Pressurized liquid extraction of six tetracyclines from agricultural soils

Veterinary antibiotics used in agriculture can be introduced into the environment through land application of animal manure, accumulating in soils and groundwaters and posing a significant risk to human health and animal well-being. As the analysis of tetracyclines in soil is challenging due to their strong interaction with soil minerals and organic carbon, the objective of this study was to develop a reliable and reproducible method for quantitative analysis of chlortetracycline and oxytetracycline, and their respective metabolites in soils. A method based on pressurized liquid extraction (PLE) with in-cell clean-up was developed for the extraction of chlortetracycline and oxytetracycline and four likely metabolites from a set of four soils. Optimized conditions included a cell size of 22?mL, soil loading of 5?g, pH of 8.0, methanol:water ratio of 3:1, 50?°C, and two cycles. Soil extracts were analysed by high-performance liquid chromatography (HPLC) coupled with ion trap mass spectrometry (MS). Recoveries of seven tetracyclines from soil ranged from 41% to 110%. The limits of detection for tetracyclines were 0.08–0.3 µg g^?1 soil, and intra- and inter-day variation ranged from 0.12–0.34%. The proposed PLE method is suitable for quantification of tetracyclines in agricultural soils at typical concentrations expected in contaminated environments.     

基于小流域尺度的目标抗生素排放量估算方法——以梅江流域为例

抗生素的大量使用和排放造成的环境污染和生态风险问题日益突出,抗生素排放量的估算是评价流域内抗生素污染程度的重要指标,但目前抗生素排放量估算方法尚不完善。本研究以梅江流域为例,建立了适合小流域尺度的典型抗生素排放量估算方法,计算了四环素类抗生素(TCs)向不同环境相的排放量并分析了其主要来源。结果表明:2016年梅江流域TCs排放量为8 558.1 kg,不同行政区抗生素排放量差异较大,其中梅江镇受人口密度及养殖密度影响,抗生素排放量最大,高达1 224.4 kg;同时流域内不同抗生素的排放量也有所不同,其大小顺序为强力霉素(DXC)土霉素(OTC)四环素(TC)金霉素(CTC);TCs受排放源、排放途径等因素影响,以进入环境水相及土壤相为主,其中进入水相中的抗生素主要来自人类及生猪粪便,进入土壤相中的抗生素主要来自生猪及三黄鸡粪便。梅江小流域抗生素具有潜在的生态风险,应加强抗生素使用管理。该研究为我国小尺度流域目标抗生素排放量的估算提供了十分有效的方法。     

四环素类抗生素和铜复合污染对猪粪厌氧消化的影响

兽用抗生素和矿物元素添加剂可起到预防动物疾病、促进动物生长、提高饲料转化率等作用,因此被广泛应用于畜禽养殖业。本研究以猪粪中温厌氧消化为研究目标,采用全自动甲烷潜势测试系统,考察了一定浓度的四环素(TC:30 mg·kg~(-1)dry weight,DW)、土霉素(OTC:50 mg·kg~(-1)DW)和金霉素(CTC:15 mg·kg~(-1)DW)对厌氧累积产甲烷量和日产甲烷速率的影响。结果表明,TC、OTC和CTC对猪粪中温厌氧消化累积产甲烷量和日产甲烷速率均有促进作用(累积产甲烷总量提高比例分别为7.9%、0.4%和5.4%)。另外,采用超高效液相色谱-四极杆串联质谱对猪粪厌氧消化前后样品中四环素类抗生素及其代谢产物进行了分析。结果表明,液相中的四环素类抗生素在猪粪厌氧消化过程中得到了明显的去除,去除率达到90%~100%;而固相中只有金霉素和差向异构金霉素有明显的去除效果,去除率分别为41.69%和41.58%。采用Tessier连续提取法对猪粪厌氧消化前后样品中5种形态的铜包括可交换态、碳酸盐结合态、铁锰氧化结合态、有机物结合态、残渣态进行了分析,结果表明,猪粪厌氧消化后,可交换态、碳酸盐结合态和铁锰结合态的铜浓度比厌氧消化前分别降低了1%~9%、0.1%~3%、12%~19%,而有机态和残渣态的铜浓度却在厌氧消化后分别增加了15%~35%、1%~2%。厌氧消化后,70%~80%的铜都是以有机铜的形态存在。铜逐渐从不稳定态转化为相对稳定的有机态和残渣态铜,因此,厌氧消化过程使铜从可生物利用态转变为不可生物利用态,趋于稳定化。     

城市污泥中温厌氧消化过程中厌氧耐药菌的分布与去除研究

城市污泥中含有大量的耐药菌,具有向环境传播耐药菌与抗性基因的潜在危险,但目前缺乏对城市污泥厌氧消化过程中耐药菌的研究.为此对某城市污水处理厂卵形消化池污泥进行了为期1 a的调查研究,考察了中温厌氧消化过程中四环素类与β-内酰胺类抗生素厌氧耐药菌的污染特征与去除效果,并分析了耐药菌的季节性变化规律.结果表明,厌氧进泥中四环素类耐药菌浓度与耐药率均低于β-内酰胺类.中温厌氧消化过程可以去除1.48~1.64 log单位的耐药菌(P<0.05),但经过厌氧消化后氨苄西林与头孢噻吩耐药率有显著增长(分别增加了12.0%与14.3%,P<0.05).厌氧进泥中耐药菌分布有明显的季节特征,除金霉素耐药菌外,其它耐药菌的浓度均为寒冷季显著高于温暖季(P<0.05).     

牛粪堆肥系统环境因子对抗性基因的影响

本研究利用实时定量PCR技术检测牛粪60 d好氧堆肥过程中典型四环素类抗性基因(tet Q、tet W、tet M、tet G、tet A和tet X)和大环内酯类抗性基因(erm35、erm36、erm B、erm F、erm T和erm X)的数量变化规律,系统研究了温度、含水率、挥发性有机质(VS)含量、碳氮比(C/N)、pH值、氧化还原电位(ORP)等堆肥系统环境因子和细菌数量(TCB)对这两类典型抗生素抗性基因的影响.结果表明,堆肥升温阶段牛粪中tet Q、tet W、tet G和tet X数量分别增加1. 7、0. 3、84. 8和4. 5倍,而tet M丰度降低了83. 1%;堆肥腐熟阶段牛粪中tet G和tet X数量分别增加23. 8和11. 5倍,而tet W、tet Q和tet M数量均降低90%.整个牛粪堆肥过程中erm35、erm36、erm B、erm F、erm T和erm X丰度分别增加2. 1、430. 4、0. 6、11. 5、2. 1和49. 1倍. RDA分析表明,牛粪堆肥过程中对四环素与大环内酯类抗性基因数量影响较大的3个环境因子分别为:ORP(54. 8%)、温度(34%)和VS(11. 3%),其中ORP与erm X和erm B、温度与tet Q和erm F以及VS与tet W数量均呈明显地正相关.     

土壤环境中四环素类抗生素残留及抗性基因污染的研究进展(Tetracycline Residues and Tetracycline Resistance Gene Pollution in Soil: A Review)

近年来,致病菌耐药性的增加和扩散已成为全世界关注的热点问题,而人类医疗和畜禽养殖业抗生素的滥用正不断加剧这一问题.众多研究表明土壤环境作为一个巨大的抗性基因储存库,在抗性微生物和抗性基因的传播中起重要的作用,具有潜在的生态与健康风险.在总结国内外最新研究基础上,对土壤环境中典型抗生素-四环素类抗生素的主要污染源以及其在土壤中的基本环境行为等进行了分析,并探讨了土壤中四环素类抗性基因的来源、迁移和扩散及分子检测手段等问题.我国作为抗生素的生产和消费大国,抗生素污染问题较其他国家更为严重,而国内相关研究才刚刚起步,迫切需要开展有关环境中抗生素和抗生素抗性基因污染的系统研究.     

Development of an efficient extraction method for oxytetracycline in animal manure for high performance liquid chromatography analysis

Oxytetracycline (2-(amino-hydroxy-methylidene)-4-dimethylamino-5,6,10,11,12a-pentahydroxy-6-methyl-4,4a,5,5a-tetrahydrotetracene-1,3,12-trione) is a major member of the tetracycline antibiotics family of which are widely administered to animals in concentrated animal feeding operations for purposes of therapeutical treatment and health protection. With the disposal of animal manure as fertilizer into agricultural land, tetracyclines enter the environment. However, tetracyclines chelate with multivalent cations and proteins, resulting in low extraction efficiencies from animal manure for tetracycline residue analysis. In this study an efficient extraction method for oxytetracycline from steer manure using methanol/water solution amended with chelating organic acid was developed for the analysis of high performance liquid chromatography. The effect of species and amount of amendment acids, shaking time, methanol/water ratio, manure weight, and repeated times of extraction was investigated. It was optimized to amend 2.5 g citric acid and 1.1 g oxalic acid with 10.0 g manure sample in a 50-ml centrifuge tube and extract with 15 ml methanol/water (9:1 in volume) by vigorously shaking for 30 min in a reciprocating shaker. After centrifugation at 11,000 rpm, supernatant is collected. Sample was extracted for a total of 3 times. The developed extraction method was further applied to extract oxytetracycline from fresh and aged cow manure, swine and poultry manure, and soil. Satisfactory recoveries ranging from (84.1 ± 2.4) % to (102.0 ± 3.1) % were obtained, demonstrating that the optimized extraction method is robust for oxytetracycline from different manure sample matrixes.     

微波-Fenton对沼液中抗生素和激素的高级氧化

采用微波强化Fenton氧化处理系统,研究H2O2浓度、Fe2+浓度、初始pH、微波辐射时间和微波辐射功率对沼液中喹乙醇、土霉素、四环素及金霉素降解效果的影响.结果发现,采用微波强化Fenton氧化降解沼液中抗生素与激素的最优条件是:H2O2浓度为40 mg/L、Fe2+浓度为12 mg/L、初始pH为4、微波辐射时间为2 min、微波辐射功率为中火(445W),沼液中喹乙醇、土霉素、四环素、金霉素和COD的去除率分别达到67％、93％、91％、88％和46％.在水浴条件下,与单独微波辐射和单独Fenton相比,微波强化Fenton氧化有明显的优越性.     

Study of spatial and temporal distribution of antimicrobial in water and sediments from caging fish farms by on-line SPE-LC-MS/MS

An on-line solid phase extraction liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method for the determination of 12 antimicrobials in sediment and surface water was developed and validated. Furthermore, the spatial and temporal antimicrobials distributions in the sediment and in the water of four fish farms located in the hydroelectric dam of Ilha Solteira Reservoir in Brazil were investigated over four seasons in three sampling sites: at the fish cages, 100 and 1,000 m downstream far from the cages. The method was performed using an Agilent Zorbax 80 SB-C8 column (9.4 × 15 mm, 5 µm) as the loading column, and the Agilent Zorbax Eclipse Plus C18 column (3.0 × 100 mm, 3.5 µm) as a separation column within a run time of 13 min. The limits of quantification were less than 9 ng·L^?1 for the antibiotics in water and 16 µg·kg^?1 in sediment; the recovery ranged from 80 to 119%, with a variation coefficient less than 11%, and the repeatability was lower than 15%. Oxytetracycline was found in the water in all sample seasons. However, florfenicol was identified in April and October 2013 and January 2014, and tetracycline was present in July 2013. Regarding the sediment, oxytetracycline and tetracycline were found in all sampling periods, but chlortetracycline was only identified in January 2014. The spatial distribution of antimicrobials showed that the main pollution source came from the fish farms. This study demonstrated that the proposed method is reliable for the monitoring of antimicrobials in water and sediments and it showed contamination in both matrices from Ilha Solteira Reservoir.     

Fate of tetracyclines in swine manure of three selected swine farms in China

Veterinary antibiotics can enter the environment due to the common practice of land application of manure from treated animals. The environmental fate of tetracyclines in swine manure after composting and field application remains largely unknown. This study analyzed the concentrations of tetracyclines in manure, manure-based compost and compost amended soil in selected swine farms from Beijing, Jiaxing and Putian, China to determine the dilution effects of antibiotics when released into the soil environment. The results demonstrate that residues of antibiotics were detected in all samples and chlortetracycline as well as its degradation products should be regarded critically concerning their potential ecotoxicity. Application of manure-based compost to soil could reduce the possible risk posed by antibiotic contamination, but the trigger value of 100 μupg/kg was still exceeded in soil samples (776.1 μupg/kg dw) from Putian City after application of compost. Field studies such as the present one can help to improve the routine administration of antibiotic-containing composted manure.