Methamphetamine

SUPPORTING INFORMATION FOR:

Reflection of socio-economic changes in wastewater: licit and illicit

drug use patterns

Νikolaos S. Τhomaidis1,*, Pablo Gago-Ferrero1, Christoph Ort2, Niki C. Maragou1,

Nikiforos A. Alygizakis1, Viola L. Borova1, Marilena Dasenaki1

1 Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens,

Panepistimiopolis Zografou, 15771 Athens, Greece

2 Eawag, Swiss Federal Institute of Aquatic Science and Technology, Überlandstrasse 133,

CH-8600 Dübendorf, Switzerland

Pages: 61 Tables: 13 Figures: 3

*Corresponding author: Tel: +30 210 7274317 Fax: +30 210 7274750 E-mail: [email protected]

For Submission to: Environmental Science & Technology

CONTENTS

SI-1: Detailed substance information……………………………………………………………………………… S3

SI-2: Analytical method………………………………………………………………………………………………….. S9

SI-3: Pharmacokinetics and other relevant data used for back-calculated use of the selected substances……………………………………………………………………………………………………… S15

SI-4: Concentration values and loads of the target compounds in the analyzed influent wastewaters………………………………………………………………………………………………………………… S23

SI-5: Greek socio-economic indicators from 2010 to 2014……………………………………………. S58

SI-1: Detailed substance information

All drug standards were of high purity grade (>90%) and were purchased from Sigma-Aldrich (Athens, Greece) except sulfadoxine (SDX) and sulfaclozine (SClZ), which were donated by the National Laboratory of Residue Analysis of Food of Animal Origin of the Hellenic Ministry of Rural Development and Food. Regarding psychotropic drugs and illicit drugs and their metabolites, all target analytes were of high purity (˃ 98%). Solutions or solids were purchased from LGC Promochem (Molsheim, France), except topiramate and lamotrigine, which were obtained from Glenmark (Mahwah, NJ, USA) and Sigma Aldrich Chemie GmbH (Steinheim, Germany), respectively. Acetonitrile (ACN) and methanol (MeOH) LC–MS grade were purchased from Merck (Darmstadt, Germany) as well as hydrochloric acid (37%). Formic acid (FA) 99% was purchased from Fluka (Buchs, Switzerland). Ammonia 25% was purchased from Panreac (Barcelona, Spain) and ammonium formate from Fluka (Buchs, Switzerland). Distilled water was provided by a MilliQ purification apparatus (Millipore Direct-Q UV, Bedford, MA, USA). Strata-X cartridges (200 mg / 6 mL) and RC syringe filters (4 mm diameter, 0.2 μm pore size) were provided from Phenomenex (Torrance, CA, USA), while the glass fiber filters used (GFF, pore size 0.7 μm) in wastewater filtration were obtained from Millipore (Cork, Ireland). Individual stock solutions were prepared in either acetone or MeOH, at concentrations varying between 10 mgL-1 and 1250 mgL-1. Only penicillins, cephalosporines, and macrolides were dissolved in MilliQ-water. In quinolone standard solutions, 50 μL of formic acid were added to enhance solubility. Stock solutions of each compound were obtained and stored at −20 °C. A multi component solution of all the compounds was obtained by diluting the stock solutions in MeOH to a final concentration of 1 μgmL-1 and was also stored at −20 °C. From this multi-analyte solution all working solutions and calibration standards were prepared by gradient dilution in concentrations varying from 1 μgmL-1 to 1 ngmL-1. Infusion solutions of individual standards and internal standards (IS) were prepared at concentrations of 2 mgL-1 in ACN:water (50:50, v/v), just before infusion experiments.

Table SI-1: Target analytes: Class, name, CAS number, molecular formula, molecular weight

(MW), pKa and logKow values. Pharmaceuticals detected at >80% of the samples and presented in Figure 1 are highlighted in grey background.

Molecular MW Class Compound CAS number pK logK formula (g/mol) a ow a b c MDMA 42542-10-10 C11H15NO2 193.2 9.4 /10.3 2.28 b c Amphetamine-like MDEA 82801-81-9 C12H17NO2 207.3 10.3 2.77 a b c compounds and MDA 4764-17-5 C10H13NO2 179.2 9.67 /9.9 1.84 metabolites Methamphetamine 537-46-3 C H N 149.2 10.1a/10.4b 2.22c (Stimulants) (MA) 10 15 a b c Amphetamine 300-62-10 C9H13N 135.2 10.1 /9.9 1.76 a c Caffeine 58-08-2 C8H10N4O2 194.1 10.4 -0.07 a c Analgesics Paracetamol 103-90-2 C8H9NO2 151.1 9.4 0.46 a c Tramadol 27203-92-5 C16H25NO2 263.2 9.4 2.51 a c Lidocaine 137-58-7 C14H22N2O 234.3 8.0 1.66 a b c Fentanyl 437-38-8 C22H28N2O 336.5 9.0 /8.9, 0.3 3.89 b c Norfentanyl 1609-66-2 C14H20N2O 232.3 9.8, 0.3 1.70 Anesthetics a b c Thiopental 76-75-6 C11H18N2O2S 242.3 7.4 /10.7 2.87 Ketamine 1867-66-10 C13H16ClNO 237.7 7.5a/6.5b 3.12c a c Norketamine 35211-10-1 C12H14ClNO 223.7 6.7 2.66 b c Chloramphenicol 56-75-7 C11H12Cl2N2O5 322.0 7.5 1.14 Antibiotics C H Cl FNO Florfenicol 73231-34-2 12 14 2 4 357.0 6.8b -0.04c (Amphenicols) S b c Thiamphenicol 15318-45-3 C12H15Cl2NO5S 355.0 7.7 -0.27 Antibiotics Trimethoprim 738-70-5 C H N O 290.1 7.1a 0.91c (Bacteriostatic) 14 18 4 3 b c Cefaclor 53994-73-3 C15H16ClN3O4S 367.0 3.0, 7.4 0.35 b c Antibiotics Cefadroxil 66592-87-8 C16H19N3O6S 363.1 3.5, 7.4 -0.06 b c (Cefalosporins) Cefalexin 15686-71-2 C16H17N3O4S 347.1 3.5, 7.4 0.65 b c Cefazolin 25953-19-9 C14H14N8O4S3 454.0 3.0 -0.58 Antibiotics Tiamulin 55297-95-5 C H NO S 493.3 9.5b 4.75c (Diterpene) 28 47 4 a c Azithromycin 83905-01-5 C38H72N2O12 748.5 8.7 4.02 a c Antibiotics Clarithromycin 81103-11-9 C38H69NO13 747.5 9.0 3.16 b c (Macrolides) Lincomycin 154-21-2 C18H34N2O6S 406.2 8.0, 12.4 0.56 b Tylosin 1401-69-0 C46H77NO17 915.5 7.2 - Antibiotics Metronidazole 443-48-1 C H N O 171.1 3.1b 0.02c (Nitroimidazole) 6 9 3 3 Amoxicillin 26787-78-0 C H N O S 365.1 2.4, 7.4b 0.87c Antibiotics 16 19 3 5 C H Cl N O (Penicillins) Dicloxacillin 3116-76-5 19 17 2 3 5 469.0 3.8b 2.91c S b c Antibiotics Ciprofloxacin 85721-33-1 C17H18FN3O3 331.1 5.8, 8.6 0.28

b c (Quinolones) Difloxacin 98106-17-3 C21H19F2N3O3 399.1 5.6, 6.5 1.28 b c Enrofloxacin 93106-60-6 C19H22FN3O3 359.2 5.7, 6.7 0.70 b c Flumequine 42835-25-6 C14H12FNO3 261.1 6.0 2.6 b c Marbofloxacin 115550-35-1 C17H19FN4O4 362.1 5.4, 6.2 -2.92 b c Norfloxacin 70458-96-7 C16H18FN3O3 319.1 5.8, 8.7 -1.03 b c Ofloxacin 82419-36-1 C18H20FN3O4 361.1 5.5, 6.2 -2.00 b c Oxolinic acid 14698-29-4 C13H11NO5 261.1 5.6 0.94 b c Sarafloxacin 98105-99-8 C20H17F2N3O3 385.1 5.8, 8.6 1.07 Sulfachloro- 80-32-0 C H ClN O S 284.0 2.0, 6.5b 0.31c pyridazine 10 9 4 2 b c Sulfaclozine 102-65-8 C10H9ClN4O2S 284.0 2.0, 6.5 0.31 a c Sulfadiazine 68-35-9 C10H10N4O2S 250.1 6.36 -0.09 b c Sulfadimethoxine 122-11-2 C12H14N4O4S 310.1 2.0, 6.9 1.36 b c Sulfadimidine 57-68-1 C12H14N4O2S 278.1 2.0, 7.0 0.89 b c Sulfadoxine 2447-57-6 C12H14N4O4S 310.1 2.3, 6.1 0.70 b c Sulfaguanidine 57-67-0 C7H10N4O2S 214.1 1.0, 3.4 -0.99 Sulfamerazine 127-79-7 C H N O S 264.1 2.0, 7.0b 0.14c Antibiotics 11 12 4 2 b c (Sulfonamides) Sulfamethizole 144-82-1 C9H10N4O2S2 270.0 1.9, 5.5 0.54 b c Sulfamethoxazole 723-46-6 C10H11N3O3S 253.0 2.0, 7.6 0.89 Sulfamethoxypyrida 80-35-3 C H N O S 280.1 2.0, 6.8b 0.32c zine 11 12 4 3 Sulfamonomethoxin 1220-83-3 C H N SO 280.1 2.0, 6.5b 0.31c e 11 12 4 3 b c Sulfamoxole 729-99-7 C11H13N3O3S 267.1 2.0, 6.8 1.03 a c Sulfapyridine 144-83-2 C11H11N3O2S 249.1 8.43 0.35 a c Sulfathiazole 72-14-0 C9H9N3O2S2 255.0 7.2 0.05 a c Sulfisoxazole 127-69-5 C11Χ13Ν3Ο3S 267.1 5.0 1.01 b c Chlortetracycline 57-62-5 C22H23ClN2O8 478.1 4.4 -0.62 b c Antibiotics Doxycycline 564-25-0 C22H24N2O8 444.1 4.6 -0.02 a b c (Tetracyclines) Oxytetracycline 79-57-2 C22H24N2O9 460.1 3.27 /4.6 -0.90 a b c Tetracycline 60-54-8 C22H24N2O8 444.1 3.3 /4.6 -1.30 a c Carbamazepine 298-46-5 C15H12N2O 236.3 4.17,12.5 2.25 a b c Lamotrigine 84057-84-2 C9H7Cl2N5 256.1 5.7 /19.2 0.99 a c Topiramate 97240-79-5 C12H21NO8S 339.4 8.7 -0.33 a c Antiepileptic Levetiracetam 102767-28-3 C8H14N2O2 170.2 <-2 -0.49 a c Phenytoin 57-41-1 C15H12N2O2 252.3 8.3 2.16 a c Primidone 125-33-8 C12H14N2O2 218.3 11.6 0.73 a c Valproic acid 99-66-2 C8H16O2 144.2 4.6 2.96 a c Antihypertensive Atenolol 29122-68-7 C14H22N2O3 266.2 9.6 0.16 a c drugs Propranolol 525-66-6 C16H21NO2 259.2 9.4 3.48

b c Valsartan 137862-53-4 C24H29N5O3 435.2 4.4 3.65 b c Metoprolol 51384-51-1 C15H25NO3 267.2 9.7 1.88 b Atorvastatin 134523-00-5 C33H35FN2O5 558.2 4.3 - b Antilipidemic Simvastatin 79902-63-9 C25H38O5 418.3 4.2 - b c Agents Gemfibrozil 25812-30-0 C15H22O3 250.2 4.4 4.77 b c Clofibric acid 882-09-7 C10H11ClO3 214.0 3.4 2.57 Antiprotozoal Ronidazole 7681-76-7 C H N O 200.0 1.6b -0.38c Agent 6 8 4 4 a c Chlorpromazine 50-53-4 C17H19ClN2S 318.9 9.3 4.32 a c Clozapine 5786-21-1 C18H19ClN4 326.8 3.6, 7.9 3.35 c Norclozapine 6104-71-9 C17H17ClN4 312.8 - 3.01 a c Antipsychotics Olanzapine 132539-06-2 C17H20N4S 312.4 5.0, 7.4 2.56 a c Risperidone 106266-06-3 C23H27FN4O2 410.5 8.3 3.49

c 9-OH-Risperidone 147687-18-2 C23H27FN4O3 426.5 - 1.95

a c Cimetidine 51481-61-9 C10H16N6S 252.1 6.8 0.57 b c Antiulcer Drugs Omeprazole 73590-58-6 C17H19N3O3S 345.1 4.8, 9.7 2.23 b c Ranitidine 66357-35-5 C13H22N4O3S 314.1 8.1 0.27 a c Phenobarbital 50-06-7 C12H12N2O3 232.2 7.5 1.33 Barbiturates a c Pentobarbital 76-74-5 C11H18N2O3 226.3 7.9 2.00 a c Alprazolam 92623-85-4 C17H13ClN4 308.8 8.2 3.87 a c Bromazepam 1812-30-3 C14H10BrN3O 316.2 2.5, 5.2, 11.8 1.93 a b c Chlordiazepoxide 58-25-4 C16H14ClN3O 299.8 4.8 / 8.6, 6.5 -1.57 a c Clobazam 22316-47-9 C16H13ClN2O2 300.7 1.6 1.94 a c Diazepam 439-14-6 C16H13ClN2O 284.7 3.3 2.70 12.0, 3.5a/11.7, Nordiazepam 1088-11-6 C H ClN O 270.7 3.89c 15 11 2 3.2b a c Flunitrazepam 1622-62-5 C16H12FN3O3 313.3 1.71 1.91 Benzodiazepines 7-amine- 34084-50-10 C H FN O 283.3 - 0.78c (Tranquilizers) flunitrazepam 16 14 3 a c Lorazepam 846-49-2 C15H10Cl2N2O2 321.2 1.3, 11.5 3.98 3.2, 10.8a/11.4, Nitrazepam 146-22-6 C H N O 281.3 2.45c 15 11 3 3 2.6b a c Midazolam 59467-70-9 C18H13ClFN3 325.8 6.15, 1.7 4.33 11.6, 1.7a/12.8, Oxazepam 604-75-2 C H ClN O 286.7 3.34c 15 11 2 2 10.9, 1.2b

a b c Temazepam 846-50-5 C16H13ClN2O2 300.7 1.6 /11.7, 1.6 2.15

a b c Bronchodilators Theophylline 58-55-9 C7H8N4O2 180.1 8.8, 2.4 /11.4 -0.02 a b c Cocaine and Cocaine (COC) 50-36-3 C17H21NO4 303.2 8.6 /8.9 2.17 metabolites Benzoylecgonine 519-09-6 C H NO 289.3 11.8a/10.8, 3.3b -1.32c (Stimulants) (BECG) 16 19 4

Ecgonine 02/09/7143 C H NO 199.3 9.16a -0.29c methylester (EME) 10 17 3 b c Furosemide 54-31-9 C12H11ClN2O5S 330.0 4.3 2.03 b c Indapamide 26807-65-8 C16H16ClN3O3S 388.8 6.7 1.54 Diuretics a b c Hydrochlorothiazide 58-93-5 C7H8ClN3O4S2 297.0 7.9 , 9.1 -0.07 b c Triamterene 396-01-0 C12H11N7 253.1 3.1 0.98 a c Hallucinogenic Δ9-THC 04/08/1972 C21H30O2 314.5 10.6 7.60 a c (Cannabinoids) 11-nor-Δ9-THC acid 56354-06-5 C21H28O4 344.4 4.7 6.36 a b c Hallucinogenic LSD 50-37-4 C20H25N3O 323.4 7.5 /7.4 2.26 (LSD and b metabolite) 2-oxo-3-hydroxy-LSD - C20H25N3O3 355.4 11.7, 6.8 - b c Hypnotic Zopiclone 43200-80-2 C17H17ClN6O3 365.8 8.8 2.66 a c Diclofenac 15307-86-5 C14H11Cl2NO2 295.0 4.2 4.51 a c Ketoprofen 22071-15-4 C16H14O3 254.1 4.5 3.12 b c Niflumic acid 4394-00-7 C13H9F3N2O2 282.1 1.9, 5.5 4.43 b c Acetylsalicylic acid 50-78-2 C9H8O4 180.0 3.4 1.19 a c NSAIDs Ibuprofen 15687-27-1 C13H18O2 206.1 4.9 3.97 a c Meloxicam 71125-38-7 C14H13N3O4S2 351.0 4.1 3.43 a c Mefenamic Acid 61-68-7 C15H15NO2 241.1 4.2 5.12 a c Naproxen 22204-53-1 C14H14O3 230.1 4.2 3.18 b c Salicylic acid 69-72-7 C7H6O3 138.0 2.97 2.24 8.0, 9.9a/ Morphine (MOR) 57-27-3 C H NO 285.3 0.72c 17 19 3 13.5, 9.5, 8.3b b c Heroin 561-27-3 C21H23NO5 369.4 7.95 1.80 6- c Monoacetylmorphin 2784-73-9 C19H21NO4 327.4 - 1.72 e (6-MAM) Opiates, opioids a b c Methadone (METH) 76-99-34 C H NO 309.5 8.94 /9.1 4.17 and metabolites 21 27 b c EDDP 30223-73-6 C20H23N 277.4 8.4 4.94 a b c Codeine (COD) 76-57-4 C18H21NO3 299.4 8.2 /13.4, 8.2 1.28 a b c Oxycodone (OC) 76-42-7 C18H21NO4 315.4 8.9 /13.1, 7.6 0.66 8.5, 10.0a/ 9.5, Buprenorphine (BN) 52485-79-8 C H NO 467.6 4.90c 29 41 4 8.3b SNRIs (Serotonin– Norepinephrine Venlafaxine 93413-69-6 C H NO 277.4 9.3b 3.28c Reuptake 17 27 2 inhibitors) a c Citalopram 59729-33-9 C20H21FN2O 324.4 9.4 3.74 b c SSRIs (Selective Fluoxetine 54910-89-4 C17H18F3NO 309.3 10.1 4.65 Serotonin Paroxetine 61869-08-8 C H FNO 329.4 9.5a 2.57c Reuptake 19 20 3 a c Inhibitors) Sertraline 79617-96-3 C17H17Cl2N 306.2 9.5 5.29 c Norsertraline 87857-41-9 C16H15Cl2N 292.2 - 4.82

b c Betamethasone 378-44-9 C22H29FO5 392.2 12.4 1.83 b c Cortisone 53-06-5 C21H28O5 360.2 12.6 1.47 Steroids b c Methylprednisolone 83-43-2 C22H30O5 374.2 12.6 1.82 a c Progesterone 57-83-0 C21H30O2 314.2 18.9 3.87 a b c Ephedrine 299-42-4 C10H15NO 165.2 9.6 /9.5 0.68 Sympathomimetics b c Norephedrine 492-41-2 C9H13NO 151.2 12.1, 8.5 0.22 a c Tetracyclic Mirtazapine 61337-67-6 C17H19N3 265.4 7.1 3.03 Antidepressants (TeCAs) 8-OH mirtazapine - C17H19N3O 281.4 - - a c Amitriptylline 50-48-7 C20H23N 277.4 9.4 4.95 Nortriptyline 894-71-4 C H N 263.4 10.1a 4.74c Tricyclic 19 21 a c Antidepressants Clomipramine 303-49-2 C19H23ClN2 314.9 9.4 5.25 a c (TCAs) Imipramine 50-49-8 C19H24N2 280.4 9.6 5.01 a c Doxepine 1668-19-6 C19H21NO 279.4 8.0 3.86

a experimental pka; b theoretical pka; c KOWWIN v1.67 estimate, (EPI: Estimation Program Interface (EPI) Suite) (from the United States Environmental Protection Agency (USEPA) http://www.epa.gov/opptintr/exposure/pubs/episuite.htm (-): Data not available

SI-2: Analytical method.

SPE was carried out using the polymeric sorbent Strata-X (hydrophilic- lipophilic reversed phase), which offered the best results for most compounds. In the optimized procedure, Strata-X cartridges were conditioned with 6 mL of MeOH and 6 mL of ultrapure water. Samples of 50 mL (0.1% w/v EDTA) were loaded under gravity. The addition of EDTA improves the extraction recovery of some antibiotics, especially tetracyclines.1-3 Cartridges were washed with 3 mL of ultrapure water and subsequently dried under vacuum (1 hour, 8 psi). Analytes were eluted with 6 mL of MeOH and taken to dryness under constant stream of nitrogen. Reconstitution of the analytes was performed in 500 μL of water (FA 0.05%) and MeOH (75:25 v/v). Finally, the extracts were filtered through a 0.2 mm RC syringe filter and then transferred to the vial for immediate UPLC-MS/MS analysis. The cocaine metabolite ecgonine methylester (EME) was analyzed following the procedure described by Borova et al.4

Instrumental analysis was performed with a Thermo UHPLC Accela system connected to a TSQ Quantum Access triple-200 quadrupole mass spectrometer from Thermo Electron Corporation (San Jose, CA, USA) equipped with an electrospray ionization source (Thermo IonMAX) in both positive and negative mode. Chromatographic separation was achieved on an Atlantis T3 C18 (100 mm x 2.1mm, 3 μm) column from Waters corporation (Milford, MS, USA) at a constant flow rate of 100 μL min-1. The mobile phase for the positive detection mode consisted of water (0.01% FA (v/v)) and MeOH for the positive detection mode and water (1 mM ammonium formate), MeOH and ACN (constant in a proportion of 5%) for the negative detection mode. Gradient elution programs are presented in Table SI-2A along with other relevant ESI parameters, which were obtained as a compromise using the optimum values for most compounds. The injection volume was set to 10 μL. The optimized Ionization mode, fragmentation voltages and collision energies for each compound have been previously described in detail by Borova et al. and Dasenaki et al.1, 4 Instrument control and data acquisition and evaluation were performed with Xcalibur software (Thermo Electron Corporation). Identification and quantification were performed under selected reaction monitoring (SRM) mode, recording the transitions between the precursor ion and the two most abundant product ions for each target analyte, thus achieving 4 identiﬁcation points per compound (2002/657/EC). The performance of the analytical method was assessed and the quality control parameters obtained for each target analyte are shown Table SI-3B.

Table SI-2A: Gradient elution program and ESI parameters in positive and negative ionization modes.

Positive Ionization Mode

GRADIENT PROGRAM ESI PARAMETERS Time (min) % B Spray Voltage 3500V 0 2 Capillary temperature 270 °C 3 2 Sheath gas 30 psi 20 100 Auxiliary (drying) gas 10 a.u. 29 100 30 2 B: MeOH 45 2 Negative Ionization Mode GRADIENT PROGRAM ESI PARAMETERS Time (min) % B Spray Voltage 2500V 0 25 Capillary temperature 270 °C 10 95 Sheath gas 20 psi 23 95 Auxiliary (drying) gas 10 a.u. 24 25 B: MeOH 37 25 C: ACN (constant 5%)

Table SI-2B: Analytical method performance.

Absolute RSD MLOD MLOQ Compounds Recovery (n = 6) (%) (ng L-1) (ng L-1)

Positive Ionization Mode 6-monoacetylmorphine 107.9 13.7 5.5 18.2 7-aminoflunitrazepam 70.4 6.7 7.0 23.1 8-OH mirtazapine 99.5 11.2 6.5 21.3 9-OH Risperidone 89.8 15.4 1.7 5.6 Alprazolam 93.5 12.8 2.3 7.5 Amitriptyline 97.8 10.9 0.7 2.4 Amoxicillin 90.5 10.2 4.6 15.1 Amphetamine 97.3 15.3 1.6 5.4 Atenolol 95.5 6.9 6.2 20.3 Atorvastatin 45.9 19.3 4.5 14.7

S10

Azithromycin 92.1 14.8 19.3 63.7 Benzoylecgonine 102.4 11.7 1.0 3.2 Betamethasone 98.4 13.0 30.4 100 Bromazepam 92.4 9.8 2.7 9.0 Buprenorphine 89.7 18.3 3.6 12.0 Caffeine 107.4 11.0 7.0 23.0 Carbamazepine 106.5 17.8 1.1 3.6 Cefaclor 110.1 13.0 3.8 12.5 Cefadroxil 113.4 17.1 8.3 27.2 Cefalexine 111.7 10.6 7.5 24.6 Cefazolin 76.2 3.2 4.4 14.5 Chlordiazepoxide 90.2 12.8 1.5 5.0 Chlorpromazine 67.0 12.7 3.6 12.0 Chlotetracycline 83.1 12.4 7.4 24.5 Cimetidine 68.5 5.9 15.1 49.8 Ciprofloxacin 103.7 9.8 5.4 17.8 Citalopram 107.4 13.5 1.2 4.0 Clarithromycin 89.4 11.2 1.9 6.3 Clobazam 100.6 24.8 3.4 11.4 Clomipramine 81.9 12.9 2.1 6.9 Clozapine 95.4 6.6 2.1 6.9 Cocaine 101.5 12.1 1.5 5.0 Codeine 100.7 11.7 4.5 14.9 Cortisone 106.3 19.8 10.0 33.0 Diazepam 85.6 10.9 1.1 3.5 Diclofenac 79.8 15.6 21.0 69.3 Dicloxacillin 90.0 18.7 34.9 115 Difloxacin 92.4 15.8 9.9 32.5 Doxepin 95.6 19.8 1.6 5.1 Doxycycline 87.1 9.8 14.9 49.2 EDDP 97.3 11.8 2.1 6.8 EME 79.0 0.9 1.4 4.5 Enrofloxacin 94.3 11.2 7.4 24.5 Ephedrine 95.4 11.5 10.3 34.1 Fentanyl 98.8 15.6 1.4 4.6 Flumequine 101.6 11.3 2.5 8.2 Flunitrazepam 95.0 7.0 24.5 80.8 Fluoxetine 100.6 4.5 1.6 5.3 Heroin 97.6 16.2 3.7 12.3 Imipramine 100.4 5.8 1.1 3.6 Ketamine 104.9 15.9 3.1 10.2

S11

Ketoprofen 95.0 13.2 3.8 12.7 Lamotrigine 108.3 12.2 12.2 40.2 Levetiracetam 103.3 16.2 4.4 14.5 Lidocaine 99.3 8.0 2.9 9.5 Lincomycin 95.0 5.5 5.9 19.5 Lorazepam 93.3 9.5 3.8 12.4 LSD 92.2 11.2 2.3 7.5 LSD-OH 101.6 15.4 15.5 51.2 Marbofloxacin 95.9 8.4 5.1 16.9 MDA 96.2 16.5 2.4 7.8 MDEA 100.3 19.0 3.3 10.9 MDMA 103.1 9.6 1.5 5.0 Methadone 106.0 12.4 3.1 10.4 Methamphetamine 99.9 13.6 1.8 5.8 Methylprednisolone 107.4 8.2 18.6 61.4 Metronidazol 99.4 12.2 2.4 8.1 Metropolol 101.0 6.7 53.9 178 Midazolam 95.5 11.4 2.0 6.6 Mirtazapine 95.9 16.5 1.3 4.2 Morphine 85.5 12.6 3.6 11.9 Niflumic Acid 83.6 11.5 5.3 17.4 Nitrazepam 79.8 11.4 6.4 21.2 Norclozapine 92.5 7.8 1.5 4.8 Nordiazepam 93.5 14.6 5.4 17.7 Norephedrine 40.1 9.3 5.1 16.9 Norfentanyl 98.8 15.2 1.4 4.6 Norfloxacin 98.3 10.0 7.2 23.7 Norketamine 98.4 12.5 0.9 2.8 Norsertraline 93.2 9.9 0.7 2.4 Nortriptyline 93.2 13.1 1.0 3.3 Ofloxacin 96.5 11.9 1.7 5.6 Olanzapine 81.3 10.5 1.3 4.3 Omeprazole 114.5 18.7 1.1 3.5 Oxazepam 100.7 8.3 1.3 4.1 Oxolinic Acid 96.1 9.3 2.4 7.9 Oxycodone 100.1 6.8 1.5 5.0 Oxytetracycline 89.5 12.3 7.0 23.2 Paracetamol 102.7 8.1 22.6 74.6 Paroxetine 88.4 6.5 10.1 33.3 Primidone 106.6 14.8 7.0 23.1 Progesterone 90.5 10.4 8.2 27.1

S12

Propanolol 100.4 7.3 5.2 17.3 Ranitidine 78.8 15.3 7.7 25.3 Risperidone 90.5 5.0 0.3 1.0 Ronidazol 103.3 14.5 1.1 3.8 Sarafloxacin 98.2 11.1 1.9 6.1 Sertraline 86.6 6.4 5.4 17.8 Simvastatin 54.5 17.3 27.8 91.7 Sulfachloropyridazine (SCP) 82.5 7.3 19.0 62.7 Sulfaclozine (SCIZ) 70.8 19.4 21.6 71.3 Sulfadiazine (SDZ) 85.9 12.1 14.0 46.2 Sulfadimethoxine (SDM) 81.1 12.7 10.1 33.3 Sulfadimidine (SDD) 77.9 8.4 12.2 40.3 Sulfadoxine (SDX) 75.2 11.4 18.9 62.4 Sulfaguanidine (SGN) 51.6 17.7 8.6 28.3 Sulfamerazine (SMR) 84.5 3.5 11.0 36.3 Sulfamethizole (SMT) 71.8 17.9 22.0 72.6 Sulfamethoxazole (SMTX) 88.1 8.1 15.7 51.8 Sulfamethoxypyridazine (SMP) 74.6 13.6 6.6 21.7 Sulfamonomethoxine (SMM) 69.2 9.6 7.7 25.3 Sulfamoxole (SMX) 74.2 8.6 17.3 57.1 Sulfapyridine (SPD) 79.4 9.8 9.6 31.7 Sulfathiazole (STZ) 74.2 12.0 18.3 60.4 Sulfisoxazole (SΙΧ) 71.6 10.0 13.6 44.9 Temazepam 91.0 12.2 1.3 4.4 Tetracycline 92.0 14.6 23.0 75.9 THC 28.5 15.2 70.8 233 THCA 50.5 14.4 79.6 263 Theophylline 96.3 4.1 5.5 18.2 Tiamulin 96.4 8.7 9.8 32.2 Topiramate 99.7 6.6 21.3 70.4 Tramadol 96.8 4.0 6.2 20.5 Triamterene 100.5 14.1 4.3 14.2 Trimethoprim 103.0 11.0 1.7 5.7 Tylosin 94.1 15.9 28.0 92.4 Valsartan 76.4 4.7 8.9 29.2 Venlafaxine 102.0 10.6 0.8 2.6 Zopiclone 94.1 12.2 2.8 9.2 Negative Ionization Mode Acetylsalicylic Acid 114.0 5.0 50.0 165 Chloramphenicol 93.6 9.7 5.0 16.5 Clofibric Acid 80.1 7.3 6.0 19.8

S13

Diclofenac 101.2 14.1 20.6 68.5 Florfenicol 101.8 12.7 1.4 4.7 Furosemide 107.0 12.6 21.4 70.6 Gemfibrozil 67.1 14.5 2.6 8.6 Hydrochlorothiazide 95.3 11.1 9.2 30.4 Ibuprofen 94.3 22.3 15.5 51.2 Indapamide 100.4 7.1 71.2 235 Meloxicam 73.0 7.5 6.5 21.6 Mefenamic Acid 69.5 28.1 66.6 220 Naproxen 102.4 11.8 8.0 26.4 Pentobarbital 93.3 14.2 180.1 594 Phenobarbital 95.6 9.4 15.5 51.0 Phenytoin 102.7 11.7 104.7 346 Salicylic acid 101.4 10.2 3.10 10.2 Thiamphenicol 100.0 15.4 5.00 16.5 Thiopental 57.2 7.3 77.0 254 Valproic Acid 105.4 14.9 17.5 57.6 RSD: relative standard deviation; MLOD: method limit of detection; MLOQ: method limit of quantification.

S14

SI-3: Pharmacokinetics and other relevant data used for back-calculated use of the selected substances.

Table SI-3A: Pharmacokinetics of selected licit drugs (pharmaceuticals) used for back-calculated use.

Pharmacokinetics（%）

Bioavailability (%Rabs) Excreted unchanged (%Rexcreted) Biotransformation Typical oral dose Drugs Protein Binding rate (D, mg)[1][8] Values used for Values used for Literature data Literature data back-calculation back-calculation

Amoxicillin 20[1][2] 75-90[3],85-90[1] 88.3 60[4][3][5] 60.0 1.67 500

Atenolol 6-16[2],3[1] 40-50[6][4][3][1] 45.0 85-100[6][3] 92.5 1.12 50

Atorvastatin 98[2] 14[4],12[3] 13.0 2[4][2] 2.00 1.24 10

Azithromycin 7-51[2] 37[3][2],38[4] 37.5 12[4][3] 12.0 1.61 500

Carbamazepine 76[2] 85-100[4][3] 92.5 2[4],3[3][2] 2.50 11.02 400

Cefaclor 23,5[2] 50-75[4][3] 87.5 60-85[4][3][1] 72.5 1.42 500

[48-75 (45 oral, 75 I.M., Cimetidine 15-20[2] 60[3] 60.0 61.5 1.41 400 I.V.)][4][1]

[2] [4] [3][1] [3] [4][2] Ciprofloxacin 20-40 70 ,70-80 72.5 44.7 ,40-50 44.85 1.80 500

S15

[6][1] [6][4][3] [4][7] [3] Citalopram 80 80 80.0 10-13 ,12 11.75 3.68 20

Clarithromycin - 50[1] 50.0 20-30[1][2] 25.0 1.73 500

Clomipramine 96,7[6][1] 50[6][1] 50.0 2[6][3][1] 2.00 2.12 25

[Little or no free Diclofenac 99[2],100[8] 50[8], 60-70%[6] 57.5 unchanged diclofenac is 1.00 2.51 100 excreted in the urine][4][8]

Doxycycline 82-93[3],90[2] >90[2] 95.0 29-55,4[4] 42.2 2.40 200

Ephedrine - 85[2] 85.0 70-80%[6] 75.0 1.37 50

[1(unchanged) + 65- Ketoprofen 99[2] 90[8] 90.0 75(glycuronide)][3], 75.0 1.39 200 [80(glycuronide)][4][2]

Lamotrigine 55[1][2] 98[4][2] 98.0 10[4][3][1] 10.0 9.16 200

Levetiracetam 10[6][1][2] nearly 100[6][9][3][1] 100 66[6][4][9][1][2] 66.0 1.64 250

Lidocaine 60-80[6][2] 30-35[6][3] 32.5 10[6][4][3] 10.0 1.53 8

[10–47% I.M., 13–72% Lincomycin 28-86[8][2] 20-30[2] 25.0 26.3 1.33 500 I.V., 3–13% oral][8] 3

74.5[10],70- Lorazepam 85[1] 90[4] 90.0 75[1][6](glucuronide and 73.5 1.42 3 free)

S16

Metronidazole 20[1][2] <20[2] 20.0 12[6] 12.0 1.31 500

Metoprolol 10[6][1] 12[6] 12.0 5[6][4][1] 5.00 1.22 100

Mirtazapine 85[6][1][2] 50[6][4][3][1] 50.0 25[6] 25.0 1.73 15

8OH-Mirtazapine - - - 40[11] 40.0 - - (metabolite)

Niflumic Acid 90[1][2] - - 40[12] 40 - 250

Norfloxacin 10-15[3][2] 40[4] 40.0 25-40[8] 32.5 1.48 400

Ofloxacin 32[2] 98[4][2] 98.0 65-80[4][2] 72.5 1.48 200

58-76[4], 40[3], Omeprazole 95[1][2] 52.2 0[4][3][1] 0 2.26 20 35[1] 5

Oxazepam 80-99[6][2] 92.8[6][13] 92.8 1[13],(trace amounts)[6] 1.00 13.30 15

Paracetamol 20-50[1] 25[2] 25.0 2-3[3],<5[1] 3.75 1.42 500

[Completely absorbed from GI, but extensive Paroxetine 95[1][2] 100 2[6][3][1] 2.00 54.05 20 first-pass metabolism in the liver][6]

Primidone 20[8] 60-80[8] 70.0 40[3] 40.0 1.86 250

S17

Progesterone 96-99[2] - - 5[8] 5.00 - 200

Propranolol 90[6][2] 26[6] 26.0 0.5[6][14] 0.50 1.46 50

[35(oral),70(I.V.)][1], Ranitidine 15[2] 50[3][1][2] 50.0 51.25 1.43 150 30[8][3]

Ronidazole ------

Sertraline 98[1] 44[6] 44.0 0.2[6][3][1] 0.20 1.93 100

Simvastatin 95[2] 5[1] 5.00 - - - 40

Sulfadiazine (SDZ) 50[3] - - 44[15] 44.0 - 500

Sulfamethoxazole 70[4][2] 70[4],90[6] 80.0 20[4],15-30[1] 21.2 2.92 800 (SMTX) 5

Almost completed eliminated by hydroxylation, Sulfapyridine (SPD) 50[2] 92.7[16] 92.7 1.00 13.14 800 glucuronidation and polymorphic acetylation[17]

[40%, primarily Theophylline 100[18] 100 10[4][3][2] 10.0 10.81 300 to albumin][2]

S18

13-17[1]

15- 41(depending [4][2] [3][1] [6][4][2] 1.42 100 Topiramate blood 80 ,81 80.0 70 70.0 concentration range of 0.5 - 250 mg/mL)[2]

[70 (absolute mean Bioavailability), Tramadol 20[6][1][2] 70.0 30[6][4][2] 30.0 2.12 150 90 (oral Bioavailability)][6][ 3][1]

Trimethoprim 45[1],42-46[2] 100[19] 95.0 40-50[3],40-48[2] 44.5 2.29 160

Venlafaxine 27[6][2] 40-45[6][3][1][2] 42.5 5[6][4][20][3][1][2] 5.00 1.81 37.5

[Concentration- dependent, from 90% at 40 Valproic Acid 100[6][1] 100 3[6][4][1][2] 3.00 36.04 2000 µg/mL to 81.5% at 130 µg/mL][6][2]

Valsartan 94-97[1][2] 23[3][1][2],25[4] 24.0 81[4][21][1] 81.0 1.13 320

S19

[66-98(depending Acetylsalicylic Acid 99,5[2] 82.0 10[8] 10.0 4.13 500 concentration)][1]

52(mean Furosemide 95[2] 52.0 80[14] 80.0 1.21 40 Bioavailability)[3]

Gemfibrozil 95[2] 100[3] 100 6[3],2[4][2] 4.00 27.03 600 hydrochlorothiazide 67,9[2] 65-75[4],60[3] 65.0 95[8] 95.0 1.12 25

15 (free and Ibuprofen 90-99[2] <80[7] 80.0 15.0 3.38 400 conjugated)[4]

Mefenamic Acid 90[2] 86[23] 86.0 6[4][3] 6.00 5.64 500

Naproxen 99[6][2] 95[6][2] 95.0 1[6][4] 1.00 18.17 250

Pentobarbital 20-45[6] 70-90[6] 80.0 1[6][22] 1.00 5.20 100

Phenobarbital 20-45[6][2] >95[6] 95.0 25-50[6][4] 37.5 2.66 54

70-100 (oral Phenytoin 90[6][2] 85.0 5[6][1] 5.00 5.62 200 Bioavailability) [6]

References

[1] Galinos medication guide http://www.galinos.gr/ [last visit 17/12/2013]. [2] DrugBank: a comprehensive resource for in silico drug discovery and exploration. http://www.drugbank.ca /[last visit 15/12/2013]. [3] electronic Medicines Compendium (eMC)

S20

http://www.medicines.org.uk [last visit 15/12/2013]. [4] RxList The Internet Drug Index http://www.rxlist.com/drugs/alpha_a.htm [last visit 15/12/2013]. [5] New Zealand Data Sheet for APO-AMOXI http://www.medsafe.govt.nz/profs/datasheet/a/alphamoxcap.pdf [last visit 15/12/2013]. [6] Baselt, R. C. Disposition of Toxic Drugs and Chemicals in Man 7th edition; Biomedical Publications: Foster City, California, 2004. [7] Oyehaug, E.; Ostensen, E. T.; Salvesen, B. High performance liquid chromatographic determination of citalopram and four of its metabolites in plasma and urine samples from psychiatric patients. Journal of Chromatography. 1984, 308,199-208. [8] Online Pharmaceutical Encyclopedia Drugs.com [last visit 20/12/2013]. [9] Patsalos, P. N. Pharmacokinetic profile of levetiracetam: toward ideal characteristics. Pharmacol. Therap. 2000, 85 (2), 77-85. [10] Elliott, H.W. Metabolism of lorazepam. Br. J. Anaesth. 1978, 48 (10), 1017-1023. [11] Störmer, E.; von Moltke, L. L.; Shader, R. I.; Greenblatt, D. J. Metabolism of the antidepressant mirtazapine in vitroQ Contribution of cytochrimes P-450 1A2, 2D6, and 34E. Drug Metab. and Dispos. 2010, 28 (10), 1168-75. [12] Lan, S. J.; Chando, T. J.; Weliky, I.; Schreiber, E. C. Metabolism of niflumic acid-14C: absorption, excretion and biotransformation by human and dog. J. Pharmacol. Exp. Ther. 1973, 186 (2), 323-330. [13] Sonne, J.; Loft, S.; Døssing, M.; Vollmer-Larsen, A.; Olesen, K. L.; Victor, M.; Andreasen, F.; Andreasen, P.B. Bioavailability and pharmacokinetics of oxazepam. Eur. J. Clin. Pharmac. 1988, 35 (4), 385-389. [14] IPCS Health and Safety Guide http://www.inchem.org/ [last visit 15/12/2013]. [15] Stachowska, B.; Seńczuk, W. Studies on kinetics of sulfadiazine and trimethoprim excretion in man. Int. J. Clin. Pharmacol. Ther. Toxicol. 1987, 25 (2), 81-85. [16] Pieniaszek, H. J..; Resetarits, D. E.; Wilferth, W. W.; Blumenthal, H. P.; Bates, T. R. Relative systemic availability of sulfapyridine from commercial enteric-coated and uncoated sulfasalazine tablets. J. Clin. Pharmacol. 1979, 19 (1), 39-45. [17] Klotz, U. Clinical pharmacokinetics of sulphasalazine, its metabolites and other prodrugs of 5-aminosalicylic acid. Clin. Pharmacokinet. 1985, 10 (4), 285-302. [18] Rovei, V.; Chanoine, F.; Strolin, B. M.; Pharmacokinetics of theophylline: a dose-range study. Br. J. Clin. Pharmacol. 1982, 14(6), 769-7. [19] [Chin, T. W. F.; Vandenbroucke A.; Fong I. W.; Pharmacokinetics of Trimethoprim-Sulfamethoxazole in Critically Ill and Non-Critically Ill AIDS Patients. Antimicrob. Agents Chemother. 1995, 39(1), 28-33. [20] da Fonseca, P.; Bonato, P. S. Chiral HPLC analysis of venlafaxine metabolites in rat liver microsomal preparations after LPME extraction and application to an in vitro biotransformation study. Anal. Bioanal. Chem. 2010, 396 (2), 817–824. [21] Nakashima, A.; Kawashita, H.; Masuda, N.; Saxer, C.; Niina, M.; Nagae, Y.; Iwasaki, K. Identification of cytochrome P450 forms involved in the 4-hydroxylation of valsartan, a potent and specific angiotensin II receptor antagonist, in human liver microsomes. Xenobiotica. 2005, 35 (6), 589–602. [22] Tang, B. K.; Inaba, T.; Kalow, W. N-hydroxylation of pentobarbital in man. Drug. Metab. Dispos. 1977, 5 (1), 71-74. [23] Hamaguchi, T.; Shinkuma, D.; Yamanaka, Y.; Mizuno, N.; Bioavailability of mefenamic acid: influence of food and water intake. J Pharm Sci. 1986, 75(9), 891-893. .

S21

Table SI-3B: Selected drug residues targeted for the back-calculation of illicit drug use.

Percentage of the Drug Target Conversion Compound substance excreted Dosage (mg) Residue (DTR) factor as DTR (Rexcreted) Cocaine BEGC 45[1] 2.33 100[4] Heroin Morphine 42[1] 3.1 30[4] Amphetamine Amphetamine 30[1] 3.3 50[4] ΜDMA (ecstasy) MDMA 65[1] 1.5 100[4] Methamphetamine Methamphetamine 43[1] 2.3 30[4] THC (Cannabis) THCA 0.6[1] 152 125[4] Methadone EDDP 3-25[2] 3.6 - Codeine Codeine 70[3] 1.4 - DTR: Drug target residue

[1] Zuccato, E.; Chiabrando, C.; Castiglioni, S.; Bagnati, R.; Fanelli, R. Estimating Community Drug Abuse by Wastewater Analysis. Environ. Health Perspect. 2008, 116 (8), 1027-1032. [2] Baselt, R. C. Disposition of Toxic Drugs and Chemicals in Man 7th edition; Biomedical Publications: Foster City, California, 2004. [3] Thorn, C. F.; Klein, T. E.; Altman, R.B. Codeine and morphine pathway. Pharmacogenet. Genomics. 2009, 19 (7), 556-558. [4] Zuccato, E.; Castiglioni, S. Assesing illicit drug consumption by wastewater analysis: history, potential and limitation of a novel approach. In Illicit drugs in the environment, Occurrence, Analysis, and Fate using Mass Spectrometry, Castiglioni, S.; Zuccato, E.; Fanelli, R.; John Wiley & Sons: New Jersey 2011; pp 296.

S22

SI-4: Concentration values and loads of the target compounds in the analyzed influent wastewaters and statistical analysis

Table SI-4A: Mean, median, minimum and maximum concentrations in influent wastewater for the studied substances during the sampling campaign carried out in December 2010 during 7 consecutive days (compounds determined in negative and positive ionization mode).

LODs [N]>LOD mean median Min max 6-monoacetylmorphine 5.5 0

S23

Ketamine 3.1 0

S24

Thiopental 77.0 0

Table SI-4B Mean, median, minimum and maximum concentrations in influent wastewater for the studied substances during the sampling campaign carried out in April 2011 during 8 consecutive days (compounds determined in negative and positive ionization mode).

LODs [N]>LOD mean median min max 6-monoacetylmorphine 5.5 2 6.1

S25

Citalopram 1.2 8 121.1 82.8 37.0 244.8 Clarithromycin 1.9 8 1377 1004 671.0 2683 Clobazam 3.4 0

S26

MDA 2.4 0

S27

Ronidazol 1.1 8 13.4 15.4 3.1 22.4 Sarafloxacin 1.9 5 12.6 15.3

S28

Acetylsalicylic Acid 50.0 8 11657 8367 5908 25900 Chloramphenicol 5.0 1

Table SI-4C: Mean, median, minimum and maximum concentrations in influent wastewater for the studied substances during the sampling campaign carried out in April 2012 during 8 consecutive days (compounds determined in negative and positive ionization mode).

LODs [N]>LOD mean median min max 6-monoacetylmorphine 5.5 4 6.9

S29

Benzoylecgonine 1.0 8 291.2 262.1 214.9 409.5 Betamethasone 30.4 0

S30

Lamotrigine 12.2 8 190.4 187.0 144.1 264.1 Levetiracetam 4.4 8 2687 2791 2093 3254 Lidocaine 2.9 8 216.8 234.8 108.1 307.3 Lincomycin 5.9 8 27.9 27.9 12.2 42.8 Lorazepam 3.8 8 65.0 59.7 53.2 87.2 LSD 2.3 0

S31

Ranitidine 7.7 8 1233 1803 32.5 2137 Risperidone 0.3 8 2.2 2.1 1.6 3.1 Ronidazol 1.1 8 15.0 16.4 3.9 21.3 Sarafloxacin 1.9 0

S32

Gemfibrozil 2.6 8 121.5 113.7 9.1 207.2 Hydrochlorothiazide 9.2 8 1469 1410 1363 1650 Ibuprofen 15.5 8 1245.5 1245.8 1018.4 1397.1 Indapamide 71.2 0

Table SI-4D: Mean, median, minimum and maximum concentrations in influent wastewater for the studied substances during the sampling campaign carried out in March 2013 during 11 consecutive days (compounds determined in negative and positive ionization mode).

LODs [N]>LOD mean median min max 6-monoacetylmorphine 5.5 10 9.4 9.2

S33

Cefalexine 7.5 0

S34

MDA 2.4 6 2.4 2.4

S35

Sulfadiazine (SDZ) 14.0 11 62.3 59.0 35.1 110.4 Sulfadimethoxine (SDM) 10.1 1

S36

Phenobarbital 15.5 0

Table SI-4E: Mean, median, minimum and maximum concentrations in influent wastewater for the studied substances during the sampling campaign carried out in March 2014 during 8 consecutive days (compounds determined in negative and positive ionization mode).

LOD [N]>LOD mean median min max 6-monoacetylmorphine 5.5 8 16.5 16.3 6.7 28.9 7-aminoflunitrazepam 7.0 7 23.0 18.2

S37

Chlorpromazine 3.6 0

Doxycycline 14.9 8 239.5 178.0 127.3 725.6 EME 1.4 8 202.9 203.0 174.2 245.2 EDDP 2.1 8 50.4 50.7 47.3 54.5 Enrofloxacin 7.4 2

S38

Lincomycin 5.9 7 9.7 9.1

S39

Paroxetine 10.1 8 40.9 39.1 20.8 73.5 Primidone 7.0 8 77.7 78.2 48.4 110.0 Progesterone 8.2 8 314.8 275.5 243.8 442.4 Propanolol 5.2 8 229.1 209.0 196.4 291.1 Ranitidine 7.7 8 2041 2006 1174 3085 Risperidone 0.3 8 5.9 6.1 3.7 7.2 Ronidazol 1.1 7 19.9 20.5

S40

Triamterene 4.3 8 106.0 100.2 84.6 138.6 Trimethoprim 1.7 8 106.4 100.1 84.6 138.7 Tylosin 28.0 0

S41

Figure SI-4A: Changes in mean loads (loads (g day-1)) in the analyzed influent samples. Only the concentration in the dissolved phase are used for the calculation of loads. The only compounds that the loads were underestimated are Azithromycin (46% in the particulate phase), Sertraline (53%), EDDP (27%), and Amitriptylline (39%). In the preparation of these graphs only substances that were detected in over 80% of the samples in at least two of the sampling periods were considered (the most relevant compounds).

S42

S43

S44

S45

S46

S47

Figure SI-4B: Daily use (g day-1) of drugs of abuse through the five years investigation (2010- 2014).

Ecstasy 70

1 1

30 g day g 20

Dec 2010 Apr 2011 Apr 2012 Mar 2013 Mar 2014

Cocaine

1050

900

750

600

1 1

450 g day g 300

150

Dec 2010 Apr 2011 Apr 2012 Mar 2013 Mar 2014

S48

Amphetamine 900

750

600

1 1 - 450

g day g 300

150

Dec 2010 Apr 2011 Apr 2012 Mar 2013 Mar 2014

Cannabis

120000

105000

90000

75000

1 1 - 60000

g day g 45000

30000

15000

Apr 2011 Apr 2012 Mar 2013 Mar 2014

S49

Heroin

1500

1350

1200

1050

1 -

900 g day g 750

600

450

Dec 2010 Apr 2011 Apr 2012 Mar 2013 Mar 2014

Methamphetamine

1 1 -

30 g day g 20

Dec 2010 Apr 2011 Apr 2012 Mar 2013 Mar 2014

S50

Methadone 160

140

120

100

1 1

80 g day g 60

Dec 2010 Apr 2011 Apr 2012 Mar 2013 Mar 2014

Statistical Analysis: The mean values of two years (pairwise) were compared statistically with t-test at probability level of 0.05. In order to perform the appropriate t-test, assuming equal or not equal variances between the tested data sets, F-test was preceded. This statistical analysis was applied for all the pairs of annual data sets.

S51

Table SI-4F: Mean use of illicit drug (g day-1) for each year; n: number of samples for each year; SD: standard deviation of the n measurements.

2010 2011 2012 2013 2014 Compound n= 7 n=8 n = 8 n=11 n=8 Mean SD Mean SD Mean SD Mean SD Mean SD

Cocaine 473 (a) 103 488 (a) 54 474 (a) 115 529 (a) 78.7 690 (b) 166

Heroin 892 (a) 124 1050 (a) 268 560 (b) 40 888 (a) 103 1311 (c) 103

Amphetamine 390 (a) 194 200 (b) 65 64 (c) 10 196 (b) 34 206 (b) 74

Methamphetamine 20.5 (a) 5.0 20.2 (a) 3.2 39.7 (b) 3.6 41.1 (b) 4.1 45.1 (b) 6.9

MDMA (Ecstasy) 5.6 (a) 4.3 6.2 (a) 3.9 9.0 (a) 6.4 19.1 (b) 13.5 24.4 (b) 17.6

THC (cannabis) - - 30222 (a) 19319 33482 (a) 11383 54704 (a) 42309 37935 (a) 19257

Methadone 20.3 (a) 8.3 34.0 (a) 15.1 72.2 (b) 11.6 116.9 (c) 18.8 138.6 (d) 6.8

Values in the same line with the same letter(s) (a, b, c and d) are not statistically different at P = 0.05.

Compounds names in blue show an overall increase between 2010 and 2014.

S52

Table SI-4G: Mean use of pharmaceuticals (doses day-1) for each year; n: number of samples for each year; SD: standard deviation of the n measurements.

Compound 2010 2011 2012 2013 2014 n= 7 n=8 n = 8 n=11 n=8

Mean SD Mean SD Mean SD Mean SD Mean SD

NSAIDs

Acetylsalicylic Acid - - 81062 (a) 9231 84268 (a) 9304 80618 (a) 16553 55221 (b) 12898

Diclofenac - - 15617 (a) 1746 25540 (b) 2681 21843 (c) 3621 18183 (d) 2066

Ibuprofen - - 6021 (a) 1547 5943 (a) 747 4870 (ab) 662 4774 (b) 761

Ketoprofen - - 1152 (a) 121 1685 (b) 349 1862 (b) 385 936 (c) 29

Mefenamic Acid - - 607949 (a) 39304 59212 (b) 7289 31824 (c) 2686 21986 (d) 1578

Naproxen - - 57767 (a) 5301 65074 (ab) 8453 64521 (b) 7729 68924 (b) 3167

Sum of NSAIDs - - 769568 (a) 40787 241723 (b) 14800 205538 (c) 18832 170024 (d) 13555

Antihypertensives

Atenolol - - 24482 (a) 2861 38757 (b) 2215 23514 (a) 2990 35047 (c) 1326

Propranolol - - 1127 (a) 127 2293 (b) 315 919 (c) 132 5319 (d) 385

S53

Valsartan - - 25988 (a) 3025 51805 (b) 7928 625189 (c) 92764 385263 (d) 38287

Metoprolol - - 3422 (a) 513 7203 (b) 726 7172 (b) 814 7892 (c) 372

Sum of Antihypertensives - - 55018 (a) 4197 100059 (b) 8270 656795 (c) 92816 433521 (d) 38314

Antibiotics

Azithromycin - - 639 (a) 69 646 (a) 96 682 (a) 103 272 (b) 44

Cefaclor - - 471 (a) 50 570 (b) 101 643 (b) 115 639 (b) 52

Clarithromycin - - 4019 (a) 479 1569 (b) 318 506 (c) 98 624 (c) 97

Doxycycline - - 2040 (a) 493 1083 (b) 135 1633 (a) 246 1681 (a) 242

Ciprofloxacin - - 4827 (a) 505 3183 (b) 558 3376 (b) 529 3558 (b) 332

Norfloxacin - - 892 (a) 47 706 (b) 104 582 (c) 92 283 (d) 35

Ofloxacin - - 819 (a) 45 799 (a) 124 641 (b) 54 992 (c) 30

Sulfamethoxazole (SMTX) - - 670 (a) 105 718 (a) 204 451 (b) 107 488 (b) 41

Sulfapyridine (SPD) - - 619 (a) 114 455 (b) 115 485 (b) 60 462 (b) 26

Lincomycin - - 39 (a) 10 62 (b) 9 62 (b) 5 23 (c) 3

Trimethoprim - - 2344 (a) 316 2955 (b) 420 1321 (c) 209 1250 (c) 80

S54

Metronidazole - - 1784 (a) 1302 2632 (a) 270 2770 (a) 401 3834 (b) 285

Sum of Antibiotics - - 19163 (a) 1599 15379 (b) 884 13152 (c) 778 14107 (d) 524

Antiepileptics

Carbamazepine 23512 (a) 2671 25579 (a) 1690 16967 (b) 2029 9591 (c) 1275 12445 (d) 484

Lamotrigine - - 3375 (a) 625 7412 (b) 1037 6093 (c) 959 15181 (d) 796

Topiramate - - 3066 (a) 528 3361 (a) 493 3601 (a) 822 1290 (b) 122

Levetiracetam 19184 (a) 2026 18930 (a) 1640 14891 (b) 1653 20046 (ac) 2584 20688 (c) 1099

Phenytoin 595 (a) 46 604 (a) 11 1248 (b) 77 1282 (b) 104 904 (c) 38

Primidone 474 (a) 74 493 (a) 66 665 (b) 129 1078 (c) 171 749 (b) 72

Valproic Acid 11494 (a) 464 16821 (b) 5673 33390 (c) 7471 44154 (d) 6282 36761 (c) 6208

Sum of Antiepileptics 55258 (a) 3386 68868 (b) 6197 77935 (c) 8000 85846 (d) 7028 88018 (d) 6375

Antilipidemics/Diuretics

Atorvastatin - - 20289 (a) 3422 17003 (a) 3144 39128 (b) 5148 36243 (b) 1699

Gemfibrozil - - 13203 (a) 1325 4641 (b) 1743 7978 (c) 1101 20824 (d) 4365

Hydrochlorothiazide - - 26620 (a) 3008 55805 (b) 4855 22293 (c) 1543 108330 (d) 3027

S55

Sum of - - 60112 (a) 4744 77448 (b) 6041 69399 (c) 5486 165397 (d) 5577 Antilipidemics/Diuretics

Benzodiazepines/ Antidepressants

Lorazepam 8538 (a) 1144 17309 (b) 2117 25771 (c) 3124 35201 (d) 4128 36866 (d) 2708

Oxazepam 9417 (a) 1733 20649 (b) 2951 49135 (c) 7460 60638 (d) 8156 203638 (e) 17245

Mirtazapine 497 (a) 46 2005 (b) 234 5264 (c) 783 10469 (d) 1883 14309 (e) 769

Citalopram 5153 (a) 1098 18748 (b) 4065 38401 (c) 5791 94135 (d) 7103 116416 (e) 3929

Paroxetine 1130 (a) 21 3338 (a) 3751 41390 (b) 17325 15641 (c) 602 96833 (d) 7916

Venlafaxine 11481 (a) 1474 13877 (b) 1260 27776 (c) 2722 23108 (d) 2419 34269 (e) 1697

Gastric/Ulcer drugs

Cimetidine - - 221 (a) 6 315 (b) 47 110 (c) 15 97 (e) 8

Omeprazole - - 2921 (a) 344 4440 (b) 829 3050 (a) 634 5724 (c) 476

Ranitidine - - 7607 (a) 795 9213 (b) 760 20556 (c) 2711 20265 (c) 2608

S56

Sum of Gastric/Ulcer - - 10749 (a) 866 13969 (b) 1126 23717 (c) 2784 26087 (c) 2651 drugs

Values in the same line with the same letter(s) (a, b, c, d and e) are not statistically different at P = 0.05

Compounds’ names in blue show an overall increase between 2010 and 2014, while compounds’ names green show an overall decrease between 2010 and 2014.

S57

SI-5: Greek socio-economic indicators from 2010 to 2014.

Figure SI-5: Variation in Gross Domestic Product per capita (GDP), unemployment and young unemployment from 2010 to 2014 and variation in health expenditure from 2009 to 2013.

Source: World Bank / www.tradingeconomics.com

Health Expenditure in Greece

15000

13000

11000

9000

7000

Expenditure(Billion Euros) 5000

3000 2009 2010 2011 2012 2013 Years Private Sector General Government Total Expenditure

Source: National Statistical Service of Greece http://www.statistics.gr/

S58

th th 5 Sampling 4 Sampling

rd 3 Sampling

nd 2 Sampling st 1 Sampling

Source: National Statistical Service of Greece / www.tradingeconomics.com

th 4 Sampling th 5 Sampling rd 3 Sampling

nd 2 Sampling st 1 Sampling

Source: National Statistical Service of Greece / www.tradingeconomics.com

S59

Table SI-5: Spearman Rank Order Correlations. Marked correlations in red are significant at p <0.050.

Healt Youth SUM SUM SUM SUM SUM SUM SUM GDP expend Unempl. Cocaine MDMA AMPH Cannabis Heroin METHAMPH Methadone Unempl. NSAIDs Antibiotics antihypertensives antiulcer antilipidemics antiepileptics psychiatrics (bn€) GDP 1,00000 1,00000 -0,90000 -0,90000 1,00000 0,80000 -0,80000 -1,00000 -0,800000 -1,00000 -1,00000 -0,90000 -1,0000 0,30000 -0,80000 -0,20000 -0,90000 -1,00000 Healt expend 1,00000 -1,00000 -1,00000 1,00000 1,00000 -1,00000 -1,00000 -0,500000 -1,00000 -1,00000 -0,80000 -1,0000 0,800000 -1,00000 0,600000 -0,80000 -1,00000 (bn€) Unemployment 1,00000 1,00000 -0,8000 -1,00000 1,00000 0,80000 0,400000 0,90000 0,90000 0,800000 0,90000 -0,50000 1,00000 -0,10000 0,80000 0,90000 Youth Unempl. 1,00000 -0,8000 -1,0000 1,00000 0,80000 0,400000 0,90000 0,90000 0,800000 0,90000 -0,50000 1,00000 -0,10000 0,80000 0,90000 SUM NSAIDs 1,00000 0,80000 -0,80000 -1,00000 -0,800000 -1,00000 -1,00000 -0,80000 -1,0000 -0,40000 -0,80000 -0,40000 -1,00000 -1,00000 SUM Antibiotics 1,00000 -1,00000 -0,80000 -0,400000 -0,80000 -0,80000 -0,60000 -0,8000 0,000000 -1,00000 0,000000 -0,80000 -0,80000 SUM 1,00000 0,80000 0,400000 0,80000 0,80000 0,600000 0,80000 0,000000 1,00000 0,000000 0,80000 0,80000 antihypertensives SUM antiulcer 1,00000 0,800000 1,00000 1,00000 0,800000 1,00000 0,400000 0,80000 0,400000 1,00000 1,00000 SUM 1,000000 0,80000 0,80000 0,400000 0,80000 0,200000 0,40000 0,200000 0,80000 0,80000 antilipidemics SUM 1,00000 1,00000 0,900000 1,00000 -0,30000 0,80000 0,200000 0,90000 1,00000 antiepileptics SUM psychiatrics 1,00000 0,900000 1,00000 -0,30000 0,80000 0,200000 0,90000 1,00000 Cocaine 1,000000 0,90000 -0,10000 0,60000 0,500000 0,70000 0,90000 MDMA 1,00000 -0,30000 0,80000 0,200000 0,90000 1,00000 AMPH 1,000000 0,00000 0,700000 -0,10000 -0,30000 Cannabis 1,00000 0,000000 0,80000 0,80000 Heroin 1,000000 0,10000 0,20000 METHAMPH 1,00000 0,90000 Methadone 1,00000

S60

References

1. Dasenaki, M. E.; Thomaidis, N. S., Multianalyte method for the determination of pharmaceuticals in wastewater samples using solid-phase extraction and liquid chromatography-tandem mass spectrometry. Anal. Bioanal. Chem. 2015, 407, (15), 4229-45. 2. Lee, H. B.; Peart, T. E.; Svoboda, M. L., Determination of endocrine-disrupting phenols, acidic pharmaceuticals, and personal-care products in sewage by solid-phase extraction and gas chromatography-mass spectrometry. J. Chromatogr. A 2005, 1094, (1-2), 122-9. 3. Lin, W.-C.; Chen, H.-C.; Ding, W.-H., Determination of pharmaceutical residues in waters by solid-phase extraction and large-volume on-line derivatization with gas chromatography–mass spectrometry. J. Chromatogr. A 2005, 1065, (2), 279-285. 4. Borova, V. L.; Maragou, N. C.; Gago-Ferrero, P.; Pistos, C.; Thomaidis, N. S., Highly sensitive determination of 68 psychoactive pharmaceuticals, illicit drugs, and related human metabolites in wastewater by liquid chromatography-tandem mass spectrometry. Anal. Bioanal. Chem. 2014, 406, (17), 4273-85.

S61