**4. Mortality, growth impairment an other classic tools**

Despite their traditional use for the toxicity assessment of a large number of substances, mortality and growth impairment endpoints have been extensively used to describe the toxicity of pharmaceutical substances towards wild biota. In fact, more than reflecting real scenarios of contamination, the validity of calculating and interpreting mortality data relies on the establishment of sublethal toxicity criteria and to analyse in comparative terms, the ecotoxicity of substances that act by unknown (or largely uncharacterised) mechanisms of toxic action. In fact, LC50 values of pharmaceutical drugs are well suited to establish rankings of ecotoxicity among very distinct compounds, and these criteria are straightforward to imple‐ ment and interpret. However, these endpoints are not a first choice if one requires the establishment of complex mechanisms, or even effects at extremely low levels, which are likely to cause alterations other than death, immobilization, or impairment of the population growth. These responses are not fine tuned alterations, and may be seen as blunt tools for toxicity characterization, with poor of even null ecological relevance.

The number of ecotoxicity studies analysing lethality or growth impairment is thus consider‐ able. The study published by Carlsson et al. [45], showed that antiparasitic drugs could exert extreme toxicity, when compared to antibiotics, thus threatening the survival of zebrafish embryos. The crustacean species *Daphnia magna* was very sensitive to the antidepressant sertraline, as shown by Minagh et al. [46]; this compound was capable of inducing strong alterations in the population of this crustacean species, after 21 days of exposure. Population changes were also observed after chronically exposing *D. magna* to the compounds testosterone and 4-hydroxyandrostenedione, even for ecologically relevant levels [47], reinforcing the possibility of exertion of deleterious effects in the wild. Another crustacean, namely *Neocaridina denticulate*, demonstrated to be extremely sensitive in terms of lethality to a combination of pharmaceutical drugs (paracetamol and ibuprofen) commonly found in the aquatic environ‐ ment [48]. The ecotoxicological effects of mefenamic acid on *D. magna* and *Moina macrocopa* were evaluated by Collard et al. [49]. This study showed that lethal effects were not likely to occur for already reported levels of this compound; however, the chronic exposure to this compound showed that changes in reproduction are possible, especially in the case of *M. macrocopa*.

On the other hand, the absence of potential risk posed by the presence of clofibric acid was evidenced by the study by Nunes et al. [50] and by Emblidge and DeLorenzo [51], in multi‐ species assessments that focused on mortality and growth impairment as toxicity endpoints. Similarly, the work by Ferreira et al. [52] evidenced the absence of potential lethal effects posed by antibiotics (oxytetracycline and florfenicol) on the crustacean species *Artemia parthenogene‐ tica*. Despite not exerting lethal effects, several combinations of pharmaceutical drugs (includ‐ ing an ecologically relevant mixture) were capable of causing significant alterations in zebrafish embryos, as shown by Madureira et al. [53]. The antibiotic oxytetracycline was shown to be reasonably safe towards the fish species *Labeo rohita*, since the lethal levels were well above concentrations that are not likely to be found in the wild, as shown by Ambili et al. [54].

Growth of autothrophic organisms is another endpoint likely to respond to exposure to human use drugs; the study conducted by Berninger et al. [55] showed the refractivity of the aquatic plant species *Lemna minor* to the drug diphenhydramine. Somewhat similarly, the work conducted by Nunes et al. [56] evidenced the occurrence of an oxidative-based response of two species of the genus *Lemna* (*L. minor* and *L. gibba*) elicited by the drug paracetamol, which resulted in significant growth alterations. On the other hand, the study conducted by Ferreira et al. [52] presented evidences concerning the toxicity of the antibiotics oxytetracycline and florfenicol on the microalgae species *Tetraselmis chuii*.
