Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

The input of environmental stressors into natural water bodies can affect the health of the aquatic ecosystem¹. Exposure to these environmental stressors can affect the survival or fitness of aquatic organisms through different toxicity mechanisms, including direct exposure (both short- and long-term)². Hence, standardized laboratory bioassays to assess toxicological endpoints related to fitness and survival may be an unreliable estimate of the many indirect effects of stress in the field. Furthermore, alterations in normal physiological levels and effects on individuals, such as in terms of prey capture, may be better long-term indicators of the impact on survival and reproductive fitness in organisms and, ultimately, on the health of the ecosystem^1,3. Predicting changes in ecosystem composition and function, as well as organism health, based on a known set of environmental parameters and contaminant concentrations, is important for improving pollution management¹.

Biomarkers are defined as biochemical, physiological, or histological changes due to either exposure to or the effects of xenobiotic chemicals^4,5. Biomarkers have proven to be very useful as early warning signals^4,5. An important question that biomarkers help answer is whether certain stressors are present in high enough concentrations in the environment to cause adverse effects. This information contributes to the assessment of whether it is worth investigating the nature and extent of the damage and the causative agents or whether no more resources should be invested in that case^6,7,8. Moreover, since the concept of evaluating a single biomarker as a bioindicator may not be adequate^5,7,8,9,10, there is a growing trend toward performing a comprehensive evaluation of multiple biomarkers in order to detect early warning signs and, thus, prevent irreversible effects on ecosystems.

It is very important to note that all toxic effects begin with the interaction of a stressor with biomolecules. In this sense, effects can cascade through the biochemical, subcellular, cellular, tissue, organ, individual, population, community, ecosystem, landscape, and biospheric levels of organization. Cells are the primary site of interaction between environmental stressors and biological systems. Thus, understanding molecular and genetic effects allows researchers to associate low and high levels of ecological organization and helps them to predict the effect of environmental pollutants, for example, on human health, that have not yet been tested⁵. Moreover, due to the high specificity of cells, they are not only useful for evaluating environmental pollutants but also human health^5,11. Therefore, understanding the effects of stressors at the biochemical level may provide insights into the causes of the observed effects and allow them to be connected with those at the next higher level⁵. In addition, by understanding the biochemical mechanisms of stressors, the effects of new stressors that have not yet been toxicologically evaluated may be predicted with respect to other well-known contaminants based on their similarities in function. In the presence of various environmental stressors, genetic and biochemical biomarkers may provide valuable information on the specific effects observed. In addition to this, histochemical evaluations related to biochemical changes can provide information on toxicodynamics⁵. In short, a comprehensive analysis of cellular, biochemical, and histological biomarkers is necessary^10,12, and this type of analysis, in turn, should be included in biomonitoring programs for local species^5,13,14.

The study of biomarkers under laboratory conditions may, nonetheless, present some difficulties, including difficulties in the detection of sublethal effects and chronic impacts after exposure to pollutants and in the validation and standardization of the methods employed, as well as the complex time- or dose-dependent responses, the unclear or undetermined links to fitness, and the lack of integrated mechanistic models^1,4. To solve these problems, the solution is not to increase the number of biomarkers measured but to carefully design studies and testable hypotheses that contribute to explaining the mechanistic bases of chemical effects on whole organisms⁴.

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers to generate ecotoxicological predictions that improve the interpretation of the effects of environmental stressors on organisms, as well as decision-making about their possible impact. Moreover, the importance of combining both concepts-biomarkers and bioindicators-in environmental risk assessments and biomonitoring is that this will allow researchers to determine whether organisms in a specific environment of interest are physiologically normal or stressed. The approach taken in this study resembles that of the biochemical analysis that is carried out in humans. In this sense, a battery of biomarkers may be analyzed to see if an organism is healthy both in the field and in the laboratory⁶. Finally, biomarkers will contribute to ecological risk assessments in two ways: (1) assessing the exposure of rare and/or long-lived species, and (2) testing hypotheses about the mechanisms of chemical impacts at different levels of biological organization⁴.

In the last decade, biomarkers have been used in anurans for biomonitoring the exposure to cytotoxic and genotoxic contaminants. Among these, the techniques that have been used most frequently are the micronucleus (MN) assay and the comet assay or the induction of single-stranded DNA breaks by single-cell gel electrophoresis (SCGE) assay. In addition, those techniques have been successfully used to estimate the DNA damage induced by various environmental stressors in several neotropical anurans^{14,15,16,17,18,19}. Other biomarkers can used to examine changes in the oxidative status in organisms exposed to environmental pollutants^16,17,18,19. Oxidative stress is a response to exposure to different xenobiotics, leading to several detrimental effects, including on the antioxidant capacity of the exposed individuals^5,6,7,19,20.

In ecotoxicological studies, bioindicator species are used because they are organisms that identify the long-term interactions and adverse effects of environmental stressors at higher organizational levels (e.g., organism, population, community, and ecosystem levels)^10,20,21. By integrating the two concepts-biomarkers and bioindicators-species can be screened to broadly define biochemical, physiological, or ecological structures or processes that are correlated or linked with measured biological effects at one or more levels of biological organization. Finally, the great challenge of utilizing both concepts to improve the estimates of the toxicity of a stressor relates to analyzing biomarkers and bioindicators that have high utility in the evaluation of ecological risks²⁰. In this sense, there is consensus on the relevance of employing biomarkers and bioindicators as early warning signs, as they offer relevant information about the response of a test organism to environmental stressors^12,20,21.

Amphibians are one of the most threatened and rapidly declining groups of organisms worldwide. One of the main reasons for this decline is pollutants that enter their habitat, such as pesticides, metals, and emerging pollutants^22,23,24,25. Anurans have several characteristics that make them useful as bioindicator species, such as their permeable skin, close relationship with water, and sensitivity to environmental pollution^2,23,24. These characteristics make amphibians effective bioindicators of environmental health^{7,8,22,23,24,26}.

Nevertheless, it is necessary not only to consider the optimization of basic procedures and the generation of historical data in control groups but also to employ specific bioassays to evaluate responses in organs and tissues to elucidate the nature and variation of effects observed in bioindicators. In this sense, the present work aims to describe several ecotoxicological methodologies to be employed in all stages of neotropical anurans at different ecological levels and validate them as useful biomarkers to be employed both in wildlife and laboratory conditions. This work presents a battery of biomarkers that may be integrated and that have been proven for laboratory and wildlife biomonitoring in anurans exposed to environmental stressors.