【作者】 巫杨；

【导师】 陈东辉； Rai Kookana；

【作者基本信息】 东华大学 ， 环境科学与工程， 2011， 博士

【摘要】 自1928年青霉素的发现到现在短短的几十年内,抗生素药物的生产和使用量在全球范围内一直维持在持续增长的势头中。抗生素在给人类和牲畜疾病的治疗带来积极作用的同时,也引发了不可忽视的负面效应。磺胺甲恶唑(SMX)、磺胺吡啶(SPY)是人类和牲畜常用的一类磺胺类抗生素药物,甲氧苄氨嘧啶(TRM)作为磺胺类药物增效剂通常被与磺胺类药物结合使用,特别是与SMX常以1：5的质量比联合使用。同其他抗生素药物一样,这些药物残留通过各种途径排放进入到水体环境及土壤环境中,给环境安全造成威胁。当SMX、SPY及TRM排放进入水体后,光降解和水解是其在水体中的重要环境行为。当前对于这三种药物在水体环境中的光降解报道还较少,更缺乏在不同光源条件下的相关行为比较,因此全面比较这三种药物在不同环境水体条件下的光降解行为十分有必要。另一方面土壤也是这些药物在环境中的迁移转化的重要承载体,而目前对于这些药物在土壤中的累积及降解动力学报道仍然较少,其原因一方面可能是由于无标准统一的检测方法,另一方面是对这些药物引起污染的重视程度不够。因此当前建立起有效灵敏的在土壤等复杂基质中的检测方法,进一步了解其在土壤中发生的微生物降解行为,探究其是否对土壤微生物造成毒性的研究近在眉睫。论文研究中,首先确定了三种药物在水体及土壤环境中的检测方法。在水相中使用Agilent 1100 HPLC进行检测。结合前人文献报道及预试验,选用了乙腈(A)和含有0.3%的乙酸水溶液(B)做为流动相,采用梯度洗脱。通过对梯度洗脱条件的反复比较,确定了试验中所采用的洗脱条件,该梯度能很好地实现三种药物的分离,且峰形尖锐、对称。HPLC具体测定的色谱条件为：紫外检测器下的检测波长280 nm,C18柱(4.6 mm×250 mm,5μm,10 mm, Alltech)。柱温为28℃,流动相起始比例为A：B=20：80,总的运行时间为19分钟,流速1mL min-1,注射体积20μL。在土壤复杂基质中的检测,由于SPE-HPLC无法较好地去除基质干扰,检测灵敏度不高,因而在土壤降解实验中采用了SPE-LC-ESI-MS/MS检测方法。土壤降解样品加入10 ml的混合溶剂(methanol/EDTA-Mcllvaine 1:1,pH=4).通过超声,离心,洗脱等一系列步骤后进样检测。HPLC色谱分离使用Thermo Scientific Surveyor分析仪器。色谱分离柱选用Hypersil ODS-2μm,150×2.6 mm(Thermo Scientific).流动相A为乙腈,流动相B为0.1%甲酸溶于10 mM的乙酸铵溶液(1 L)。采用的梯度为,A起始于20%,持续3分钟,然后在2分钟后增加到50%,再在0.1分钟内增加到90%,持续2.9分钟,然后在0.1分钟内降低到20%,接着3.9分钟用来平衡。整个流动速率为0.2 ml min-1.土壤中回收率检测作三次平行,在三种实验所用的单一土壤(5 g)和添加牛粪的土壤中(5g+0.2 g)注入1 mg kg-1或200μg kg-1的SMX.TRM,回收率分别为71±5%～80±8%(SMX,干重),60±9%～72±7%(TRM,干重)。总之,QA/QC验证结果表明了上述在水相及土壤中的检测方法灵敏度较高,检测方法准确可行。在确定了实验中所用的检测方法后,接着展开研究了三种药物SMX,SPY及TRM在不同水体环境介质中及土壤中的环境行为。在光降解实验中,考察了SMX,SPY及TRM在不同水体条件下的光降解行为,比较了三种药物在高压汞灯、自然光及紫外灯下的降解差异。研究结果表明：光源、水体pH值、温度、光照时间及各种不同的水体介质都会影响药物的光降解效率。SMX和SPY比较容易发生光降解,但在某些水体条件下降解率较高,而在有些条件下则降解率相对较低,这与药物在不同pH条件下的离子形态影响到对光源波长的吸收可能有一定关系。在降解过程中二者均出现了明显的降解产物,同时实验过程中对控制样品的监测发现SMX和SPY一般情况下不容易发生水解。对于TRM,其在各种不同光降解条件下表现十分稳定,甚至在类Fenton反应这类高级氧化过程中(女口UV+Fe2+, UV+Fe3+)也基本未发生降解,在8小时内的降解率低于2%,呈现出持久稳定状态。然而在自然光照下,对包裹的试验控制样品中的浓度及对应温度、光强监测对比发现,在pH=4和pH=7的TRM溶液在白天吸收热量,夜间放热的过程中促进了其发生热水解的能力,并出现了明显的水解产物,水解发生率大于10%。药物在土壤中的吸附解吸行为也是重要的环境行为之一。为考察这些药物在各种不同性质土壤中的流动性和累积性,参照OECD(?)旨导方针选择了5种具有不同理化性质的土壤进行了试验研究。同时考察了土壤溶液pH值的变化对吸附造成的影响进行了选择性实验。实验结果发现,SMX和SPY在吸附过程中受土壤有机碳含量影响较大,通常含量越高的土壤,吸附能力随之提高,这和前人对其他磺胺类药物吸附实验的报道相一致。总体来说,SMX和SPY在土壤中的吸附系数较小,因而在土壤中具有高度的流动性。而对于TRM,在吸附过程中受到有机碳含量的影响不明显,而与土壤的阳离子交换容量关联程度较高。相比SMX和SPY,在各种类型的土壤中都呈现出较高的吸附系数,呈现出较强的吸附性能。解吸实验计算得出的迟滞系数表明,三种药物在各种土壤中都呈现出不同程度的迟滞行为,这为进一步评价药物在土壤中的流动性及累积性提供了有利的参考数据。在土壤溶液pH值调节对吸附影响的实验发现,SMX随着土壤溶液pH值的提高,吸附能力呈显著下降超势,而SPY随着pH值的提高,吸附能力未呈现出明显的波动,而TRM则呈现出两段变化,在前一阶段,吸附能力随着pH的提高而呈现上升趋势,但在后一阶段,吸附能力随着pH的提高则呈现下降趋势。这充分表明土壤溶液的pH值在药物吸附过程起到相当重要的作用。吸附实验结果发现,磺胺类药物(SMX和SPY)在土壤中呈现低吸附能力,而TRM呈现高吸附能力,通常低吸附的化合物在土壤中易发生生物降解,高吸附的化合物则不易发生生物降解。为考察它们实际的生物降解能力,接着进行了生物降解实验。实验中主要以SMX和TRM这两种常结合在一起使用的药物作为研究主体,考察了在三种具有不同理化性质的土壤中,有无添加牛粪修复的情况下,在好氧及厌氧培养条件下的降解行为。实验结果表明,在好氧培养条件下,在具有生物活性的土壤中,在前20天内,>50%的SMX在粘壤土和壤质砂土中消失,在壤土中则观察到有>80%的损失出现。而TRM则在三种土壤中表现出较强的持久性。在100天后,残留在具有生物活性的壤质砂土、粘壤土及壤土中的含量比分别61±6%,31±7%,和26±6%。土壤中牛粪的添加(37.8％C,2.3％N,pH 8.19,添加比例为4%)仅仅轻微地影响到两种化合物的起始降解速率,即在前20天内的降解速率要快于未添加牛粪的土壤。但20天过后,降解速率已无差别显现,实验结束后,在牛粪修复和未修复的土壤中未发现降解效率的显著差别。在厌氧培养条件下,两种化合物的降解同好氧培养条件下相比迅速的多。在20天内,SMX几乎损失殆尽,而TRM也出现了85%以上的损失。同时在厌氧环境体系中,实时监测了体系的pH,N03-,S042-的变化,进一步验证了厌氧环境体系的存在。由于在降解实验中发现了SMX和TRM在灭菌控制样品中的损失出现,推测可能有部分原因是因为随着时间的推移,微生物活性逐渐复苏造成。因而为进一步验证灭菌及未灭菌体系的微生物活性,同时考察土壤中药物单体或混合物,不同浓度下的添加对微生物活性是否造成影响,在这部分研究中,采用基质诱导呼吸法考察了药物在土壤中对微生物呼吸的影响。实验结果表明,灭菌后的样品经历长时间实验过程中,均呈现出不同程度的复苏迹象,因而这也是药物在灭菌样品中浓度损失的原因之一。对于微生物呼吸的影响比较发现,同控制土壤样品对比,药物对土壤微生物呼吸的短暂抑制作用出现在前2天,随后快速恢复。100天后,在添加不同浓度,单体或混合物的抗生素土壤中,总的矿化程度无较大差异。这表明无论是在二种不同浓度下,添加单体或混合药物的土壤中(10mg kg-1 SMX+2 mg kg-1 TRM或100 mg kg-1 SMX+20 mg kg-1 TRM),在100天后对微生物呼吸造成的影响较小。更多还原

【Abstract】 Since the discovery of penicillin in 1928, just in a few decades, the production and use of pharmaceutical antibiotics has been increasing over the world. The introduction of antibiotics posed on positive effects for curing human and livestock’s deseases, but also led to negative effects which could not be ignored. Sulfamethoxazole (SMX), sulfapyridine (SPY) are commonly used in human and livestocks which belonged to a class of sulfonomides. As a synergist, trimethoprim (TRM) is often used in combination with sulfa-drugs, expecially with sulfamethoxazole, in a ratio of 1:5. Like other antibiotics, these drug residues were emitted into the aquatic environment and soil environment by various pathways, posing part of threat to the environmental safety as well. When SMX, SPY and TRM are discharged into aquatic environment, photodegradation and hydrolysis become important environmental behaviors for them. However, there are little reports on these drugs currently, especially lack of comparisons under different conditions. So it is necessary to fully understand the photodegradation behaviors of these antibiotics in aquatic environment. On the other hand, soil is also an important carrier, but there is still little report on the accumulation and degradation kinetics in soil. The reasons might be related to two aspects, one is no standard detection method has been established until now, and the other aspect is the poor emphasis on the drug-induced pollution. So it is imminent to make the establishment of detection method with high efficacy and sensitivity in soil and other complex matrix, to further understand biodegradation behaviors and explore whether they are toxic to soil microorganisms.In this project, determination of three pharmaceuticals in aquatic and soil environment was first identified. Agilent 1100 HPLC was used in aquatic phase. Based on previous literatures and preliminary tests, acetonitrile (A) and 0.3% acetic acid in ultra-pure water (B) are used for mobile phase and gradient elution was adopted. Comparing with different elution conditions, optimized condition was identified. The gradient was well to achieve the separation of three compounds, and the peaks were sharp and symmetry. Analysis of samples was undertaken by HPLC (Agilent Technologies, series 1100) with C-18 column (4.6 mm×250 mm,5μm,10 mm, Alltech). A UV detection wavelength of 280 nm was used and the oven temperature was set at 28℃. The initial ratio of mobile phase is A:B=20:80. The run time was 19 min with a flow rate of 1mL min-1 and an injection volume of 20μL. In the complex matrix like soil, it could not well remove matrix interferences,and with low sensitivity via using SPE-Agilent 1100 HPLC, so SPE-LC-ESI-MS/MS method was developed to use in soil degradation experiment. An aliquot of 10mL of the extractant (methanol/EDTA-Mcllvaine 1:1, pH=4) was added to each degradation samples. Samples were ultrasonicated, centrifuged and eluted before LC-ESI-MS/MS analysis. A Thermo Scientific Surveyor HPLC system was used for liquid chromatography with a Thermo Hypersil ODS column (150×2.6 mm,2μm) used as a stationary phase. Gradient elution was achieved with acetonitrile (A) and formic acid 0.1% in 10 mM ammonium acetate (B).The gradient program began at 20% A for 3 min, increased to 50% A in 2 min, then increase to 90% A in 0.1 min, hold for 2.9min, then decrease to 20% A in 0.1 min followed by 3.9 min of equilibration time. The flow rate was 0.2 mL min-1. The recovery for spiked concentration of at 1 mg kg-1 SMX or 200μg kg-1 TRM and in three experimental soils with and without manure amendment (dry weight) was ranged from 71±5% with manure to 80±8% without manure for SMX and 60±9% with manure to 72±7% without manure for TRM.QA/OC validation demonstrates that the analysis methods mentioned above in aquatic and soil phase are highly sensitive, accurate and feasible.Soon after detection methods were identified, environmental behavior of three drugs-SMX, SPY and TRM in different aquatic and soil environment was studied. In the photodegradation experiments, photogegradation of SMX, SPY and TRM under different conditions was investigated and degradation differences of the three drugs under high-pressure mercury lamp, natural light and UV light with different water matrix was compared. The results show that:light source, pH of aquatic phase, temperature, exposure time and various water media, are important factors for photodegradation. SPY and SMX are more prone to be degraded under photodegradation. The degradation rates were higher under some aquatic conditions, while the degradation rates were relatively lower under other conditions. This could be related with the speciation of phamarceuticals under different pH conditions, which might affect the absorption of light wavelength. Significant degradation products were observed. Meantime, hydrolysis did not easily occur under normal conditions by control samples tests.For TRM, it’s very stable under different light conditions, even low response to the advanced oxidation process like simulated Fenton reaction (such as UV+Fe2+, UV+Fe3+). Degradation rates of TRM were less than 2% within 8 hours, showing a persistent stable state. However, monitoring of concentration changes of control samples with responding temperature and light intentisity found that, thermal hydrolysis occurred during the heat release from day to night in pH 4 and pH 7 solutions. Hydrolysis rates were greater than 10%.Sorption-desorption behavior is also an important environmental behavior of these drugs. To investigate mobility and accumulation in variety of soil properties, five types of soils were selected and studied according to the reference of OECD 106. Meantime, selected soils were conducted to investigate the pH effect on sorption. The results that sorption of SMX and SPY are highly dependent on the organic carbon content. The more content in soil, the higher sorption potential displayed. This is consistent with the previous reports on other sulfa-drugs. Overall, sorption coefficient of SMX and SPY are low in soil, thus they have high mobility in soil. With regard to TRM, it’s not obviously affected by organic carbon content, but highly related with CEC of soil.TRM is readily sorbed onto different types of soils compared with SMX and SPY. The high sorption potential of TRM is displayed. Hysteresis index calculated by the results of desorption experiment indicated that hysteresis was present in soils for these compounds to some extent and it is a helpful reference for further accessing the mobility and accumulation. Selective pH adjustment on sorption found the extent of sulfamethoxazole sorption on selected soils was highest at low pH and decreased with increasing pH. An increase in pH was found to have no significant affect on the sorption of sulfapyradine, while sorption was found to have increased initially and decreased beyond a certain pH value for trimethoprim with changes of two phases. Therefore, pH in suspended soil solution plays an important role on sorption.Sorption exprements showed that low sorption of SMX and SPY in soil, while high sorption for TRM. In general, biodegradation was easy to occur for compounds with low sorption potential and hard for compounds with high sorption potential. To investigate the real biodegradability of them, biodegradation experiment was conducted next. SMX and TRM were mainly studied, which commonly used in combination with each other.This evaluation was undertaken in three soils of varying characteristics and included amendment of the soil with manure. The inhibition of microbial respiration of these antibiotics in soils was also investigated using substrate-induced respiration. Under aerobic conditions, sulfamethoxazole (SMX) dissipated rapidly mainly through microbial degradation. Within first 20 days in biologically active soils,> 50% of the SMX was lost from the clay loam and loamy sand soils, and> 80% loss was noted in the loam soil. In contrast, trimethoprim (TRM) was more persistent under aerobic conditions. After 100 days, the TRM residue remaining in biologically active aerobic soil was 61±6%,31±7%, and 26±6% in loamy sand, loam, and clay loam soils, respectively. Addition of manure to soil (37.8% C,2.3%N, pH 8.19) at a rate of 4% (w w-1) to soil only slightly affected the initial dissipation rate of the two compounds (faster rate in manure amended soils). At the end of experiment there was no significant difference between amended and unamended soils. Anaerobic dissipation of both compounds was more rapid than that under aerobic conditions, with nearly total dissipation of SMX, and over 85% dissipation of TRM within 20 days. At the same time, real-time monitoring of the system pH, NO3-, SO42-’changes was established, to further validate the existence of an anaerobic system.As it was found that the concentration loss of SMX and TRM in sterile control samples in the degradation experiment, suggesting that the recovery of microbial activity might occur with time. To further investigate the variation of microbial activity in non-sterile and sterile system, and also investigate the effect of on soil in the presence of antibiotics as a mixture and individually, substrate induced respiration via measuring the mineralisation of 14C labelled glucose was introduced to answer these questions. The results indicate that in longer term experiments, contribution of low level of biological activity may occur, and this is one reason contributing to the loss in sterile samples. From the comparison on microbial respiration, it showed obvious suppress respiration of these antibiotics occurred in the first 2 days compared to control soils, but a quick recovery appeared later. There was no significant concentration-dependent inhibition on the total mineralization of soils following spiking with sulfamethoxazole or sulfamethoxazole combined with trimethoprim after 100 days, indicating the less influence on microbial respiration no matter two different concentrations, single compound or mixture added in soils (10 mg kg-1 SMX+2 mg kg-1 TRM or 100 mg kg-1 SMX+20 mg kg-1 TRM).更多还原