| Antibiotics are one of the most important pharmaceuticals used extensively for preventing and/or treating bacterial infections. Large amounts of antibiotics and their metabolites are continuously released into environmental compartments such as surface water, ground water and soil, due to overuse and abuse in these substances. Although the exposed concentration of these compounds in the environment is normally low, they could create a potential risk for aqueous and terrestrial organisms due to their persistence, bio-reactivity, non-biodegradability, etc. And worse, the evolution and dissemination of antibiotic resistance in bacteria developed rapidly due to the persistent selective pressure resulted from these low concentrations of antibiotics, which posed unpredictable threats to public health. In recent year, these new micropollutants have attracted a lot of attention owing to the rapid development and applications of environmental analytical techniques.The existing studies indicated that sewage effluent is the principal entry source of antibiotics and antibiotic resistant bacteria into the environment. Since most of the sewage treatment plants can only partially remove these persistent antibiotics from the wastewater, in some cases with no removal, many studies reported that different ranges of concentrations of antibiotics and antibiotic resistant bacteria were detected in sewage effluents and sludge. It is very important and urgent to systematically investigate the fate, transport and distribution of these micropollutants in the wastewater treatment systems.In this work, the analytical method which can detect fifteen trace pharmaceuticals belonging to multiple classes using accelerated solvent extraction, solid-phase extraction (SPE) and liquid chromatography tandem mass spectrometry (ASE-SPE-LC-MS/MS) was established. Concentrations of these pharmaceutical compounds in both wastewater and sludge collected along each treatment unit were measured in East Lansing wastewater treatment plant (WWTP) applying conventional activated sludge treatment process in Michigan in United States. Detailed mass balance calculations were performed to identify the partitioning behaviors and removal mechanisms of these micropollutants. In the meantime, culture-based approach and real-time quantitative polymerase chain reaction (qPCR) were used to detect the concentrations of antibiotic resistant bacteria and antibiotic resistance genes, respectively, in wastewater and sludge from different treatment units. Finally, the selective pressure resulted from the sulfonamides and tetracyclines on the evolution of antibiotic resistant bacteria and antibiotic resistance genes, as well as their relationships were studied. The results are as follows:(1) Determination of low concentrations of pharmaceuticals in sludge using ASE-SPE-LC-MS/MSIn this chapter, the established method for fifteen different pharmaceuticals in sludge was of satisfactory extraction efficiency, sensitivity and reproducibility, and its limits of detection (LOD) were between the range of 0.6μg/kg dw for sulfamethazine (SMZ) and 146μg/kg dw for tetracycline (TC). Moreover, the matrix effects of impurities in sludge caused a minimal impact on the effective extraction and analysis of pharmaceuticals.During the optimization of operating parameters, the best extraction efficiency of fifteen pharmaceuticals is sludge was achieved using acetonitrile/water mixture (v/v=7:3), and their recoveries were in the range of 52～96.4%. Decreasing pH in extraction solvent cannot significantly improve the extraction efficiencies of pharmaceuticals. Furthermore, the ASE parameters of extraction pressure, extraction temperature and extraction time had varying impacts on the extraction of pharmaceuticals. In the end, the optimal operative condition was determined and selected with an extraction pressure of 10.0 MPa, an extraction temperature of 100 C and three extraction cycles with 15 min static time in each cycle.(2) Distribution behaviors and removal mechanisms of pharmaceuticals in activated sludge treatment processBasing on the results obtained, it was found that demeclocycline (DMC), sulfamerazine (SMR), erythromycin (ERY) and tylosin (TYL) were not detected in all of the wastewater and sludge samples, while other eleven pharmaceuticals of chlortetracycline (CTC), doxycycline (DOC), oxytetracycline (OTC), tetracycline (TC), sulfadiazine (SDZ), sulfamethazine (SMZ), sulfamethoxazole (SMX), lincomycin (LCM), carbamazepine (CBZ), acetaminophen (AMP) and caffeine (CAF) were detected above their LOD at least once. The concentrations of CTC in raw influent and primary sludge were 178 ng/L and 61μg/kg dw, respectively, and was not detected in final effluent and dewatered sludge. Except CTC, the concentrations of other ten pharmaceuticals in raw influent varied significantly, in the range of 26 ng/L for SMZ and 61683 ng/L for AMP. After the biological treatment, the average concentrations of these pharmaceuticals in final effluent were between the range of nd (not detected) for SMZ and 370 ng/L for DOC. In the meantime, the residual concentrations of these compounds in dewatered sludge were in the range of 3.5μg/kg dw for SMZ and 568μg/kg dw for DOC. According to the analysis of mass balance calculation, biodegradation was found to be the primary mechanism for the removal of these pharmaceuticals, whereas the impact of sludge sorption was minor, as low as 7.1%. However, it was found that DOC, OTC, SDZ, SMX and LCM can only be partially removed during the biological wastewater treatment process, and the residual percentages were 50%,61%,73%,11% and 60%, respectively.It is noteworthy that a net increase for CBZ in final effluent compared with that in raw influent after biological treatment was observed. This is probably due to the presence of metabolites of CBZ in raw influent, which can be retransformed to CBZ during wastewater treatment process, resulting in the increase of CBZ concentration in the wastewater.(3) Distribution and removal of tetracycline and sulfonamide antibiotic resistance genes and antibiotic resistant bacteria in wastewater treatment processAccording to the results obtained, activated sludge wastewater treatment process can effectively reduce the amounts of TC and SMX antibiotic resistant bacteria in wastewater, and their concentrations in final effluent were 1.05x101CFU/mL and 3.09x103 CFU/mL, respectively. In dewatered sludge, the concentrations were 7.08x106 CFU/g and 3.09x108 CFU/g, respectively. Meanwhile, the concentrations of antibiotic resistance genes of tetO, tetW and sull in final effluent were 1.05x106 copies/mL,9.12x105 copies/mL and 5.13x105 copies/mL, respectively, while in dewatered sludge, their concentrations were 1.78x109 copies/g,5.62x108 copies/g and 1.00x108copies/g.It can be seen that biological treatment step was basically responsible for the removal of antibiotic resistance genes in wastewater, whereas chlorination was of minimal impact for their removal. This is because that the inactivation of bacteria with chlorination disinfectants was primarily achieved by inhibition of bacterial activity rather than destruction of their DNA structures. Additionally, bacteria encoded with tetO, tetW and sull genes are mainly exiting in Gram-positive bacterial strains, which can resist inactivation caused by chlorines via synthesis of unique proteins as a response to stress or formation of bacterial aggregates, leading to the poor removal efficiencies of these antibiotic resistance genes.Basing on the fitting curve analysis between antibiotic concentrations and corresponding resistance genes and bacteria, no significant linear correlation could be found between the abundance of tet genes (sum of tetO and tetW) and concentration of tetracyclines. This is probably because only two types of tet genes were quantified and analyzed, and other tet genes did present in the wastewater that were not accounted for. By contrast, a significant correlation was observed between the abundance of sull genes and total concentration of sulfonamides, suggesting that the presence of sulfonamide antibiotic in wastewater was responsible for the development of sull genes. Meanwhile, significant linear correlations were found between total numbers of TC and SMX resistant bacteria and concentrations of tetracycline and sulfonamide antibiotics in wastewater, respectively. However, no significant relationship was observed between abundance of antibiotic resistance genes and bacteria and concentrations of corresponding antibiotics in sludge. |
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