**8. The effects of drugs in the wild**

importance to define rankings of comparative toxicity for several substances, independently from their mode of action and toxicity. Data from acute tests can also be useful to determine ranges of concentrations representing sublethal levels to which organisms may be exposed. Finally, the entire set of information potentially gathered from this type of test may serve to prioritise compounds to be studied under chronic conditions of exposure. However, and if one considers the need to increase the ecological relevance of data obtained from ecotoxicity tests, chronic assays are likely to represent a more credible simulation of what happens in the wild. Organisms are frequently subjected to contamination during considerable periods of their entire life cycle, or may even contact with chemical pollution of anthropogenic origin for different generations. It is thus important to prioritise a testing strategy that simulates these conditions, and the most adopted type of bioassay, despite its inherent difficulties, is the chronic exposure. Chronic exposures can more easily mimic real events, occurring under realistic low levels of contaminants, and can consequently increase the ecological relevance of the obtained data. Furthermore, the selection of chronic exposures can permit the proposal of multispecies assessments (e.g. mesocosms), which are obviously advantageous if one intends to simulate real environmental conditions. On the other hand, multispecies assessments are a valid approach, since the sensitivity of distinct organisms towards pharmaceuticals is fre‐

From the majority of the cited studies, it is possible to conclude that a biomarker-based approach is valid to obtain information regarding specific pathways involved in the toxic response. This does not necessarily imply that more traditional approaches (including mortality, or growth/population effects) are fully inadequate to assess the ecotoxicological effects of drugs. Nevertheless, the low levels of exposure make difficult the exertion of such effects, and the resultant toxicity often occurs by impairment of specific, subtler, biochemical pathways. It is thus important to analyse the sub-individual level, and more biomarkers must be proposed and fully validated. Effects at the molecular level, including enzymes, must be interpreted as signalling tools for effect or damage in biological systems. Given their overall importance, specific pathways must be primary sources of analytical tools. It is possible to suggest that novel biomarkers can derive from analysis of the enzymatic machinery involved in the energetic metabolism, gene expression and epigenetics, and damage (e.g. of oxidative nature) repair. These will be the most likely biomarkers of contamination of the future.

The next step will be transposing laboratory biomarker-based assays, with a combination of chronic-acute exposure of multiple species to other alternative models, namely under field conditions. It is now mandatory to propose new test species, well adapted to conditions that do not represent standard settings: species from tropical/polar (or otherwise extreme) regions must be analysed following the above-described strategy, and their use for ecotoxicological purposes validated. This will be of crucial importance to transfer bioassays from the laboratory

Finally, a last step will combine the simultaneous analysis of complex mixtures of drugs. Frequently, environmental matrices are contaminated by a large number of distinct pharma‐ ceuticals; any analytical procedure based on the quantification of effects caused by a single chemical will always be unsatisfactory, and will underestimate the actual toxicity of complex

to the field, increasing the validity of data and of the conclusions drawn.

quently very diverse.

142 Toxicology Studies - Cells, Drugs and Environment

Despite the considerable number of cited research articles so far, the present chapter would not be satisfactorily summarized without a critical evaluation of the potential ecological damages posed by pharmaceutical residues. The major drawbacks for the analysis of effects caused by pharmaceuticals are also the most important defence against their risks: their extremely low levels. Being present in residual amounts, sometimes below the ng/l range, the majority of drugs do not attain levels capable of exerting effects. However, the simultaneous presence of compounds that act via a similar pathway may favour the exertion of effects. Still, few are the examples of substances for which this behaviour has been described. Despite the lack of reported effects, and the impossibility so far of establishing a direct, unequivocal relationship between pharmaceutical exposure and deleterious effects in wildlife, an increas‐ ing number of studies has brought the issue of ecological relevance of data to the discussion. By testing already reported levels of contaminants, some researchers have already claimed having demonstrated the putative effects of drugs in exposed organisms that are also likely to occur in the wild. This has been the case of several fibrates [7], synthetic hormones [93], ivermectin [94], paracetamol [1, 57, 59], ibuprofen [95], neostigmine and pyridostigmine [15, 30, 31], fluoxetine and other antidepressants [93, 96]. However, the majority of studies published so far do not demonstrate this intrinsic association between pharmaceuticals contamination and deleterious effects. Nevertheless, research studies on this matter have clearly demonstrated the responsiveness of a large number of species towards drugs, evi‐ dencing the potential for toxic effects if a threshold level is attained and surpassed. This is of the uttermost importance, since it clearly shows that some of the highly conserved pathways used and activated by pharmaceutical drugs in humans, are also present in a significant number of wild organisms; this increases the possibility of biological-chemical interaction, with sometimes totally unpredicted overall effects. The protection created against the effects of human drugs by their low levels can thus be simply temporary, considering the everincreasing amount of drugs and their residues in the wild.
