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 following techniques include the previous sacrifice of the animal, which was carried out in accordance with international ethical standards46,47,48, and the subsequent dissection and ablation of the organs. The animals were captured under the authorization of the Ministry of Environment, Agriculture and Production of San Luis Province (Resolution 49-PMA2019). The methods of sacrifice and euthanasia of the animals were duly a...

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 s...

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.

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	<tbody>
		<tr>
			<td>Analytical scale</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis power supply</td>
			<td>&#160;Enduro</td>
			<td>&#160;E0203-250V&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin&#160;</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence photomicroscope</td>
			<td>&#160;Olympus</td>
			<td>&#160;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&#160;</td>
			<td></td>
			<td></td>
			<td>&#160;&#62;16 megapixels</td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal electrophoresis chamber</td>
			<td>&#160;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>&#160;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&#8211;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 addit...

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 ...

While the techniques for evaluating biochemical biomarkers are extensively utilized, this protocol present crucial details for standardizing and replicating the methodology in neotropical anurans. The main advantage of this technique is its low cost, replicability, and its application. This technique can be applied to obtain quantitative data to evaluate alterations in the internal melanin.The comet assay is a sensitive, versatile, and simple technique that allows the determination of genotoxic effects induced by a specific compound. It can be applied to any type of eukaryotic cells to monitor DNA damage, DNA repair, and assess the antioxidant status of cells. To begin, open the software Image-Pro Plus 6.0 and select menu and toolbar as biological.To quantify the area occupied by melanin, select measure, then select the spatial calibration of magnification used to take the photo micrographs. After opening the histological image, select tool count and then measure objects. Mark the coloring to be measured in the image using the select colors and dropper tool, and then close the window.Visualize the results by clicking on count, view, and statistics. Incubate a mixture of PBS, hydrogen peroxide, and a sample in a three milliliter quartz cuvette with a one centimeter path length. Then remove the sample kept in a 500 microliter centrifuge tube and add it to the quartz cuvette.Read the catalase kinetics at 240 nanometers for two minutes in a UV spectrophotometer and calculate the enzyme activity using the equation shown on the screen. Dilute the collected blood sample with one milliliter of PBS. Centrifuge it at 381 G for nine minutes, and resuspend the pellet with 50 milliliters of PBS.Mix 30 microliters of the blood sample in 70 microliters of low melting point agarose or LMPA at a 0.5%concentration of agarose to form layer two. Place 50 microliters of layer two on a slide previously coated with layer one containing 100 microliters of 0.5%normal melting point agarose. Cover the slide with a coverslip and place it in the cold at four degrees Celsius for 10 minutes to solidify layer two.After solidification, remove the coverslip. Form the third layer by laying down 100 microliters of 0.5%LMPA and cover the slide again. After the third layer solidifies, remove the coverslip and immerse each slide in a 100 milliliter coplin jar containing the lysis solution prepared on the same day.Place the coplin jars in a cold bath at four degrees Celsius for one hour in the dark for lysis to occur. At the end of the lysis, remove the slides and immerse them in the electrophoresis buffer solution in the electrophoresis tank for 15 minutes at four degrees Celsius to allow the DNA to unwind. After nuclear DNA unwinding, perform electrophoresis on the same buffer at four degrees Celsius for 10 minutes at 25 volts and 250 milliamperes.At the end of the electrophoresis, neutralize the samples in the slides by adding two milliliters of tris hydrochloric solution three times for five minutes. Place the samples in 100 milliliter coplin jars containing 95%alcohol at four degrees Celsius to dehydrate the samples. Stain the samples by adding 10 microliters of DAPI to the center of the slide.Then spread the dye throughout the samples with a coverslip. Examine the slides under a fluorescence photomicroscope with an NU filter. Quantify the DNA damage by evaluating the length of DNA migration from the nucleoid.In this case, visually determine it on 100 randomly selected cells without overlap. Then classify the DNA damage into four classes where zero to one is for no damage, two is for minimum damage, three is for medium damage, and four is for maximum damage. Express the data as the mean number of damaged cells and the mean comet score for each treatment group.Finally, from these data, calculate the genetic damage index or GDI for each test organism using the equation shown on the screen. The scaled mass index of Boana pulchella and Leptodactylus luctator adults are shown in this figure. For the analysis of the scaled mass index, a non-linear regression analysis was performed that demonstrates the power function relationship between the SVL and body mass for the species to determine the scaling exponent.The scaled mass index is useful for comparing groups of adults or juveniles from the same species. The histological sections of the liver of Boana raniceps are presented here. A greater amount of c and greater dimensions of the hepatocyte and nucleus of the hepatocytes can be observed from these sections.Different and complementary responses of cytogenetic biomarkers in an herbicide scenario exposure in Boana pulchella are shown in this figure. Here, the black bars indicate 48 hours, and the gray bars indicate 96 hours. The time of exposure and the concentration should always be considered as a factor here since a particular time or concentration may not be enough to induce damage.The representative image describes how the biomarkers are related to the biomarker response index. Here, the blue indicates the control group, while the orange, yellow, and green colors indicate the exposure scenarios to BDE 209. This data indicates that catalase is an effective biomarker for stress situations in which the stressor is present at the highest concentrations.When calculating the body condition index, consistently take photographs from the same distance. In tadpoles, measure the SVL body length up to the cloacal tube, excluding the collar fin. This approach safely addresses amphibian physiology and the impact of the environmental changes.While alternative potentially invasive biomarkers exist, these methods offer insights into oxidative stress, toxicodynamics, and toxicogenetics. Additional enzyme measurements related to oxidative stress can serve as alternative methodologies. The color difference analysis technique evaluates tissue responses in photomicrographs based on color disparities.For the pigmentary system, ensure the program's calibration aligns with the photomicrograph. This advanced technique is instrumental in investigating whether xenobiotics directly affect DNA in diverse cell types, addressing queries regarding genetic material oxidative damage and repair processes that other cytogenetic techniques are unable to address.

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186 Görüntüleme.  Universidad Nacional de San Luis (UNSL). Biyokimyasal biyobelirteçleri değerlendirme teknikleri yaygın olarak kullanılsa da, bu protokol neotropik anuranlarda metodolojiyi standartlaştırmak ve çoğaltmak için çok önemli ayrıntılar sunmaktadır. Bu tekniğin temel avantajı, düşük maliyeti, tekrarlanabilirliği ve uygulamasıdır. Bu teknik, iç melanindeki değişiklikleri değerlendirmek için kantitatif veriler elde etmek için uygulanabilir.Kuyruklu yıldız testi, belirli b...