While the prudent and reasonable use of veterinary antimicrobial agents in food-producing animals is necessary, researchers over the decades have shown that these antimicrobial agents can spread into the environment through livestock manure and wastewater. The analysis of the occurrence of antimicrobial compounds in soil samples is of a great importance to determine potential impacts on human and animal health and the environment. In this study, an affordable, rugged and simple analytical method has been developed for the determination of twenty-nine antimicrobial compounds from five different classes (tetracyclines, fluoro(quinolones), macrolides, sulfonamides and diaminopirimidines). Liquid-liquid extraction (LLE) with extract filtration combined with ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was the best strategy for the simultaneous determination of all analytes. The developed method was validated according to the Commission Implementing Regulation (EU) 2021/808. The limit of detections (LODs) ranged from 0.5 to 2.0 µg/kg, while the limit of quantitation (LOQ) was established at 1.0 to 20.0 µg/kg. The developed method was successfully applied for the determination of antimicrobial residues in one hundred and eighteen soil samples obtained from four European countries (Austria, Czech Republic, Estonia and Portugal). Doxycycline in the concentration levels of 9.07 µg/kg-20.6 µg/kg was detected in eight of the analysed samples. Samples were collected from areas where natural fertilizers (swine or cow manure) were applied. Our method can be efficiently used to monitor anti-microbial compounds in soil samples.

Associate Laboratory for Animal and Veterinary Sciences AL4AnimalS 1300 477 Lisbon Portugal

Austrian Agency for Health and Food Safety 1220 Vienna Austria

Centre for the Studies of Animal Science Institute of Agrarian and Agri Food Sciences and Technologies University of Porto 4050 453 Porto Portugal

CIISA Center for Interdisciplinary Research in Animal Health Faculty of Veterinary Medicine University of Lisbon 1300 477 Lisbon Portugal

Department of Pharmacology and Toxicology National Veterinary Research Institute Al Partyzantow 57 24 100 Pulawy Poland

Faculty of Engineering and Physical Sciences School of Chemistry and Chemical Engineering University of Surrey Guildford GU2 7XH UK

Institute for Land and Water Management Research Federal Agency for Water Management 3252 Petzenkirchen Austria

Institute of Technology University of Tartu 50411 Tartu Estonia

National Institute of Public Health 10042 Prague Czech Republic

National Reference Laboratory of Antibiotic Resistances and Healthcare Associated Infections Department of Infectious Diseases National Institute of Health Dr Ricardo Jorge 1649 016 Lisbon Portugal

See more in PubMed

World Organisation for Animal Health . Annual Report on Antimicrobial Agents Intended for Use in Animals 7th Report. World Organisation for Animal Health; Paris, France: 2019.

European Medicines Agency . Sales of Veterinary Antimicrobial Agents in 31 European Countries in 2018—10th ESVAC Report. European Medicines Agency; Amsterdam, The Netherlands: 2020. pp. 1–104.

Wei R., Ge F., Zhang L., Hou X., Cao Y., Gong L., Chen M., Wang R., Bao E. Occurrence of 13 veterinary drugs in animal manure-amended soils in Eastern China. Chemosphere. 2016;144:2377–2383. doi: 10.1016/j.chemosphere.2015.10.126. PubMed DOI

Robles-Jimenez L.E., Aranda-Aguirre E., Castelan-Ortega O.A., Shettino-Bermudez B.S., Ortiz-Salinas R., Miranda M., Li X., Angeles-Hernandez J.C., Vargas-Bello-pérez E., Gonzalez-Ronquillo M. Worldwide traceability of antibiotic residues from livestock in wastewater and soil: A systematic review. Animals. 2022;12:60. doi: 10.3390/ani12010060. PubMed DOI PMC

Eurostat Statistics|Eurostat. [(accessed on 5 June 2023)]. Available online: https://ec.europa.eu/eurostat/databrowser/view/tps00202/default/map?lang=en.

EC Regulation (EU) 1069/2009 Animal By-Products. Off. J. Eur. Union. 2019;53:1689–1699.

Zubair M., Wang S., Zhang P., Ye J., Liang J., Nabi M., Zhou Z., Tao X., Chen N., Sun K., et al. Biological nutrient removal and recovery from solid and liquid livestock manure: Recent advance and perspective. Bioresour. Technol. 2020;301:122823. doi: 10.1016/j.biortech.2020.122823. PubMed DOI

Vaneeckhaute C., Meers E., Michels E., Buysse J., Tack F.M.G. Ecological and economic benefits of the application of bio-based mineral fertilizers in modern agriculture. Biomass Bioenergy. 2013;49:239–248. doi: 10.1016/j.biombioe.2012.12.036. DOI

Li J., Xin Z., Zhang Y., Chen J., Yan J., Li H., Hu H. Long-term manure application increased the levels of antibiotics and antibiotic resistance genes in a greenhouse soil. Appl. Soil Ecol. 2017;121:193–200. doi: 10.1016/j.apsoil.2017.10.007. DOI

Li C., Chen J., Wang J., Ma Z., Han P., Luan Y., Lu A. Occurrence of antibiotics in soils and manures from greenhouse vegetable production bases of Beijing, China and an associated risk assessment. Sci. Total Environ. 2015;521–522:101–107. doi: 10.1016/j.scitotenv.2015.03.070. PubMed DOI

Karci A., Balcioǧlu I.A. Investigation of the tetracycline, sulfonamide, and fluoroquinolone antimicrobial compounds in animal manure and agricultural soils in Turkey. Sci. Total Environ. 2009;407:4652–4664. doi: 10.1016/j.scitotenv.2009.04.047. PubMed DOI

Li Y.W., Wu X.L., Mo C.H., Tai Y.P., Huang X.P., Xiang L. Investigation of sulfonamide, tetracycline, and quinolone antibiotics in vegetable farmland soil in the pearl river delta area, Southern China. J. Agric. Food Chem. 2011;59:7268–7276. doi: 10.1021/jf1047578. PubMed DOI

Kim J.W., Hong Y.K., Ryu S.H., Kwon O.K., Lee Y.B., Kim S.C. Development of analytical method for veterinary antibiotics and monitoring of residuals in agricultural environment. Appl. Biol. Chem. 2023;66:20. doi: 10.1186/s13765-023-00777-3. DOI

Qasim B., Razzak A.A., Motelica-Heino M., Kamil G.M., Morabito D. Quantitative determination of fluoroquinolones in contaminated soils by HPLC with solid-phase extraction. Baghdad Sci. J. 2020;17:48–566. doi: 10.21123/bsj.2020.17.1.0048. DOI

Cycoń M., Mrozik A., Piotrowska-Seget Z. Antibiotics in the soil environment—Degradation and their impact on microbial activity and diversity. Front. Microbiol. 2019;10:338. doi: 10.3389/fmicb.2019.00338. PubMed DOI PMC

Boxall A.B.A., Johnson P., Smith E.J., Sinclair C.J., Stutt E., Levy L.S. Uptake of veterinary medicines from soils into plants. J. Agric. Food Chem. 2006;54:2288–2297. doi: 10.1021/jf053041t. PubMed DOI

Tasho R.P., Cho J.Y. Veterinary antibiotics in animal waste, its distribution in soil and uptake by plants: A review. Sci. Total Environ. 2016;563–564:366–376. doi: 10.1016/j.scitotenv.2016.04.140. PubMed DOI

Madikizela L.M., Ncube S., Chimuka L. Uptake of pharmaceuticals by plants grown under hydroponic conditions and natural occurring plant species: A review. Sci. Total Environ. 2018;636:477–486. doi: 10.1016/j.scitotenv.2018.04.297. PubMed DOI

Zhang H., Zhou Y., Huang Y., Wu L., Liu X., Luo Y. Residues and risks of veterinary antibiotics in protected vegetable soils following application of different manures. Chemosphere. 2016;152:229–237. doi: 10.1016/j.chemosphere.2016.02.111. PubMed DOI

Jechalke S., Heuer H., Siemens J., Amelung W., Smalla K. Fate and effects of veterinary antibiotics in soil. Trends Microbiol. 2014;22:536–545. doi: 10.1016/j.tim.2014.05.005. PubMed DOI

Aga D.S., O’Connor S., Ensley S., Payero J.O., Snow D., Tarkalson D. Determination of the persistence of tetracycline antibiotics and their degradates in manure-amended soil using enzyme-linked immunosorbent assay and liquid chromatography-mass spectrometry. J. Agric. Food Chem. 2005;53:7165–7171. doi: 10.1021/jf050415+. PubMed DOI

Hamscher G., Pawelzick H.T., Höper H., Nau H. Different behavior of tetracyclines and sulfonamides in sandy soils after repeated fertilization with liquid manure. Environ. Toxicol. Chem. 2005;24:861–868. doi: 10.1897/04-182R.1. PubMed DOI

Grenni P., Ancona V., Barra Caracciolo A. Ecological effects of antibiotics on natural ecosystems: A review. Microchem. J. 2018;136:25–39. doi: 10.1016/j.microc.2017.02.006. DOI

Sonola V.S., Katakweba A.S., Misinzo G., Matee M.I.N. Occurrence of Multi-Drug-Resistant Escherichia coli in Chickens, Humans, Rodents and Household Soil in Karatu, Northern Tanzania. Antibiotics. 2021;10:1137. doi: 10.3390/antibiotics10091137. PubMed DOI PMC

Bengtsson-Palme J. Antibiotic resistance in the food supply chain: Where can sequencing and metagenomics aid risk assessment? Curr. Opin. Food Sci. 2017;14:66–71. doi: 10.1016/j.cofs.2017.01.010. DOI

Fang H., Han Y., Yin Y., Pan X., Yu Y. Variations in dissipation rate, microbial function and antibiotic resistance due to repeated introductions of manure containing sulfadiazine and chlortetracycline to soil. Chemosphere. 2014;96:51–56. doi: 10.1016/j.chemosphere.2013.07.016. PubMed DOI

Góchez D., Raicek M., Ferreira J.P., Jeannin M., Moulin G., Erlacher-Vindel E. OIE annual report on antimicrobial agents intended for use in animals: Methods used. Front. Vet. Sci. 2019;6:1–9. doi: 10.3389/fvets.2019.00317. PubMed DOI PMC

Graham D.W., Bergeron G., Bourassa M.W., Dickson J., Gomes F., Howe A., Kahn L.H., Morley P.S., Scott H.M., Simjee S., et al. Complexities in understanding antimicrobial resistance across domesticated animal, human, and environmental systems. Ann. N. Y. Acad. Sci. 2019;1441:17–30. doi: 10.1111/nyas.14036. PubMed DOI PMC

Samreen, Ahmad I., Malak H.A., Abulreesh H.H. Environmental antimicrobial resistance and its drivers: A potential threat to public health. J. Glob. Antimicrob. Resist. 2021;27:101–111. doi: 10.1016/j.jgar.2021.08.001. PubMed DOI

Kumar Mehata A., Lakshmi Suseela M.N., Gokul P., Kumar Malik A., Kasi Viswanadh M., Singh C., Selvin J., Muthu M.S. Fast and highly efficient liquid chromatographic methods for qualification and quantification of antibiotic residues from environmental waste. Microchem. J. 2022;179:107573. doi: 10.1016/j.microc.2022.107573. DOI

Chan C.L., Wai H.K.F., Wu P., Lai S.W., Chan O.S.K., Tun H.M. A Universal LC-MS/MS Method for Simultaneous Detection of Antibiotic Residues in Animal and Environmental Samples. Antibiotics. 2022;11:845. doi: 10.3390/antibiotics11070845. PubMed DOI PMC

Kokoszka K., Kobus A., Bajkacz S. Optimization of a method for extraction and determination of residues of selected antimicrobials in soil and plant samples using HPLC-UV-MS/MS. Int. J. Environ. Res. Public Health. 2021;18:1159. doi: 10.3390/ijerph18031159. PubMed DOI PMC

Schlüsener M.P., Spiteller M., Bester K. Determination of antibiotics from soil by pressurized liquid extraction and liquid chromatography-tandem mass spectrometry. J. Chromatogr. A. 2003;1003:21–28. doi: 10.1016/S0021-9673(03)00737-4. PubMed DOI

Hoff R., Pizzolato T.M., Diaz-Cruz M.S. Trends in sulfonamides and their by-products analysis in environmental samples using mass spectrometry techniques. Trends Environ. Anal. Chem. 2016;9:24–36. doi: 10.1016/j.teac.2016.02.002. DOI

Huygens J., Rasschaert G., Heyndrickx M., Dewulf J., Van Coillie E., Quataert P., Daeseleire E., Becue I. Impact of fertilization with pig or calf slurry on antibiotic residues and resistance genes in the soil. Sci. Total Environ. 2022;822:153518. doi: 10.1016/j.scitotenv.2022.153518. PubMed DOI

Mishra A., Chhonker Y.S., Bisen A.C., Prasad Y.D., Tulsankar S.L., Chandasana H., Dey T., Verma S.K., Bala V., Kanojiya S., et al. Rapid and Simultaneous Analysis of Multiple Classes of Antimicrobial Drugs by Liquid Chromatography-Tandem Mass Spectrometry and Its Application to Routine Biomedical, Food, and Soil Analyses. ACS Omega. 2020;5:31584–31597. doi: 10.1021/acsomega.0c03863. PubMed DOI PMC

Chen G., Li M., Liu X. Fluoroquinolone Antibacterial Agent Contaminants in Soil/Groundwater: A Literature Review of Sources, Fate, and Occurrence. Water. Air. Soil Pollut. 2015;226:418. doi: 10.1007/s11270-015-2438-y. DOI

Franklin A.M., Andrews D.M., Williams C.F., Watson J.E. Simultaneous Extraction of Four Antibiotic Compounds from Soil and Water Matrices. Separations. 2022;9:200. doi: 10.3390/separations9080200. DOI

Gbylik-Sikorska M., Posyniak A., Mitrowska K., Gajda A., Błądek T., Şniegocki T., Zmudzki J. Occurrence of veterinary antibiotics and chemotherapeutics in fresh water, sediment, and fish of the rivers and lakes in Poland. Bull. Vet. Inst. Pulawy. 2014;58:399–404. doi: 10.2478/bvip-2014-0062. DOI

Chopra I., Roberts M. Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiol. Mol. Biol. Rev. 2001;65:232–260. doi: 10.1128/MMBR.65.2.232-260.2001. PubMed DOI PMC

Askari Rizvi S.F. Tetracycline: Classification, Structure Activity Relationship and Mechanism of Action as a Theranostic Agent for Infectious Lesions-A Mini Review. Biomed. J. Sci. Tech. Res. 2018;7:5787–5796. doi: 10.26717/BJSTR.2018.07.001475. DOI

Commission E. Commission Implementing Regulation (EU) 2021/808 of 22 March 2021 on the performance of analytical methods for residues of pharmacologically active substances used in food-producing animals and on the interpretation of results as well as on the methods to. Off. J. Eur. Union. 2021;180:84–109.

Holmes N.E., Charles P.G.P. Safety and Efficacy Review of Doxycycline. Clin. Med. Ther. 2009;1:CMT.S2035. doi: 10.4137/CMT.S2035. DOI

Nahler G., Nahler G. Committee for Veterinary Medicinal Products (CVMP) Dict. Pharm. Med. 2009:32. doi: 10.1007/978-3-211-89836-9_245. DOI

EMA Categorisation of antibiotics for use in animals for prudent and responsible use. Eur. Med. Agency. 2019:1–73.

SUNU WHO List of Critically Important Antimicrobials for Human Medicine (WHO CIA List) [(accessed on 12 June 2023)]. Available online: http://who.int/foodsafety/publications/antimicrobials-fifth/en/

Wenzl T., Haedrich J., Schaechtele A., Robouch P., Stroka J. Guidance Document on the Estimation of LOD and LOQ for Measurements in the Field of Contaminants in Feed and Food 2016. Publications office of the EU; Luxembourg: 2016. EUR 28099 EN. DOI