Survived the Glaciations, Will They Survive the Fish? Allochthonous Ichthyofauna and Alpine Endemic Newts: A Road Map for a Conservation Strategy.

The Calabrian Alpine newt (Ichthyosaura alpestris inexpectata) is a glacial relict with small and extremely localised populations in the Catena Costiera (Calabria, Southern Italy) and is considered to be "Endangered" by the Italian IUCN assessment. Climate-induced habitat loss and recent fish introductions in three lakes of the Special Area of Conservation (SAC) Laghi di Fagnano threaten the subspecies' survival in the core of its restricted range. Considering these challenges, understanding the distribution and abundance of this newt is crucial. We surveyed the spatially clustered wetlands in the SAC and neighbouring areas. First, we provide the updated distribution of this subspecies, highlighting fish-invaded and fishless sites historically known to host Calabrian Alpine newt populations and two new breeding sites that have been recently colonised. Then, we provide a rough estimate of the abundance, body size and body condition of breeding adults and habitat characteristics in fish-invaded and fishless ponds. We did not detect Calabrian Alpine newts at two historically known sites now invaded by fish. Our results indicate a reduction in occupied sites and small-size populations. These observations highlight the need for future strategies, such as fish removal, the creation of alternative breeding habitats and captive breeding, to preserve this endemic taxon.

Tissues injury and pathological changes in Hyla intermedia juveniles after chronic larval exposure to tebuconazole.

Tebuconazole (TBZ), an azole pesticide, is one of the most frequently detected fungicides in surface water. Despite its harmful effects, mainly related to endocrine disturbance, the consequences of TBZ exposure in amphibians remain poorly understood. Here, we investigated the adverse and delayed effects of TBZ chronic exposure on a native anuran species, often inhabiting cultivated areas, the Italian tree frog (Hyla intermedia). To disclose the multiple mechanisms of action through which TBZ exerts its toxicity we exposed tadpoles over the whole larval period to two sublethal TBZ concentrations (5 and 50 µg/L), and we evaluated histological alterations in three target organs highly susceptible to xenobiotics: liver, kidney, and gonads. We also assessed morphometric and gravimetric parameters: snout-vent length (SVL), body mass (BM), liver somatic index (LSI), and gonad-mesonephros complex index (GMCI) and determined sex ratio, gonadal development, and differentiation. Our results show that TBZ induces irreversible effects on multiple target organs in H. intermedia, exerting its harmful effects through several pathological pathways, including a massive inflammatory response. Moreover, TBZ markedly affects sexual differentiation also by inducing the appearance of sexually undetermined individuals and a general delay of germ cell maturation. Given the paucity of data on the effects of TBZ in amphibians, our results will contribute to a better understanding of the environmental risk posed by this fungicide to the most endangered group of vertebrates.

Exposure and post-exposure effects of chlorpyrifos on Carassius auratus gills: An ultrastructural and morphofunctional investigation.

Widespread environmental contamination from chlorpyrifos (CPF) is well acknowledged and has led to the proposal to ban or limit its use in agricultural and domestic, within the regulatory context of both America and Europe. Furthermore, great concerns arise as to whether exposure to CPF represents a potential risk to human health. In the present study, by subjecting the goldfish model to three environmentally realistic concentrations of CPF (1, 4, and 8 µg/L) for 96 h, we demonstrated that this pesticide has the potential to induce severe morphological, ultrastructural and functional alterations in gills, even at very low concentrations. The degree of pathological effects was dose-dependent, and the main morphological alterations recorded were: regression of interlamellar cellular mass (ILCM), hypertrophy, and hyperplasia of epithelial cells, degeneration of both chloride cells and pillar cells. CPF exposure resulted in a decrease of Na+/K+-ATPase expression and the induction of iNOS, as revealed by immunohistochemical analysis. In order to determine the overall toxicity of CPF, we also investigated the recovery capability of goldfish gills following a period of 7 days in pesticide-free water. Our results clearly showed that there exists a threshold of CPF dose below which the effects on gills are reversible and beyond which the ability of gills to recover their typical features is completely lost. The information presented in this paper emphasises the importance of evaluating the recovery ability of organisms after chemical input and enhances our knowledge of the potential hazard of organophosphorus pesticides (OPs) on freshwater ecosystems.

Lead toxicity in seawater teleosts: A morphofunctional and ultrastructural study on the gills of the Ornate wrasse (Thalassoma pavo L.).

Lead is one of the most alarming toxic pollutants for the environment due to its acute toxicity and high bioaccumulation tendency. Despite legislative efforts, world lead production has more than doubled since the early 1970s to 2016. Due to extensive exploitation and human activity, the coastal and estuarine regions belong to marine environments that are mostly and more rapidly deteriorated by pollutants including lead. A limited number of studies examined the effects of lead in fishes, compared to other aquatic models and even fewer studies have been dedicated to seawater fishes especially regarding Pb adsorption and accumulation in specific organs. Fish gills, key compartments involved in several crucial functions such as gas exchange, osmoregulation, and excretion, are also the organs in which Pb is mainly accumulated. Herein, we investigated the morphofunctional and ultrastructural modifications in the gills of a marine teleost (Thalassoma pavo) after acute exposure (48, 96, 192 h) to three lead concentrations. We showed that pathological alterations can be detected in all the examined samples. The most common modifications observed were: the curling of the lamellae and the dilation of their apical tips, the lamellar edema, the proliferation and the hypertrophy of CCs, the progressive epithelial disorganization with detachment of the epithelium from connective tissue. This study also demonstrates that there is a weak influence on the expression pattern of Na+/K+-ATPase and AQP3 biomarker enzymes while high metallothioneins expression has been observed. The described alterations may adversely affect gas exchange and ionic balance, with a long chain of cascading effects. This is the first evidence of the effects exerted by lead on gills of seawater fishes that highlights the harmful properties of this metal, even at low concentration.

Chronic exposures to fungicide pyrimethanil: multi-organ effects on Italian tree frog (Hyla intermedia).

Amphibian habitats are easily contaminated by several pollutants, and in agricultural landscapes the likely exposure scenario is represented by pesticides. Many of these substances are known or suspected to act as endocrine disrupting chemicals (EDCs). The goal of the present study was to assess the effects of pyrimethanil, a common-used but also overlooked fungicide, on liver, kidney and gonadal differentiation of Hyla intermedia. Through a multi-organ evaluation, we demonstrated that a long term exposure to two environmentally relevant concentrations of pyrimethanil (5 and 50 µg/L) elicits a range of toxic responses. First we showed that pyrimethanil induces underdevelopment of ovaries and interferes with normal sexual differentiation, thus revealing the endocrine disruption potential of this fungicide. Moreover we revealed that all considered organs are seriously affected by this fungicide and both necrosis and apoptosis contribute to the histological response. This is the first report on the effects of pyrimethanil on gonads, liver and kidney histology of a non-model species and it demonstrates that the hazardous properties of this fungicide can result from several pathological processes affecting different key compartments of amphibian.

Effects of Two Sublethal Concentrations of Mercury Chloride on the Morphology and Metallothionein Activity in the Liver of Zebrafish (Danio rerio).

Mercury (Hg) is a highly hazardous pollutant widely used in industrial, pharmaceutical and agricultural fields. Mercury is found in the environment in several forms, elemental, inorganic (iHg) and organic, all of which are toxic. Considering that the liver is the organ primarily involved in the regulation of metabolic pathways, homeostasis and detoxification we investigated the morphological and ultrastructural effects in Danio rerio liver after 96 h exposure to two low HgCl2 concentrations (7.7 and 38.5 µg/L). We showed that a short-term exposure to very low concentrations of iHg severely affects liver morphology and ultrastructure. The main effects recorded in this work were: cytoplasm vacuolization, decrease in both lipid droplets and glycogen granules, increase in number of mitochondria, increase of rough endoplasmic reticulum and pyknotic nuclei. Pathological alterations observed were dose dependent. Trough immunohistochemistry, in situ hybridization and real-time PCR analysis, the induction of metallothionein (MT) under stressor conditions was also evaluated. Some of observed alterations could be considered as a general response of tissue to heavy metals, whereas others (such as increased number of mitochondria and increase of RER) may be considered as an adaptive response to mercury.

Effects of long-term exposure to two fungicides, pyrimethanil and tebuconazole, on survival and life history traits of Italian tree frog (Hyla intermedia).

Over the last few years, the hazards associated with the extensive use of fungicides have become an issue of great concern but, at present, the effects of these substances on amphibians remain poorly understood. The goal of the present study was to assess the effects of two commonly used fungicides, tebuconazole and pyrimethanil, on Italian Tree Frog (Hyla intermedia), a species frequently found in agricultural areas. Tadpoles were exposed to fungicides from developmental Gosner stage 25 (GS 25) to completion of metamorphosis (GS 46) and the whole exposure period lasted 78 days. For both tested fungicides we used two concentrations (5 and 50µg/L) that are comparable to those detected in surface waters, near agricultural fields. A variety of sublethal effects-on growth, development, behavior, and physiology-may be used for evaluating alterations induced by pollutants in amphibians. We estimated whether pyrimethanil and tebuconazole exposure impacted on H. intermedia life history traits. For this purpose, survival, growth, development, initiation of metamorphosis, success and size at metamorphosis, time to metamorphosis, and frequency of morphological abnormalities were evaluated. We showed, for all considered endpoints, that the exposure to tebuconazole exerts more harmful effects on H. intermedia than does exposure to pyrimethanil. Before the onset of metamorphic climax we showed, for both fungicides, that the low concentrations (5µg/L) induced significantly greater effects than the higher ones (50µg/L) on survival and deformity incidence. During the metamorphic climax, a complete reversal of this nonlinear trend takes place, and the percentage of animals initiating metamorphosis was reduced in fungicide-exposed groups in a concentration-dependent manner. Furthermore, a strong correlation emerged between fungicide exposure and the incidence of morphological abnormalities such as tail malformations, scoliosis, edema, mouth and limb deformities. Exposure to tested fungicides also caused a reduction in developmental rates just prior to the onset of metamorphic climax, which translated to a significant delay in timing of metamorphosis. We detected a drastic decrease in the success at metamorphosis in all exposed groups, compared to control group (86.25%). In fact, the percentage of survived larvae to GS 46, in the high and low concentrations, respectively, was only 22.5% and 36.25% in tebuconazole-exposed groups and 43.75% (50µg/L) and 56.25% (5µg/L) in pyrimethanil-exposed groups. Our findings underscore the hazardous properties of these two fungicides for non-target species in the context of ecotoxicological risk assessment. No published studies have addressed the long-term effects of tebuconazole and pyrimethanil on amphibians. To date, this is one of only a few studies documenting the effects of fungicide exposure over the whole larval development.

Liver histology and ultrastructure of the Italian newt (Lissotriton italicus): normal structure and modifications after acute exposure to nonylphenol ethoxylates.

We examined, from a morphological and ultrastructural point of view, the liver of the Italian newt (Lissotriton italicus), under basal conditions and after exposure to nonylphenol ethoxylates (NPEs). Nonylphenol ethoxylates are surfactants widely used in a variety of industrial and agricultural processes that may pose a significant risk to aquatic fauna. NPEs, and their degradation intermediates, are known to affect reproductive biology acting as endocrine disruptors; besides estrogenic effects, nonylphenolic compounds may induce organ toxicity, particularly in liver and gonads. We investigated the effects of a nonylphenol ethoxylate (NPE10-ETO) on L. italicus liver using two low concentrations, consistent with the environmental concentrations. For this purpose, animals were exposed to nominal concentrations of 50 and 100 µg/L in a short-term experiment (96 h). A morpho-functional analysis was performed in order to investigate the amphibian responses to NPEs thus contributing to elucidate other potential mode of action of these compounds; indeed very little attention has been dedicated to amphibians though they are often exposed to such contaminants in aquatic ecosystems. Pathological alterations on liver histology and ultrastructure were observed at both tested concentrations; the main effects recorded were: increase of intercellular spaces, accumulation of large lipid droplets, increase in melanin content, and a degeneration phenomenon. We also detected, through confocal analysis, the induction of caspase-3, a key mediator of apoptosis, and an up-regulation of cytochrome P450-1A. By using both ultrastructural and a morpho-functional approach, we found that a short-term exposure to NPEs negatively affected the amphibian liver.

Exposure and post-exposure effects of endosulfan on Bufo bufo tadpoles: morpho-histological and ultrastructural study on epidermis and iNOS localization.

Endosulfan is a persistent organic pollutant (POP) that has lethal and sublethal effects on non-target organisms, including amphibians. In a laboratory study, we investigated direct and post-exposure effects of endosulfan on Bufo bufo tadpoles. For this purpose we exposed the tadpoles to a single short-term contamination event (96 h) at an environmentally-realistic concentration (200 µg endosulfan/L). This was followed by a recovery period of 10 days when the experimental animals were kept in pesticide-free water. The endpoints were assessed in terms of mortality, incidence of deformity, effects on behavior, and the morpho-functional features of the epidermis. We found that a short-term exposure to the tested concentration of endosulfan did not cause mortality but induced severe sublethal effects, such as hyperactivity, convulsions, and axis malformations. Following relocation to a pesticide-free environment, we noted two types of response within the experimental sample, in terms of morphological and behavioral traits. Moreover, by using both ultrastructural and a morpho-functional approach, we found that a short-term exposure to endosulfan negatively affected the amphibian epidermis. We also observed several histo-pathological alterations: increased mucous secretion, an increase in intercellular spaces and extensive cell degeneration, together with the induction of an inducible isoform of nitric oxide synthase (iNOS). Following the post-exposure period, we found large areas of epidermis in which degeneration phenomena were moderate or absent, as well as a further increase in iNOS immunoreactivity. Thus, after 10 days in a free-pesticide environment, the larval epidermis was able to partially replace elements that had been compromised due to a physiological and/or a pathological response to the pesticide. These results highlight the need for both exposure and post-exposure experiments, when attempting to assess pollutant effects.

Effects of salinity stress on Bufo balearicus and Bufo bufo tadpoles: Tolerance, morphological gill alterations and Na(+)/K(+)-ATPase localization.

Freshwater habitats are globally threatened by human-induced secondary salinization. Amphibians are generally poorly adapted to survive in saline environments. We experimentally investigated the effects of chronic exposure to various salinities (5%, 10%, 15%, 20%, 25%, 30% and 35% seawater, SW) on survival, larval growth and metamorphosis of tadpoles from two amphibian populations belonging to two species: the green toad Bufo balearicus and the common toad Bufo bufo. In addition, gill morphology of tadpoles of both species after acute exposure to hypertonic conditions (20%, 25%, and 30% SW) was examined by light and electron microscopy. Tadpoles experienced 100% mortality above 20% SW in B. balearicus while above 15% SW in B. bufo. We detected also sublethal effects of salinity stress on growth and metamorphosis. B. bufo cannot withstand chronic exposure to salinity above 5% SW, tadpoles grew slower and were significantly smaller than those in control at metamorphosis. B. balearicus tolerated salinity up to 20% SW without apparent effects during larval development, but starting from 15% SW tadpoles metamorphosed later and at a smaller size compared with control. We also revealed a negative relation between increasing salt concentration and gill integrity. The main modifications were increased mucous secretion, detachment of external layer, alteration of epithelial surface, degeneration phenomena, appearance of residual bodies, and macrophage immigration. These morphological alterations of gill epithelium can interfere with respiratory function and both osmotic and acid-base regulation. Significant variations in branchial Na(+)/K(+)-ATPase activity were also observed between two species; moreover an increase in enzyme activity was evident in response to SW exposure. Epithelial responses to increasing salt concentration were different in the populations belonging to two species: the intensity of histological and ultrastructural pathology in B. bufo was greater and we noticed the appearance in exposed samples of the tubular vesicle cells (TVCs). Taken together, our results demonstrated that increased salinity of freshwater may give cause for concern and must be considered a stressor for amphibians as well as other pollutants.

Endosulfan effects on Rana dalmatina tadpoles: quantitative developmental and behavioural analysis.

Endosulfan is an organochlorine pesticide that was recently labeled as a persistent organic pollutant, but it is still widely employed, particularly in developing countries. The goal of this study is to evaluate the acute (LC(50)) and chronic effects (developmental and behavioural traits) of this insecticide on Rana dalmatina tadpoles after exposure to ecologically relevant concentrations (0.005, 0.01, and 0.05 mg/L) by applying video-tracking techniques to evaluate the quantitative effect of endosulfan on amphibian behavioural patterns. The 96 h LC(50) value was 0.074 mg endosulfan/L. Tadpoles chronically exposed to 0.01 and 0.05 mg endosulfan/L underwent high mortality rate, decreased larval growth, delayed development, and increased incidence of malformations, and they did not reach metamorphosis by the end of the experiment. Moreover, tadpoles exposed to these concentrations exhibited several abnormalities in swimming patterns, such as shorter distance moved, swirling, resting, and unusual use of space. The exposure to 0.005 mg endosulfan/L did not cause any significant effects on behaviour, larval growth, or development, but we observed a significant decrease in both survival and time to metamorphosis. We showed that developmental abnormalities are dose-dependent and that the pesticide effects could differ depending on the endosulfan concentration and the species tested. We also validated the hypothesis that behavioural analysis, along with the use of new analytical methods, could be a useful tool in amphibian ecotoxicological studies.

The shift from aquatic to terrestrial phenotype in Lissotriton italicus: larval and adult remodelling of the skin.

Morphology and ultrastructure of the skin of Lissotriton italicus (previously named Triturus italicus) have been described in different phases of its biological cycle: larval stage, metamorphic stage and adult stage with emphasis on modifications occurring between aquatic and terrestrial adults. In the present study, light microscopy and both scanning and transmission electron microscopy were employed to analyze the histological and cytological remodelling that occurs in the skin of L. italicus during metamorphosis. The ultrastructure of the larval epidermis is arranged into three principal layers comprising an external layer of pavement cells, a basal layer and 1-3 intermediate layers consisting of Leydig cells along with accessory cells and mitochondria-rich cells. By the onset of metamorphosis, morphological changes of the skin include stratification and flattening of epidermal layers and disappearance of typical larval cells. In both aquatic and terrestrial adult phases the thin, cornified epidermis shows the same general arrangement as found in other vertebrates with an external stratum corneum and a variable number of intermediate cell layers. During the terrestrial adult phase, the skin is characterized by the presence of numerous tubercles; moreover, the lower epithelium is thicker than in the aquatic phase. Ultrastructural analysis revealed no substantial differences in the cellular composition of the skin between aquatic and terrestrial phases.

Survival, development, and gonadal differentiation in Rana dalmatina chronically exposed to chlorpyrifos.

Chlorpyrifos is an organophosphate pesticide among the most widely used in the world, which is suspected to be an endocrine-disrupting compound. To determine the capacity of chlorpyrifos to affect gonadal differentiation in Rana dalmatina, tadpoles were exposed to this pesticide during larval development until completion of metamorphosis at ecologically relevant concentrations (0.025 and 0.05 mg/L). No effects of chlorpyrifos exposure on survival, development, or metamorphosis were observed. After a 1 month metamorphosis, the gonadal phenotype was determined by gross morphology and histological examination. Morphological and histological analysis revealed normal ovaries or testes in froglets belonging to control group, whereas testes from several froglets exposed to chlorpyrifos were interspersed with testicular oocytes in histological sections. Chlorpyrifos exposure during the entire larval period did not affect sex ratio, but reduced the percentage of males with histologically normal testes. The findings suggest that chlorpyrifos exposure has significant effects on gonadal differentiation in some animals by inducing an intersex condition and alterations to testicular morphology, and that R. dalmatina is sensitive to endocrine disruption. Thereby, this study provides evidence that the ecologically relevant concentrations of chlorpyrifos, although not adversely affect the survival, development, or metamorphosis, may interfere with sex differentiation and reproductive development of R. dalmatina via endocrine-disrupting mechanisms.

Toxicity of chlorpyrifos to larval Rana dalmatina: acute and chronic effects on survival, development, growth and gill apparatus.

Chlorpyrifos [O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl)phosphorothioate] is a widely used non-systemic organophosphorus insecticide frequently detected in surface waters around the world. The goal of this study is to evaluate the acute and chronic effects of this insecticide on Rana dalmatina tadpoles. To assess the sensitivity of this species, the LC50 value (i.e. the concentration at which 50% of tadpoles die) was determined after 96 h. Our results showed that 5.174 mg L(-1) chlorpyrifos caused 50% mortality in tadpoles at Gosner stage 25. Chronic toxicity tests were also conducted to evaluate the sublethal effects of chlorpyrifos; tadpoles were exposed to three ecologically relevant concentrations (0.025, 0.05 and 0.1 mg L(-1)) in static renewal tests from Gosner stage 25 (tadpoles shortly after hatching) until completed metamorphosis (Gosner stage 46). No significant reduction was observed in survival, larval growth (mass), snout-vent length, stage development or number metamorphosed. In contrast, chlorpyrifos exhibited significant chronic toxic effects on larval development, manifested as the appearance of abnormalities, including tail flexure, skeletal and muscle defects in later stages of development in tadpoles exposed to all tested concentrations. We also evaluated the chronic effects of chlorpyrifos on gill morphology and ultrastructure. Tadpoles were sacrificed after 8 days and 30 days of exposure. Observations by both scanning (SEM) and transmission electron microscopy (TEM) showed considerable morphological and ultrastructural changes. The main gill effects recorded were mucous secretion, epithelium detachment and a degeneration phenomenon. Comparing these results with our previous findings, we demonstrate that the first effect of chlorpyrifos on R. dalmatina is gill alteration, thus supporting the role of a morphological approach in toxicological studies.

Ultrastructural and immunohistochemical investigation on the gills of the teleost, Thalassoma pavo L., exposed to cadmium.

An investigation was conducted to determine the effects of the heavy metal, cadmium (Cd), on the gills of the teleost fish, Thalassoma pavo Linnaeus, 1758. The fishes were exposed to several sublethal concentrations of cadmium (10, 40, 60 and 120 µM (mg/L)) for a period of 48, 96 and 192 h. The value of the LC50 after 96 h of cadmium exposure, determined using the System of Finney, was equal to 128.3 µM. The gills of the fishes were examined by light and electron microscopy. Toxic, apoptotic and cadmium effects were analyzed using some neuropeptides, metallothioneins (MT), caspase 3, PCNA and calmodulin, as bioindicators, respectively. The results showed that the alterations in the gills were proportional to the exposure periods and concentrations of the metal, which were found to be both dose and time dependent. The biological responses in the gills of the tested animals are discussed in relation to results obtained by analysis of the biomarkers. These data may be used for the planning of a model to determine biological risk in the marine environment and may be particularly useful to investigate organisms exposed to cadmium.

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.

Endosulfan acute toxicity in Bufo bufo gills: ultrastructural changes and nitric oxide synthase localization.

Endosulfan is an organochlorine pesticide used in agriculture for a wide range of crops. Endosulfan concentrations of up to 0.7 mg/L can be found in ponds and streams near sprayed agricultural fields. We investigated the short-term toxicity of endosulfan in common toad (Bufo bufo) tadpoles after 24, 48, and 96 h of exposure. Acute toxicity was evaluated at nominal concentrations ranging from 0.01 to 0.6 mg/L: concentrations that could be found after the application of pesticide. Our results show that 0.43 mg/L of endosulfan caused 50% mortality (LC(50)). The effects of a sublethal endosulfan concentration (0.2mg/L) on gill apparatus morphology were evaluated by scanning and transmission electron microscopy. Immunohistochemical methods were also applied to detect the expression pattern of the inducible isoform of nitric oxide synthase (iNOS) in the gills using the confocal laser scanner microscope. Exposure to 0.2mg/L of endosulfan caused an apparent increase in mucus production, the occurrence of secretory vesicles and lamellar bodies, a widening of intercellular spaces and additionally there was evidence of an inflammatory response in the gill apparatus. The morphological alterations occurred after 24h and were more pronounced after 48 and 96 h of exposure. Altered morphology and increased mucus secretion indicate impaired gas exchange and osmoregulation in the gills. In addition, there was an increase of iNOS expression after 24 and 48 h which may reflect hypoxia and inflammation in the gill epithelium. Our results clearly indicate that short-term exposure to a sublethal concentration of endosulfan, near the high end of the environmental range, disrupts gill morphology and function in B. bufo tadpoles.