**6. Alternative tools to assess the environmental effects of drugs: Toxicity, pharmacology and other effects**

Several toxicity assessment projects have relied in the development and validation of new tools to quantify the extent of the toxic response. As previously stated, known pharmacological properties can serve as a comprehensive source of biomarkers to be used in ecotoxicity assessments. However, some of the responses of wild organisms to drugs may be based on physiological mechanisms that are not directly related (activated or impaired) following patterns included in the pharmacology of pharmaceutical substances. Some of these responses are purely paradoxical, while others are only the reflex of the activation of mechanisms and receptors in wild organisms that were never studied and/or identified in common experimen‐ tal models.

This is the case of behavioural alterations in several wild organisms. The work by Berninger et al. [55] showed that the fish species *Pimephales promelas* was highly sensitive to the antihistaminic drug diphenhydramine in terms of feeding behaviour. The feeding behaviour was also modulated after exposure of the fish *Perca fluviatilis* to the antidepressant sertraline, as evidenced by Hedgespeth et al. [63]. Behaviour is also a trait that can be significantly changed after exposure to pharmaceuticals, both in fish [64], and in crustacean species [65]. Strong behavioural alterations were also reported by Nunes et al. [13] after the exposure of the fish *Gambusia holbrooki* to the neuroactive compound diazepam, with impairment of the swimming capability. An opposite pattern has been presented by crustacean species, which seem not to be equally responsive to pharmaceutical drugs. As evidenced by Nieto et al [66], the food ingestion behaviour of the freshwater crustacean *Atyaephyra desmarestii* was not affected by ecologically relevant levels of several therapeutic drugs, such as diclofenac, ibuprofen, and carbamazepine. Food ingestion was also affected following exposure of *Xenopus laevis* to fluoxetine, as demonstrated by Conners et al. [67], thus conditioning the development of this organism during its early life stages. Antidepressants that exert their therapeutic activity through the selective inhibition of serotonin reuptake are likely to be adequate candidates to alter the behaviour of a large number of aquatic organisms, considering that the most promi‐ nent pathway involved in their activity is highly conserved. According to the editorial by Ford [68], specific compounds including sertraline and fluoxetine, can dramatically alter the feeding behaviour profile of a large number of aquatic organisms, from fish to crustaceans.

increased in aquatic organisms by exposure to pharmaceutical drugs [7, 8, 13]. Being routinely quantified in modern ecotoxicological laboratories, the activities of such enzymes are intrins‐ ically interesting to quantify not only the level of exposure to drugs, but also the likely consequences of drug exposure. This may be justified since these enzymatic forms are not only involved in the bioactivation/detoxification of drugs, but do also participate in numerous endogenous processes, which can be altered following an environmentally drug-induced chemical insult. The broad spectrum of these analytical tools is also a factor to consider, being highly unspecific, effects both phase I and II metabolic enzymes can indeed respond to a

**6. Alternative tools to assess the environmental effects of drugs: Toxicity,**

Several toxicity assessment projects have relied in the development and validation of new tools to quantify the extent of the toxic response. As previously stated, known pharmacological properties can serve as a comprehensive source of biomarkers to be used in ecotoxicity assessments. However, some of the responses of wild organisms to drugs may be based on physiological mechanisms that are not directly related (activated or impaired) following patterns included in the pharmacology of pharmaceutical substances. Some of these responses are purely paradoxical, while others are only the reflex of the activation of mechanisms and receptors in wild organisms that were never studied and/or identified in common experimen‐

This is the case of behavioural alterations in several wild organisms. The work by Berninger et al. [55] showed that the fish species *Pimephales promelas* was highly sensitive to the antihistaminic drug diphenhydramine in terms of feeding behaviour. The feeding behaviour was also modulated after exposure of the fish *Perca fluviatilis* to the antidepressant sertraline, as evidenced by Hedgespeth et al. [63]. Behaviour is also a trait that can be significantly changed after exposure to pharmaceuticals, both in fish [64], and in crustacean species [65]. Strong behavioural alterations were also reported by Nunes et al. [13] after the exposure of the fish *Gambusia holbrooki* to the neuroactive compound diazepam, with impairment of the swimming capability. An opposite pattern has been presented by crustacean species, which seem not to be equally responsive to pharmaceutical drugs. As evidenced by Nieto et al [66], the food ingestion behaviour of the freshwater crustacean *Atyaephyra desmarestii* was not affected by ecologically relevant levels of several therapeutic drugs, such as diclofenac, ibuprofen, and carbamazepine. Food ingestion was also affected following exposure of *Xenopus laevis* to fluoxetine, as demonstrated by Conners et al. [67], thus conditioning the development of this organism during its early life stages. Antidepressants that exert their therapeutic activity through the selective inhibition of serotonin reuptake are likely to be adequate candidates to alter the behaviour of a large number of aquatic organisms, considering that the most promi‐ nent pathway involved in their activity is highly conserved. According to the editorial by Ford [68], specific compounds including sertraline and fluoxetine, can dramatically alter the feeding

behaviour profile of a large number of aquatic organisms, from fish to crustaceans.

multiplicity of therapeutic classes.

138 Toxicology Studies - Cells, Drugs and Environment

tal models.

**pharmacology and other effects**

The endocrine disrupting effects of several human use drugs has been the subject of research for several years, and quite a few studies report the occurrence of significant effects caused by drugs (e.g. anti-inflammatories) on fish [69]. Endocrine disruptive effects caused by pharma‐ ceuticals are not exclusive to fish, since invertebrates, such as crustaceans, are also prone to be affected in their endocrine functions by exposure to pharmaceutical drugs, as reviewed by Hutchinson [70]. Neuroendocrine effects are another aspect of this issue. Considering that a large number of pharmaceutical drugs act by altering the expression and effects of biological compounds of high physiological importance (e.g. neuropeptides, neurotransmitters, or neurohormones), it is with no surprise that similar mechanisms can be impaired in non-target species environmentaly exposed to these same drugs. The consequences are not only so far uncharacterised, but also, unpredictable. Consequences to be expected will naturally include alterations in the physiology of exposed wildlife, affecting behavioural traits, or the hormonal homeostasis, which are of fundamental importance to the organisms and to the ecosystem. It is thus expectable to observe impairments at several levels, such as reproduction, develop‐ ment, growth, response to chemical aggression or other sources of stress [71, 72]. The neuro‐ endocrine effects of specific compounds, such as sertraline, were shown by Conners et al. [67] in tadpoles of the species *Xenopus laevis*. This antidepressant substance caused significant developmental impairments during the early life stages of this organism, which occurred for ecologically relevant levels. Another antidepressant drug, such as fluoxetine, was also capable of inducing strong alterations in the reproductive physiology of the fish species, *Carassius auratus*. Another antidepressant, mianserin, was also related to estrogenic activity in fish (*Danio rerio*) by inducing molecular biomarkers of estrogenicity (such as vitellogenin1 and zona pellucida proteins), as evidenced by van der Ven et al. [73]. The study conducted by Mennigen et al. [74]concluded that exposure to relevant levels of this substance could alter the expression and release of several physiological hormones, thus compromising the sexual behaviour of this fish species. Therapeutic drugs such as paracetamol and lincomycin are also involved in endocrine disruption effects. The study conducted by Kim et al. [75] showed that these two pharmaceuticals could affect the steroidogenic pathway and increase estrogenicity, in crusta‐ ceans (*D. magna* and *Moina macrocopa*), but also in fish (*Oryzias latipes*). These effects were translated into a significant reduction in juvenile survival of fish, and on a significant increase in the vitellogenin levels in male fish. Other substances, such as furosemide and several fibrates (e.g. bezafibrate, fenofibrate and gemfibrozil) can also exert this type of endocrine effects. According to the data obtained by Isidori et al. [76], these substances were shown to activate the human estrogenic receptor α, thus favouring estrogenic responses in wild organisms. Mefenamic acid is another example of an endocrine compound whose pharmacology in most experimental organisms does not include this aspect. However, the data compiled by Collard et al. [49] showed its involvement in endocrine effects in fish (*D. rerio*), evidenced by alterations in vitellogenin and its mRNA expression, overexpression of genes of the hypothalamus– pituitary–gonad axis, and histological changes in ovaries of exposed females.

Epigenetic effects can also derive from the environmentally-driven impact of specific com‐ pounds; exposure to persistent organic pollutants (including pharmaceuticals) or endocrine disrupting chemicals are examples of classes of chemicals that have been related to alterations in epigenetic marks, including in fish and cladocerans (Vandegehuchte and Janssen, 2011) [77]. Several published papers refer that deleterious effects of transient chemical exposure (namely, via environment) of *D. magna* can result in the transference to nonexposed generations through epigenetic inheritance [78, 79, 80], which is a decisive factor to link ecotoxicological effects observed at the levels of communities to alterations at the ecosystem levels [81]. The effects of chemical pollutants on the epigenetics of fish is also significant, as shown by the screening of pollution resistance of north American fish species [82]. Alteration of gene expression is also another factor to consider after environmental exposure to chemical stressors; several papers show the responsiveness of aquatic organisms to environmental pharmaceuticals, demon‐ strating the validity of this approach [83, 84].

Specific drugs, not anticholinesterasic by nature, can also impair neurotransmission, by cholinesterasic inhibition [85]. One of the most significant examples is the one represented by zinc pyrithione. According to the work developed by Sánchez-Bayoa and Goka [86], this antidandruff compound is extremely toxic to several aquatic organisms, including the crustacean *D. magna*. Despite being photodegradable, recent studies show that zinc pyrithione may exert important toxic effects on aquatic organisms (e.g. *Paracentrotus lividus* and *Mytilus edulis*), even at extremely low levels [87]. Effects of zinc pyrithione are not restricted to invertebrates, since fish species are also extremely sensitive to the presence of this compound [88]. The products of degradation of zinc pyrithione can be of great environmental concern per se, since the effects of such compounds on several marine organisms are well known. The toxicity of zinc pyri‐ thione has been documented for organisms such as the algae species *Skeletonema costatum*, the crustacean *Tigriopus japonicus*, and the fish *Pagrus major* [89]. The mechanism of toxic action of zinc pyrithione metabolites includes AChE inhibition, as shown by Mochida et al. [90].

The energy metabolism of wild organisms is a putative target for pharmaceutical toxicity. As shown by Mennigen et al. [91], exposure to the drug fluoxetine could result in significant alterations in the fish species *Carassius auratus*, namely in terms of energy metabolism. Low levels of exposure were causative of anorectic effects, while higher levels could directly compromise the hepatic glucose metabolism, by means of depressing the activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase. Chronic exposure of marine mussels (*Mytilus* sp.) to two therapeutic drugs, genfibrozil and diclofenac, showed the interference of these substances on several parameters, including energy metabolism features [92]. The respiratory activity of exposed organisms is another function that can be altered after exposure to anthropogenic compounds, which interfere with metabolic pathways used by organisms to obtain energy (anaerobiosis vs. aerobiosis) [7]. This study evidenced the roles of both clofibrate and clofibric acid, hypolipidemic fibrates used in human therapeutics, in the increase in muscle lactate dehydrogenase activity, thus favouring the less energetically efficient anaerobiotic pathway.
