Developmental stages of Rhinella arenarum (Anura, Bufonidae) in toxicity studies: AMPHITOX, a customized laboratory assay

PERÉZ COLL C.S.; ARONZON C.M; SVARTZ G.

Ecotoxicology and genotoxicology: Non-traditional aquatic models

The Royal Society of Chemistry

Lugar: Cambridge; Año: 2017; p. 407 - 424

Increasing negative impacts on natural resources and life support systems result from continuing population growth and anthropic activities. This situation is worsened by increasing environmental pollution and inappropriate environmental management.These facts are of major concern because the decline of environmental quality and biodiversity could severely affect ecosystem services that are essential to the quality of the biosphere and human health. In present day conditions, it is well-documented that environmental degradation could result in lethal effects while sublethal impacts, from teratogenesis to endocrine disruption,could also be a threat to wildlife and human health. The aquatic environment is particularly important as a recipient, transporting medium and sink for the majority of xenobiotic chemicals.In 1999, in order to cope with an increasing diversity of chemicals from different matrices and processes,AMPHITOX was developed15 to cover the need for a sensitive, cost-effective and customized assay for different exposure times and developmental stage toxicity tests. The usefulness of the AMPHITOX test for toxicity assessment of environmental samples was shown in those early contributions. As stewardship for environmental and human health protection improves,the development of a more flexible and customized toxicity test for amphibians seems to be of high priority. FETAX, Frog Embryo Teratogenesis Assay-Xenopus, represented the most important antecedent of toxicity test employing amphibian embryos for assessing lethal and teratogenic effects of xenobiotics within an acute exposure period. AMPHITOX hasintroduced additional possibilities to this test, such as: (1) The extension of the exposure time from acute to short-term chronic and chronic exposures, for the assessment of toxicity in adjusted bioassays, according to the characteristics of the sample.(2) The use of two different developmental periods, embryos and larvae, for toxicity tests. (3) The plot of the LC10 (or LOEC), LC50 and LC90 (or LC100), to describe the toxicity profile (TOP) curves from acute to chronic exposure, allowing the visualization of concentrationand time-exposure thresholds, as well as the range of concentrations that exert adverse effects in each case. These TOP curves provide, within a systemic toxicity approach, a more appropriate set of data for hazard and risk assessment purposes.(4) Additionally, the use of Rhinella arenarum (ex Bufo arenarum) as a native amphibian specie distributed in a large area of South America also provides relevant information on the local impact of pollutants. This species is highly sensitive to toxic agents and at the same time is placed among the ??least concern?? species according to the International Union for Conservation of Nature. AMPHITOX is a standardized test employing amphibian embryos that can be used to evaluate toxicity for acute, short-term chronic, chronic, and early life stage exposure to hazardous chemicals and complex mixtures. By means of AMPHITOX the toxicity of both environmental samples and physicochemical agents singly or in combination can be evaluated by adjusting the exposure period to the toxicity of the sample and/or the assessment purpose. The growing concern about chronic effects of xenobiotics at environmentally relevant concentrations led us to expand the exposure conditions of classic, acute (up to 96 h of exposure), short-term chronic (168 h) and chronic (14 days) AMPHITOX tests with young larvae to evaluate noxious effects on specific stages of the early life cycle (pulse exposures) and exposure periods covering whole development (embryo?larval and metamorphosis). This last test has even included post-exposure effects e.g. 60 days post-metamorphosis, to assess adverse effects on gonadogenesis and sexual differentiation. It is useful to highlight that a worldwide decline of amphibian populations linked to the increasing environmental degradation has been widely documented. Amphibians play a key role in food webs, living near or in water reservoirs, were they can be direct or indirectly affected by xenobiotic exposure. Moreover, as amphibians are considered keystone members of ecosystems and vital links in food chains, contaminants might be magnified through them. Furthermore, contaminants might be transported by metamorphic amphibians to terrestrial communities. This taxonomic group includes extremely sensitive species because they have permeable skin and eggs that readily absorb chemicals from the environment. Moreover,many species complete their life cycle in shallow or ephemeral water bodies in which pollutants might be concentrated by seasonal temperature increments, coincident with breeding and larval development. In this contribution, the basic conditions of the AMPHITOX test and some case studies were included to illustrate both the basic and customized potential of this toxicity test. TOP curves, scanning electron microscopy (SEM) studies and histological analysis accompanying the toxicity assessment of different matrices were added.