**2. Effects to be expected from drug exposure**

conventional STPs is reducing the amount of organic pollution, and not the elimination of often-recalcitrant compounds such as drugs. This results frequently in extremely low removal rates in STPs when it concerns to pharmaceuticals [16], requiring the implementation of novel and usually expensive technologies. This results in the continuous release of drugs an their metabolites into the receiving waters. Given that the amount introduced into the wild generally equals the sum of drug that is naturally degraded by natural pathways, it is possible to sustain that pharmaceuticals are environmentally pseudopersistent [17, 18]. Other alternative routes can also explain the presence of pharmaceuticals in the aquatic compartment, but in a lesser extent, such as release from manufacturing industries, and leachates from landfills [19]. Despite the existence of distinct routes by which pharmaceutical substances reach water bodies, it is important to stress that the majority of the residues result from human use and release, and from the inefficacy of treatments systems. Consequently, the issue of aquatic contamination by drugs is intrinsically connected to the personal use made by human consumers, which cannot be stopped or prevented, even if more advanced solutions to mitigate

As a consequence of human use, several classes of drugs are routinely detected and quantified in the most varied water matrices. The most prominent classes of drugs found in the wild include non steroidal anti-inflammatories, antibiotics, anticonvulsants, antidepressants and oral contraceptives, which are systematically reported in monitoring surveys [20, 21] of the aquatic environment. However, this corresponds to a generic assumption and the reality shows that almost all substances (or their metabolites/degradation products) used in human thera‐ peutics can be virtually detected, mainly in sewage or even in receiving waters. It is also noteworthy to observe that representatives of all these pharmacotherapeutic classes co-occur, simultaneously, in the same sample or matrix. Despite co-occurring in extremely low amounts, usually ranging from the ng to the µl per litre, it is not possible to discard the possibility of exertion of effects, mediated or not by the same receptors activated during human therapy. This poses important challenges, not only in analytical terms (which are out of the scope of this chapter), but especially in terms of the toxicological deleterious outcomes resulting from exposure to such complex mixtures, in individual terms (altered physiology of exposed organisms) and to the ecosystem [22]. Given these main topics, the major scientific question addressed here can be described as an interconnected two-tier approach: do pharmaceutical drugs, or metabolites/residues, exert deleterious effects in wildlife? If so, what is the type of effects to be expected, and what the extent to be considered? To answer these two issues, it will be necessary to adopt new strategies to surpass the usual difficulties in obtaining responses or measurable biological effects. Until the present day, few studies clearly showed the relationship between realistic conditions of exposure and deleterious effects caused by pharmaceuticals in non-target organisms. Considering the most frequently adopted toxico‐ logical endpoints (e.g. death, growth impairment) and the levels, concentrations or dosages required to elicit such effects, it is possible to state that traditional approaches are not suited, for most cases, to address the effects of drugs in aquatic organisms. Thus, it is mandatory to select an additional set of tools that may address the issues initially raised, and constitute future testing guidelines for pharmaceuticals in the wild. The combination of standardized methods, well-established analytical techniques, and new biochemical strategies (including gene

the presence of drugs are developed and implemented.

132 Toxicology Studies - Cells, Drugs and Environment

It is impossible to predict the effects of drugs in the wild, given their sheer number and the possible interactions among them in the wild. The mere quantity of different drugs in use in modern human therapeutics is overwhelming, and prevents the establishment of any plausible prediction in terms of toxicity of complex mixtures, such as urban effluents. The human use of pharmaceutical drugs in the European Union is vast, and approximately 3000 distinct substances are used, including substances from different pharmaco-therapeutic classes such as anti-inflammatories, β-blockers, oral contraceptives, blood lipid regulators, antibiotics and others [23]. This is a brief, albeit comprehensive summary of the therapeutic classes one can find in the aquatic compartment, from an empirical perspective of only considering classes of drugs that are used in extremely high amounts. However, this is a criterion that is not exempt of drawbacks or criticisms, since the mentioned classes, those that are used and dispersed in the highest amounts, are not necessarily representative of drugs with the highest biological or toxicological activity. For instance, cytostatic or anticancer drugs are extremely active and biologically aggressive, despite not being extensively used [24]; however, these substances are among the most active environmental drugs [25]. It is thus extremely difficult to prioritise substances only based on simple criteria of use and consumption; more difficult is the task to develop and validate markers of toxicological interest to be used in routine analysis.

Furthermore, the presence of an even larger number of metabolites [26], and products of degradation by natural or anthropogenic means (photodegradation, hydrolysis, microbial degradation, chemical treatment processes at STPs, chemical reaction among drug residues and with other substances) implies the need to include the possibility of toxicological interac‐ tions among all compounds that may be present in a given environmental (especially water) sample; these interactions, that may result in increased toxicological activity, has been already shown to be a possibility in the wild [27, 28]. It is not just a matter of selecting a biological response, but to choose the one most likely to respond to this vast group of compounds, in a specific organism that may be successfully analysed, both in field surveys and in laboratorybased bioassays. It is not just feasible to study all pharmaceutical compounds, on all putative model organisms.
