Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

Summary

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.

Abstract

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.

Introduction

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 l....

Protocol

The following techniques include the previous sacrifice of the animal, which was carried out in accordance with international ethical standards^46,47,48, 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.......

Discussion

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.......

Materials

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