Study On The Contamination Of Tetracycline Antibiotics In Livestock Manure-Soil-Vegetable System

In recent years,the residual of new organic pollutant antibiotics in the environment and the risk to the ecological environment have become the focus of research at home and abroad.China is the largest producer and consumer of antibiotics,and a large amount of antibiotics are used in animal husbandry.Due to antibiotics could not completely absorbed or metabolized by animals,most of them would excreted as the maternal form through feces and urine.Tetracyclines?TCs?are one of the most widely used antibiotic variaty.A large number of studies have reported the detection of tetracycline residues in livestock and poultry manure and farmland soil.The direct application of livestock and poultry manure into farmland as fertilizer is one of the main way for antibiotics enter the soil environment.On the one hand,the residual of tetracyclines in soil disturb the soil microbial community,leading to the spread of resistance genes,on the other hand,they will be uptake by vegetable crops and remain in agricultural products,thereby threatening human health and ecological security.In this paper,the investigation combined laboratory analysis was conducted to objectively evaluate the environmental and ecological risk of three tetracycline antibiotics?TCs?,including oxytetracycline?OTC?,tetracycline?TC?and chlorotetracycline?CTC?of livestock and poultry manure in pig and chicken farms and the soil in vegetable bases in the west of Chongqing.Pot culture experiment?root bag method?was conducted to study the effect of exogenous addition of different concentration gradients OTC,TC and CTC on the growth of lettuce.The law of uptake,accumulation and tranportation of OTC,TC and CTC by lettuce,and soil enzyme activity and microorganisims in the rhizosphere and non-rhizosphere response to TCs,and the degradation characteristics of TCs in soil were researched.Field experiments were used to study the effects of different amounts,types and treatments of livestock and poultry manure on vegetable yield and quality,as well as soil TCs residues,soil enzyme activity,soil TCs resistant bacteria and resistance genes abundance.With a view to accumulate data for the study of environmental and ecological toxicology of antibiotics,and provide a theoretical basis for rational use of livestock and poultry manure and safe production of vegetables.The main results are as follows:1.The investigation combined with the analysis were conducted to evaluate the environmental ecological risks of three agricultural tetracycline antibiotics?oxytetracycline,tetracycline,and chlortetracycline?in livestock manure of pig and chicken farms and soil of vegetable base in the west of Chongqing.The average contents of OTC,TC,CTC,and TCs??TCs?of pig and chicken manure were 3.39,4.82,5.92,15.95 mg·kg^-1 and 1.10,1.35,4.22,5.16 mg·kg^-1,respectively.The TCs ecological risk in chicken manure was lower than that in pig manure,and the risk quotient was in order of CTC>OTC>TC.The average contents of OTC,TC,CTC,and?TCs in the soil of the vegetable bases and surrounding areas in Chongqing were 18.92,39.10,21.80 and 79.81?g·kg^-1,respectively.The TCs content was in order of vegetable field near the pig and chicken farm>pollution-free vegetable base>common vegetable field.The content of?TCs in soil was in order of leaves>melons>solanoids>legumes.The HQ values of the three TCs were in order of CTC>OTC>TC.Compared with other regions of China,the content of TCs in the west of Chongqing was at a medium level.The ecological risk was at a medium and low risk level.2.Pot experiments in greenhouse soil showed that TCs could inhibit chlorophyll content and Photosynthesis of plants at high concentration levels,and then inhibit plant growth.At the same time,TCs stress could stimulate the increase of antioxidant enzyme activity of plants.Biomass and Pn decreased with the increase of TCs applied levels.SOD,POD and CAT increased with the increase of TCs applied levels.The antioxidant enzyme activity of roots was more sensitive than that of shoots.Different variety of TCs have different toxicity effects at different concentration levels.Lettuce has the ability to intake TCs,and the amount of TCs uptake increased with the increase of TCs applied levels in soil.TCs content in root>in shoot,the bioaccumulation coefficient of TCs in shoots of lettuce is between 0.0029 and 0.0161,and 0.0149-0.0552 in root.The enrichment coefficient of TCs to lettuce showed a decreasing trend with the increase of soil TCs applied levels.The translocation factor of TCs to lettuc ranged from 6.1%to 30.5%.The translocation factor decreased significantly with the increase of soil TCs concentration.The transport coefficient of different kinds of TCs varied at the concentration level.TCs degraded naturally in potted soils.The degradation rate of OTC in different soils was significantly lower than that of TC and CTC,in control soils>rhizosphere soils>rhizosphere soils.The types and concentrations of TCs and lettuce roots have a common effect on soil enzyme activity and soil microbial abundance.High concentration of TCs(150-1350 mg·kg^-1)significantly inhibited soil urease activity,reduced the number of soil bacteria and fungi,catalase activity and the number of culturable bacteria and actinomycetes in rhizosphere soil were significantly higher than those in non-rhizosphere soil and control soil.3.A fixed field experiment was carried out to study the influence of different amounts of rotted and fresh chicken manure(low chicken manure:300 kg·667m^-2,high chicken manure:600 kg·667m^-2)on the concentration of TCs;number of culturable microbes,including antibiotic resistant bacteria;organic matter content;and enzyme activity in soil.After a one-year application of chicken manure,the amount of soil culturable bacteria was 3.2-4.3 times higher than that of the control soil.The amount of soil culturable bacteria and actinomycetes in rotted chicken manure?CCM?treatments was significantly higher than in fresh chicken manure?FCM?treatments by 17.0%-33.9%and 201.2%-271.2%,respectively.The application of low rotted chicken manure?L-CCM?significantly reduced the number of fungus,which was 83.8%lower than the control.The application of CCM significantly increased the activities of soil urease and catalase by 81.9%-103.0%and 7.9%-17.9%,respectively,compared with the control.After applying chicken manure in soil where Ipomoea aquatic Forsk was planted,the soil antibiotic residues increased by 3.6%-27.5%.When applying chicken manure to soil where Lactuca sativa L.or Brassica juncea var.gemmifera was planted,the soil antibiotic residues decreased by 19.4%-52.2%and 22.6%-26.6%,respectively.After one year,the antibiotic residues in soil treated with FCM and CCM were higher than that in the control by 176.5%-217.9%and 168.5%-191.5%,respectively.The number of resistant bacteria in the CCM treatments was significantly higher than that in the FCM treatments and in the control.The number of resistant bacteria in the L-CCM treatment was the highest,wherein the quantity of resistant bacteria to oxytetracycline,tetracycline and chlortetracycline was higher than that in other treatments by 11.6%-339.6%,127.0%-635.4%and 32.2%-130.9%,respectively.The drowned planting mode may worsen soil tetracycline antibiotic residues,when compared to drought planting.Long-term application of FCM may cause residue and accumulation of tetracycline antibiotics in soil;hence the hazard to the environment is worthy of further study.Compared with control,tetA,tetC,tetG and tetX increased by 29.76,1.64,69.30 and 6.43times respectively when treated with L-FCM,increased by 38.38,1.17,82.22 and 9.71 times respectively when treated with H-FCM,and by 5.82,2.79,35.57 and 4.54 times respectively when treated with L-CCM,increased by 7.68,3.95,52.49 and 3.87 times respectively when treated with H-CCM.TetX was the most abundant gene(2.44×10⁹ copies·g^-1),followed by tetG(9.88×10⁸ copies·g^-1),tetA(2.07×10⁸ copies·g^-1)and tetC(8.39×10⁷ copies·g^-1),respectively,the lowest was the ribosomal protective protein gene tetW(4.68×10⁷ copies g^-1).In the treatments of chicken manure,the abundance of ARGs in soil was in order of H-FCM>L-FCM>H-CCM>L-CCM>CK.4.Field experiments were conducted to study the effects of different applied levels and types of pig manure and chicken manure on the yield and quality of brussel mustard,and the content of tetracycline antibiotic in vegetables and soil.The medium chicken manure and medium pig manure increased the yield of vegetables by 58.90%and 54.12%,respectively,compared with the control.The application of organic fertilizer improved the edible quality of Brussel Mustard by increase the contents of vitamin C?VC?,amino acid and reduce soluble sugar as compared with the control.Compared with chemical fertilizer treatment,organic fertilizer application increased the amount of antibiotic residue in the edible part of Brussel Mustard and soil,With the pig manure and chicken manure application,the contents of TC,OTC and CTC increased by 191.68?143.20?101.96,26.99?22.11?22.45?g·kg^-1?plant edible part?,640.35?339.67?703.39,333.75?245.11?76.82?g·kg^-1?soil after harvest?.TCs residues of plants and soil with the biogas residue treatment was lower than that with the medium amount application of pig manure treatments.In general,the best yield and quality of vegetables were found in the medium amount application of pig manure and chicken manure treatments(2700kgp 667m^-2 and 900kg·667m^-2,respectively).The increase of organic fertilizer applied had no obvious effect on yield and quality of vegetables,but increased the residue of TCs in vegetables and soil.The biogas residue after fermentation effectively reduced the risk of antibiotic residues.Based on 16S rRNA sequence analysis,it was found that organic fertilizer treatment could change the richness and diversity of soil bacterial community,however,TCs did not play a significant role in this change.