Assessing antibiotic sorption in soil: a literature review and new case studies on sulfonamides and macrolides

The increased use of veterinary antibiotics in modern agriculture for therapeutic uses and growth promotion has raised concern regarding the environmental impacts of antibiotic residues in soil and water. The mobility and transport of antibiotics in the environment depends on their sorption behavior, which is typically predicted by extrapolating from an experimentally determined soil-water distribution coefficient (Kd). Accurate determination of Kd values is important in order to better predict the environmental fate of antibiotics. In this paper, we examine different analytical approaches in assessing Kd of two major classes of veterinary antibiotics (sulfonamides and macrolides) and compare the existing literature data with experimental data obtained in our laboratory. While environmental parameters such as soil pH and organic matter content are the most significant factors that affect the sorption of antibiotics in soil, it is important to consider the concentrations used, the analytical method employed, and the transformations that can occur when determining Kd values. Application of solid phase extraction and liquid chromatography/mass spectrometry can facilitate accurate determination of Kd at environmentally relevant concentrations. Because the bioavailability of antibiotics in soil depends on their sorption behavior, it is important to examine current practices in assessing their mobility in soil.

To study the influence of HA concentrations on the sorption of ERY, SUL, and TYL, the HA levels were varied by spiking the soil with 1, 10 and 50 ppm HA prepared in 0.01 M CaCl 2 , while maintaining the pH at 7 and ionic strength at 30mM. Control and blank set-ups were prepared for all tests. Control set-ups contained the analyte solution in 0.01M CaCl 2 only (no soil), while the blank set-up contained soil suspended in 0.01M CaCl 2 without any spiked analyte. The control set-ups were used to check for container sorption. All results were corrected accordingly. All set-ups were performed in duplicates. soil:solution ratio) followed by a 98-h equilibration time. A second experiment with SUL-14 C was performed using the same experimental setup with the exception of a wider concentration range that include 1, 3, 10, 20, 30, 50, 100, and 300 ng/mL. The solid and liquid portions were separated by centrifugation at 1500 rpm. One mL aliquots were taken from the liquid phase, transferred to a 5-mL scintillation vial to which 3-mL scintillant were added for measurement of the radioactivity remaining in solution. The resulting solutions were measured against 0.01M CaCl 2 background. Samples were quantified using 5 standards: 4.62, 6.93, 9.25, 23.1, and 46.2 pg/mL for ERY-3 H, and 10.6, 17.7, 35.4, 70.8, and 106 ng/mL for SUL-14 C. The radioactivity of the sorbed compound in soil was calculated by subtraction, based on the original radioactivity spiked into the system.

Mass Spectrometer
Because no commercial radiolabelled TYL was available, non-radiolabelled TYL (tylosin tartate was purchased from Sigma Chemical Co., St. Louis, MO.) was used at 10, 100, 500, and 1000 ng/mL spiking concentrations. The use of higher concentrations of TYL was necessary because of the detection limit on the HPLC-MS instrument. The soil:solution ratio of 1 g:40 mL 0.01M CaCl 2 solution was determined to fall within 20-80% sorption of TYL in sediment based on preliminary experiments. A 24-h equilibration time was determined to be sufficient to reach equilibrium. Aliquots from the supernatant were then pre-concentrated by solid phase extraction (SPE), using OASIS™ HLB cartridges from Waters (Milford, MA). Samples for SPE were first treated with 20mL 0.05M citric acid buffer (pH 3.8), 20 mL 0.01M Na 2 EDTA then diluted to 250 mL (final solution pH ~4). SPE cartridges were conditioned using the following scheme: 3mL ACN, 3mL ethyl acetate (EtOAc), 3mL ACN, and 3 mL H 2 O. The samples were then loaded onto conditioned cartridges at an average flow rate of about 7mL/min (equivalent to SPE box pressure of ~8 torr). After 5 minutes of drying, the cartridges were eluted using 3 mL acetonitrile followed by 3 mL ethyl acetate. The percent recovery of TYL using these conditions was 107 ± 5%. Eluates were dried under a slow stream of air, and reconstituted in 1000 uL 10% methanol/90% water. All samples were spiked with 100 ng/mL roxithromycin as an internal standard prior to injection.
Liquid chromatography mass spectrometry (Thermo Fisher Scientific Surveyor coupled to Thermo Fisher Scientific LCQ Advantage, San Jose, CA) was employed for the analysis of TYL from the batch equilibrium experiments. To separate TYL-A from its known hydrolysis forms, TYL-B (Desmycosin), TYL-C (Macrosin) and TYL-D (Relomycin), a mobile phase with a 15-min gradient elution was applied. Separation was performed on a C-18 column (Thermo BetaBasic 100 x 2.1mm C-18 column with 3-μm particle diameter) using 20:80 acetonitrile:water (with 0.1% formic acid). The flow rate of the mobile phase was 0.300 mL/min. From the initial conditions, the percentage of acetonitrile was increased to 90% from 0-4 min, and was held constant 4 min. Finally, the mobile phase was brought back to initial conditions in 2 min, which was then maintained for 5 minutes. Detection of TYL was achieved using electrospray ionization in positive mode. Mass spectrometric acquisition was performed by single ion monitoring MS parameters for this method are listed in Table A1. Samples were quantified based on an external calibration curve constructed using 10, 40, 100, 400 and 1000 ng/mL TYL, normalized by the signal of an internal standard containing 100 ppb roxithromycin.

Method 3: Quantification of Tylosin by Liquid Chromatography Tandem Mass Spectrometry
A second set of sorption experiments for TYL were performed using a wider concentration range that includes 1, 5, 10, 100, 200, 500, and 1000 ng/mL spiking concentrations. The soil:solution ratio of 0.5 g:400 mL Nanopure™ water (1:1000) was determined in a preliminary study, at which approximately 40-50% of the TYL will sorb in sediment. A 24-h equilibration time was also determined to be sufficient to reach equilibrium.
Aliquots from the supernatant were collected and filtered with 0.45 µm polypropylene membrane syringe filters (VWR) for direct analysis (without SPE) using a more sensitive HPLC  Table A2. Samples were quantified based on single-point standard addition (by adding TYL to reach a final concentration of 25 ng/mL standard added to each sample) to reduce matrix effects associated with calibration by an external curve.