**9. Conclusions**

The need to understand the potential effects of a large number of biologically active substances in the wild, has driven researchers to the development of new assessment methodologies. Unlike other substances with human origin, pharmaceutical drugs are active, and may pose significant risks even after their elimination and for long periods. Being excreted in extremely high amounts and on a daily basis, such substances are pseudopersistent and recalcitrant. Not even the implementation of dedicated sewage treatment systems endured to reduce the global amounts of drugs entering especially the aquatic environment. Consequently, the presence of such substances is now a global reality, needing to be dully characterized. Non-target species are the most likely and vulnerable targets for the exertion of deleterious effects. The study of putative toxic effects requires the development, implementation and validation of novel analytic tools, specifically devoted to the particularities of drug contamination. From the revised literature, it is possible to anticipate that ecotoxicological analysis, in the future, will require the combination of distinct tools, on a complementary basis. The tools to be used in the future will not only respond to extremely low levels of contamination, but will include signalling responses well suited to diagnose exposure to specific classes of drugs. Despite not being entirely adapted to the issue of contamination by low levels of pharmaceutical drugs, standardized bioassays can be a valuable tool, if complemented with adequate molecular and subindividual endpoints. Being impossible to characterize the entire set of toxic responses elicited by single, individual compounds, it will be important to know the most important toxic responses of common pharmacotherapeutic classes, to allow the prediction of potential interactions. The use of multispecies assessments will also be important, since the sensitivity towards specific compounds is not necessarily comparable among distinct test organisms. Long-term studies, favouring phenomena of bioaccumulation during important periods of the organisms' life cycles will allow knowing in detail the potential endocrine effects elicited by specific compounds. Behaviour is another feature likely to be altered after pharmaceuticals exposure, thus requiring the development of new testing methodologies to address this issue.

#### **Acknowledgements**

This work was supported by funding FEDER through COMPETE- Programa Operacional Factores de Competitividade, and by National funding through FCT-Fundação para a Ciência e Tecnologia.
