# Absorption-Translocation of Veterinary Antibiotics in Rice Plants Introduced with Irrigation Water
## 2 Materials and Methods
### Experimental Design, Agricultural Activity, and Sample Collection  
The background levels of VAs in previous studies detected in the water ecosystem in the Republic of Korea were as follows: AMX: 0.1 mg L\({}^{-1}\), CTC: 0.3 mg L\({}^{-1}\), and OTC: 0.05 mg L\({}^{-1}\)[13; 14; 15]. The different combinations for each treatment are detailed in Table 2. In brief, three different doses of VAs were used in this study: background level, ten-fold background level, and fifty-fold background level. The experiments were performed in a randomized complete block design with three replicates. The three VAs used in the experiment were obtained from Sigma Aldrich (Seoul, Republic of Korea; 99%). The VAs were dissolved in a watering can and uniformly watered into each plot. The plot size was 4 m in length and 2.5 m in width. Rice cultivation was carried out in all of the experimental plots under the same conditions as in the rest of the field. The Japonic cultivar 'Shindongjin' was chosen for this experiment. Organic farmyard manure was not used to avoid unexpected antibiotic contamination. Spring blowing was conducted on April 30. The experimental paddy plots received basal fertilization and were plowed on May 25 and maintained under flooded conditions. Approximately four rice seedlings (35 days old) per hill were transplanted by hand on June 1. Additional fertilizers were applied June 20 (maximum tilling stage), July 25 (panicle formation stage), and August \begin{table}
\begin{tabular}{l c c c} \hline \hline
**Item** & **Chemical Properties** & **Particle Size Fraction (\%)** \\ \hline Soil organic matter (g 100 g\({}^{-1}\)) & 2.59 & Sand & 50.3 \\ pH (1.5 H\({}_{2}\)O) & 5.68 & Silt & 40.2 \\ Total-N (mg kg\({}^{-1}\)) & 921.4 & Clay & 9.5 \\ Total-P (mg kg\({}^{-1}\)) & 336.2 & & \\ CEC (cmol\({}^{+}\) kg\({}^{-1}\)) & 8.6 & & \\ Electrical conductivity (\(\mu\)S cm\({}^{-1}\)) & 0.3 & & \\ Ca\({}^{++}\) (cmol\({}^{+}\) kg\({}^{-1}\)) & 1.6 & & \\ Mg\({}^{++}\) (cmol\({}^{+}\) kg\({}^{-1}\)) & 1.2 & & \\ K\({}^{+}\) (cmol\({}^{+}\) kg\({}^{-1}\)) & 1.5 & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Physical and chemical properties of the test plot soil. 10 (booting stage). Inorganic fertilizers (N-P2O5-K2O = 90-45-57 kg ha\({}^{-1}\)) were applied equally to all plots according to the guidelines of the Rural Development Administration (RDA) [16]. The water level in the experimental plots was maintained at 5-10 cm until 25 days after transplantation. The experimental plots were then drained of all water for 3 days at the late tilling stage, and the water level was maintained thereafter at 2 to 3 cm until 10 days prior to harvest. The rice plants were harvested on October 15. Borders were constructed between plots to prevent the exchange of irrigation water and VAs. Each plot had an independent water inlet and outlet. The rice plants were kept healthy via prophylactic sprays of recommended insecticides and fungicides as necessary. The fields were kept weed-free by hand weeding conducted at 2-week intervals [17]. About 500 g of soil and two hills of rice plant samples (including underground and upper ground parts) were collected at 15, 30, 45, 60, 75, 90, and 105 days after transplantation (DAT). The samples were collected from five random sites per experimental plot. Soil samples were taken from a depth of 0-10 cm. All samples were lyophilized and stored at \(-80\)\({}^{\circ}\)C until further analysis. Brown rice samples were collected at harvesting and subsequently lyophilized and stored at \(-80\)\({}^{\circ}\)C after threshing and removing the outer hulls. After air-drying the soil samples in the shade at room temperature for a week, they were ground by a pestle and sieved into 2 mm particles. About 5 g of sample was poured into a glass funnel (1000 mL volume) joined to a vacuum filter flask (bore filter size: 11 \(\upmu\)m). Then, 50 mL acetone (Sigma Aldrich; 98%) was poured over the soil sample.