**7. Future directions: A combination of tools**

From the previous sections, it was made clear that it is extremely difficult to search and define without any shadow of doubt the biological effects to be expected from a large number of extremely different chemical substances, exerted on a vast multiplicity of organisms. Not only are the substances very different per se, thus exerting distinct effects, but also the organisms can have alternative pathways and receptors that make them more or less prone to the exertion of those same effects. It is not possible always to rely on the well-described human pharma‐ cology, despite the large number of studies that sustain the most common effects, since humans are not environmentally exposed to the majority of these substances or to their residues. However, some of the effects are shared both by humans and by other organisms in the wild, a decisive factor when one tries to select an adequate tool to quantify an effect elicited by a pharmaceutical drug in the environment.

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‐

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

From the previous sections, it was made clear that it is extremely difficult to search and define without any shadow of doubt the biological effects to be expected from a large number of

strating the validity of this approach [83, 84].

140 Toxicology Studies - Cells, Drugs and Environment

**7. Future directions: A combination of tools**

pathway.

The present scenario shows us that we are halfway between the total lack of data concerning pharmaceutical effects in the wild, and a full and comprehensive knowledge about their faith and ultimate consequences. A large effort has been undertaken and toxicity of several pharmaceutical classes is nowadays already characterized in a vast number of organisms. Despite the validity of this effort, other pharmaceutical classes are not fully understood in their interaction with biota, thus requiring the development of additional attempts until a definite light is shed on this issue. Presently, environmental scientists dealing with this issue still have to face a significant array of drawbacks, from the simple lack of data for some drugs/organisms, unpredictability of data and, of the biological responses in somewhat exotic species, con‐ founding factors that already occur for other studies, but whose influence is exponentially increased in this specific area, lack of analytical tools, such as biomarkers with enough sensitivity to face the extremely low levels found in the wild and that are able to understand highly subtle biological responses, lack of test protocols or species well adapted to be used in ecotoxicological testing under conditions of brackish water, tropical or artic climates, or extreme environments, in which drugs are also likely to occur.

Despite not being the core of this chapter, the quantification of the levels of pharmaceutical drugs in the wild, especially in the aquatic environment, is crucial. It is important to know in detail the compounds that may exist (or co-exist) in the same matrix, since these compounds are important to select an analytical tool/biomarker that will allow the prediction of biological effects. It is also of fundamental importance to know which are the most representative compounds (or pharmacotherapeutic classes) in a given sample also to establish causal relationships between their levels and the extent of the observed biological response. Only with a complimentary approach comprising hydrology, water analytical chemistry and biological assessment of effects it will be possible to fully characterize the impact of drugs on the wildlife.

The typology of exposure is also a matter of concern. From the previously mentioned studies, two main types of exposure were adopted in the majority of studies: acute (short-term) and chronic (long term). The use of short-term exposure periods is somewhat neglected, but this is not a totally invalid strategy. Despite not having the relevance of a long period of exposure, which reflects the most likely conditions of exposure in the wild, short exposure periods are also of extreme importance, and must be included in bioassays for the assessment of the effects of pharmaceutical drug. Acute assays are important since they allow researchers to test the responsiveness of a specific test species towards a given drugs, Acute exposure can also be of 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‐ quently very diverse.

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

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 but realistic mixtures. To avoid this underestimation, it will be of the uttermost importance to know the general interaction profiles that may occur for a large number of test species, caused not by isolated single compounds, but by the main representatives of known therapeutic classes: not being possible to test the potential interactions occurring by the simultaneous presence of hundreds or even thousands of compounds in the same matrix, a more systematic approach may involve the definition and characterization of putative biological relations of pharmacotherapeutic classes among each other.
