Effetti dell’endosulfan sugli Anfibi: uno studio morfologico e comportamentale

Abstract

The declining amphibian population is a global phenomenon in conservation biology that has attracted the attention of many scientists (Russell et al., 1995; Stallard, 2001; Sparling e Fellers, 2009). As for the majority of recent reductions in Earth biodiversity, the amphibian decline can also be attributed to direct human impact on the environment such as the use of pesticides and other chemical pollutants. Therefore, understanding the impacts of agrochemicals on amphibians is a challenging proposition. Amphibians are sensitive to the action of pollutants and they are considered excellent bioindicators of the environmental quality due to their biological, ecological and anatomical characteristics. For these reasons, amphibians are broadly used as typical targets in evaluating the effects of chemicals on aquatic and agricultural ecosystems (Schuytema e Nebeker, 1996; Pollet e Bendell-Young, 2000; Venturino et al., 2003; Bernabò et al., 2008). Decreased species richness, reduced populations, and high deformity incidences have been reported in agroecosystems, which might be linked to the extensive use of pesticides (Berrill et al., 1994, 1997; Bonin et al., 1997; Knutson et al., 2002; Davidson, 2004; Bridges et al., 2004; Relyea, 2005). As outlined by Boone et al. (2005) there is a lack of data for many commonly used pesticides with regard to their effects on amphibians. We chose endosulfan, an organochlorine globally used on a wide variety of crops and other sectors. The environmental persistence and toxicity of endosulfan forced several national authorities to ban it and to propose its inclusion as a persistent organic pollutant in the Stockholm convention (Kelly et al., 2007; EPA, 2010). Endosulfan reaches aquatic systems through direct application, as well as spray drift and runoff from agricultural areas (Leonard et al., 1999, 2000, 2001; Broomhall, 2002; Jergentz et al., 2004) and levels in nature pose a risk to aquatic ecosystems. The last EPA (2010) report on endosulfan, highlights the need for further work to consider amphibians as model species. Previous studies on the effects of endosulfan on amphibians showed detrimental effects on survival, growth, development and metamorphosis, and even caused deformity, severe changes in gill morphology and behavioural alterations (Bernabò et al., 2008; Brunelli et al., 2009, 2010; Jones et al., 2009; Shenoy et al., 2009). Therefore, we proposed to assess the effects of chronic exposure to ecologically relevant concentrations of endosulfan on two anuran species during larval development. In particular, we focused attention on the morphological endpoint evaluating the effects on Bufo bufo skin, to validate the hypothesis of our research group using a morphological approach in a toxicological study. Recently we demonstrated that environmentally relevant endosulfan concentrations (0.1, 0.05 and 0.01 mg/L) may affect Bufo bufo gills morphology and ultrastructure (Brunelli et al., 2010). On this basis we proposed completing the study on endosulfan toxicity in Bufo bufo tadpoles evaluating the effects of the same sublethal concentrations on skin. In fact, the thin and permeable skin of amphibians directly interacts with all environmental constituents, including toxicants. Moreover there is a gap in literature on endosulfan effects on this target organ. We also analyzed the effects of endosulfan on Rana dalmatina tadpoles. Firstly, we examined the short-term toxicity of endosulfan determining the LC50 value and after this preliminary analysis we conducted a classical toxicological study evaluating the effects of sublethal concentrations of endosulfan (0.005, 0.01 and 0.05 mg/L) on survival, growth, development, metamorphosis, deformities presence in Rana dalmatina tadpoles and, in collaboration with the University of Liege we applied new tools for evaluating behavioural patterns. For the first time we used video-tracking analyses to test the quantitative effect of endosulfan on the locomotion. We exposed Bufo bufo tadpoles from Gosner stage 25 until stage 42 to a chronic static renewal system according to the experimental design and conditions of our previous studies (according to Brunelli et al., 2010). and the epidermis was removed after 96 h, 14 and 20 days and analyzed by Trasmission Electron Microscopy (TEM). Exposure to endosulfan caused a general defensive response by mucous secretion and the appearance of large secretory vescicles in Bufo bufo larval epidermis. The exposure to all concentrations, already after 96 h, caused an increase of intercellular spaces, alteration of the cell-cells interactions, cellular degeneration, in particular of the typical larval bufonidae cells (Riesenzellens). The epidermal-dermal junctions and the figures of Eberth in the basal layer, were completely lost at the end of exposure. Our results were successful in showing that environmentally relevant concentrations of endosulfan negatively affected the epidermis of Bufo bufo tadpoles after long-term exposure, resulting in ultrastructure modification in the treated animals compared to controls and that epidermal damage was strongly correlated with exposure time. These results are consistent with our previous findings (Bernabò et al., 2008; Brunelli et al., 2010) concerning the morphological alteration pattern of the gill epithelium during long term exposure. We confirmed that the morphological approach is an useful tool in evaluating the effects of environmental contamination at sublethal levels in sensitive organisms like amphibians, and we supported the role of amphibian skin as a good biomarker following pesticide exposure. Rana dalmatina tadpoles were exposed to several nominal concentrations of endosulfan to assess the sensitivity of the species by estimating the LC50 value at 96 h in a static exposure system. We found that the nominal 96 h LC50 value was 0.074 mg/L. After this preliminary analysis, Rana dalmatina tadpoles were exposed to sublethal concentrations of endosulfan (0.005, 0.01, 0.05 mg/L) in a chronic static renewal system from Gosner stage 25 for the entire course of larval development. Mortality, larval growth (mass), body length, development, time to metamorphosis and deformities presence were monitored regularly over the entire course of larval development. The behaviour (swimming activity) was recorded after 7, 14, 21 and 28 days of exposure and analyzed by a video-tracking software Ehovision 7 XT (Noldus Information Technology, The Netherlands). We analyzed behavioural traits such as: distance moved, velocity, immobility and space use. Our results showed that long-term exposure to environmentally relavant concentrations of endosulfan impaired survival, larval growth, development, metamorphosis, swimming activity and caused severe morphological alteration in tadpoles exposed to 0.01 and 0.05 mg endosulfan/L (medium and high concentrations). With regards to the inhibition of metamorphosis in the highest concentrations, this has a profound implication in the light of amphibian decline. Instead, the lowest concentration of endosulfan (0.005 mg/L) did not cause any significant effects on growth, development and on swimming activity, although we have observed a slightly significant reduction in survival without decreasing the success of metamorphosis. In natural environments, increased incidences of deformities, a reduced larval growth could increase susceptibility to predation (Rohr et al., 2003), delay metamorphosis or result in metamorphosis at a smaller size having negative effects on future fitness, growth and overwinter survival (Brodie and Formanowicz, 1983; Semlitsch et al., 1988; Smith, 1987; Boone and Semlitsch, 2002; Altwegg and Reyer, 2003; Boone and James, 2003). We used video-tracking analyses for the first time to test the quantitative effects of a pesticide on the behaviour of amphibians. And we observed that behavioural effects occurred from 7 days of exposure and tadpoles exposed to the medium and high concentrations of endosulfan exhibited several anomalies in swimming activity such as: shorter distance moved, slower speed, swirling, resting and different space use. Our findings are in agreement with the previous studies of our research group on endosulfan effects on Bufo bufo (Brunelli et al., 2009) and, with the use of video-tracking analysis, we had advantages over standard visual methods. In fact we precisely quantified swimming activity anomalies. We demonstrated that quantitative ecotoxicology is thus a valuable tool to assess conservation concerns when other techniques cannot detect detrimental effects. Our study thus confirms and extends the results of previous studies on the toxicity of this organochlorine pesticide in showing its possible role in amphibian decline.