**3. Typology of toxic effects elicited by drugs and its relation with pharmacology**

The toxicity of pharmaceutical drugs in exposed aquatic biota is frequently determined by their intrinsic pharmacology and toxicology, and outcomes that are already described for other species are also possible to occur in aquatic organisms [29]. Additionally, other toxic effects may not derive at all from the known pharmacology of these substances, and can indeed result from specific biochemical and physiological pathways that are over-stimulated in highly responsive species. Thus, it is possible to expect a wide range of toxic effects, which difficult the process of selection of adequate and responsive toxicological endpoints to be observed and studied, both in monitoring and in laboratory based assays.

Despite the multiplicity of effects drugs can cause on non-target aquatic organisms, the selection of a marker of toxicity is sometimes extremely difficult. It is impossible to select a single biomarker that will be equally responsive to all drugs, considering the diversity of pharmacological mechanisms involved in the activities of such a large number of substances. Consequently, it must be emphasized that a thorough process of selection of adequate markers of toxicity is mandatory. However, the quest for a putative toxic effect of a drug can be directly connected to its mode of action, in pure pharmacological terms. This was the case of anticho‐ linesterasic compounds of therapeutic use, such as pyridostigmine and neostigmine, as shown by Rocha et al. [30]. This study showed that the most likely effects of environmental contam‐ ination by these two substances could be related to cholinesterasic impairment, a key factor encompassing other effects at the individual and population level, such as reduced feeding behaviour and decrease offspring production. However, this study was not followed by other successful attempts to establish relationships between drug exposure and potential ecotoxi‐ cological effects, and very scarce is the number of published papers that point this possibility. The study conducted by Rodrigues et al. [15] evidenced that the same drug, pyridostigmine, could elicit similar results in the fish species *Lepomis gibbosus*, in terms of cholinesterasic inhibition; however, this pattern of response was not reflected by behavioural modifications, despite the occurrence of neurotoxicity. Drugs can act by pathways that are also shared by other classes of compounds, which share with drugs the same aquatic matrix. Cholinesterasic inhibition was again the target of the study by Nunes et al. [31], when studying the combination effects of a pharmaceutical drug, pyridostigmine, and two common environmental contami‐ nants (the metal copper, and the organophosphate pesticide chlorfenvinphos), well known for their ability to impair cholinesterasic activity of exposed aquatic organisms. This study showed that the combination of the three compounds, even for realistic levels, could result in a toxicological outcome that constitutes the exacerbation of the pharmacological pathway activated by the drug pyridostigmine.

The environmental effects of other substances of therapeutic use are also related with their intrinsic effects. One of the most thoroughly characterized examples is the one of antibiotics. By being discharged into the aquatic environment, often maintaining intact their pharmaco‐ logical properties, antibiotics still exert their effects on wild bacterial species [32]. This favours the selection of resistant strains, by means of dispersing resistance genes among susceptible bacteria. This phenomenon has been extensively characterized, and derives from the anthro‐ pogenic pressure exerted by human residues containing drugs that are released into the environment, favouring the dispersion of ancient natural genes that encode resistance mechanisms [33, 34]. To address the issue of antibiotic presence and effects in the environment, namely those that can increase gene transfer among bacterial strains, a common strategy is to analyse where these genes can be found, especially in the water compartment, which is the most vulnerable to this issue. The review by Zhang et al. [35] summarizes the efforts recently devoted to the development of new methodologies to characterize the dissemination of resistance genes in the wild following antibiotic release from human activities. It is thus possible to conclude that the mere analysis of genes encoding resistance factors can be considered an effective tool to analyse the effects of antibiotics in the wild.

from specific biochemical and physiological pathways that are over-stimulated in highly responsive species. Thus, it is possible to expect a wide range of toxic effects, which difficult the process of selection of adequate and responsive toxicological endpoints to be observed and

Despite the multiplicity of effects drugs can cause on non-target aquatic organisms, the selection of a marker of toxicity is sometimes extremely difficult. It is impossible to select a single biomarker that will be equally responsive to all drugs, considering the diversity of pharmacological mechanisms involved in the activities of such a large number of substances. Consequently, it must be emphasized that a thorough process of selection of adequate markers of toxicity is mandatory. However, the quest for a putative toxic effect of a drug can be directly connected to its mode of action, in pure pharmacological terms. This was the case of anticho‐ linesterasic compounds of therapeutic use, such as pyridostigmine and neostigmine, as shown by Rocha et al. [30]. This study showed that the most likely effects of environmental contam‐ ination by these two substances could be related to cholinesterasic impairment, a key factor encompassing other effects at the individual and population level, such as reduced feeding behaviour and decrease offspring production. However, this study was not followed by other successful attempts to establish relationships between drug exposure and potential ecotoxi‐ cological effects, and very scarce is the number of published papers that point this possibility. The study conducted by Rodrigues et al. [15] evidenced that the same drug, pyridostigmine, could elicit similar results in the fish species *Lepomis gibbosus*, in terms of cholinesterasic inhibition; however, this pattern of response was not reflected by behavioural modifications, despite the occurrence of neurotoxicity. Drugs can act by pathways that are also shared by other classes of compounds, which share with drugs the same aquatic matrix. Cholinesterasic inhibition was again the target of the study by Nunes et al. [31], when studying the combination effects of a pharmaceutical drug, pyridostigmine, and two common environmental contami‐ nants (the metal copper, and the organophosphate pesticide chlorfenvinphos), well known for their ability to impair cholinesterasic activity of exposed aquatic organisms. This study showed that the combination of the three compounds, even for realistic levels, could result in a toxicological outcome that constitutes the exacerbation of the pharmacological pathway

The environmental effects of other substances of therapeutic use are also related with their intrinsic effects. One of the most thoroughly characterized examples is the one of antibiotics. By being discharged into the aquatic environment, often maintaining intact their pharmaco‐ logical properties, antibiotics still exert their effects on wild bacterial species [32]. This favours the selection of resistant strains, by means of dispersing resistance genes among susceptible bacteria. This phenomenon has been extensively characterized, and derives from the anthro‐ pogenic pressure exerted by human residues containing drugs that are released into the environment, favouring the dispersion of ancient natural genes that encode resistance mechanisms [33, 34]. To address the issue of antibiotic presence and effects in the environment, namely those that can increase gene transfer among bacterial strains, a common strategy is to analyse where these genes can be found, especially in the water compartment, which is the most vulnerable to this issue. The review by Zhang et al. [35] summarizes the efforts recently devoted to the development of new methodologies to characterize the dissemination of resistance genes in the wild following antibiotic release from human activities. It is thus

studied, both in monitoring and in laboratory based assays.

134 Toxicology Studies - Cells, Drugs and Environment

activated by the drug pyridostigmine.

Effects of other drugs can involve alterations in the regulation of the endocrine system. The use of synthetic oral contraceptives, glucorticoids and others is one of the main causes for the ubiquitous presence of these classes in most aquatic matrices, where they maintain their biological effects [36]. Some of these compounds have already been demonstrated to exert potential endocrine effects in exposed wildlife, even in the ranges of concentrations in which they were found [37]. Despite their present levels of contamination, which are indisputably low, the large use and liberation of these compounds into the aquatic compartment will not exempt this endocrine disrupting substances from exerting deleterious effects on exposed biota, as reviewed by Runnalls et al. [38]. In fact, the effects caused by this class of compounds have already been documented, especially in terms of development, metamorphosis, and sexual dimorphism, in amphibians [39], fish [40, 41], molluscs [42], and also crustaceans [43]. It is not surprising that these compounds, even after excretion, maintain their initial pharma‐ cological properties. However, effects elicited are well distinct from those caused in humans, and can include feminization and significant alterations in individual features and population structures, which constitutes a major effect in ecological terms. So, it is possible to conclude that the assessment of endocrine disrupting effects is another valid example on how to use the pharmacological properties of a given class of chemical drugs to search for their effects in the environment. The study conducted by Velasco-Santamaría et al. [44] evidenced that the presence of such compounds in the wild can also be potentiated by the concomitant presence of other substances with similar pharmacological properties and therapeutic use. The synthetic oestrogen ethinylestradiol can exert increased toxic endocrine effects in the presence of another endocrine active pharmaceutical, such as trenbolone, in the marine fish species *Zoarces viviparous*.
