## 2 Materials and Methods  
### Sampling of Environmental Waters and Sediments  
The question of primary importance is of how to select the sampling field to determine the distribution of pharmaceuticals in the environmental waters. For this purpose, considering the spreading out of the geographical region to core cities throughout Japan, the sampling areas in the Kansai district of Japan were selected; the locations were on the right bank of the middle-to-downstream region of the Yodo River [32] and on two other rivers, the Kanzaki and the Ai, and their tributaries. This area, known as the Kanzaki-Ai River basin, covers an important commercial and urban area of about 790 km\({}^{2}\) in Osaka and Hyogo prefectures and is home to 2 million people [33; 34].  
To collect three different types of waters (river, tributary, and sewage treatment plant (STP)), 13 sampling sites consisting of six river sites (R1 to R6), four tributary sites (T1 to T4), and three STP sites (S1 to S3) (Figure 1) were set in the Kanzaki-Ai River basin. The sampling site at the Suita Bridge (R5) was set as the farthest downstream boundary. One sample was basically taken per site, but two effluents, S3(1) and S3(2), were sampled at S3, compiling 14 samples per each sampling time. Names of the sampling sites were used for identification (ID) of the samples. A conventional activated sludge (CAS) process followed by chlorination for disinfection was used in all STPs except S3 where ozonation (8.6 mg/L ozone) was used after partial CAS. The properties of the STPs including treatment process are shown in Table 1[35]. Annual flow rates and BOD (biological oxygen demand, mg/L) at the sampling sites in the Kanzaki-Ai River basin are listed in Table 2[30; 36]. Stainless-steel pails were used to collect water samples, while a stainless-steel bottom sampler was used for river sediment samples collected at R5 (Suita Bridge) and R6 (Hirakata Bridge). All samples were transferred in separate glass bottles, transported to the laboratory within 2 h and kept at 4 \({}^{\circ}\)C under dark. Analysis of the concentration of the target anticancer drugs in each sample was started within 24 h after collection by filtration through a GF/B glass fiber filter (pore size, 1-\(\upmu\)m) for separation of liquid from the suspended solids [37].  
The surveying anticancer drugs was conducted once in the four seasons in 2013 and 2014; on 4 April (spring), 29 July (summer), 18 December (late autumn), and 4 February (winter). Sampling days were selected on rain-less days accompanied by 2 more ahead fine days (rainfall \(\leq\) 1 mm) [38].  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Sample-ID** & **Service Area (ha)** & \begin{tabular}{c} **Service Population** \\ **(Person)** \\ \end{tabular} & \begin{tabular}{c} **Flow Rate (m\$\$/day)** \\ **Mean** \\ \end{tabular} & \begin{tabular}{c} **Treatment Process** \\ **SD** \\ \end{tabular} \\ \hline S1 & 5459 & 494,974 & 256,110 & 31,285 & \begin{tabular}{c} CAS + Chlorine disinfection \\ A2O + Chlorine disinfection \\ disinfection \\ \end{tabular} \\ \hline S2 & 453 & 50,732 & 17,050 & 2355 & \begin{tabular}{c} CAS + Chlorine disinfection \\ disinfection \\ \end{tabular} \\ \hline S3(1) & 3550 & 415,364 & 128,497 & 11,876 & \
\begin{tabular}{c} CAS + Chlorine disinfection \\ Stop AO + Chlorine \\ disinfection \\ \end{tabular} \\ \hline S3(2) & 2152 & 18 &
\begin{tabular}{c} CAS + Oxonation \\ Stop AO + \\ Oligonation \\ \end{tabular} \\ \hline \hline \end{tabular}  
CAS: Conventional activated sludge; AO: Anaerobic/aerobic; A2O: Anaerobic/anoxic/aerobic.  
\end{table}
Table 1: Information of sewage treatment plants (STPs) located in the Kanzaki–Ai River basin (Figure 1) (reproduced from [30]). (reproduced from the data in [30]).  
Figure 1: Locations of sampling sites in the Kanzaki–Ai River basin (reproduced from [30]). (Information of the sampling sites of S1–S3 are shown in Table 1, while those of a combined group of R1–R6 and T1–T4 are shown in Table 2.).