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.

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.

The ultrastructure of the book lungs of the Italian trap-door spider Cteniza sp. (Araneae, Mygalomorphae, Ctenizidae).

The fine structure of book lungs is not homogeneous across Arachnids and is considered phylogenetically informative, however few reports on the ultrastructural features of this organ have been published. In this study, we examined the general morphology and ultrastructure of adult spiders of the genus Cteniza. The respiratory system of Cteniza sp. consists of two pairs of well-developed book lungs, which is considered indicative of primitive spiders. The general organization of the book lungs is similar to that described for other arachnids and consists of leaves of alternating air and hemolymph channels. The air channels are lined with cuticle and open to an atrium that leads to a slit-like spiracle. The air channels are held open by cuticular trabeculae. The space holders in the hemolymph channels are pillar trabeculae formed by two cells from the opposed walls. The pillar cells have a complex ultrastructure that includes an interdigitating connection, gap junctions, microtubules and hemidesmosomes. These features apparently help strengthen the pillar cells and their interconnections with each other and the underlying cuticle. The cytoskeleton resembles that of arthropod tendon cells where substantial structural support is needed.

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.