于抗生素的认识及其风险意识具有重要意义。本研究采集了上海市老港和黎明城市生活垃圾填埋场固体废弃物和渗滤液样品,采用SAX-HLB串联固相萃取和超高效液相色谱三重四级杆质谱联用仪(UPLC-3QMS)检测分析填埋场固体废弃物和渗滤液中15种抗生素的检出浓度及其浓度分布特征;采用荧光定量PCR分析填埋场固体废弃物和渗滤液中7种抗生素抗性基因。探讨了填埋场环境中抗生素及其抗性基因之间的相关性,为城市生活垃圾填埋场中抗生素的迁移转化、风险评估提供参考依据。研究发现,在填埋场渗滤液中,四环素类(TCs)、喹诺酮类(FQs)、磺胺类(SAs)、大环内脂类(MLs)、β-内酰胺类(BLs)5类15种抗生素在渗滤液中均有不同浓度检出,其中磺胺二甲基嘧啶(SMZ)、诺氟沙星(NOR)、氧氟沙星(OFL)、罗红霉素(RTM)和去水红霉素(ETM-H_2O)5种抗生素检出率达到100%。去水红霉素在渗滤液中检出浓度最高(20200.0 ng/L),平均检出浓度达到5695.7 ng/L,其次分别为OFL、NOR,最高检出浓度分别为6950.0 ng/L、3225.0 ng/L,平均检出浓度分别为1870.9 ng/L、1101.2 ng/L,阿莫西林在渗滤液中检出浓度较低,平均检出浓度仅为8.6 ng/L,检出率22.0%。抗生素在老龄渗滤液(3075.8±4004.0ng/L)中浓度均低于新鲜(15063.6±2475.9ng/L)和黎明渗滤液(13628.5±8792.5 ng/L)中浓度(pp0.01),老港新鲜和黎明渗滤液中抗生素检出浓度无明显差异(p=0.107)。三种渗滤液(老港老龄、新鲜和黎明渗滤液)在雨季的抗生素浓度相比旱季渗滤液中的浓度均存在不同程度降低,其中老龄渗滤液和黎明渗滤液中抗生素浓度下降程度分别达到93%(5737.9±4229.4 ng/L vs 413.6±38.0ng/L)和 78%(22398.6±887.1 ng/L vs 4858.4±24.4ng/L);而新鲜渗滤液中抗生素浓度仅下降28%(17135.0±1931.4 ng/L vs 13003.4±322.6 ng/L),浓度下降程度较低。在填埋场固体废弃物中,TCs、QNs、MLs三类9种抗生素的检出率均达到100%,SAs和BLs两类抗生素的检出率为23.4-93.6%;喹诺酮类抗生素NOR和OFL最高检出浓度分别达到1497.5 μg/kg和1250.0 μg/kg,平均检出浓度分别为260.1 μg/kg和215.8 μg/kg,阿莫西林(AMOX)在所有固体废弃物样品中的检出浓度普遍较低,平均检出浓度小于0.1 μg/kg。相比黎明填埋场固体废弃物中的抗生素检出浓度,TCs、QNs和MLs三类抗生素在老港填埋场固体废弃物中的抗生素检出浓度均普遍降低(p0.05),而SAs和BLs在两个填埋场固体废弃物中的检出浓度无显著变化。在老港填埋场填埋表层(0.5-1.5m)的固体废弃物样品中,呈现出抗生素浓度在深度上的变化特征,除BLs外其它四类抗生素(TCs、QNs、SAs、MLs)在1.5 m深度处的抗生素检出浓度均高于其在0.5 m深度处的检出浓度(p0.05),而黎明填埋场填埋表层固体废弃物中则无此浓度分布特征。这可能是由于老港填埋场中固体废弃物已处于填埋成熟阶段,且抗生素在雨水、渗滤液等作用向下迁移扩散所致。研究选择检测了 7种抗生素抗性基因(tetQ、tetW、mefA、ermB、sul1、sul2和mexF)以及整合子int1。检测结果显示,在填埋场渗滤液中,7种ARGs的检出浓度为2.91×10~2～4.88×10~6 copies/ngDNA,整合子int1检出浓度为4.55×10~4～8.31×10~7 copies/ngDNA;在填埋场固体废弃物中,tetQ、tetM、mefA三种ARGs检出浓度低于标准曲线定量范围,ermB、sul1、sul2和mexF四种ARGs的检出浓度为1.85×1 02～5.07×1 06 copies/ngDNA,整合子int1检出浓度为6.90×105～1.86×108 copies/ngDNA。分析抗生素与其抗性基因之间相关性结果表明,在填埋场渗滤液中,四环素类抗生素均与抗性基因tetQ之间呈现显著相关性,ETM-H_2O、MLs与mefA存在显著相关性,磺胺甲恶唑(SMX)、SAs则与sul1和sul2呈现显著负相关性,而多重抗性基因mexF与所检测抗生素之间均无相关性;在老港填埋表层固体废弃物中,仅RTM与ermB具有显著正相关性,与多重抗性基因mexF呈现显著负相关性;在黎明填埋场表层固体废弃物中,仅sul2与磺胺嘧啶(SD)、磺胺二甲基嘧啶(SMZ)以及SAs呈现显著负相关性。这可能是因为城市生活垃圾填埋场环境介质理化性质(温度、光照、pH等)复杂,污染物质(重金属、有毒物质等)浓度高,影响ARGs在填埋场中的诱导产生、衰减和传播扩散。在渗滤液中,整合子intl与sul1、sul2和mexF之间存在极显著正相关性,在老港填埋场固体废弃物中整合子int1与sul1和sul2之间极显著相关,但其在黎明填埋场固体废弃物中与ermB、sul1、sul2以及mexF均无相关性,这可能表明在固体废弃物填埋后期,抗性基因sul1、sul2以水平基因转移为重要途径在固体废弃物中的传播扩散。综上所述,本研究中15种常用抗生素在城市生活垃圾填埋场表层固体废弃物和渗滤液中均能高浓度检出,且不同种类的抗生素检出浓度之间存在差异。抗性基因与部分种类的残留抗生素之间显著相关,但填埋场环境介质复杂的理化性质和高浓度的污染物质影响ARGs在填埋场中的诱导产生、衰减和传播扩散。
[Abstract]:The abuse of antibiotics (Antibiotics, ABs) is an important problem in many countries and regions are currently facing, the residue in the environment to accelerate the environment induced by antibiotic resistant bacteria and antibiotic resistance genes (Antibiotic, Resistance Genes, ARGs) generation, propagation, potential ecological and health risk to the surrounding environment. China is a large country in the field of antibiotic production and use. Because of the imperfect system of recovery, most of the drugs are mixed into daily life garbage to enter the municipal solid waste landfill site. However, the research on the residual and concentration characteristics of antibiotics in MSW landfill is rarely reported. Concerned about the correlation between antibiotic residues and their resistance genes in municipal solid waste landfill sites, it is of great significance to improve the awareness and risk awareness of antibiotics. This study collected in Shanghai city and the old port city dawn landfill of solid waste and leachate samples by SAX-HLB solid-phase extraction and ultra performance liquid chromatography three four quadrupole mass spectrometry (UPLC-3QMS) detection concentration and concentration distribution of 15 kinds of antibiotics in solid waste landfill and leachate detection analysis; using fluorescence quantitative PCR analysis of 7 kinds of antibiotic resistance genes and solid waste landfill leachate. The correlation between antibiotics and their resistance genes in the landfill environment is discussed, so as to provide references for the migration, transformation and risk assessment of antibiotics in municipal solid waste landfill. The study found that in the landfill leachate, tetracyclines and quinolones (TCs) (FQs) (SAs), sulfonamides, macrolides (MLs), beta lactam (BLs) 5 categories of 15 kinds of antibiotics in the leachate were detected at different concentrations, the sulfamethazine (SMZ), norfloxacin (NOR), ofloxacin (OFL), roxithromycin (RTM) and erythromycin (ETM-H_2O) water to 5 kinds of antibiotics detection rate reached 100%. Go to the water in the leachate were erythromycin the highest concentration (20200 ng/L), the average detection concentration reached 5695.7 ng/L, followed by OFL, NOR, the highest concentration were 6950 ng/L, 3225 ng/L, the average detection concentration were 1870.9 ng/L, 1101.2 ng/L, Amos Lin in the leachate were detected in low concentrations, the average detection the concentration is 8.6 ng/L, the detection rate of 22%. The concentration of antibiotics in the aged leachate (3075.8 + 4004.0ng/L) was lower than that in fresh (15063.6 + 2475.9ng/L) and leachate (13628.5 + 8792.5 ng/L) concentration (pp0.01). There was no significant difference in the concentration of antibiotics in fresh port and leachate in old port (p=0.107). Three kinds of leachate (Hong Kong, fresh and old aged dawn leachate) concentration in leachate infiltration compared to dry season there are different degrees of reduction in the concentration of antibiotics during the rainy season, the aging of leachate and leachate infiltration dawn antibiotic concentration decreased respectively 93% (5737.9 + 4229.4 ng/ L vs 413.6 + 38.0ng/L and 78% (22398.6) 887.1 + ng/L vs 4858.4 + 24.4ng/L); while the fresh leachate concentration of antibiotics decreased only 28% (17135 + 1931.4 + 322.6 ng/L vs 13003.4 ng/L), decreased the concentration of low degree. In the landfill of solid waste, TCs, QNs, MLs three categories of 9 kinds of antibiotics detection rate reached 100%, the detection of SAs and BLs two kinds of antibiotics was 23.4-93.6%; NOR and OFL highest quinolone antibiotics detection concentration reached 1497.5 g/kg and 1250 g/kg, the average detection concentration were 260.1 g/kg and 215.8 g/kg, amoxicillin (AMOX) in all solid waste samples the detection concentration is generally low, the average detection concentration of less than 0.1 g/kg. Compared with the solid waste landfill in the dawn of antibiotics detection concentration of antibiotics TCs, QNs, and MLs three kinds of antibiotics in solid waste Laogang Landfill in detected concentrations were generally lower (P0.05), SAs and BLs in solid waste landfill in two detected concentration of no significant change. In the landfill surface Laogang landfill (0.5-1.5m) of solid waste samples, showing a variation in the depth of the antibiotic concentration, except BLs the other four kinds of antibiotics (TCs, QNs, SAs, MLs) in 1.5 m depth of antibiotic detection concentration was higher than that in the depth of 0.5 m at the concentration of detection (P0.05), and the surface of solid waste landfill landfill is no dawn characteristics of the concentration distribution. This may be due to the mature stage of solid waste in the old port landfill and the downward migration and diffusion of antibiotics in rainwater and leachate. The study selected 7 antibiotic resistance genes (tetQ, tetW, mefA, ermB, sul1, sul2 and mexF) and integron INT1. Test results showed that in the landfill leachate, were detected in 7 ARGs concentration was 2.91 * 10~2 to 4.88 * 10~6 copies/ngDNA, INT1 integron detection concentration was 4.55 * 10~4 to 8.31 * 10~7 copies/ngDNA; in the landfill of solid waste, tetQ, tetM, mefA three ARGs concentration is lower than the standard curve range of quantitative detection the detection rate of ermB, sul1, sul2, and mexF four concentration of 1.85 ARGs * 102 ~ 5.07 * 106 copies/ngDNA integron INT1 detection concentration is 6.90 * 105 ~ 1.86 * 108 copies/ngDNA. Analysis of antibiotic resistance genes and the correlation between the results show that in the landfill leachate, showed a significant correlation between tetracyclines and resistance genes tetQ, ETM-H_2O, MLs and mefA have significant correlation, sulfamethoxazole (SMX), SAs and sul1 and sul2 showed a significant negative correlation, and multiple resistance genes and mexF there was no correlation between the detection of antibiotics; in Laogang surface of solid waste, only RTM and ermB have significant positive correlation, shows a significant negative correlation with multidrug resistance gene mexF; on the surface of solid waste landfill in the dawn of only sul2, and sulfadiazine (SD), sulfamethazine (SMZ) and SAs showed a significant negative correlation. This may be due to the physical and chemical properties of the environmental medium of municipal solid waste landfill (temperature, light, pH, etc.).
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X799.3

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