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Environmental Science: Processes & Impacts

Electronic Supplementary Information

Baseline toxicity and ion-trapping models to describe the pH-dependence of bacterial toxicity of pharmaceuticals

Andreas Baumer,a,b Kai Bittermann,a Nils Klüver,a and Beate I. Eschera,c

S1. Additional information on the Microtox assay

Phenol pH 5.5 pH 6 pH 7 pH 8 pH 9 -1.5 100

80 A.fischeri [%] 60 -2.0 40

20 (M) for inhibition

0 50 -2.5 Aliivibrio fischeri

in -20 logEC Inhibition of bioluminescence -40 of bioluminescence in -3.0 -5 -4 -3 -2 -1 -4 -3 -2 -1 -4 -3 -2 -1 -4 -3 -2 -1 -4 -3 -2 -1

log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] pH 6 pH 7 pH 8 pH 9 pH 5.5

Fig. S1 A. Reference compound phenol as a measure of repeatability and robustness of the 384-well plate set up of the Microtox assay.

S2. Additional information on the chemicals

Table S1 Additional information on the pharmaceuticals tested; CAS numbers, purity supplier and counterion if a salt was used. All calculations are done in molar, i.e., the counterion of the salt was not considered. The pharmaceuticals are sorted in groups of acidic, basic, multiprotic chemicals and alphabetically within the groups.

Name CAS Batch Purity Supplier Salt

Caffeine 58-08-2 1378801 >99% Sigma-Alrich -

Carbamazepine 298-46-4 0455124-10 >98% Sigma-Aldrich -

Acetyl-sulfamethoxazole 21312-10-7 BCBQ9726V >98,5% Sigma-Aldrich -

Bezafibrate 41859-67-0 BCBQ1675V >98% Fluka -

Clofibric acid 882-09-7 BCBK9475V >99% Fluka - Diclofenac 15307-86-5 0457729-34 >99% Cayman Chemical Company Sodium

Furosemide 54-31-9 0473175-1 >98% Cayman Chemical Company - Ibuprofen 15687-27-1 015K0586 n.b. Sigma-Aldrich Sodium Ketoprofen 22071-15-4 BCBP4500V >98% Sigma-Aldrich - Losartan 114798-26-4 0443154-54 >98% Cayman Chemical Company Potassium

Mefenamic acid 61-68-7 BCBM5220V >99% Fluka - Naproxen 22204-53-1 0430295-34 >99% Cayman Chemical Company - Paracetamol 103-90-2 1061901 n.b. - -

Electronic Supplementary Information S1 Electronic Supplementary Information Environmental Science: Processes & Impacts

Name CAS Batch Purity Supplier Salt

Salicylic acid 69-72-7 MKBG8728V >99% Sigma-Aldrich -

Sulfadimidine 57-68-1 065M4186V >99% Sigma-Aldrich - Sulfamethoxazole 723-46-6 BCBN0016V n.b. Fluka -

Theophylline 58-55-9 10148004 n.b. Alfa Aesar -

Torasemide 56211-40-6 1340038 n.b. abcr GmbH - Triclosan 3380-34-5 LRAA1072 n.b. Fluka -

Warfarin 81-81-2 MKBS1257V n.b. Fluka -

Atenolol 29122-68-7 BCBN4825V >98% Sigma-Aldrich -

Cimetidine 51481-61-9 MKBV2403V n.b. Sigma-Aldrich -

Cinnarizine 298-57-7 BCBP4836V n.b. Sigma-Aldrich - Citalopram 59729-33-8 WG0121801 n.b. Fluorochem Ltd. Hydrobromide

Diphenhydramine 58-73-1 MKBQ9569V >98% Sigma-Aldrich Hydrochloride

Doxylamine 469-21-6 109K1438V n.b. Sigma-Aldrich Succinate Fluoxetine 54910-89-3 129K2148 n.b. Sigma-Aldrich Hydrochloride

Lamotrigine 84057-84-1 0455123-14 >98% Cayman Chemical Company -

Lidocaine 137-58-6 SLBJ7206V 99.70% Sigma-Aldrich Hydrochloride Loratadine 79794-75-5 LRAA9165 n.b. Sigma-Aldrich -

Metoprolol 37350-58-6 BCBH0239V >98% Sigma-Aldrich Tartrate

Propranolol 525-66-6 BCBL8710V >99% Sigma-Aldrich Hydrochloride Tramadol 27203-92-5 n.b. n.b. n.b. Hydrochloride

Venlafaxine 93413-69-5 2593609 99.10% LKT Laboratories Hydrochloride

Verapamil 52-53-9 SLBH9660V >99% Sigma-Aldrich Hydrochloride

Candesartan 139481-59-7 F17X009 >98% Alfa Aesar -

Cetirizine 83881-51-0 BCBK5848V >98% Sigma-Aldrich Dihydrochloride

Enalapril 75847-73-3 0458574-16 >98% Cayman Chemical Company - Fexofenadine 83799-24-0 G0K303 n.b. Twinbrook Pkwy Hydrochloride

Genistein 446-72-0 Bertin Pharma -

Irbesartan 138402-11-6 0440822-9 >98% Cayman Chemical Company -

Labetalol 36894-69-6 077K1824V >98% Sigma-Aldrich Hydrochloride

Telmisartan 144701-48-4 0437940-22 >98% Cayman Chemical Company -

Valsartan 137862-53-4 LRAA0634 n.b. Fluka -

Table S2 Additional information on the physicochemical properties (some data are reprinted from (Escher et al., 2017)). The data are sorted in groups of acidic, basic, multiprotic chemicals and alphabetically within the groups.

log Klipw

Name log Kow Reference pKa Reference (neutral) Reference log Klipw (ion) Reference (25°C)

S2 Electronic Supplementary Information Environmental Science: Processes & Impacts Electronic Supplementary Information

log Klipw

Name log Kow Reference pKa Reference (neutral) Reference log Klipw (ion) Reference (25°C)

NEUTRAL PHARMACEUTICALS

a PhysProp (Newton and Kluza, Caffeine -0.07 0.6; 14 0.28 PP-LFERb - - exp. 1978) Carbama- PhysProp 2.45 - - 3.10 PP-LFER - - zepine exp. ACIDIC PHARMACEUTICALS Acetyl- sulfametho- 1.18 PP-LFER 5.54 (Prankerd, 2007) 1.74 PP-LFER 1.61 COSMOmicd xazole Bezafibrate 4.07 PP-LFER 3.6 (Huber et al., 2003b) 4.25 PP-LFER 1.81 COSMOmic PhysProp Clofibric acid 2.57 2.5 (Drillia et al., 2005) 2.62 PP-LFER 1.83 COSMOmic exp. PhysProp Diclofenac 4.51 3.99 (Avdeef et al., 1998) 4.45 (Avdeef et al., 1998) 2.64 (Avdeef et al., 1998) exp. PhysProp (Newton and Kluza, Furosemide 2.03 3.9 2.40 PP-LFER 2.46 COSMOmic exp. 1978) PhysProp Ibuprofen 3.97 4.45 (Avdeef et al., 1998) 3.80 (Avdeef et al., 1998) 1.81 (Avdeef et al., 1998) exp. PhysProp Ketoprofen 3.12 4.22 (Geiser et al., 2005) 3.78 PP-LFER 1.69 COSMOmic exp. Losartan 5.06 PP-LFER 4.9 (Williams et al., 1996) 5.41 PP-LFER 2.40 COSMOmic Mefenamic PhysProp 5.12 4.55 (Prankerd, 2007) 5.39 PP-LFER 2.67 COSMOmic acid exp. PhysProp Naproxen 3.18 4.18 (Bouchard et al., 2002) 3.84 PP-LFER 1.92 COSMOmic exp. Paracetamol 0.48 PP-LFER 9.38 PhysProp, exp. 1.02 COSMOmic 1.35 COSMOmic PhysProp (Ottiger and Wunderli- (Ottiger and Wunderli- Salicylic acid 2.26 2.93 (Geiser et al., 2005) 2.50 1.04 exp. Allenspach, 1997) Allenspach, 1997) PhysProp PhysProp, exp. Sangster Sulfadimidine 0.19 7.59 1.53 PP-LFER 1.61 COSMOmic exp. (1994) Sulfametho- PhysProp 0.89 5.70 (Huber et al., 2003a) 1.26 PP-LFER 1.56 COSMOmic xazole exp. PhysProp Theophylline -0.04 8.81 (Kortüm et al., 1961) -0.37 COSMOmic 1.06 COSMOmic exp. Torasemide 3.54 PP-LFER 6.68 (Masereel, 1993) 3.48 PP-LFER 2.23 COSMOmic PhysProp Triclosan 4.76 7.9 (Lindström et al., 2002) 5.43 PP-LFER 3.41 COSMOmic exp. PhysProp (Ottiger and Wunderli- (Ottiger and Wunderli- (Ottiger and Wunderli- Warfarin 2.7 4.9 3.39 1.40 exp. Allenspach, 1997) Allenspach, 1997) Allenspach, 1997) BASIC PHARMACEUTICALS PhysProp (Betageri and Rogers, Atenolol 0.16 9.55 -0.08 pp-LFER 1.79 (Escher et al., 2006) exp. 1987) Cimetidine 0.19 PP-LFERb 7.11 (Prankerd, 2007) 0.46 COSMOmic 0.22 COSMOmic PhysProp 3.5e/ Cinnarizine 5.77 (Belsner et al., 1993) 5.73 pp-LFER 3.55 COSMOmic exp. 7.47 Citalopram 3.58 PP-LFER 9.59 (Vasskog et al., 2006) 3.30 PP-LFER 1.31 COSMOmic Diphen- PhysProp 3.27 9.02 (Prankerd, 2007) 4.80 COSMOmic 2.11 COSMOmic hydramine exp. Doxylamine 2.46 PP-LFER 9.2 (Prankerd, 2007) 4.46 COSMOmic 1.24 COSMOmic Fluoxetine 4.62 PP-LFER 9.62 (Balon et al., 1999) 4.28 pp-LFER 3.84 (Neuwoehner et al., 2009) Lamotrigine 2.25 PP-LFER 5.34 (Ishihama et al., 2002) 2.74 PP-LFER 1.74 COSMOmic PhysProp PhysProp, exp. Sangster Lidocaine 2.44 8.01 2.39 (Avdeef et al., 1998) 1.22 (Avdeef et al., 1998) exp. 1994 Loratadine 5.2 PhysProp 4.75 ACD/pKa GALAS 4.31 COSMOmic 1.76 COSMOmic

Electronic Supplementary Information S3 Electronic Supplementary Information Environmental Science: Processes & Impacts

log Klipw

Name log Kow Reference pKa Reference (neutral) Reference log Klipw (ion) Reference (25°C)

exp.

PhysProp (Newton and Kluza, Metoprolol 1.88 9.68 1.27 PP-LFER 1.43 (Escher et al., 2006) exp. 1978) PhysProp Propranolol 3.48 9.53 (Avdeef et al., 1998) 3.40 (Barzanti et al., 2007) 2.60 (Barzanti et al., 2007) exp. PhysProp (Azodi-Deilamia et al., Tramadol 2.51 9.41 2.44 PP-LFER 1.55 COSMOmic exp. 2010) Venlafaxine 3.40 PP-LFER 9.5 (Prankerd, 2007) 2.76 PP-LFER 1.80 COSMOmic PhysProp Verapamil 3.79 8.92 (Hasegawa et al., 1984) 3.82 PP-LFER 1.27 COSMOmic exp. COMPLEX & MULTIPROTIC PHARMACEUTICALS 2.61(A);3.7 3.13 (anion), 3.13 Candesartan 4.21 ppLFER 3(A); ACD/pKa GALAS 4.91 pp-LFER COSMOmic (dianion) 4.85(B) 2.2 (B), 2.9 (Plemper van Balen 2.3 (zwitterion and (Plemper van Balen et al., Cetirizine 3.96 PP-LFER 3.60 pp-LFER (A), 8.0 (B) et al., 2001) anion), 3.2 (cation) 2001) 3.0 (A); 5.5 (Loftsson et al., 1.39 (anion), 0.34 Enalapril 0.07 PP-LFER 2.66 pp-LFER COSMOmic (B) 2010) (zwitterion) 4.25 (A); 4.31 (cation), 3.31 (Yasui-Furukori et Fexofenadine 7.21 PP-LFER 9.53 (B) 6.58 pp-LFER (zwitterion), 5.1 COSMOmic al., 2005) (anion) 7. (A)2; (Zielonka et al., 3.30 (anion), 3.7 Genistein 3.08 PP-LFER 10.0 (A); 3.65 PP-LFER COSMOmic 2003) (dianion) 13.1 (A) 3.7 (A); 1.2 (zwitterion), Irbesartan 6.38 PP-LFER ACD/pKa GALAS 6.54 pp-LFER COSMOmic 10.2 (B) 2.13 (anion) 7.35 (A), 2.32 (cation), 1.84 Labetalol 2.55 PP-LFER 9.11 (B) ACD/pKa GALAS 2.20 pp-LFER (anion), 3.01 COSMOmic (zwitterion) 3.65 (A); 1.59 (zwitterion), Telmisartan 7.74 PP-LFER 4.78 (B); (Chemaxon, 2016) 7.81 pp-LFER COSMOmic 5.01 (zwitterion) 6.54 (B) 3.9 (A); Valsartan 3.84 PP-LFER (Flesch et al., 1997) 5.00 pp-LFER 2.99 (dianion) COSMOmic 4.73 (A)

a b PhysProp database accessed via Episuite (U.S. EPA, 2009). Calculated with the PP-LFER equation for log Klipw = 0.48 + 0.55L – 0.95 S – 0.05 A -4.02 B + 1.65 V(Endo et al., 2011) and chemical parameters taken from the UFZ-LSER database v 3.1 (Endo et al., 2015). cACD/Percepta (ACD/Percepta, 2015). dCalculated withCOSMOmic extended for the e description of charged organic chemicals via the implementation of the membrane bilayer potential (Bittermann et al., 2014). first pKa value (3.5) not relevant for the speciation of the molecule, therefore listed as monoprotic base even though it is in reality a diprotic base.

S4 Electronic Supplementary Information Environmental Science: Processes & Impacts Electronic Supplementary Information

S3. Speciation of pharmaceuticals

pK1 " " " """""""pK2 " "" + " """ " + " The speciation of diprotic acids and bases for the reaction A " """B"+"H C"+"2H " is as follows:

1 f = A pH−pK 2⋅pH−pK −pK 1+10 1 +10 1 2 (S1)

1 f = B pK −pH pH−pK 1+10 1 +10 2 (S2)

1 f = C pK +pK "2⋅pH pK −2⋅pH 1+10 1 2 +10 2 (S3)

Electronic Supplementary Information S5 Electronic Supplementary Information Environmental Science: Processes & Impacts

Table S3 Fraction of neutral species and liposome-water distribution ratios log Dlipw(pH) at the pH values of the experiments.

fneutral fneutral fneutral fneutral fneutral log Dlipw log Dlipw log Dlipw log Dlipw log Dlipw Name a a a a a b b b b b (pH 5.5) (pH 6) (pH 7) (pH 8) (pH 9) (pH 5.5) (pH 6) (pH 7) (pH 8) (pH 9)

ACIDIC PHARMACEUTICALS

Acetyl- sulfametho- 52.3% 25.7% 3.4% 0.346% 0.035% 1.68 1.65 1.62 1.61 1.61 xazole Bezafibrate 1.2% 0.4% 0.04% 0.004% 0.000% 2.46 2.13 1.86 1.81 1.81

Clofibric acid 0.0999% 0.0316% 0.0032% 0.0003% 0.00001% 1.83 1.83 1.83 1.83 1.83 Diclofenac 3.0% 1.0% 0.10% 0.010% 0.001% 3.10 2.85 2.67 2.64 2.64

Furosemide 2.5% 0.8% 0.08% 0.008% 0.001% 2.46 2.46 2.46 2.46 2.46 Ibuprofen 8.2% 2.7% 0.3% 0.028% 0.003% 2.76 2.37 1.91 1.82 1.81 Ketoprofen 5.0% 1.6% 0.2% 0.017% 0.002% 2.54 2.17 1.77 1.70 1.69 Losartan 20.1% 7.4% 0.8% 0.079% 0.008% 4.71 4.28 3.36 2.66 2.43 Mefenamic 10.1% 3.4% 0.4% 0.035% 0.004% 4.40 3.95 3.12 2.74 2.68 acid Naproxen 4.6% 1.5% 0.2% 0.015% 0.002% 2.60 2.27 1.97 1.93 1.92

Paracetamol 100.0% 100.0% 99.6% 96.0% 70.6% 1.02 1.02 1.02 1.04 1.15

Salicylic acid 0.27% 0.085% 0.00851% 0.00085% 0.00009% 1.07 1.05 1.04 1.04 1.04 Sulfadimidine 99.2% 97.5% 79.6% 28.01% 3.75% 1.53 1.53 1.55 1.59 1.61 Sulfa- 61.3% 33.4% 4.8% 0.499% 0.050% 1.40 1.48 1.55 1.56 1.56 methoxazole Theophylline 100.0% 99.8% 98.5% 86.6% 39.2% -0.36 -0.35 -0.22 0.28 0.85

Torasemide 93.8% 82.7% 32.4% 4.57% 0.476% 3.45 3.40 3.04 2.48 2.26

Triclosan 99.6% 98.8% 88.8% 44.3% 7.36% 5.43 5.42 5.38 5.08 4.34

Warfarin 20.1% 7.4% 0.8% 0.079% 0.008% 2.71 2.31 1.65 1.43 1.40

BASIC PHARMACEUTICALS

Atenolol 0.01% 0.03% 0.28% 2.74% 22.0% 1.79 1.79 1.79 1.77 1.68

Cimetidine 2.40% 7.20% 43.7% 88.6% 98.7% 0.23 0.24 0.34 0.44 0.46

Cinnarizine 1.05% 3.27% 25.30% 77.21% 97.13% 3.96 4.32 5.14 5.62 5.72 Citalopram 0.01% 0.03% 0.26% 2.51% 20.45% 1.31 1.32 1.41 1.84 2.63 Diphen- 0.03% 0.10% 0.95% 8.72% 48.8% 2.17 2.28 2.86 3.75 4.49 hydramine Doxylamine 0.02% 0.06% 0.63% 5.94% 38.69% 1.36 1.55 2.30 3.24 4.05

Fluoxetine 0.01% 0.02% 0.24% 2.34% 19.35% 3.84 3.84 3.84 3.86 3.97

Lamotrigine 59.1% 82.0% 97.9% 99.8% 100.0% 2.54 2.66 2.73 2.74 2.74 Lidocaine 0.31% 0.97% 8.90% 49.42% 90.72% 1.24 1.27 1.57 2.11 2.35

Loratadine 84.9% 94.7% 99.4% 99.9% 100.0% 4.24 4.29 4.31 4.31 4.31

Metoprolol 0.01% 0.02% 0.21% 2.05% 17.28% 1.43 1.43 1.43 1.43 1.41 Propranolol 0.01% 0.03% 0.29% 2.87% 22.79% 2.60 2.60 2.61 2.66 2.94 Tramadol 0.01% 0.04% 0.39% 3.74% 28.01% 1.55 1.55 1.56 1.65 2.01

Venlafaxine 0.01% 0.03% 0.32% 3.07% 24.03% 1.80 1.80 1.81 1.90 2.27 Verapamil 0.04% 0.12% 1.19% 10.73% 54.59% 1.32 1.42 1.99 2.86 3.56

COMPLEX & MULTIPROTIC PHARMACEUTICALS

Candesartan No neutral species 3.13 3.13 Cetirizine No neutral species 2.31 2.30 2.30 2.30 2.30

Enalapril No neutral species 1.13 1.28 1.38 1.39 1.39

Fexofenadine No neutral species 4.31 4.31 4.32 4.37 4.65

Genistein 98.0% 94.1% 61.3% 13.6% 1.4% 3.65 3.65 3.61 3.46 3.27

S6 Electronic Supplementary Information Environmental Science: Processes & Impacts Electronic Supplementary Information

fneutral fneutral fneutral fneutral fneutral log Dlipw log Dlipw log Dlipw log Dlipw log Dlipw Name a a a a a b b b b b (pH 5.5) (pH 6) (pH 7) (pH 8) (pH 9) (pH 5.5) (pH 6) (pH 7) (pH 8) (pH 9)

1.20 1.20 1.20 1.22 1.26 Irbesartan No neutral species

Labetalol No neutral species 2.34 2.39 2.66 2.92 2.78 Telmisartan No neutral species 4.23 3.54 2.55 2.50 2.51

Valsartan No neutral species 3.42 3.17 3.01 2.99 2.99 aCalculated with eq. 3 and 4. bCalculated with eq. 12.

Electronic Supplementary Information S7 Electronic Supplementary Information Environmental Science: Processes & Impacts

S3. Concentration-effect curves

Caffeine

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio Aliivibrio fischeri Aliivibrio 20 in in 0 Inhibition of bioluminescence of Inhibition Inhibition of bioluminescence of Inhibition 0

-5 -4 -3 -2 -1 -5 -4 -3 -2 -1 -5 -4 -3 -2 -1 -5 -4 -3 -2 -1 -5 -4 -3 -2 -1 -5 -4 -3 -2 -1 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Carbamazepine

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio 20 fischeri Aliivibrio in in 0 Inhibition of bioluminescence of Inhibition 0 bioluminescence of Inhibition

-7 -6 -5 -4 -3 -2 -1 -6 -5 -4 -3 -2 -1 -6 -5 -4 -3 -2 -1 -6 -5 -4 -3 -2 -1 -6 -5 -4 -3 -2 -1 -7 -6 -5 -4 -3 -2 -1 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Fig. S2 Concentration-effect curves of the neutral pharmaceuticals. The different symbols in each of the single concentration-effect curves relate to independent repeats of the experiment on different days. On the right for comparison all curves overlaid, with one symbol per pH value.

Acetyl-sulfamethoxazole

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio 20 fischeri Aliivibrio in in 0 Inhibition of bioluminescence of Inhibition 0 bioluminescence of Inhibition

-6 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -6 -5 -4 -3 -2 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Bezafibrate

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri 20 Aliivibrio fischeri in in 0 Inhibition of bioluminescence 0 Inhibition of bioluminescence

-5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -6 -5 -4 -3 -2 -1 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Clofibric Acid

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio Aliivibrio fischeri Aliivibrio 20 in in 0 Inhibition of bioluminescence of Inhibition 0 bioluminescence of Inhibition

-5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -1 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

S8 Electronic Supplementary Information Environmental Science: Processes & Impacts Electronic Supplementary Information

Diclofenac

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri 20 Aliivibrio fischeri in in 0 Inhibition of bioluminescence 0 Inhibition of bioluminescence

-6 -5 -4 -6 -5 -4 -6 -5 -4 -6 -5 -4 -6 -5 -4 -6 -5 -4 -3 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Furosemid

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri 20 Aliivibrio fischeri in in 0 Inhibition of bioluminescence 0 Inhibition of bioluminescence

-5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Ibuprofen

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

Ketoprofen

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

Losartan

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 precipitation pH 7 40 40 - data not used pH 8 for derivation pH 9 20 Aliivibrio fischeri Aliivibrio fischeri of EC 20 50 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

Mefenamic Acid

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

Electronic Supplementary Information S9 Electronic Supplementary Information Environmental Science: Processes & Impacts

Naproxen

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-6 -5 -4 -3 -2 -1 -5 -4 -3 -2 -1 -5 -4 -3 -2 -1 -5 -4 -3 -2 -1 -5 -4 -3 -2 -1 -6 -5 -4 -3 -2 -1 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Paracetamol

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-6 -5 -4 -3 -2 -1-6 -5 -4 -3 -2 -6-1 -5 -4 -3 -2 -6-1 -5 -4 -3 -2 -6-1 -5 -4 -3 -2 -1 -6 -5 -4 -3 -2 -1 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Salicylic Acid

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri 20 Aliivibrio fischeri in in 0 Inhibition of bioluminescence 0 Inhibition of bioluminescence

Sulfadimidine

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-6 -5 -4 -3 -2 -1-6 -5 -4 -3 -2 -6-1 -5 -4 -3 -2 -1 -5 -4 -3 -2 -6-1 -5 -4 -3 -2 -1 -6 -5 -4 -3 -2 -1 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Sulfamethoxazole

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -7 -6 -5 -4 -3 -2 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Theophylline

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

S10 Electronic Supplementary Information Environmental Science: Processes & Impacts Electronic Supplementary Information

Torasemide

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-6 -5 -4 -3 -6 -5 -4 -3 -6 -5 -4 -3 -6 -5 -4 -3 -6 -5 -4 -3 -6 -5 -4 -3 -2 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Triclosan

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri 20 Aliivibrio fischeri in in 0 inhibition of bioluminescence 0 inhibition of bioluminescence

-7 -6 -5 -4 -7 -6 -5 -4 -7 -6 -5 -4 -7 -6 -5 -4 -7 -6 -5 -4 -7 -6 -5 -4 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Warfarin

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 not well pH 9 20 Aliivibrio fischeri Aliivibrio fischeri dissolved 20 in in (not used for derivation 0 Inhibition of bioluminescence Inhibition of bioluminescence 0 of EC50) -7 -6 -5 -4 -3 -5 -4 -3 -5 -4 -3 -5 -4 -3 -5 -4 -3 -6 -5 -4 -3 -2 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Fig. S3 Concentration-effect curves of the acidic pharmaceuticals. The different symbols in each of the single concentration-effect curves relate to independent repeats of the experiment on different days. On the right for comparison all curves overlaid, with one symbol per pH value.

Atenolol Atenolol

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri 20 Aliivibrio fischeri in in 0 Inhibition of bioluminescence 0 Inhibition of bioluminescence

-5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -7 -6 -5 -4 -3 -2 -1 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Cimetidine

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

Electronic Supplementary Information S11 Electronic Supplementary Information Environmental Science: Processes & Impacts

Cinnarizine

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100 not well 80 dissolved 80 [%] [%] pH 5.5 (not used 60 pH 6 60 for derivation pH 7 of EC50) 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-7 -6 -5 -4 -6 -5 -4 -6 -5 -4 -7 -6 -5 -4 -7 -6 -5 -4 -7 -6 -5 -4 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Citalopram

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri 20 Aliivibrio fischeri in in 0 Inhibition of bioluminescence 0 Inhibition of bioluminescence

-6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -7 -6 -5 -4 -3 -2 -1 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Diphenhydramine

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-6 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -7 -6 -5 -4 -3 -2 -1 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Doxylamine

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

Fluoxetine

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri 20 Aliivibrio fischeri in in 0 Inhibition of bioluminescence 0 Inhibition of bioluminescence

-7 -6 -5 -4 -7 -6 -5 -4 -7 -6 -5 -4 -7 -6 -5 -4 -7 -6 -5 -4 -7 -6 -5 -4 -3 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Lamotrigine

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

S12 Electronic Supplementary Information Environmental Science: Processes & Impacts Electronic Supplementary Information

Lidocaine

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

Loratadine

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

Propranolol

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -1 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Metoprolol

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

Tramadol

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-5 -4 -3 -2 -5 -4 -3 -5 -4 -3 -5 -4 -3 -5 -4 -3 -6 -5 -4 -3 -2 -1 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Venlafaxine

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-6 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -5 -4 -3 -2 -6 -5 -4 -3 -2 -1 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Electronic Supplementary Information S13 Electronic Supplementary Information Environmental Science: Processes & Impacts

Verapamil

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-6 -5 -4 -3 -5 -4 -3 -5 -4 -3 -5 -4 -3 -5 -4 -3 -6 -5 -4 -3 -2 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Fig. S4. Concentration-effect curves of the basic pharmaceuticals. The different symbols in each of the single concentration-effect curves relate to independent repeats of the experiment on different days. On the right for comparison all curves overlaid, with one symbol per pH value.

Candesartan

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-6 -5 -4 -3 -5 -4 -3 -5 -4 -3 -5 -4 -3 -5 -4 -3 -6 -5 -4 -3 -2 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Cetirizine

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri 20 Aliivibrio fischeri in in 0 Inhibition of bioluminescence 0 Inhibition of bioluminescence

Enalapril

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence 0 Inhibition of bioluminescence

Fexofenadine

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-6 -5 -4 -3 -6 -5 -4 -3 -6 -5 -4 -3 -6 -5 -4 -3 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Genistein

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-7 -6 -5 -4 -3 -6 -5 -4 -3 -6 -5 -4 -3 -6 -5 -4 -3 -6 -5 -4 -3 -7 -6 -5 -4 -3 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

S14 Electronic Supplementary Information Environmental Science: Processes & Impacts Electronic Supplementary Information

Irbesartan

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri 20 Aliivibrio fischeri in in 0 Inhibition of bioluminescence 0 Inhibition of bioluminescence

-6 -5 -4 -3 -5 -4 -3 -5 -4 -3 -5 -4 -3 -5 -4 -3 -6 -5 -4 -3 -2 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Labetalol

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence 0 Inhibition of bioluminescence

Telmisartan

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 precipitation 40 pH 8 40 at high pH 9 concentrations 20 Aliivibrio fischeri Aliivibrio fischeri 20 (not used for in in derivation of EC50) 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-7 -6 -5 -4 -3 -2 -6 -5 -4 -3 -6 -5 -4 -3 -6 -5 -4 -3 -6 -5 -4 -3 -7 -6 -5 -4 -3 -2 -1 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Valsartan

100 pH 5.5 pH 6 pH 7 pH 8 pH 9 100

80 80 [%] [%] pH 5.5 60 pH 6 60 pH 7 40 40 pH 8 pH 9 20 Aliivibrio fischeri Aliivibrio fischeri 20 in in 0 Inhibition of bioluminescence Inhibition of bioluminescence 0

-6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 -6 -5 -4 -3 -2 log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M] log concentration [M]

Fig. S5 Concentration-effect curves of the complex and multiprotic pharmaceuticals. The different symbols in each of the single concentration-effect curves relate to independent repeats of the experiment on different days. On the right for comparison all curves overlaid, with one symbol per pH value.

Electronic Supplementary Information S15 Electronic Supplementary Information Environmental Science: Processes & Impacts

Table S4. Experimental effect concentrations EC50 in the Microtox assay derived from the concentration effect curves depicted in Figure S2 to S4 using the log-logistic concentration effect model:

100% inhibition!%#= slope logEC log(concentration " $ ⋅( 50− ) 1+10 (S4)

Name logEC50 (pH 5.5) log EC50 (pH 6) log EC50 (pH 7) log EC50 (pH 8) log EC50 (pH 9)

NEUTRAL PHARMACEUTICALS

Caffeine -2.39±0.01 -2.43±0.01 -2.44±0.02 -2.45±0.01 -2.46±0.02

Carbamazepine -3.18±0.03 -3.13±0.03 -3.13±0.04 -3.32±0.03 -3.30±0.03

ACIDIC PHARMACEUTICALS

Acetyl-sulfamethoxazole -3.30±0.01 -2.94±0.01 no effect no effect no effect

Bezafibrate -4.20±0.01 -3.76±0.01 -2.59±0.02 no effect no effect

Clofibric acid -3.80±0.01 -3.50±0.01 -2.68±0.01 -2.00±0.02 no effect

Diclofenac -5.08±0.01 -4.64±0.01 -3.89±0.01 no effect no effect

Furosemide -3.35±0.01 -2.55±0.02 no effect no effect no effect

Ibuprofen -5.23±0.05 -4.03±0.01 -3.26±0.02 -2.60±0.03 -2.14±0.02

Ketoprofen -4.51±0.01 -4.03±0.01 -3.16±0.01 -2.32±0.02 -1.90±0.27

Losartan (solubility issues, excluded) -3.12±0.04 -2.54±0.05 no effect no effect no effect

Mefenamic acid -3.79±0.03 -3.60±0.04 -3.45±0.02 -3.50±0.02 -3.29±0.01

Naproxen -4.54±0.01 -3.86±0.01 -3.10±0.01 -2.34±0.02 no effect

Paracetamol no effect -1.86±0.03 -2.66±0.02 -3.04±0.01 -3.66±0.01

Salicylic acid -3.85±0.01 -3.29±0.01 -2.35±0.01 no effect no effect

Sulfadimidine -2.56±0.02 -2.59±0.02 no effect no effect no effect

Sulfamethoxazole -3.83±0.02 -3.73±0.02 -2.82±0.02 no effect no effect

Theophylline -2.04±0.03 -2.01±0.03 -1.99±0.03 -1.98±0.02 -2.00±0.03

Torasemide -3.67±0.02 -3.53±0.03 -2.88±0.05 no effect no effect

Triclosan -5.12±0.01 -4.83±0.01 -4.52±0.01 -4.62±0.01 -4.59±0.01

Warfarin -4.10±0.01 -3.96±0.02 -3.37±0.01 -2.89±0.02 -2.72±0.03

S16 Electronic Supplementary Information Environmental Science: Processes & Impacts Electronic Supplementary Information

Table S4. continued

Name logEC50 (pH 5.5) log EC50 (pH 6) log EC50 (pH 7) log EC50 (pH 8) log EC50 (pH 9)

BASIC PHARMACEUTICALS

Atenolol no effect no effect no effect no effect -2.59±0.02

Cimetidine no effect no effect -2.01±0.02 -2.19±0.02 -2.26±0.02

Cinnarizine -4.66±0.02 -4.41±0.01 -4.39±0.02 no effect no effect

Citalopram -2.62±0.04 -2.20±0.08 -2.86±0.02 -3.93±0.02 -4.63±0.02

Diphenhydramine -2.80±0.02 -2.42±0.02 -2.45±0.03 -3.31±0.02 -4.25±0.03

Doxylamine -2.00±0.04 -1.75±0.05 -2.33±0.03 -3.17±0.03 -3.96±0.01

Fluoxetine no effect no effect no effect -4.23±0.03 -4.92±0.01

Lamotrigine -2.88±0.05 -3.05±0.04 -2.96±0.03 -2.98±0.03 -3.03±0.02

Lidocaine no effect no effect no effect -2.27±0.01 -2.64±0.01

Loratadine -4.90±0.02 -4.59±0.02 -4.14±0.05 -4.13±0.05 -4.32±0.05

Metoprolol no effect no effect no effect -2.44±0.01 -3.27±0.01

Propranolol -2.68±0.03 -2.40±0.02 -2.65±0.01 -3.46±0.01 -4.18±0.01

Tramadol no effect no effect no effect no effect -2.91±0.01

Venlafaxine no effect no effect no effect -2.51 -3.45±0.01

Verapamil -2.82±0.02 -2.26±0.05 -2.62±0.01 -2.54±0.02 -2.32±0.06

COMPLEX & MULTIPROTIC PHARMACEUTICALS

Candesartan -2.90±0.01 -2.39±0.01 no effect no effect no effect

Cetirizine -3.37±0.01 -3.32±0.01 -3.16±0.01 -3.04±0.01 -2.73±0.01

Enalapril -2.62±0.01 no effect no effect no effect no effect

Fexofenadine no effect no effect no effect no effect -2.82±0.03

Genistein -3.86±0.2 -4.26±0.06 no effect -3.76±0.02 -3.35±0.03

Irbesartan -4.09±0.02 -3.48±0.01 -2.56±0.01 no effect no effect

Labetalol -2.57±0.03 -2.42±0.01 -2.58±0.01 -2.88±0.02 -2.86±0.02

Telmisartan -4.44±0.02 no effect no effect no effect -2.96±0.02

Valsartan -3.15±0.01 -2.51±0.01 no effect no effect no effect

Electronic Supplementary Information S17 Electronic Supplementary Information Environmental Science: Processes & Impacts

Table S5. Slopes of the concentration effect curves (eqn. S4) depicted in Figure S2 to S4.

Name slope (pH 5.5) slope (pH 6) slope (pH 7) slope (pH 8) slope (pH 9)

NEUTRAL PHARMACEUTICALS

Caffeine 1.49 1.42 1.31 1.198 1.51

Carbamazepine 1.21 0.99 0.84 Set to 1 1.11

ACIDIC PHARMACEUTICALS

Acetyl-sulfamethoxazole 1.67 1.51 no effect no effect no effect

Bezafibrate 1.55 1.59 1.43 0.89 no effect

Clofibric acid 1.80 1.67 1.71 2.02 no effect

Diclofenac 2.57 2.69 2.42 no effect no effect

Furosemide 2.32 2.06 no effect no effect no effect

Ibuprofen 1.00 1.12 1.12 1.00 2.93

Ketoprofen 1.75 1.71 1.81 2.37 2.64

Losartan (solubility issues, excluded) 1.57 1.92 no effect no effect no effect

Mefenamic acid 1.31 1.20 1.88 2.51 3.71

Naproxen 1.29 1.24 1.41 1.55 no effect

Paracetamol no effect 2.29 2.04 2.58 2.61

Salicylic acid 1.06 1.10 1.20 no effect no effect

Sulfadimidine 1.01 1.34 no effect no effect no effect

Sulfamethoxazole 0.92 1.00 1.17 no effect no effect

Theophylline 1.62 2.06 2.72 2.69 fixed to 3

Torasemide 1.13 1.15 1.15 no effect no effect

Triclosan 1.17 2.59 3.28 2.61 3.02

Warfarin 1.12 1.76 1.61 1.28 1.71

S18 Electronic Supplementary Information Environmental Science: Processes & Impacts Electronic Supplementary Information

Table S5. continued

Name slope (pH 5.5) slope (pH 6) slope (pH 7) slope (pH 8) slope (pH 9)

BASIC PHARMACEUTICALS

Atenolol no effect no effect no effect no effect 1.56

Cimetidine no effect no effect 2.14 1.77 2.13

Cinnarizine 2.10 2.29 2.39 no effect no effect

Citalopram fixed to 1 0.96 0.94 0.89 0.97

Diphenhydramine 0.92 0.87 1.06 1.04 0.77

Doxylamine 0.99 0.90 1.21 1.07 1.33

Fluoxetine no effect no effect no effect 1.88 2.04

Lamotrigine 1.39 1.14 1.32 1.01 1.18

Lidocaine no effect no effect no effect 2.11 2.99

Loratadine 1.81 1.96 1.48 1.55 1.90

Metoprolol no effect no effect no effect 0.94 1.11

Propranolol 0.71 1.04 1.69 1.17 1.36

Tramadol no effect no effect no effect no effect 1.53

Venlafaxine no effect no effect no effect 1.21 1.33

Verapamil 0.87 0.98 2.00 1.78 1.24

COMPLEX & MULTIPROTIC PHARMACEUTICALS

Candesartan 2.64 2.91 no effect no effect no effect

Cetirizine 1.76 1.72 1.99 2.26 5.17

Enalapril 2.37 no effect no effect no effect no effect

Fexofenadine no effect no effect no effect no effect 1.53

Genistein 0.72 0.65 no effect 1.49 3.61

Irbesartan 1.38 1.77 3.06 no effect no effect

Labetalol 0.91 1.96 2.35 1.19 1.43

Telmisartan 1.68 no effect no effect no effect 2.20

Valsartan 2.31 2.51 no effect no effect no effect

Electronic Supplementary Information S19 Electronic Supplementary Information Environmental Science: Processes & Impacts

Acidic pharmaceuticals Basic pharmaceuticals

-2

(M)) -4 50

-6 log (EC

neutral species, model 1 -8

Atenolol IbuprofenLosartan TriclosanWarfarin Tramadol Diclofenac Naproxen Cimetidine FluoxetineLidocaineLoratadineMetoprolol Verapamil BezafibrateFurosemideKetoprofen Torasemide CinnarizineCitalopramDoxylamineLamotrigine PropranololVenlafaxine Clofibric acid ParacetamolSalicylicSulfadimidine acidTheophylline Mefenamic acid Sulfamethoxazole Diphenhydramine

Acetyl-sulfamethoxazole

Fig. S6 Model 1 (eq. 1) applied to each data point individually and summary of resulting EC50 values for the neutral species.

Acidic pharmaceuticals Basic pharmaceuticals 0

-2

-4 model 3 (cytoplasm, pH 7) (M))

50 -6

log (EC -8

Atenolol Ibuprofen TriclosanWarfarin Tramadol Diclofenac Naproxen Cimetidine FluoxetineLidocaineLoratadineMetoprolol Verapamil Bezafibrate FurosemideKetoprofen Torasemide CinnarizineCitalopramDoxylamineLamotrigine PropranololVenlafaxine Clofibric acid ParacetamolSalicylicSulfadimidine acidTheophylline Mefenamic acid Sulfamethoxazole Diphenhydramine

Acetyl-sulfamethoxazole

Fig. S7 Range of EC50(cytoplasm, pH7.7) derived by model 3 (eq. 10).

S20 Electronic Supplementary Information Environmental Science: Processes & Impacts Electronic Supplementary Information

6 6 6 6 6 6 6 6 6 6 Acetyl-sulfamethoxazole Bezafibrate Clofibric acid Diclofenac Furosemide TR=10 5 TR=10 TR=1 5 5 TR=1 5 5 TR=10 TR=1 5 5 TR=10 TR=1 5 5 TR=10 TR=1 5 log(1/IEC log(1/IEC log(1/IEC log(1/IEC log(1/IEC 4 4 4 4 4 4 4 4 4 4 TR=0.1 TR=0.1 TR=0.1 TR=0.1 TR=0.1 (M)) (M)) (M)) (M)) (M)) 50 50 50 50 50 3 3 3 3 3 3 3 3 3 3 50 50 50 50 50 (M)) (M)) (M)) (M)) (M)) 2 2 2 2 2 2 2 2 2 2 log(1/EC log(1/EC log(1/EC log(1/EC log(1/EC

1 1 1 1 1 1 1 1 1 1

0 0 0 0 0 0 0 0 0 0 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6

logDlipw(pH) logDlipw(pH) logDlipw(pH) logDlipw(pH) logDlipw(pH)

6 6 6 6 6 6 6 6 6 6 Ibuprofen Ketoprofen Losartan Mefenamic acid Naproxen 5 TR=10 TR=1 5 5 TR=10 TR=1 5 5 TR=10 TR=1 5 5 TR=10 TR=1 5 5 TR=10 TR=1 5 log(1/IEC log(1/IEC log(1/IEC log(1/IEC log(1/IEC 4 4 4 4 4 4 4 4 4 4 TR=0.1 TR=0.1 TR=0.1 TR=0.1 TR=0.1 (M)) (M)) (M)) (M)) (M)) 50 50 50 50 50 3 3 3 3 3 3 3 3 3 3 50 50 50 50 50 (M)) (M)) (M)) (M)) (M)) 2 2 2 2 2 2 2 2 2 2 log(1/EC log(1/EC log(1/EC log(1/EC log(1/EC

1 1 1 1 1 1 1 1 1 1

0 0 0 0 0 0 0 0 0 0 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6

logDlipw(pH) logDlipw(pH) logDlipw(pH) logDlipw(pH) logDlipw(pH)

6 6 6 6 6 6 6 6 6 6 Paracetamol Salicylic acid Sulfadimidine Sulfamethoxazole Theophylline 5 TR=10 TR=1 5 5 TR=10 TR=1 5 5 TR=10 TR=1 5 5 TR=10 TR=1 5 5 TR=10 TR=1 5 log(1/IEC log(1/IEC log(1/IEC log(1/IEC log(1/IEC 4 4 4 4 4 4 4 4 4 4 TR=0.1 TR=0.1 TR=0.1 TR=0.1 TR=0.1 (M)) (M)) (M)) (M)) (M)) 50 50 50 50 50 3 3 3 3 3 3 3 3 3 3 50 50 50 50 50 (M)) (M)) (M)) (M)) (M)) 2 2 2 2 2 2 2 2 2 2 log(1/EC log(1/EC log(1/EC log(1/EC log(1/EC

1 1 1 1 1 1 1 1 1 1

0 0 0 0 0 0 0 0 0 0 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6

logDlipw(pH) logDlipw(pH) logDlipw(pH) logDlipw(pH) logDlipw(pH)

6 6 6 6 6 6 6 6 Torasemide Triclosan Warfarin Genistein 5 TR=10 TR=1 5 5 5 5 5 5 5 log(1/IEC log(1/IEC log(1/IEC log(1/IEC 4 4 4 4 4 4 4 4 TR=0.1 (M)) (M)) (M)) (M)) 50 50 50 50 3 3 3 3 3 3 3 3 50 50 50 50 (M)) (M)) (M)) (M)) 2 2 2 2 2 2 2 2 log(1/EC log(1/EC log(1/EC log(1/EC

TR=10 TR=10 TR=10 1 1 1 1 1 1 1 1 TR=1 TR=1 TR=0.1 TR=1 TR=0.1 TR=0.1 0 0 0 0 0 0 0 0 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 logD (pH) logD (pH) logD (pH) logD (pH) lipw lipw lipw lipw

Fig. S8 QSAR analysis for model 2 (eq. 8) and model 3 (eq. 10) for the acidic pharmaceuticals and genistein (diprotic acid).

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6 6 6 6 7 7 6 6 6 6 Atenolol Cimetidine Cinnarizine Citalopram Diphenhydramine 5 TR=10 TR=1 5 5 TR=10 TR=1 5 6 6 5 TR=10 TR=1 5 5 5 log(1/IEC log(1/IEC log(1/IEC log(1/IEC log(1/IEC 4 4 4 4 5 5 4 4 4 4 TR=0.1 TR=0.1 TR=0.1 (M)) (M)) (M)) (M)) (M)) 50 50 50 50 50 3 3 3 3 4 4 3 3 3 3 50 50 50 50 50 (M)) (M)) (M)) (M)) (M)) 2 2 2 2 3 3 2 2 2 2 log(1/EC log(1/EC log(1/EC log(1/EC log(1/EC

TR=10 1 1 1 1 2 2 1 1 1 1 TR=10 TR=1 TR=0.1 TR=1 TR=0.1 0 0 0 0 1 1 0 0 0 0 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6

logDlipw(pH) logDlipw(pH) logDlipw(pH) logDlipw(pH) logDlipw(pH)

6 6 6 6 6 6 6 6 6 6 Doxylamine Fluoxetine Lamotrigine Lidocaine Loratidine 5 TR=10 TR=1 5 5 TR=10 TR=1 5 5 TR=10 TR=1 5 5 TR=10 TR=1 5 5 5 log(1/IEC log(1/IEC log(1/IEC log(1/IEC log(1/IEC 4 4 4 4 4 4 4 4 4 4 TR=0.1 TR=0.1 TR=0.1 TR=0.1 (M)) (M)) (M)) (M)) (M)) 50 50 50 50 50 3 3 3 3 3 3 3 3 3 3 50 50 50 50 50 (M)) (M)) (M)) (M)) (M)) 2 2 2 2 2 2 2 2 2 2 log(1/EC log(1/EC log(1/EC log(1/EC log(1/EC

TR=10 1 1 1 1 1 1 1 1 1 1

TR=1 TR=0.1 0 0 0 0 0 0 0 0 0 0 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6

logDlipw(pH) logDlipw(pH) logDlipw(pH) logDlipw(pH) logDlipw(pH)

6 6 6 6 6 6 6 6 6 6 Metoprolol Propranolol Tramadolol Venlafaxine Verapamil 5 TR=10 TR=1 5 5 TR=10 TR=1 5 5 TR=10 TR=1 5 5 TR=10 TR=1 5 5 TR=10 TR=1 5 log(1/IEC log(1/IEC log(1/IEC log(1/IEC log(1/IEC 4 4 4 4 4 4 4 4 4 4 TR=0.1 TR=0.1 TR=0.1 TR=0.1 TR=0.1 (M)) (M)) (M)) (M)) (M)) 50 50 50 50 50 3 3 3 3 3 3 3 3 3 3 50 50 50 50 50 (M)) (M)) (M)) (M)) (M)) 2 2 2 2 2 2 2 2 2 2 log(1/EC log(1/EC log(1/EC log(1/EC log(1/EC

1 1 1 1 1 1 1 1 1 1

0 0 0 0 0 0 0 0 0 0 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 -2 -1 0 1 2 3 4 5 6 logD (pH) logD (pH) logD (pH) logD (pH) logD (pH) lipw lipw lipw lipw lipw

Fig. S9 QSAR analysis for model 2 (eq. 8) and model 3 (eq. 10) for the basic pharmaceuticals

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