Phalangeal bone anomalies in the European common toad Bufo bufo from polluted environments.

Every spring, many of amphibians are killed by motor vehicles on roads. These road-killed animals can be used as valuable material for non-invasive studies showing the effect of environmental pollution on amphibian populations. The aims of our research were to check whether the phalanges of road-killed toads may be useful as material for histological analysis, and whether various degrees of human impact influence the level in bone abnormalities in the common toad. We also examined whether the sex and age structure of toads can differ significantly depending in the different sites. We chose three toad breeding sites where road-killed individuals had been observed: near the centre of a city, the outskirts of a city, and a rural site. We collected dead individuals during spring migration in 2013. The sex of each individual was determined and the toes were used to determine age using the skeletochronology method. While performing age estimates, we looked for abnormalities in relation to normal bone tissue structure. In urban site, females dominate males (sex ratio 2.6:1), but in populations from rural and semi-urban sites, sex ratio was reverse (1:2.2 and 1:1.4, respectively). However, we did not find any significant differences between age structure of all populations (average age of each population: approximately 4 years). We observed abnormalities in more than 80 % of all toads from the city, compared to approximately 20 % from the rural and semi-urban sites. In particular, we found hypertrophic bone cells, misaligned intercellular substance, and irregular outer edges of bones. We suggest that these malformations are caused by different pollution, e.g. with heavy metals.

[Individual variation in the development of the common toad, Bufo bufo (Anura, Bufonidae): I. Timing of development and anomalies of external structure].

Parameters of development have been analyzed in all offspring (1633 ind.) from the same spawn of one pair of common toads under conditions of laboratory rearing, with natural elimination being reduced to a minimum. The results show that the range of developmental options for the species is much wider than it follows from the standard tables of development. "Background anomalies" (Kovalenko, 2003) in the external structure of developing larvae are described and their probable frequencies under nearly optimal conditions are indicated.

[Individual variation in the development of the common toad, Bufo bufo (Anura, Bufonidae): 2. Diagnostic characters of the axial skeleton].

Analysis of individual variation in diagnostic characters of the axial skeleton has been performed in all offspring (1633 ind.) from the same spawn of one pair of common toads under conditions of laboratory rearing, with natural elimination being reduced to a minimum. More than 50 skeletal anomalies have been recorded. Factual data are presented that characterize deviations from the species norm in the frequency of "background anomalies" (Kovalenko, 2003). Correlations between manifestations of different variants of character states are considered. The problem of norm and anomaly in individual variation is discussed.

[Individual variation in the development of the common toad, Bufo bufo (Anura, Bufonidae): 3. Limitations of individual variation and their causes].

The results of studies on the axial skeleton of the common toad (a model species) have been used to analyze factual limitations of individual variations. The results show that the states of the studied characters do not freely combine with each other but are subject to certain morphogenetic limitations. The causes of most these limitations have been revealed during the study. Classification of the main factors limiting individual variation in the course of development is presented.

Gill alterations as biomarkers of chronic exposure to endosulfan in Bufo bufo tadpoles.

Endosulfan sprayed on agricultural fields accumulates in temporary pools due to surface runoff or sediment transport and may result in high water concentrations in spring and summer, coinciding with breeding and crucial stages of amphibian larval development. In the present study, Bufo bufo tadpoles were exposed to three different concentrations of endosulfan (0.01, 0.05 and 0.1 mg/L) until they reached complete metamorphosis. The aim of the study was to investigate the effects of endosulfan, at environmentally relevant concentrations on gill morphology and ultrastructure. Modifications in ultrastructure and cell composition were observed at all concentrations after 96 h. The main gill effects recorded in treated animals were: mucous secretion, the appearance of tubular vesicles cells (TVC) and a degeneration phenomenon. Comparing these results with our previous findings in which we used growth, developmental rate and behaviour as endpoints, we also demonstrated that the first effect of endosulfan on Bufo bufo was gill alteration, thus supporting the role of a morphological approach in toxicological studies. This study provides additional information on the role of morphological studies in demonstrating the effects of exposure to environmental pollutants. In this context, the use of amphibian gills, as effective biomarkers, is a valuable approach in evaluating exposure to agrochemicals.

Environmentally relevant concentrations of endosulfan impair development, metamorphosis and behaviour in Bufo bufo tadpoles.

Endosulfan is a widely used organochlorine pesticide with well-documented neurotoxic effects in both humans and laboratory animals (mammals and fish). Neurotoxicity has been implied also in amphibians after short-term exposure to endosulfan. Little is known about effects of chronic exposure of endosulfan in amphibians. Previously, we examined the short-term toxicity of endosulfan in common toad (Bufo bufo) tadpoles and determined the LC50 value to 0.43 mg/L. In the present study, we investigated the effects of endosulfan on B. bufo tadpoles after chronic exposure to ecologically relevant concentrations. Tadpoles were exposed in a static renewal test, from shortly after hatching (Gosner stage 25) to completed metamorphosis, to 0.01, 0.05 and 0.1mg endosulfan/L (nominal). The exposure period lasted 43-52 days. Mortality, larval growth (mass), development (reached Gosner stage at various times and deformities presence), metamorphosis and behaviour (swimming activity) were monitored regularly over the entire course of larval development. Our results show that 0.05 and 0.1mg endosulfan/L caused impaired behaviour, prolonged time to metamorphosis, increased incidences of mouth and skeletal malformations as well as mortality, and reduced body weight (observed also at 0.01 mg/L) in B. bufo tadpoles. Behavioural effects occurred at exposure day 4, before any other effects occurred, indicating a neurotoxic effect. Endosulfan levels found in groundwater and surface water range from 0.1 to 100 microg/L and after extraordinary runoff events, concentrations exceed 0.5 mg/L in surface water. Our results indicate that endosulfan may negatively affect wild frog populations in agricultural areas.