| 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).
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