Research On Determination Of Veterinary Antibiotics In Manure And Their Seasonal Pollution Characteristics

In recent years,China's livestock and poultry breeding industry has developed rapidly and has become an important industry for agricultural and rural e conomic development.In the process of pig breeding,antibiotics are often used to treat diseases.However,after entering animal body,30%~90% of antibiotics are excreted in the form of original drugs with manure or urine.Antibiotic residues are often detected in pig manure.Antibiotics in pig manure can enter into the soil with pland application,thus may endanger human health.In order to understand the level of contamination of antibiotics in pig manure,it is urgent to establish a method suitable for detecting antibiotic residues in pig manure.At present,the method for antibiotic detection are mainly focuse on food,feed and organic fertilizers.There is no suitable method for antibiotic detection in fresh manure.Therefore,how to accurately detect the level of antibiotic residues in pig manure and study the pollution characteristics of antibiotics in pig manure has become a hot issue in the field of environmental science.This paper mainly carries out the following research contents:A solid phase extraction-liquid chromatography-tandem mass spectrometry(SPE-UPLC-MS/MS)method was developed for the determination of 42 veterinary antibiotics in pig manure by optimizing instrumental methods(chromatography and mass spectrometry)and pretreatment methods.The study mainly compared the extraction effects of three different solid phase extraction cartridges(Oasis Waters HLB,Agela Technologies Cleanert PEP and Oasis Waters PRi ME HLB),the various extracts,the ratio of the extract,the extraction conditi ons of the extract and the amount of eluent,etc.The optimal pretreatment methods were determined by comparing recovery rates for extracting 42 antibiotics from pig manure.That is,using 10 m L of methanol: acetonitrile: citrate buffer(1:1:2)as extractant,vortex for 3 min,ultrasound for 10 min,centrifuging at 10000 r/min for 15 min,collecting the supernatant after centrifugation.The pig manure was extracted twice,and the supernatant was diluted to 200 m L.Then the supernatant purified by HLB cartridge and eluted with 6 m L of methanol.purified by HLB cartridge and eluted with 6 m L of methanol.The eluent was blown to near dryness by nitrogen,and reconstitution with 1 m L of 0.1% formic acid: methanol(1:1,V/V)into the sample vial.Then they were stored at-20 oC for the machine detecting.Acquity HSS T3 column was selected as the chromatographic column;0.1% formic acid water and acetonitrile were selected as mobile phases.Under gradient elute and optimal pretreatment,42 kinds of antibiotics were separated within 8 min,and the detection was completed in 10 min.The limit of quantitation and detection limit were low,except for penicillin G,the other substances were used,the limit of quantification was 0.03~5.13 ?g/kg;the detection limit was 0.01~1.54 ?g/kg.The method was verified by detecting antibiotics in the feces of different scale pig farms in Tianjin,and the results showed that they were consistent with the reported concentration levels,indicating that the method is suitable for simultaneous analysis of 42 veterinary antibiotics in pig manure.In order to understand the seasonal pollution characteristics of antibiotics in the manure of different scale pig farms in Tianjin.The pollution characteristics of the four seasons(spring,summer,autumn and winter)were analyzed by using the optimized methods.The monitoring results showed that the seasonal pollution characteristics of antibiotics in pig manure were that: the lowest degree of pollution was detected in summer and the highest pollution was detected in winter.The seasonal pollution level of medium-sized farms was the lowest in autumn and the highest in spring;The seasonal pollution law in large-scale farms is the lowest in autumn and the highest in winter.The overall pollution level is spring and winter> summer and autumn.Among all the monitored samples,tetracycline antibiotics(TCs)accounted for the highest proportion in different scales of pig farms and different seasons.The total concentration of TCs was up to 1260.42 mg/kg,with the highest content for OTC.?-lactams(?-lacs)have the lowest content,and the total concentration can reach 0.26 mg/kg,with highest content for penicillin G(PENG).The other three claaes,sulfonamides(SAs),quinolones(FQs),macrolides(MLs)antibiotics,the total concentrations can reach 17.05?6.32?41.95 mg/kg,respectively;the highest content of monomeric antibiotics are sulfamethazine(SDMD),ofloxacin(OFL)and tilmi cosin(TIL).For different growth stages,sows manure has the lowest level of antibiotcs and piglet manure has the highest level of pollution.In addition,the characteristics of the total amount of antibiotic contamination is large farms(3,000 in stock)> small farms(300 in stock)> medium-sized farms(1200 in stock)in different pig farms scales.