Detection of selected antibiotics and their degradation products in water and soil from Korea’s four main rivers using SPE-LC-MS/MS Ryan De Sotto, Sungpyo Kim, Yunchul Cho, Carl Medriano, and Youngja Park

1 2 3 4 5 Background Objective Methodology Results Contents Conclusions and Implications Methodology Results

Background Pharmaceuticals are continuously introduced into the environment and are prevalent at small concentrations (Kolpin et al., 2002, Envi Sci Tech). They are considered as emerging pollutants in waterbodies because they still remain unregulated or are currently undergoing a regularization process (Rivera-Utrilla, 2013, Chemosphere). The presence of antibiotics in the environment is of special concern due to their biological activity and their potential to have adverse effects on non target organisms. “Aims to improve water quality and restore river ecosystems.” Diverse veterinary pharmaceuticals and antibiotics in aquatic environments were detected in aquatic systems in South Korea (Kim et al., 2008, Environ Toxicol Pharmacol) Antibiotics are discharged in the environment from different sources such as WWTP effluent discharge, animal farming, etc. The Four Major Rivers Restoration Project 

Background However, studies on antibiotics in South Korea dealt mostly with the parent compound and not the metabolites which are also discharged in the environment. Therefore, analysis of both the antibiotics and their degradation products is necessary. These degradation products of antibiotics are sometimes more toxic than the parent compound (De Bel et al., 2009, Chemosphere) What are the concentrations of select veterinary antibiotics and their metabolites in water and soil samples in the four major rivers in South Korea?

Objective To quantify select antibiotics and their metabolites from environmental samples collected from the four major rivers of South Korea.

Sample collection Methods Han River Yeongsan River Geum River Okcheon Region (North) Nakdong River Dong-i-myeon Iwon-myeon

Shake at 250 rpm for 5 minutes Methods Treatment of Soil Samples Samples were dried naturally (20-40°C) 1 gram of soil sample +Phosphate buffer pH 2.0 +Acetonitrile +Terbutylazine-2-hydroxy 250 rpm for 5 minutes Separate supernatant +Acetonitrile Shake at 250 rpm for 5 minutes Centrifuge for 10 minutes (4°C at 12,000 rpm) Microwave digestion for 30 minutes

Treatment of Soil Samples Methods Treatment of Soil Samples Remove organic solvent in supernatant Microwave digestion for 30 minutes Centrifuge 10 minutes (4°C at 12,000 rpm) Solid phase extraction

Solid Phase Extraction Methods Solid Phase Extraction +HPLC-grade water +methanol +pH 2.0 water Water samples were kept at 4°C +adjusted to pH 2.0 +terbuthylazine +EDTA Cartridge washing Sample loading Sample evaporation Sample elution Sample storage in amber vials at -20°C prior to analysis +HPLC-grade water (washing) +methanol +methanol: acetone Gentle stream of Nitrogen gas and water bath at 60°C

Sample Analysis Methods Injection volume: 10.0 µL Flow rate: 0.30 ml/min Run time: 13 minutes Post time: 2 minutes GRADIENT: Time %B 0 10.0 0.5 10.0 4.0 90.0 6.0 90.0 6.5 10.0 13.0 10.0 Mobile phase: A: 0.1% Formic acid in water B: 0.1% FA in Acetonitrile Agilent 6460 Triple Quad LC-MS COLUMN SPECS Higgins TARGA 2.1 x 100 mm 5um particle size

Fragmentor Voltage (eV) Methods Sample Analysis: Multiple Reaction Monitoring Compounds Fragmentor Voltage (eV) Precursor Ion (m/z) Daughter Ion (m/z) Collision Energy (eV) Chlortetracycline 120 479.0 462.0 13 4-epichlortetracycline 80 479.3 444.0 19 Clarithromycin 160 748.4 158.1 30 N-desmethylclarithromycin 734.4 144.1 15 Sulfamethazine 90 279.1 186.1 10 N-acetylsulfamethazine 100 321.2 17 Sulfamethoxazole 253.9 92.1 N-Acetylsulfamethxazole 296.2 65.2 31 Terbutylazine-2-hydroxy 110 212.0 156.0

4-epichlortetracycline Results Chlortetracycline 4-epichlortetracycline

N-desmethylclarithromycin Results Clarithromycin N-desmethylclarithromycin

N-acetylsulfamethazine Results Sulfamethazine N-acetylsulfamethazine

N-acetylsulfamethoxazole Results Sulfamethoxazole N-acetylsulfamethoxazole

Terbutylazine-2-hydroxy Results Terbutylazine-2-hydroxy

Results Average concentration of the antibiotics (ng/L) and their metabolites from the two sampling events (August and November 2014). Values in red represent concentrations greater than 200 ng/L in at least one of the sampling events in that area.

Results Average concentration of the antibiotics (ng/L) and their metabolites from soil samples in Okcheon region. Values in blue are the concentrations where at least 1 of the values is not detected while the numbers in red have at least one concentration greater than 200 ng/L.

Results Water samples from the four major rivers All antibiotics and metabolites were detected in all water samples from the four rivers in both sampling events (August and November 2014) Nakdong, Han and Geum rivers had concentrations of 4-epichlortetracycline greater than 200 ng/L while Yeongsan river’s values are lower than 200 ng/L. Yeongsan river generally have lower concentrations of the compounds compared to the other three rivers. Nakdong, Han, and Geum rivers have at least 2 compounds with concentrations greater than 200 ng/L.

Results Water samples from Okcheon region The compounds were all detected in the water samples from Okcheon region. The sampling sites in Okcheon had concentrations of 4-EPI greater than 200 ng/L in at least one of the sampling events in 2014. Okcheon 1 samples had N-DES, 4-EPI, SMZ, and N-SMZ’s concentrations above 200 ng/L. The concentrations of the compounds are lower in November 2014.

Results Soil samples from Okcheon region Not all compounds are detected from the soil samples in the sampling sites in Okcheon. Okcheon 1 and 2 had 4-EPI and N-SMZ’s concentrations (both metabolites) higher than 200 ng/L during the sampling in August 2014. Concentration of these antibiotics are generally lower at Okcheon 3 and during the second sampling event in November 2014.

Results General trends in the concentration of compounds Concentration of metabolites are generally higher than the parent compounds in the water samples from the four major rivers and in Okcheon region for both sampling events. Concentration is lower in the second sampling event (November 2014, Fall) than in the first sampling (August 2014, Summer). Of the four rivers, Yeongsan had the lowest concentration of all the compounds analyzed for both sampling. Okcheon 2 had the lowest concentration of the parent compounds and the metabolites.

Conclusion Four antibiotics and their respective metabolites were detected from water and soil samples in the four major rivers in South Korea and Okcheon region. The decrease in concentration of the compounds during the second sampling may be associated to the less rainfall event in Fall as compared to summer in South Korea. Metabolites were found to be more persistent and frequently detected than the parent compounds in water and soil samples.

Implications The detection of equally biologically active metabolites in water and soil samples in South Korea magnifies the need for an immediate action to regulate antibiotic discharge from non-point sources. The presence of these compounds in the environment may trigger increase in antibiotic resistance and may promote adverse effects to aquatic organisms.

Acknowledgment This study was funded by the National Institute for Environmental Research and the Korea Institute of Science and Technology and KHIDI grant#HI14C2686.

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