*2.7. Hormones*

Although hormones like E1, E2 and EE2 are mainly used for contraception purposes, the physiological e ffects are not restricted to e ffects on reproductive and sexual development, and can target mitochondrial function, energy metabolism and cell cycle control [165].

For acute toxicity, there is only data on algae and invertebrates, and algae presented higher toxicity since the lowest concentration promoting toxic e ffects was at 162 μg <sup>L</sup>−1, lower than the 1500 μg L−<sup>1</sup> observed in invertebrates [43,78]. Nonetheless, the toxicity promoted by this therapeutic group is mainly expected to be detected through chronic toxicity, however, these data could only be obtained for invertebrates and fish. Considering chronic data, in these two trophic levels, hormones

presented higher toxicity than the other therapeutic groups, since the lowest concentrations reported were of 100 ng L−<sup>1</sup> and 0.1 ng <sup>L</sup>−1, for invertebrates and fish, respectively [81,82,123]. It should also be noted that, the highest concentration found that promoted toxicity for fish was also very low (1188 ng <sup>L</sup>−1) [102].

Individually, there were no di fferences observed between E1 and E2 toxicity, while EE2 seems the most toxic compound regarding chronic toxicity in invertebrates and especially in fish, where the 36 results available presented concentrations below 44 ng L−<sup>1</sup> [135]. Namely, when two di fferent fish species were exposed to EE2 at 3 ng L−<sup>1</sup> and 4 ng L−<sup>1</sup> they su ffered sex gender reversal, from male to female, which can strongly unbalance the aquatic ecosystem [130,136,138].

#### **3. Environmental Risk Assessment**

The data regarding occurrence and toxicity already presented is crucial in order to perform the ERA, and can be used to select the pharmaceuticals that are more prone to induce toxic e ffects in aquatic biota [166]. The risk assessment, mentioned in the EMA guideline on the ERA of medicinal products for human use [4], is performed through the risk quotient (RQ) calculation, dividing the PEC by the PNEC for each pharmaceutical, observing three di fferent trophic levels (algae, invertebrates and fish). If RQ is equal or above 1, there is a potential environmental risk situation, whereas when values are lower than 1, no risk is expected. However, a certain risk could be expected for the substances with a RQ between 0.1 and 1 [167,168]. However, this guideline is only applied for marketing authorizations and for pharmaceuticals marketed after 2006. Additionally, it does not constitute a valid criterion upon which to base the refusal of a market authorization of medicinal products for human use in the EU [6]. Our evaluation of the potential ecotoxicological risk posed for the aquatic compartment was based on a dual approach: one using the worst case scenario, as stated by the EMA guideline on the ERA [4], where the maximum individual concentrations of pharmaceuticals found in the respective aquatic compartment were used as MEC [15,169,170], and another using the median concentrations for each pharmaceutical as MEC [171]. This evaluation can also be an important tool to sugges<sup>t</sup> the inclusion or removal of pharmaceuticals in the watch list of the Directive 2013/39/EU.

As discussed, some concentrations compiled in surface water are higher than the levels that induce toxicity, not applying any uncertainty factor (UF) for the PNEC calculation. Additionally, some studies have indicated that concentrations of several pharmaceuticals belonging to di fferent therapeutic groups can promote toxic e ffects on negatively impacted aquatic biota, presenting RQ higher than 1 [11,18,139,157,172,173].

As referred, aquatic biota inhabiting the receiving environment are unintentionally exposed throughout their lifetime to a complex mixture of residual pharmaceuticals and these mixtures can exhibit a greater e ffect than individual compounds [20,174,175]. Therefore, it is a challenge to address the concerns related to the chronic e ffect, low-level exposure to these compounds, including exposure of sensitive subpopulations to pharmaceutical mixtures [17,174].

#### *3.1. Predicted No-E*ff*ect Concentration (PNECs)*

Based on the toxicity data (Figures 4–7), Table 2 presents the PNECs for the selected pharmaceuticals. These values were calculated by applying an UF of 100 and 10 to the long-term EC50 and NOEC values, and an UF of 50 and 1000 to the short-term LOEC and L(E)C50 values, respectively, available in the literature. The UF is an expression of the degree of uncertainty in the extrapolation from the test data on a limited number of species to the actual environment [4]. As referred, when no experimental data are available, L(E)C50 values were estimated through ECOSAR 2.0.


**Table 2.** Predicted no-effect concentrations of the selected pharmaceuticals for algae, invertebrates and fish for the studied pharmaceuticals.

M—metabolite; TP—transformation product; NH—natural hormone. a: EC50 was estimated with ECOSAR. b: UF = 1000. c: LC50 was estimated with ECOSAR. d: UF = 50 (uncertainty factor used for lowest observed effect concentrations (LOEC) and no observed effect concentrations (NOEC) in acute toxicity). e: long-term data. f: UF = 100. g: UF = 10 (uncertainty factor used for LOEC and NOEC in chronic toxicity).

It should be taken into account that the choice of toxicity data can obviously affect the outcome [15]. However, the results obtained for the PNECs were directly related to the toxicity data, and a similar pattern to the toxicity data was observed, with the therapeutic groups and pharmaceuticals with higher toxicity presenting the lowest PNEC values.
