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

Abstract

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.

Road-killed toads as a non-invasive source to study age structure of spring migrating population

“…They found almost no juvenile carcasses, while they were observed in adjacent live population. However, in our study, we focused on breeding migrating population of common toads, therefore we detected only sexually mature individuals (Kaczmarski et al 2016;Hemelaar 1986). Juvenile toads do not migrate to breeding ponds, nevertheless, we cannot exclude the possibility of finding roadkilled young individuals migrating to foraging habitat in different locations.…”

“…An hour after dark, we captured by hand all encountered live toads, that crossed the road which intersects their migration routes, and dead ones killed due to collision with vehicles. We determined sex of live and dead individuals directly in the field (according to Kaczmarski et al 2016). We identified males by the occurrence of the nuptial pad, whereas females by ovaries and eggs (dead individuals) and auxiliary also by body size and shape.…”

“…We stored the toes in Eppendorf tubes with 80% ethanol. We performed skeletochronological analysis according to Rozenblut and Ogielska (2005) with some modifications in protocol (see Kaczmarski et al 2016 for details). After removing the soft tissue, we decalcified phalanges in 1:1 mixture of 10% formic acid and 4% formalin for a maximum of 4 h. We froze decalcified phalanges, sliced them into 10-μm thick sections by freezing microtome, and stained in 0.05% cresyl violet.…”

“…Moreover, road carcasses can be used to study species distribution, habitat preferences, or phenology (Orłowski 2007;Sutherland et al 2010). They are also a potential non-invasive alternative to obtain material for further research, including genetic analysis (Balkenhol and Waits 2009), detection of amphibians' diseases (Martel et al 2012), trace elements in bones (Simon et al 2012), or histology and age structure (Kaczmarski et al 2016). Although this approach could limit invasive methods, the data obtained from road kills may be biased due to removal of carcasses from roads by scavengers (Antworth et al 2005).…”

Road kills of non‐human primates: a global view using a different type of data

“…Many studies have shown that roads can be barriers for different taxa, such as birds, amphibians, reptiles, and mammals (e.g. de Freitas et al , Kaczmarski et al , Kiros et al ), and may have a broad impact on wildlife biodiversity (Ibisch et al ). The loss of individuals due to vehicle collisions may influence the dynamics of populations far from the road (Roger et al , Van Der Ree et al ).…”