*3.3. Calculations*

The following parameters were calculated:

Antimicrobial removal (RE) calculated using Equations (1) and (2) according to Douziech et al. [59]:

$$\text{RE } \text{[\%]} = (1 - \exp \text{(RR)}) \times 100\% \tag{1}$$

$$\text{RR} = \text{ln} \left( \text{C}\_{\text{EWW}} / \text{C}\_{\text{IWW}} \right) \tag{2}$$

where RE is the removal of antimicrobials during treatment [%], RR (response ratio (effect size)) is measured per WWTP and antimicrobial, CEWW is the mean of the effluent wastewater concentration of the antimicrobial (*n* = 3), and CIWW is the mean of the influent wastewater concentration of the antimicrobial (*n* = 3).

PNEC (predicted no effect concentration) for resistance selection in wastewater was calculated according to Bengtsson-Palme and Larsson [28], whereas PNEC for eukaryotic species was calculated as described by Page et al. [27]. Depending on the number of long-term toxicity tests performed, i.e., no test, the test on one, two, or three trophic levels, the lowest obtained NOEC (no observable effect level) or EC50 (half maximal effective concentration) was divided by 1000, 100, 50, or 10 [60]. PNEC for cyanobacteria was obtained by dividing the NOEC or EC50 by 10. All PNEC values were presented in Table 4.

PNEC for soil (PNECsoil) and sludge (PNECsludge) was obtained by multiplying PNEC with Kd (solid/liquid partition coefficient, Table 2). Kd was calculated as the ratio of the concentration of an antimicrobial on a solid phase (sludge) divided by the equilibrium concentration in the contacting liquid phase (wastewater). PNEC values for resistance selection can be found in Table 5.

Risk quotients (RQs) were calculated using Equation (3):

$$\text{ROQ}\_{\text{wastewater}} = \text{MEC}\_{\text{wastewater}} / \text{PNEC} \tag{3}$$

$$\text{RQ}\_{\text{sludgge}} = \text{MEC}\_{\text{sludgge}} / \text{PNEC}\_{\text{sludgge}} \tag{4}$$

$$\text{RQ}\_{\text{vusetevater}} = \text{PEC}\_{\text{soil}} / \text{PNEC}\_{\text{soil}} \tag{5}$$

where PECsoil is predicted environmental concentration in soil (Table 3), MEC is measured environmental concentration, and PNEC is predicted no effect concentration.

The risk ranking criterion was RQ < 0.1, minimal risk; 1 > RQ ≥ 0.1, medium risk; and RQ > 1, high risk [61].

Predicted daily load of the antimicrobial into WWTP (PLoad) from actual consumption data of the product using Equation (6) is:

$$\text{PL} \bullet \text{ad} = \text{TOTAL} \cdot \text{WWP} / (\text{INHAB} \cdot \text{30}) \tag{6}$$

where PLoad is the predicted daily load of the antimicrobial into WWTP (mg/d), TOTAL is the total monthly consumption of the pharmaceutical in a country (mg), WWTP is an equivalent number of inhabitants in a WWTP, and INHAB is the number of inhabitants in a country (38,000,000).

Average daily load of the antimicrobial into a WWTP from the measured data using Equation (7) is:

$$\text{Load}\_{W+\mathbb{S}} = \text{Load}\_{W} + \text{Load}\_{\mathbb{S}} = \text{C}\_{W}\cdot\text{Flow} + \text{C}\_{\mathbb{S}}\cdot\text{PS} \tag{7}$$

where LoadW+<sup>S</sup> is the average total daily load of antimicrobial into WWTP (mg/d), LoadW is the load of a compound in a water phase of an influent, LoadS is the load of a compound in a primary sludge, CW is the concentration of antimicrobial in the WWTP influent (water phase) (mg/m3), Flow is the average daily flow rate in the WWTP (m<sup>3</sup>/d), CS is the concentration of antimicrobial in the WWTP primary sludge (mg/m3), and PS is the average daily volume of the WWTP primary sludge (m<sup>3</sup>/d).
