The authors gratefully acknowledge Instituto de Qu&#237;mica de San Luis &#34;Dr. Roberto Olsina&#34;- Consejo Nacional de Investigaciones Cient&#237;ficas y Tecnol&#243;gicas (INQUISAL-CONICET), Universidad Nacional de San Luis (Project PROICO 2-1914), Laborat&#243;rio de Patologia Experimental (LAPEx) - Instituto de Bioci&#234;ncias (INBIO) - Universidade Federal de Mato Grosso do Sul (UFMS), C&#225;tedra de Citolog&#237;a - Universidad Nacional de La Plata (UNLP), and Agencia Nacional de Promoci&#243;n Cient&#237;fica (FONCYT; PICT-2018-02570 and PICT-2018-01067) for financial support. We would also like to thank native speaker Lidia Unger and GAECI-UNSL (scientific writing assistance center) from the National University of San Luis for the proofreading of the manuscript.

The authors declare no competing interests.

The biomarkers at the individual level are very simple to determine and very low-cost, as examining these biomarkers requires only a few pieces of equipment that are usually available in any research laboratory. In addition, these biomarkers provide general information on the health and fitness of the animals. The number of animals employed in each protocol is critical for obtaining reliable results. Due to the variability of data, a minimum of five animals (N = 5) is necessary for each treatment. In detail, a critical step for biomarkers at the individual level is measuring the SVL for the body condition index, since variations in the angle of the camera position could generate variations in the SVL measurement. It is essential that the images of all the individuals in the same study are taken in the same way, with the same angle and at the same distance from the object to be photographed. However, measuring the SVL by the image analysis method is preferable to caliper measurement for time-saving reasons. Using this image analysis method would make it possible, therefore, to increase the number of studied animals, resulting in more robust and reliable results. 
The hepatic index is useful for comparing groups of adults, juveniles, and even larvae from the same species, and as the liver is the main detoxifying organ, significant variations would indicate alterations in the histological and biochemical levels. The gonadal index is useful for comparing groups of adult anurans from the same species, and, as could be expected, female and male comparisons must be made separately due to the scale variations between ovaries and testes. Significant variations in this index could indicate alterations in gonadogenesis, which may affect the reproduction of the organisms.
Histological biomarkers are relatively low cost, although they require laboratories that have the required equipment. Biomarkers at morphological levels are important since they integrate responses from the biochemical and physiological levels. Here, we demonstrate examples of liver histological processing. However, we emphasize that the procedure may be applied to other organs such as the kidneys, heart, gonads, skin, spleen, and intestine.
The critical points for histological biomarkers are the identification of the cells and structures that are to be evaluated. It takes some training to apply the technique correctly. When analyzing the samples, methacarn is suggested because it is a fixative that allows for the better treatment of the tissues and also prevents the rapid deterioration of the histological equipment. In addition, fixatives based on methacarn have the advantages of preserving the tissues with good hydration and have a lower chance of contaminating the sample and the operator. In addition, for the biochemical biomarkers, a critical point during the enzymatic determination is maintaining the temperature at 4 &#176;C, as well as storing enough samples in case the repetition of the enzyme determination is required. The technique modifications in the protocols depend on the species employed. It is necessary to take into consideration that for smaller species (or for tadpoles), the sacrifice of the organisms will be required to obtain the samples. Briefly, some modifications at the individual level (e.g., morphometric indexes) could be the use of mathematical transformations to improve the data interpretation.
Recently, Brodeur et al.28 compared two different methods for expressing body condition: the scaled mass index (SMI) and the residuals methods. After the comparative analysis, the authors concluded that the determination of the body condition using SMI during monitoring programs with local anurans could be of great value, since it would provide accurate information on the health status of these anurans in conjunction with estimates of the population abundance. Modifications in histological biomarkers can be principally determined by employing either specific software for histological counting or visual determination. 
All the biomarkers presented in this section have a relatively low cost, can quickly detect adverse effects caused by environmental stressors, and are highly reproducible in laboratories. In addition, they may be used with a small or large amount of sample (diluted), from 5 &#181;L to more than 100 &#181;L. Further, they have been widely applied not only for anurans but also for a large number of organisms ranging from plants to mammals. In anurans, these biomarkers are extremely important because they are the main defense system against environmental changes and, thus, reflect their health status. However, the techniques have slight modifications that depend mainly on the species and the equipment. In this sense, for example, the volume of sample added in each technique will depend on the amount of anuran protein. In tadpoles, there is little protein, so more sample volume must be added for protein determination; then, in the calculations, this volume must be considered. Modifications at a biochemical level are usually related to the use of microplate incubators, incubation at a temperature of 37 &#176;C, and the use of specific software to measure each enzyme.
Troubleshooting in the colorimetric quantifications (proteins and TBARS) will ensure that the absorbances of the samples fall within the range of the standard curve; if not, it is necessary to adjust the standard concentrations accordingly. At the cytogenetic level, a slight modification may involve the addition of a staining step in the MN assay. In the SCGE assay, modifications may involve changing the length of the lysis period, the concentrations of the agarose, the unwinding and electrophoresis times, the fluorescent dyes, and the employed analysis methodology.
The main limitation of employing biomarkers is that results usually do not bring conclusive information when analyzed separately. Therefore, it is necessary to analyze them jointly. This is because several biomarkers respond monotonously and not according to the dose-response concept in toxicology.
The comet and MN assays are rapid and sensitive methods that are commonly employed to detect genetic damage. The MN assay detects clastogenic or aneugenic damage through the examination of cells in interphase, whereas the SCGE assay detects direct DNA damage (strand breaks, DNA adducts, excision repair sites, cross-links) at the single-cell level. Both methods are fast to perform, with a relatively low cost and an easy and simple analysis. Another advantage is that both require a small sample to be performed (15 &#181;L to over 100 &#181;L). Given the characteristics of the methodology, the analysis of the frequency of MNs should be restricted to cells that have undergone the first mitotic division after treatment with the agent under study, since the induced lesions in the DNA are excluded from the nuclei of new cells as small pieces of extranuclear material after mitotic metaphase-anaphase39. This is an advantage of the SCGE assay, in which cells do not need to go through a cycle of cell duplication to manifest the damage caused to them. In addition, the number of cells analyzed per sample is lower in the SCGE assay than in the MN assay. 
Among the difficulties SCGE presents are the costs of some reagents and equipment involved (mainly for the SCGE assay) and the variations in the times of lysis, unwinding, and/or electrophoresis, which have to be determined for the species employed. In addition, the analysis procedure (qualitative or quantitative) is another source of variability in the technique. Misleading results obtained with these techniques are usually due to not knowing the basal levels of cytogenetic damage in the studied species or, during the analysis, considering undamaged cells as damaged. However, erroneous results can be obtained by not following the established criteria, for example, to determine a micronucleus. The staining technique is one factor to take into consideration, since non-specific stains can be associated with false positive and false negative results due to underestimation or overestimation of the micronucleus count41. Finally, the critical points of the MN assay are fixing the material at 4 &#176;C and ensuring the observation is performed by a trained analyst in order to avoid the underestimation or overestimation of the damage. Moreover, the critical points of the SCGE assay are the manipulation of numerous slides with delicate agarose gels and the challenge of performing the assay in a dark environment to avoid inducing extra damage in the samples.
Another challenge is employing these studies in new species and under acute exposures, which sometimes are not long enough to produce a measurable response. Moreover, the number of individuals employed (sometimes N &lt; 5) produces high variability, and statistical analysis reduces the statistical robustness. A limitation of the SCGE assay is that after the exposure, the samples must be processed within 36 h. Finally, and according to the recommendations of an ecotoxicologist, it is necessary to establish standard or control values (or normal distributions) for each biomarker and species employed.
These methods provide reliable results that are easy to interpret and contribute to explaining the mechanisms of toxicity of an environmental stressor. From a single organism, it is possible to perform a comprehensive analysis, which is necessary for biomonitoring and risk assessment5,6,44. In an organism, a stressor may produce apoptosis (detected by cytogenetic biomarkers), cause DNA damage that may be repaired (detected by cytogenetic biomarkers), trigger an altered response in the cell or organ (cytogenetic, biochemical, histological, and/or individual biomarkers), and also induce oxidative damage (biochemical and cytogenetic biomarkers), which, if not repaired, could lead to cancer or death, thus decreasing the quality of the ecosystem.
Most of the individual, biochemical, and cytogenetic biomarkers presented here are non-invasive techniques (no sacrifice of animals is required) that may be performed both in a laboratory as well as in biomonitoring studies to detect the effects induced by contaminants in bioindicator species before the damage to the ecosystem becomes irreversible. In addition, the biomarkers presented here have recently gained interest and are currently employed in investigations several Neotropical anurans exposed to different environmental pollutants. 
Finally, it should be noted that the IBR has been used in recent years51 in several stress situations for different species of neotropical anurans. This analysis allows for the evaluation of the impact of environmental pollutants in an integrated way and for performing a more exhaustive evaluation of the effects that pesticides induce on different native species. This would allow for taking measures to minimize the anthropogenic impact on non-target organisms. However, there is still a long way to go to establish an accurate and complete risk assessment and evaluation protocol with Neotropical anurans. This study aims to give an overview of several available strategies and offer a base guide to facilitate future research.

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 ecosystem1,3. 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 chemicals4,5. Biomarkers have proven to be very useful as early warning signals4,5. 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 case6,7,8. Moreover, since the concept of evaluating a single biomarker as a bioindicator may not be adequate5,7,8,9,10, 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 health5,11. 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 necessary10,12, and this type of analysis, in turn, should be included in biomonitoring programs for local species5,13,14.
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 models1,4. 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 anurans14,15,16,17,18,19. Other biomarkers can used to examine changes in the oxidative status in organisms exposed to environmental pollutants16,17,18,19. Oxidative stress is a response to exposure to different xenobiotics, leading to several detrimental effects, including on the antioxidant capacity of the exposed individuals5,6,7,19,20.
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 stressors12,20,21.
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 pollutants22,23,24,25. Anurans have several characteristics that make them useful as bioindicator species, such as their permeable skin, close relationship with water, and sensitivity to environmental pollution2,23,24. These characteristics make amphibians effective bioindicators of environmental health7,8,22,23,24,26.
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.

<table><tbody><tr><td>Analytical scale</td><td></td><td></td><td></td></tr><tr><td>Electrophoresis power supply</td><td>&amp;nbsp;Enduro</td><td>&amp;nbsp;E0203-250V&amp;nbsp;</td><td></td></tr><tr><td>Eosin&amp;nbsp;</td><td>Merck</td><td></td><td></td></tr><tr><td>Fluorescence photomicroscope</td><td>&amp;nbsp;Olympus</td><td>&amp;nbsp;BX50</td><td>Equipped with an appropriate filter combination</td></tr><tr><td>Hematoxylin of Harris</td><td>Merck</td><td></td><td></td></tr><tr><td>High resolution photo camera&amp;nbsp;</td><td></td><td></td><td>&amp;nbsp;&gt;16 megapixels</td></tr><tr><td>Homogenizer</td><td></td><td></td><td></td></tr><tr><td>Horizontal electrophoresis chamber</td><td>&amp;nbsp;Sigma</td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td>Denver Instrument</td><td></td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td>DM4000 B</td><td>Equipped with image capture system Leica DFC 280</td></tr><tr><td>Microtome</td><td>Leica</td><td>2265</td><td></td></tr><tr><td>Paraplast</td><td>Sigma</td><td>P3558</td><td></td></tr><tr><td>Personal Computer</td><td></td><td></td><td>&amp;nbsp;Eqquiped with Mac OS X, Lynux or Windows</td></tr><tr><td>Refrigerated centrifuge</td><td></td><td></td><td></td></tr><tr><td>UV&amp;ndash;Vis spectrophotometer</td><td>Rayleigh</td><td>723G</td><td>With UV-lamp</td></tr></tbody></table>

ecotoxicological methodologies to evaluate biomarkers at different scales in neotropical anurans

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. 
Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). 
Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

All the biomarker techniques presented here are simple, rapid, convenient, sensitive, low cost, and accurate methods. For each biomarker, it is important to note the following.
Individual level 
Scaled mass index 
Taking photographs on the millimeter scale is of great importance since this value will be used to calibrate the software, and this results in better objectivity with respect to the caliper measurement when taking the SVL variable. In addition, using a photograph for later analysis saves processing time during the handling of individuals, either during laboratory or field studies, where the time of sampling is always critical. In order to carry out the analysis of the scaled mass index, it is important to perform a non-linear regression analysis that demonstrates the power function relationship between the SVL and body mass for the species to determine the scaling exponent (b) (Figure 2). The scaled mass index is useful for comparing groups of adults or juveniles from the same species.
Scaled liver index 
The SLI gives an idea of the relative mass of the liver in relation to the SVL. Although it can be used both with adult individuals and with larvae, the study of the SLI with adult anurans is more convenient due to the smaller size of the larvae. Alterations in this parameter indicate possible effects at lower levels (histological, biochemical). The SLI is a useful biomarker for making comparisons between groups.
Scaled gonadal index 
The scaled gonadal index provides a measure of the gonad size relative to the SVL and can indicate the degree of gonadal development. It is an index applicable only to adult anurans. Alterations in this parameter could indicate effects on gonadogenesis, among others, and the scaled gonadal index effectively complements studies at the histological and physiological levels. This is a useful index for comparative studies.
Morphological-histological level 
Histological biomarkers are sensitive and easy to analyze. However, these markers require prior tissue processing, meaning it can take some time to obtain the results. Here, we use liver sections to exemplify the techniques (Figure 3). Figure 3 demonstrates how hepatocytes can be measured. The interpretation of the results is based on interspecific or intraspecific comparisons.
Histometric data 
Here, data from the area or length measurements of the selected structures are used. With these data, it is possible to compare effects between treatments or compare species. Table 4 shows a pattern for Boana raniceps. To calculate nucleus:cytoplasm ratio (RNC), we used the following equation: RNC = (nucleus area/cytoplasm area &#215; 100). The nucleus volume was calculated using the following equation: nucleus volume = 4/3&#960;r3. The cytoplasm volume was calculated indirectly using the following equation: cytoplasm volume = (hepatocyte area &#215; nucleus volume)/nucleus area. Here, a correct delimitation of the desired structures is important for the precision of the technique. In tissues that do not have a good delimitation of cells and/or structures, the measurements may be wrong.
Histopathology 
Histopathology allows for a comparative analysis regarding the degree of tissue alteration. When a degree of tissue change (DTC) is present, it is possible to make a table comparing groups. To apply this technique with precision, personnel training is necessary to eliminate subjectivity. The best results may be achieved through double-blind analysis.
Pigmentary system 
Tissue pigmentation is associated with the presence of melanin in a given area (Table 4). With these data, it is possible to perform a comparative analysis between the same organ of different species or between experimental groups. The difficulty of this technique is the accurate selection of the color to be measured. Here, double-blind analysis is also advisable.
Biochemical level 
The correct reading of the enzymatic activity shows a drop in the values during the 120 s of measurement time, which indicates consumption of the substrate (Figure 4A). The enzymatic reaction should preferably not be carried out at 4 &#176;C, since the cold temperature of both the reagents and the sample delays the reaction, and it will take more time for the drop to show (Figure 4B). For the correct calculation of the enzyme activity, the molar extinction coefficient must be considered, and the protein value of that sample should be referred to in order to avoid underestimating or overestimating the enzymatic values. However, it is important to use the absorbance difference as a selection criterion in this case, we select the greatest difference in absorbance in the enzymes in which the kinetics are measured.
Catalase 
The underlying principle of the method is based on the fact that catalase exerts a dual function: (1) the decomposition of H2O2 to give H2O and O2 (catalytic activity, equation [9]), and (2) the oxidation of H donors (e.g., methanol, ethanol, formic acid, phenols) with the consumption of mol of peroxide (peroxidic activity, equation [10])35:
2H2O2 &#8594; H2O + O2&#160; &#160; (9)
where &#34;&#8594;&#34; represents reacting catalase.
ROOH + AH2 &#8594; H2O + ROH + A&#160; &#160; (10)
where &#34;&#8594;&#34; represents reacting catalase.
The formation of bubbles resulting from the release of oxygen in the reaction must be taken into account. This bubble formation may interfere with the reading signal when the light beam falls on the spectrum and may generate erroneous readings (Figure 5).&#160;The quartz cell (not glass or plastic) is essential for reading catalase because it absorbs the wavelengths that are below UV-visible range, thus avoiding interference with the material.
Colorimetric methods: Proteins, TBARS, GST (Table 5), and AChE 
An important point between each measurement is to clean the cuvette as much as possible, first with 95% alcohol and then with distilled water.
Phase II enzymes conjugate glutathione with a xenobiotic or its metabolite to reduce lipid hydroperoxides. Glutathione S-transferases (GSTs) are cytosolic enzymes that attach glutathione (GSH, a tripeptide comprising cysteine, glutamate, and glycine) to the xenobiotic or its transformation products. They can also be recognized by specific transporter systems5.
The foundation of the technique is based on the decomposition of polyunsaturated fatty acids such as malondialdehyde (MDA). This process serves as a convenient index for determining the extent of the peroxidation reaction. In turn, MDA, a product of lipid peroxidation, reacts with thiobarbituric acid (TBA) to yield a red species that absorbs at 535 nm37.
The method is based on the rate of production of thiocholine during the hydrolysis of acetylthiocholine. Particularly, this reaction is accomplished by the continuous reaction of the thiol with dithiobis-nitrobenzoic acid (DTNB) (I) to produce a yellow anion (II) (equation 11). The kinetics of the reaction production are measured by a spectrophotometer at 412 nm. As the reaction is sufficiently rapid, care should be taken to avoid enzymatic hydrolysis by carefully setting the time taken to measure the enzyme l concentration.
H2O + (CH3)3N+CH2CH2SCOCH3 &#8594; (enzyme) &#8594; (CH3)3N+CH2CH2S + CH3+COO&#8722; + 2H+
(CH3)3N+CH2CH2S&#8722; + RSSR (I) &#8594; (CH3)3N+CH2CH2SSR + RS&#8722; (II)]&#160; &#160; &#160;(11)
Genetic and cellular level: Micronucleus and comet assay 
The time of exposure and the concentration should always be considered as a factor in the protocol, since a particular time or concentration may not be enough to induce damage, or on the contrary, the damage peak may have already passed, the health status of the organism may have worsened, or the organism may be employing its recovery mechanisms (Figure 6).
Correlated biomarkers 
Figure 7&#160;shows how the biomarkers are related to the IBR and indicates that catalase is an effective biomarker for stress situations in the highest concentrations of the stressor (or the stress situation). In addition, there is a response of the biomarkers at increasing ecotoxicological levels. This indicates which is the most effective biomarker to use for that evaluated stress situation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64520/64520fig01.jpg" /> 
Figure 1: Data obtained from the analysis of TBARS in tadpoles of Rhinella arenarum exposed to BDE-209. (A) Constructed curve represented by the absorbance of the reaction of MDA and TBA, (B) the calculation of the equation of the curve, (C) obtaining the values in each tadpole sample, and (D) plotting the average values of MDA in each treatment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64520/64520fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64520/64520fig02.jpg" /> 
Figure 2: Scaled mass index. (A) Boana pulchella and (B) Leptodactylus luctator adults. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64520/64520fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64520/64520fig03.jpg" /> 
Figure 3: Histological sections of the liver of Boana raniceps. A greater amount of melanomacrophages and greater dimensions of the hepatocyte (white outlines) and nucleus (N) of the hepatocytes. Scale bars = 25 &#181;m (left) and 5 &#181;m (right). Abbreviations: M = melanomacrophages; N = nucleus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64520/64520fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64520/64520fig04.jpg" /> 
Figure 4: Acetylcholine absorbance in Rhinella arenarum tadpoles. The tadpoles were exposed to polybrominated BDE-209 (unpublished data). (A) The correct decrease in the reaction with acetylcholine and (B) the delay in the AChE reaction due to an inadequate temperature. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64520/64520fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64520/64520fig05.jpg" /> 
Figure 5: Irregular reading of catalase due to the addition of too much sample and an interruption of the signal due to bubbles. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64520/64520fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64520/64520fig06.jpg" /> 
Figure 6: Different and complementary responses of cytogenetic biomarkers in an herbicide scenario exposure in Boana pulchella. The black bars indicate 48 h, and the gray bars indicate 96 h. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64520/64520fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64520/64520fig07.jpg" /> 
Figure 7: IBR responses of several biomarkers in a gradient of BDE-209 concentrations in a neotropical anuran, Leptodactylus luctator. The blue color indicates the control group, while the orange (25 &#181;g/L), yellow (50 &#181;g/L), and green (75 &#181;g/L) colors indicate the exposure scenarios to BDE-209. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64520/64520fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Compositions of the reagents and solutions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64520/63520_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Calibration curve with BSA for the quantification of proteins using the method of Bradford34. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64520/63520_Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Calibration curve for the quantification of MDA using TBA as a control. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64520/63520_Table 3.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: Histometric data of the area and volume of the hepatocytes and the liver melanin pigmentation of Boana raniceps. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64520/63520_Table 4.xlsx" target="_blank">Please click here to download this Table.</a>
Table 5: Data of GST activity in two stages (tadpoles and adults) of Neotropical anurans obtained through the technique mentioned after 96 h of exposure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64520/64520_Table 5.xlsx" target="_blank">Please click here to download this Table.</a>

Read this Scientific Article Protocol about Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans at JoVE.com

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

In this paper, standardized ecotoxicological methods for the evaluation of biomarkers in neotropical anuran species are presented. Specifically, this paper details several methodologies at different scales of ecotoxicological evaluation, such as the genetic, cellular-histological, biochemical, morphological, and individual levels.

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that ...

ecotoxicological-methodologies-to-evaluate-biomarkers-at-different

Nanyang Technological University

Research

JoVE Journal

Environment

283 Views.  Universidad Nacional de San Luis (UNSL). Hoewel de technieken voor het evalueren van biochemische biomarkers op grote schaal worden gebruikt, biedt dit protocol cruciale details voor het standaardiseren en repliceren van de methodologie bij neotropische anura's. Het belangrijkste voordeel van deze techniek zijn de lage kosten, reproduceerbaarheid en de toepassing ervan. Deze techniek kan worden toegepast om kwantitatieve gegevens te verkrijgen om veranderingen in de interne melanine te evalueren.

Video: Ecotoxicologische methodologieën om biomarkers op verschillende schalen te evalueren bij neotropische anura's

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Vegetated Treatment Systems for Removing Contaminants Associated with Surface Water Toxicity in Agriculture and Urban Runoff

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may be treated with simple systems designed to promote sorption of contaminants to vegetation and soils and promote infiltration. Two example systems are described: a bioswale treatment system for urban stormwater treatment, and a vegetated drainage ditch for treating agriculture irrigation runoff. Both have similar attributes that reduce contaminant loading in runoff: vegetation that results in sorption of the contaminants to the soil and plant surfaces, and water infiltration. These systems may also include the integration of granulated activated carbon as a polishing step to remove residual contaminants. Implementation of these systems in agriculture and urban watersheds requires system monitoring to verify treatment efficacy. This includes chemical monitoring for specific contaminants responsible for toxicity. The current paper emphasizes monitoring of current use pesticides since these are responsible for surface water toxicity to aquatic invertebrates.

This article summarizes the design attributes and the effectiveness of treatment systems that treat urban stormwater and agriculture irrigation runoff to remove pesticides and other contaminants associated with aquatic toxicity.

Urban stormwater and agriculture irrigation runoff contain a complex mixture of contaminants that are often toxic to adjacent receiving waters. Runoff may ...

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use products. Based on their chemical structure and estradiol-like biological properties, they have been classified as endocrine disrupting compounds (EDC). Long-term exposure to EDCs, even at low doses, has been linked to various health defects including cancer, behavioral disorders and infertility, with greater vulnerability indicated during early developmental periods. Cellular and molecular studies with the genetically tractable nematode model Caenorhabditis elegans have demonstrated that exposure to BPA causes apoptosis, embryonic lethality and disruption in the DNA repair mechanisms. We have previously reported that exposure of C. elegans embryos to low doses of different bisphenols decreases fecundity. In addition, we have shown that the effects of exposure during the very early stages of development persist into adulthood as assayed by quantifying habituation behavior, a form of non-associative learning. Here, we provide detailed protocols for embryonic exposure to low-dose EDCs as well as the associated fecundity and anterior touch habituation assays, along with representative results.

Exposure to environmental toxicants can acutely impact development with long-term effects. Detailed protocols are provided to illustrate a strategy using an effective lab model to study the effect of early embryonic exposure to bisphenol A. We provide fecundity and behavioral assays to monitor the effectiveness of our toxicant exposure bio-assays.

Bisphenols, such as bisphenol A (BPA) and bisphenol S (BPS) are polymerizing agents widely used in the production of plastics and numerous everyday-use ...

Developmental Biology

Hellgrammite as a Non-Conventional Biomonitor for Surface Water and Environmental Health Assessment

The larvae of dobsonflies from the genus Corydalus, commonly known as Hellgrammites, are characterized by their notable size, extensive range of occurrence, and extended period of immaturity, which can last up to one year. Hellgrammites are clearly known to exhibit sensitivity to pollution and habitat structure impacts. Given these unique features, using Corydalus texanus larvae is highly suitable as reliable biomonitoring agents to assess the ecological integrity of aquatic ecosystems. This protocol aims to provide the necessary tools for C. texanus assessment and demonstrate their efficacy through a case study. Research findings have practical implications, indicating that C. texanus larvae exhibit early-warning responses to mining pollution, bioaccumulating high amounts of heavy metals such as Zn, Fe, and Al. The presence or absence of C. texanus populations may serve as a helpful indicator for identifying potential issues related to ecosystem health. The unconventional approach has shown early warnings of pollution in mining-impacted sites, highlighting the need for timely action to protect the environment. Given their unique traits, the use of C. texanus larvae is highly suggested as a reliable non-conventional bioindicator.

This protocol describes the use of biomarkers for the early detection of deleterious impacts in aquatic ecosystems. The biomarkers are closely related to sentinel traits, and their changes aid in detecting early-warning damages.

The larvae of dobsonflies from the genus Corydalus, commonly known as Hellgrammites, are characterized by their notable size, extensive range of ...

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

We quantify epidermal cell death in frogs with chytridiomycosis using two methods. First, we use terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ histology to determine differences between clinically infected and uninfected animals. Second, we conduct a time series analysis of apoptosis over infection using a caspase 3/7 protein analysis.

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is ...

Immunology and Infection

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. ...

Neuroscience

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

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