| Recently, as the concern on "fast-growing chicken" and "milk containing antibiotics" touched the sensitive nerve of the public in the field of food safety, the new pollutants such as antibiotics and their resistance genes have attracted wide attention. Sulfonamides, the synthetic antibiotics, are among the most widely used veterinary antibiotics in livestock farming and aquaculture. Sulfonamides with high water solubility, low chelating ability and low binding constant, were excreted to the environment in parent or metabolite via animal manure, leading to a selective pressure on the environmental bacteria and inducing the formation of antibiotic resistant bacteria (ARB). Additionally, manure was shown to enhance the horizontal transfer of resistance genes through its application to agricultural soils and then significantly increase antibiotic resistance genes (ARGs) and selection of resistant bacteria populations in soil, which will threaten the human health potentially by food chain. For a long time, the valid environmental management on veterinary medicine was lack in China, due to the insufficient understanding of the environmental hazard of antibiotics. Therefore, sulfonamides were selected as the typical medicine to reveal the environment pollution characteristics, ensure the environmental behavior of sulfonamides introduced by manure and understand the prevalence and distribution of sulfonamide antibiotic-resistant bacteria and genes in soils as well as the relationship with exposure concentration of sulfonamides. This work can provide basic data and technical support for establishment of environmental safety assessment system of veterinary antibiotics. Furthermore, this is the key content of environmental management of veterinary medicine which needs to fill urgently.Five sulfonamides including sulfadiazine (SDZ), sulfamerazine (SMR), sulfamethazine (SMZ), sulfadimethoxine (SDM), and sulfamethoxazole (SMX) were selected as the study medicines. Through literature review, animal feeding farms visit, sample analysis, laboratory test and statistic analysis, we studied the exposure levels and vertical exposure characteristics, investigated the influence of manure amendment, soil pH and organic carbon (OC) content on the degratigation, adsorption and leaching of five sulfonamides in five different soils (Black soil from Heilongjiang Province, red soil from Jiangxi Province, paddy soil from Wuxi City, moisture soil from Shanxi Province and yellow brown soil from Nanjing City). The impact of manure amendent on microbial flora and ARGs as well as the distribution of ARGs in ARBs were studied then. Finally, we established a prioritization scheme for identifying veterinary medicines in China that have the potential to impact the environment and human health. Based on the scheme, we attempted to list veterinary medicines considered of relatively high priority in China.(1) Since antibiotics were generally applied as treated drug and feed additive, antibiotics were generally detected in soil, vegetables, water, sediment and fish. Compared with the pig and the cattle farms, the chicken farm yielded a high number of antibiotic types and a high exposure level in environmental matrices. After analyzing the exposure levels of typical veterinary antibiotics, it was found that their physical and chemical properties significantly affected their occurrence in soil. The model prediction and verification of migration from manure to soil and from soil to plants provided foundation for revealing the migration rule and exposure level of antibiotics. The contribution of antibiotic feed additives showed that veterinary feed additive intake in normal dose was not the main way of antibiotic utilization.(2) The degradation of sulfonamides in soils followed the bi-exponential equation. The order of degradation rate in different soils was:sandy soil≈loam> clay loam. The degradation rate of different sulfonamides was related to the nature of drug, showing that the degradation half-time in Jiangxi red soil followed the order as SMX>SMT>SDZ>SMZ>SDM. Addition of manure accelerated the degradation of sulfonamides in soils, while the influence of light was not significant. The results of adsorption and leaching study showed that sorption and transport behaviors of sulfonamides were closely related to soil properties, presence of manure, OC, and pH. The order of sorption capacity of the different soils is as follows:Black soil-Paddy soil>Red soil>Yellow-brown soil>Moisture soil. There was a positive correlation between the OC content and the soil sorption of sulfonamides, except for Soil S. However, considering the soil and soil-manure mixture together, addition of manure increased the extent of sorption, following the rule that greater polar substances leads to greater increase of sorption after manure amendment. The leaching ability of sulfonamides in different soils followed the opposite order with sorption capacity. Manure amendment increased the sorption capacity of sulfonamides in the top layer and thus decreased the mobility of sulfonamides in Soils. In the GUS assessment, which considers not only the sorption but also the degradation of pollutants, the GUS values of sulfonamides were lower in soils with manure, suggesting that manure amendment would play a role in decreasing the mobility of sulfonamides in soils.(3) The number of antibiotic resistant bacteria was related to whether animal manure fertilization occurred. The present study also indicated that Bacillus (43.88%), Pseudomonas (11.39%) and Shigella (8.02%) were the most prevalent sul-positive genera in soil, suggesting a potential human health risk. A qPCR analysis of sulfonamide resistance genes was performed on the total DNA extracted directly from the soil. The DNA from the pig-manured soils contained relatively higher copy numbers of sul1than sul2. Comparatively, the relative quantity of the sull gene in the chicken-manured soils was lower than that of the sul2gene. Additionally, the sul3genes were detected at low relative quantities. Furthermore, good positive linear correlations were observed between the relative abundance of the sul2genes and the number of culturable SR isolates in the soil. For the sul2gene and sum of the three sul genes, the correlation coefficients (R2) were0.95and0.65, respectively (p<0.05). The sull, sul2, and sul3genes were all detected at a frequency of100%in the genomic DNA and plasmids of the SR isolates from the manure sample; the sul2genes were only present in the genomic DNA of the isolates collected from forest soil and non-arable soil, attributed to the notion that the sull and sul3genotype in genomic DNA maybe associate with the amended manure; the sul1, sul2and sul3genes were all located in the plasmids of the isolates from non-arable soil, possibly due to that the isolates from the non-arable soil carried sul genes through horizontal transfer via plasmids. Further analysis suggested that the frequency distribution of the sul genes in the genomic DNA and plasmids of the SR isolates from manured soil was sul2> sul1> sul3overall (p<0.05). The detection frequency of sul gene was higher in chicken farm than in pig farm. The combination of sul1and sul2was the most frequent, and the co-existence of sull and sul3was not found either in the genomic DNA or plasmids. The expression variation of sull, sul2and sul3in typical sulfonamide resistant bacteria (Bacillus anthracis SYN201, G+and Shigella flexneri NJJN802, G-) was investigated exposed to sulfonamide solutions after different cultivating time. It was found that SYN201and NJJN802expressed sul genes after72h and36h, respectively, whether they were exposed to sulfonamides or not, showing that sulfonamides can help to increase expression level of ARGs in specific time, and the expression increase had significant dosage-effect relation.(4) This study exercised a prioritization of China’s veterinary medicines based on their potentials to enter the environment and hazard potentials including ecotoxicity and human health damage. Thirty-eight of77veterinary medicines were identified as high priority despite data shortage. The antibiotics should be addressed more especially in China’s environmental management of veterinary medicines for the antibiotics account for57.9%of top priority veterinary medicines. Furthermore, the insecticides used widely in aquaculture need to be taken into account due to their high priority rankings. The prioritization results can help to guide policy-making and regulatory concern and to determine the effective targets for China’s further detailed risk assessment for veterinary medicines. |
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