**5. The quest for new biomarker tools: Oxidative stress**

As seen before, the transformation of a pharmacological effect into a biomarker, can thus work as a reliable indicator of exposure of wild organisms to several classes of pharmaceutical drugs. Despite the presented evidences, describing successful cases in which the pharmacology of a drug (or a group of drugs) may also be used to study its toxicity, the majority of therapeutic drugs do not exert effects that can be interpreted as reliable environmental biomarkers. There are two major drawbacks of this approach: the first reason is related with the levels in which these substances are found, and the second most important, is the absence of a counterpart response in wild organisms of the response elicited in human patients.

Toxicity is a matter of dose, and can be generally described by a dose-response relationship. Assuming that these compounds are found in extremely low levels, the exertion of a clear toxic response (e.g. death, immobilization, impairment of reproduction) is not always possible. It is thus important to know in detail the mechanisms by which the drugs not only exert their pharmacological activities, but also those involved in adverse effects, metabolism and detoxification processes, and other accessory or side-effects that the drugs may cause. Only by means of knowing these fundaments of pharmacology and toxicology it is possible to select biomarkers that will respond satisfactorily at extremely low levels of exposure. Given the extremely low levels in which drugs are found in the aquatic compartment, effects are sometimes minor and negligible, or may even be considered null or absent if an adequately responsive biomarker is not employed. This can be a real challenge for ecotoxicologists, since specific biomarkers, others than the main pharmacological effect, that signal a biological alteration following an exposure to drugs in concentrations between the ng/l to µg/l are not abundant.

extreme toxicity, when compared to antibiotics, thus threatening the survival of zebrafish embryos. The crustacean species *Daphnia magna* was very sensitive to the antidepressant sertraline, as shown by Minagh et al. [46]; this compound was capable of inducing strong alterations in the population of this crustacean species, after 21 days of exposure. Population changes were also observed after chronically exposing *D. magna* to the compounds testosterone and 4-hydroxyandrostenedione, even for ecologically relevant levels [47], reinforcing the possibility of exertion of deleterious effects in the wild. Another crustacean, namely *Neocaridina denticulate*, demonstrated to be extremely sensitive in terms of lethality to a combination of pharmaceutical drugs (paracetamol and ibuprofen) commonly found in the aquatic environ‐ ment [48]. The ecotoxicological effects of mefenamic acid on *D. magna* and *Moina macrocopa* were evaluated by Collard et al. [49]. This study showed that lethal effects were not likely to occur for already reported levels of this compound; however, the chronic exposure to this compound showed that changes in reproduction are possible, especially in the case of *M.*

On the other hand, the absence of potential risk posed by the presence of clofibric acid was evidenced by the study by Nunes et al. [50] and by Emblidge and DeLorenzo [51], in multi‐ species assessments that focused on mortality and growth impairment as toxicity endpoints. Similarly, the work by Ferreira et al. [52] evidenced the absence of potential lethal effects posed by antibiotics (oxytetracycline and florfenicol) on the crustacean species *Artemia parthenogene‐ tica*. Despite not exerting lethal effects, several combinations of pharmaceutical drugs (includ‐ ing an ecologically relevant mixture) were capable of causing significant alterations in zebrafish embryos, as shown by Madureira et al. [53]. The antibiotic oxytetracycline was shown to be reasonably safe towards the fish species *Labeo rohita*, since the lethal levels were well above concentrations that are not likely to be found in the wild, as shown by Ambili et al. [54]. Growth of autothrophic organisms is another endpoint likely to respond to exposure to human use drugs; the study conducted by Berninger et al. [55] showed the refractivity of the aquatic plant species *Lemna minor* to the drug diphenhydramine. Somewhat similarly, the work conducted by Nunes et al. [56] evidenced the occurrence of an oxidative-based response of two species of the genus *Lemna* (*L. minor* and *L. gibba*) elicited by the drug paracetamol, which resulted in significant growth alterations. On the other hand, the study conducted by Ferreira et al. [52] presented evidences concerning the toxicity of the antibiotics oxytetracycline and

As seen before, the transformation of a pharmacological effect into a biomarker, can thus work as a reliable indicator of exposure of wild organisms to several classes of pharmaceutical drugs. Despite the presented evidences, describing successful cases in which the pharmacology of a drug (or a group of drugs) may also be used to study its toxicity, the majority of therapeutic drugs do not exert effects that can be interpreted as reliable environmental biomarkers. There are two major drawbacks of this approach: the first reason is related with the levels in which these substances are found, and the second most important, is the absence of a counterpart

florfenicol on the microalgae species *Tetraselmis chuii*.

**5. The quest for new biomarker tools: Oxidative stress**

response in wild organisms of the response elicited in human patients.

*macrocopa*.

136 Toxicology Studies - Cells, Drugs and Environment

On the other hand, drugs are designed to be safe for human patients, and it is possible to suggest that this somewhat harmless nature can also prevent drugs from causing extreme toxicity in other organisms. However, drugs can indeed cause multiple effects, which do not occur by activation of specific single receptors, but reflect major changes in the homeostasis of exposed organisms. The presence of pharmaceutical compounds can cause changes at many different levels, including in the redox cycle of exposed organisms; these may cause the formation of reactive chemical species, capable of inducing damages to biological structures. Therefore, the quantification of oxidative stress biomarkers is important to evaluate the redox status of the exposed organisms. Several studies have already shown that, even for realistic levels of contamination, some compounds can exert pro-oxidative effects, measurable mostly in terms of modification of the antioxidant defence mechanism of exposed aquatic organisms; in many cases, membrane lipid peroxidation is a likely outcome of oxidative damage, and this event can be also quantified [8, 13, 56, 57, 58, 59]. The exertion of oxidative stress is a factor to consider when evaluating long term, thus realistic, exposures. Considering that exposure to anthropogenic therapeutic drugs can occur during a significant portion of the entire life cycle of a given organism, it is not possible to exclude the occurrence of cumulative processes ending up in irreversible conditions. In fact, oxidative stress, if sustained for long period, can also be the cause of genotoxicity. Data from the literature sustain the exertion of deleterious effects of specific pharmaceutical compounds, including genotoxicity on fish [60], and in crustaceans (e.g. *D. magna*) [61, 62]. Consequently, it is possible to use oxidative stress not only as an indicator of exposure to a broad series of compounds, but also as a predictor of other subse‐ quent effects, that may derive from oxidative alterations and damages.

The extended knowledge concerning the metabolic pathways required for the bioactivation or detoxification of therapeutic compounds can also function as a valid source of analytic tools for the assessment of effects. The activation by over-expression of metabolic enzymes, involved in the detoxification of pharmaceuticals, is a probable event whenever a biological system is challenged by exposure to drugs in the wild. Among metabolic enzymes prone to overexpression, one can identify mainly those involved in the direct metabolism of drugs. The activities of phase I metabolic enzymes, for example (such as cytochrome P450), and phase II metabolic reactions (namely isoenzymes glutathione-S-transferases, GSTs), can also be 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 multiplicity of therapeutic classes.
