3.2.1. Wastewater Influents

The RQs observed in WWIs were the highest from all the aquatic compartments, as well as the concentrations of the selected pharmaceuticals (Figure 4). The highest value (274,816) and median (13,400) were observed for IBU in invertebrates. Anti-inflammatories were the therapeutic group with the highest RQs median, both for the maximum and median values, followed by antiepileptic and hormones.

With the exception of anxiolytics, all the other therapeutic groups presented RQs > 1 for at least two trophic levels, being SSRIs and anxiolytics the only groups that did not present risk RQs > 1 for all pharmaceuticals.

For the anxiolytics and antibiotics, the algae were clearly the most susceptible trophic level, presenting higher RQs. Another clear pattern was observed for the hormones, where for fish all the RQs (median and maximum) were higher than 1 and with the maximum values between 129 and 17 271

These results demonstrated that the concentrations reaching the WWTPs could clearly endanger all the trophic levels that might be exposed to this aquatic matrix.

#### 3.2.2. Wastewater E ffluents

This aquatic compartment presents lower RQs than the WWIs (Figure 5). Antibiotics along with antiepileptic, anti-inflammatories and hormones were the therapeutic groups with highest RQs values. When considering median values alone, antibiotics have lower RQs than the other three therapeutic groups. The values observed for antibiotics can promote an even bigger problem than the direct toxicity to aquatic organism: the emergence of bacterial resistance. Nonetheless, the ERA approach does not address this issue [155].

Anxiolytics continued to present RQs values lower than 1. Antibiotics, on the other hand, had the highest value observed for CIP in algae (100,258). As in WWI, antiepileptic continued to present the highest median RQs.

In WWI, some metabolites of the SSRIs presented similar or slightly higher RQs than the parent compounds (N-CIT and Nor-SER), in WWE a similar pattern was observed. These results highlight the fact that parent compounds and metabolites reached WWEs, and that the concentrations found in this matrix were able to promote toxic effects in the aquatic biota. This fact suggests that metabolites and transformation products should also be monitored in the environment.

Regarding the anti-inflammatories therapeutic group, DIC and IBU stand out from the other pharmaceuticals presenting clearly higher median and maximum RQs. As for hormones and observing the fish trophic level, as in the WWI, all RQs were higher than one.
