**1. Introduction**

Recent years have brought a new awareness about the potential deleterious environmental impacts of a multiplicity of anthropogenic substances. We have now reached a scientific/ technological standpoint that allows the prediction of most likely effects posed by large groups of substances, including pharmaceutical drugs, and their effects on exposed biota. However, as is the case of pharmaceutical drugs, scarce are the studies and unequivocal data that establish a direct linkage between their environmental presence and dispersal, and toxicity in exposed organisms. Several drawbacks are systematically invoked by detractors of the issue of pharmaceutical contamination when considering this issue, from the unmistakable low levels in which drug residues are found, to the absence of effects caused by metabolites being excreted from biologic systems. However, one cannot discard the evidences: drug residues are present in most environmental matrices, including the particular case of the aquatic compart‐ ment; the number of drugs, their metabolites and degradation products detected in these environmental matrices is alarmingly high, and never stopped increasing since their first detections; some of these drugs are not characterized in terms of toxicity towards the majority of exposed organisms, and their toxic outcomes are unpredictable; the use, release and presence of these substances will not end, or be decreased in a near future, a factor that should, at least, work as an additional stimulus for the development of research into this field.

Therapeutic agents, both human and veterinary, are modern commodities that make part of the developed society. These chemicals are usually developed to fulfil a series of criteria, mainly effectiveness, safety, comfort of use, therapeutic success, and low incidence of side effects. However, the issue of environmental fate of these molecules has only recently been raised, and the main approach established in international guidelines (e.g. European Medi‐

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cines Agency, EMA) takes only in consideration the expected levels in which these compounds may occur in the aquatic compartment. Thus, a consensual precautionary principle was adopted, considering only the levels of dispersion, based on estimates of consumption, that drugs may undergone. No specific guidelines or testing protocols were ever developed to analyse the biological effects of pharmaceuticals in the environment, and efforts devoted so far to this problematic have always relied on the quest, validation and proposal of more accurate and reliable analytical methodologies. However, rather than quantifying the presence or levels of drugs in the environment, an integrative approach that characterizes their toxic effects on multiple components of the ecosystem is now much required. Only with results from a comprehensive, well-suited battery of multi-species biotests, complemented by a systematic survey of published data, it will be possible to answer to the big question: are drugs in the environment real threats to biological systems, or overrated innocuous chemicals?

The main drawback of studying the potential deleterious effects of drugs in the environment is the type of compound that one might expect to find. Considering that a rough estimate of the number of distinct substances presently in use in Europe is around 3000, it is possible to sustain the complexity of this task. Taking into consideration that a considerable number of these substances share a similar pharmacology/toxicology, we can reduce this number surely to a certain hundreds; however, being metabolised and excreted, the formation of metabolites and degradation products will increase again the number of substances that one can find in the wild. An additional factor to consider is the absence of toxicity data for the majority of metabolites and degradation products. For all the given reasons, the ecotoxicological profile of most therapeutic drugs is largely unknown, and a large effort must be devoted to the proposal, validation and use of a comprehensive set of biomarker tools. This effort will be mandatory to diagnose exposure to pharmaceuticals for a vast number of species, and to predict the magnitude of the threat posed by pharmaceutical compounds to non-target organisms.

Even if this approach is satisfactorily followed, the discrepancy of experimental data can be another factor to consider. For some drugs, a considerable amount of scientific data is now available, which should facilitate the interpretation of their ecotoxicological profiles and risks. However, this is not always a simple and immediate task to perform. In fact, for some drugs, the already compiled information is sparse, contradictory and based on erroneous assump‐ tions, making extremely difficult the interpretation of data. This is the case of paracetamol, as shown by Nunes et al. [1]. According to this study, the toxicity of paracetamol is highly variable, even among species of the same phylogeny; however, this situation is even more complicated if one considers the variations of magnitude in responses obtained with standar‐ dized bioassays.

More than being an exhaustive attempt to establish a comprehensive review of what has been done in recent years regarding the diagnostic of effects of drugs in the wild, the present chapter intends to summarize the new evidences showing that therapeutic drugs and their residues/ metabolites can indeed work as environmental pollutants, and may constitute additional sources of chemical stress to already polluted areas. It is also our intention to show the linkage between exposures to low, almost vestigial, levels of pollutants, and the most significant biological deleterious effects, in several biological models, mainly from the aquatic environ‐ ment. Both field and laboratory studies will serve as case studies of particular importance to demonstrate that pharmaceuticals, despite their almost negligible concentrations, can be of environmental concern for sensitive key elements of the ecosystem. On the other hand, one of the main purposes of this review is the establishment of key guidelines, for the development, implementation and validation of toxicological biomarker tools to assess the subtle effects elicited by pharmaceuticals.

cines Agency, EMA) takes only in consideration the expected levels in which these compounds may occur in the aquatic compartment. Thus, a consensual precautionary principle was adopted, considering only the levels of dispersion, based on estimates of consumption, that drugs may undergone. No specific guidelines or testing protocols were ever developed to analyse the biological effects of pharmaceuticals in the environment, and efforts devoted so far to this problematic have always relied on the quest, validation and proposal of more accurate and reliable analytical methodologies. However, rather than quantifying the presence or levels of drugs in the environment, an integrative approach that characterizes their toxic effects on multiple components of the ecosystem is now much required. Only with results from a comprehensive, well-suited battery of multi-species biotests, complemented by a systematic survey of published data, it will be possible to answer to the big question: are drugs in the

environment real threats to biological systems, or overrated innocuous chemicals?

organisms.

130 Toxicology Studies - Cells, Drugs and Environment

dized bioassays.

The main drawback of studying the potential deleterious effects of drugs in the environment is the type of compound that one might expect to find. Considering that a rough estimate of the number of distinct substances presently in use in Europe is around 3000, it is possible to sustain the complexity of this task. Taking into consideration that a considerable number of these substances share a similar pharmacology/toxicology, we can reduce this number surely to a certain hundreds; however, being metabolised and excreted, the formation of metabolites and degradation products will increase again the number of substances that one can find in the wild. An additional factor to consider is the absence of toxicity data for the majority of metabolites and degradation products. For all the given reasons, the ecotoxicological profile of most therapeutic drugs is largely unknown, and a large effort must be devoted to the proposal, validation and use of a comprehensive set of biomarker tools. This effort will be mandatory to diagnose exposure to pharmaceuticals for a vast number of species, and to predict the magnitude of the threat posed by pharmaceutical compounds to non-target

Even if this approach is satisfactorily followed, the discrepancy of experimental data can be another factor to consider. For some drugs, a considerable amount of scientific data is now available, which should facilitate the interpretation of their ecotoxicological profiles and risks. However, this is not always a simple and immediate task to perform. In fact, for some drugs, the already compiled information is sparse, contradictory and based on erroneous assump‐ tions, making extremely difficult the interpretation of data. This is the case of paracetamol, as shown by Nunes et al. [1]. According to this study, the toxicity of paracetamol is highly variable, even among species of the same phylogeny; however, this situation is even more complicated if one considers the variations of magnitude in responses obtained with standar‐

More than being an exhaustive attempt to establish a comprehensive review of what has been done in recent years regarding the diagnostic of effects of drugs in the wild, the present chapter intends to summarize the new evidences showing that therapeutic drugs and their residues/ metabolites can indeed work as environmental pollutants, and may constitute additional sources of chemical stress to already polluted areas. It is also our intention to show the linkage between exposures to low, almost vestigial, levels of pollutants, and the most significant biological deleterious effects, in several biological models, mainly from the aquatic environ‐ The issue of pharmaceuticals as contaminants has been a hot topic in environmental sciences for more than two decades [2]. However, this is not a novel issue, and early studies conducted during the mid 70s already showed the presence of significant amounts of clofibric acid (the pharmacologically active metabolite of several fibrates that explains their activity as lipid lowering agents), in water from the sewage systems of a North American town, Kansas City [3]. This same compound was again found in water quality monitoring studies, initially aimed to quantify pesticide residues, in Germany [4], almost twenty years after their initial detection. This apparent coincidence meant that compounds such as clofibric acid might have a general and ubiquitous presence, being highly dispersed among water compartments [5]. This was then confirmed by subsequent studies, showing that the dispersion of these pharmaceutical substances was not limited in any way to sewage or even freshwater, since it could also be detected in the North Sea. Clofibric acid has an undisputable historical importance, that was not followed by the confirmation of its (eco)toxicogical significance [7, 8]; nevertheless, and from a merely retrospective analysis, its detection in several water samples was a major event that served as basis for a new area of environmental toxicology, devoted to the study of the presence and effects of therapeutic drugs in the environment.

Given the enormous body of evidence that was compiled since the mid 90s to the present day, from studies involving all possible aquatic matrices (freshwater, sea water, sewage effluents, drinking water, groundwater) it is almost impossible not to consider the issue of drugs and their ecotoxicological effects one of the most challenging scenarios for years to come. Conse‐ quently, the presence of pharmaceutical residues in the wild is nowadays a matter of interest, among the scientific community and the general public [1, 9, 10, 11]. This interest derives from the intrinsic features that these compound possess. Pharmaceuticals are biologically active, capable of exerting effects in a large number of organisms, even when in extremely low concentrations. Drugs are widely used and dispersed, being ubiquitously found in the aquatic environment, as a result of the overall low degradation efficiency of sewage treatment plants; drugs are present in surface water, groundwater, and even oceans; furthermore, these substances are refractory to biological degradation or can assume other forms after metabolism [2, 9, 12], largely toxicologically uncharacterised especially for wild organisms. An adequate lipophylicity allows drugs to be slightly water soluble, but readily absorbed by living organ‐ isms [8, 13]. Aquatic organisms are by far more exposed to pharmaceutical residues. The deleterious impact of specific therapeutic compounds on aquatic organisms has already been shown to occur, even under real scenarios of contamination [13, 14, 15].

Drugs reach the aquatic compartment mainly via sewage systems. The use of pharmaceutical drugs requires its ultimate elimination from the patients' organisms, which results in its presence in the sewage treatment system, when it is present [2]. In modern western societies, sewage treatment plants (STPs) are common and generally efficient. However, the purpose of 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 the presence of drugs are developed and implemented.

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 expression/epigenetics) might result in the establishment of a link between the low levels of exposure and biological responses in non target, environmentally exposed biota.
