## 2 Materials and Methods  
### Test Substances  
The protocol was developed for quantifying concentrations of 61 strategically selected APIs (Table 1) representing 19 therapeutic classes of medicinal chemicals approved for use in humans (\(n\) = 57) and animal husbandry (\(n\) = 4). The study APIs were selected to include: (a) compounds of high usage across the world; (b) compounds with known or suspected ecological or human health concern; and (c) compounds of expected high use due to regional disease pressures (e.g., antimalarials). Significant focus was placed on antimicrobial chemicals including antibiotics (\(n\) = 13) and antifungals (\(n\) = 6) due to implications for the selection of resistance to these medicines in bacterial communities [12; 13]. Similarly, focus was also placed on antidepressants (\(n\) = 7) due to their increasing use globally and potential ecotoxicological risk [14]. All compounds were optimised for specific quantification using direct injection HPLC-MS/MS only and compounds not suitable for quantification using this instrumentation were not included in the study. Further method development may be used to broaden the scope of the studied contaminants (e.g., [PERSON] et al. [8]).  
All test standards were purchased from Sigma Aldrich (UK) and were of \(\geq\)95% purity. Deuterated internal standards were obtained from Sigma Aldrich (UK) for 32 test APIs and atrazine-D5 was used where a labelled standard was not available. Liquid chromatography-mass spectrometry (LCMS)-grade water and methanol were obtained from VWR (UK). Polystyrene boxes (5 L, 34.5 \(\times\) 21 \(\times\) 14.5 cm, L \(\times\) W \(\times\) H) used for sample shipment were obtained from JB Packaging (Torpoint, UK), whereas 15-mLamber glass sample vials, 0.7-\(\upmu\)m glass microfiber syringe filters (Whatman) and 24-mL luer lock syringes were obtained from Fisher Scientific (UK). A ZORBAX Eclipse Plus C18 chromatography column (3.0 \(\times\) 100 mm, 1.8 \(\upmu\)m, 600 bar) was purchased from Agilent Technologies (UK) and a C18 SecurityGuard guard column was purchased from Phenomenex (UK).  
\begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Therapeutic Class** & **Compound** & **Associated Internal Standard** \\ \hline \multirow{4}{*}{Analgesic} & & Lidocaine & Lidocaine D6 \\  & & Naproven & Naproven D3 \\  & & Paracetamol & Paracetamol \\ \hline \multirow{4}{*}{Anti-epileptic} & & Carbamazepine & Carbamazepine D10 \\  & & Gabapentin & Gabapentin D10 \\  & & Pregabalin & Atrazine D5 \\ \hline \multirow{4}{*}{Antibiotics} & _Fluorquinolones_ & Ciprofloxacin & Ciprofloxacin D8 \\  & & Enrofloxacin * & Atrazine D5 \\  & _Lincosmide_ & Lincomycin * & Atrazine D5 \\  & _Macrolites_ & Clarithromycin & Atrazine D5 \\  & & Erythromycin & Atrazine D5 \\  & & Tylosin * & Atrazine D5 \\  & _Nitroimidazole_ & Metropolitanazole & Metronidazole2e D3 \\  & _Penicillin_ & Cloxacillin & Atrazine D5 \\  & _Sulfonamides_ & Sulfafiazine * & Atrazine D5 \\  & & Sulfamethoxazole & Sulfamethoxazole D4 \\  & _Tetracyclines_ & Trinehoprim & Trimethoprim D9 \\  & _Cyytterracycline_ & Atrazine D5 \\  & Tetracycline & Atrazine D5 \\ \hline \hline \end{tabular}
\end{table}
Table 1: List of monitored chemicals by therapeutic class and their associated internal standard.