Urea

The following chemical compounds were used for DES preparation:
[Chol]Cl (98% purity) was purchased from Acros Organic ® , Geel, Belgium; ethylene glycol (99.5% purity) was purchased from Sigma Aldrich ® , St. Louis, Missouri, EUA; 1,2-propanediol from Panreac ® , Barcelona, Spain; propionic acid (99% purity) from Merck ® , Darmstadt, Germany; glycerol (99,8% purity) was purchased from Fischer Chemical ® , Hampton, New Hampshire, EUA; urea (99% purity) from Panreac ® , Barcelona, Spain; and 1-propanol (99.5% purity) was acquired from Merck ® , Darmstadt, Germany.
Each DES was prepared in the following molar ratios: 1:2, 1:1, 2:1 and 4:1 (HBD:HBA). Briefly, HBD and HBA were added gravimetrically to closed vials and heated with constant agitation. After the formation of a transparent liquid, the mixture was cooled down to room temperature to obtain each DES. Since the purpose of this study was the evaluation of the ecotoxicological character of DES, some of the stock eutectic mixtures were prepared by adding a known volume of water. The water content of the starting materials and of DES was determined by Karl Fischer titration and considered in the calculation of the toxicity (Table S1).

Reline was prepared in a glovebox by mixing, in a 1:2 molar ratio, choline chloride (Acros Organics) and urea (Panreac) followed by heating at 60ºC. The reagents were previously dried under vacuum for three days to avoid water contamination, as it drastically affects Reline's properties 19 . Partially and fully deuterated mixtures were prepared in the same manner. urea-D4 (98 atom % D) was purchased from Sigma-Aldrich while [Ch]Cl-OD was prepared by repeatedly washing with D 2 O and drying under vaccum and heating. The success of isotopic exchange was confirmed by the appearance of the νOD band at 2400 cm -1 in the FTIR spectrum and the νOH/νOD intensity ratio suggests an exchange rate of 90%. Initially, three 1:2 mixtures were prepared: [Ch]ClOD+urea-H4 (total of 10 atom % D), [Ch]ClOH+urea-D4 (total of 87 atom % D) and [Ch]ClOD+urea-D4 (total of 97 atom % D). Weeks later, a fourth was made from blending equal amounts of the first two, resulting in a total of 48 % atom D.

The IP antibiotics were utilised as their corresponding sodium salts: oxacillin (OXA) 95% (from Sigma-Aldrich), cloxacillin (CLX) 92.3% (from Syntopharma S.A) and dicloxacillin (DCX) 98.2% (from Research Pharmaceutical) (see chemical structures in Table 1). Glucose, peptone, yeast extract, monobasic potassium phosphate, zinc sulphate heptahydrate, tetraborate sodium decahydrate, ammonium molybdate, sodium acetate, malt extract, calcium chloride dehydrate and ammonium chloride were bought from Carlo Erba. Ammonium L-(+)-tartrate 98% and 2,6dimethoxyphenol 99% (DMP) was obtained from Alfa Aesar. Manganese sulphate heptahydrate, iron sulphate heptahydrate, potassium chloride, ammonium sulphate, sodium chloride, formic acid, tartaric acid, hydrogen peroxide, acetic acid, sodium sulphate, acetonitrile, methanol and Mueller-Hinton agar were bought from Merck.
2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt 98% (ABTS), veratryl alcohol 96%, 1-hydroxybenzotriazol (HBT), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide > 98% (MTT), dimethyl sulfoxide (DMSO), Dulbecco's modified Eagle's medium (DMEM), ethylenediaminetetraacetic acid (EDTA) and doxorubicin were obtained from Sigma -Aldrich. Fetal bovine serum (FBS) was purchased from Invitrogen. Urea was bought from Panreac.