Exposure to microcrystallized cellulose affects the health of tadpoles and sheds light on the threat these materials pose to amphibians.

The increasing use of cellulose-based materials (CBMs) has provided beneficial applications in different sectors. However, its release into environments may represent an ecological risk, therefore demanding that ecotoxicological studies be conducted to understand the risks (current and future) of CBM pollution. Thus, we evaluated the possible effects of microcrystalline cellulose (CMs) in Physalaemus cuvieri tadpoles. After seven days of exposure to CMs (at 58.29 and 100 mg/L), the animals were subjected to behavioral evaluation, and different biomarkers (biometric and biochemical) were evaluated. Although our data do not point to a neurotoxic effect of CMs (inferred by the absence of behavioral changes and changes in AChE and BChE activity), animals exposed to CMs showed differences in body condition. Furthermore, we noticed an increase in the frequency of erythrocyte nuclear abnormalities and DNA damage, which were correlated with the ingestion of CMs. We noticed that the antioxidant activity of tadpoles exposed to CMs (inferred by SOD, CAT, and DPPH radical scavenging activity) was insufficient to control the increase in ROS and MDA production. Furthermore, exposure to CMs induced a predominant Th2-specific immune response, marked by suppressed IFN-γ and increased IL-10 levels, with a consequent reduction in NO levels. Principal component analysis and IBRv-2 indicate, in general, a primarily more toxic response to animals exposed to the highest CM concentration. Therefore, our study evidence that CMs affect the health of P. cuvieri tadpoles and sheds light on the threat these materials pose to amphibians.

Mutagenicity, hepatotoxicity, and neurotoxicity of glyphosate and fipronil commercial formulations in Amazon turtles neonates (Podocnemis expansa).

Pesticides are considered one of the main causes of the population decline of reptiles worldwide, with freshwater turtles being particularly susceptible to aquatic contamination. In this context, we investigated the potential mutagenic, hepatotoxic, and neurotoxic effects in neonates of Podocnemis expansa exposed to substrate contaminated with different concentrations of glyphosate and/or fipronil during embryonic development. Eggs collected from the natural environment were artificially incubated in sand moistened with pure water, water added with glyphosate Atar 48® at concentrations of 65 and 6500 µg/L (groups G1 and G2, respectively), water added with fipronil Regent® 800WG at 4 and 400 µg/L (groups F1 and F2, respectively) and, water added with the combination of 65 µg/L glyphosate and 4 µg/L fipronil or with 6500 µg/L glyphosate and 400 µg/L fipronil (groups GF1 and GF2, respectively). For mutagenicity analysis, we evaluated the frequency of micronuclei (MN) and other erythrocyte nuclear abnormalities (ENAs), while for evaluation of hepatotoxicity and neurotoxicity, livers and encephalon were analyzed for histopathological alterations. Exposure to pesticides, alone or in combination, increased the frequency of erythrocyte nuclear abnormalities, particularly blebbed nuclei, moved nuclei, and notched nuclei. Individuals exposed to fipronil exhibited congestion and inflammatory infiltrate in their liver tissue, while, in the encephalon, congestion, and necrosis were present. Our study confirms that the incubation of eggs in substrate polluted with glyphosate and fipronil causes histopathological damage and mutagenic alteration in P. expansa, highlighting the importance of using different biomarkers to evaluate the ecotoxicological effects of these pesticides, especially in oviparous animals.

Eggshell composition of Amazon turtle (Podocnemis expansa) is altered after incubation in substrates containing glyphosate and fipronil formulations.

Besides its crucial role during embryo development, eggshells are an essential bioindicator of environmental contaminants. However, the effects of contaminant exposure during incubation on the eggshell composition remain poorly known for freshwater turtles. Accordingly, we tested the effects of incubating the eggs of Podocnemis expansa in substrates containing glyphosate and fipronil formulations on the eggshell's level of mineral and dry matter, crude protein, nitrogen, and ethereal extract. Eggs were incubated in sand moistened with water contaminated with glyphosate Atar 48 at concentrations of 65 or 6500 µg/L, fipronil Regent 800 WG at concentrations of 4 or 400 µg/L, or the combination of 65 µg/L glyphosate and 4 µg/L fipronil and 6500 µg/L glyphosate with 400 µg/L fipronil. Exposure to the tested pesticides, alone or in association, altered the chemical composition of the eggshell of P. expansa, reducing the eggshell's moisture and crude protein content and increasing levels of ethereal extract. These changes may cause significant deficiencies in the mobilization of water and nutrients to the embryo, affecting the development and reproductive success of P. expansa.

The exposure in ovo of embryos belonging to Amazonian turtle species Podocnemis expansa (Testudines) to commercial glyphosate and fipronil formulations impairs their growth and changes their skeletal development.

Pesticides are widely used in agricultural production; moreover, they can have direct and indirect effect on both flora and fauna. Aquatic organisms, among other animals, including reptiles, are mainly susceptible to contamination effects. Accordingly, the aim of the present study is to test the hypothesis that the incubation of Podocnemis expansa eggs in substrate added with glyphosate and fipronil formulations changes their viability, interferes with their growth and induces bone alterations. Eggs collected in natural environment were artificially incubated in sand moistened with water added with glyphosate Atar 48, at concentrations of 65 or 6500 µg/L (groups G1 and G2, respectively), and with fipronil Regent 800 WG at 4 or 400 µg/L (groups F1 and F2, respectively) or, yet, with the combination of 65 µg/L glyphosate and 4 µg/L fipronil, or with 6500 µg/L glyphosate and 400 µg/L fipronil (groups GF1 and GF2, respectively). The level of exposure to the herein assessed pesticides was quantified at the end of the incubation period; it was done by dosing its concentration in eggshells. Eggs exposed to the tested pesticides did not have their viability affected by it; however, all embryos exposed to the tested pesticides showed lowered body mass at hatch, as well as impaired development. In addition, bone malformation in the scleral ossicular ring was observed in individuals in groups F1, F2 and GF1. Pesticides accumulated in eggshells at concentrations related to exposure level. Thus, the recorded results have evidenced some remarkably relevant, and previously unknown, impacts associated with the exposure of a species listed as lower risk/conservation dependent, which spends most of its life in the water, to two widely used pesticides, at a very sensitive stage of its life, namely: egg incubation on land.

Effects of egg exposure to atrazine and/or glyphosate on bone development in Podocnemis unifilis (Testudines, Podocnemididae).

This study was designed to investigate skeletal changes in Podocnemis unifilis embryos derived from artificially incubated eggs exposed to different concentrations of atrazine, glyphosate or atrazine and glyphosate mixture. Forty-two eggs were randomly allocated to one of seven trays containing vermiculite treated distilled water (control group) or the following solutions: 2 or 200 µg L-1 of atrazine (groups A1 and A2 respectively); 65 or 6500 µg L-1 of glyphosate (groups G1 and G2 respectively); 2 µg L-1 and 65 µg L-1 or 200 µg L-1 and 6500 µg L-1 of atrazine and glyphosate mixture (groups AG1 and AG2 respectively). Three eggs per tray were randomly collected on days 30 and 50 of the incubation period. Embryos were submitted to soft tissue diaphanization and stained with Alizarin red S or Alcian blue for morphological analysis of bone and cartilage tissues; histological analysis was performed to confirm ossification changes. Findings were compared between groups. Morphological changes were limited to sclerotic ring features and number of ribs. Malformations rates differed significantly (p < 0.05) between embryos in the control and treated groups A2, AG1 and AG2. Concurrent exposure to atrazine and glyphosate did not affect the presence or severity of embryonic malformations and was not associated with appendicular skeleton changes in P. unifilis embryos. However, further studies focusing on the axial skeleton with particular emphasis on rib abnormalities are warranted.

Chemical characteristics and thickness of Podocnemis expansa post-hatching eggshells (Testudines, Podocnemididae) / Aspectos químicos e espessura da casca de ovos de Podocnemis expansa pós-eclosão (Testudines, Podocnemididae)

Knowledge on chemical components of the post -hatching eggshell of reptiles may provide indicators of the quality of the diet offered to females kept in captivity. Therefore, the objective of this study was to investigate the chemical characteristics of the calcareous layer, as well as the thickness of Podocnemis expansa post-hatching eggshells. Eggshell thickness was 183±1.405 µm. This value is similar to that of the eggs of other Testudines with flexible eggshells. As for the chemical composition, the following percentages were observed: nitrogen 7.983 ± 0.054; crude protein 49.91 ± 0.324; crude fat 0.068 ± 0.002; mineral matter 20.302 ± 0.807; calcium 13.374 ± 0.647; and phosphorus 0.176 ± 0.003. Knowledge on chemical composition of the eggshell may aid the nutrition of P. expansa raised in commercial facilities, once this species is an alternative and promising source of exotic meat.

O conhecimento sobre os constituintes químicos da casca de ovos pós-eclosão de répteis pode ser um referencial de qualidade da dieta fornecida para as fêmeas que vivem em cativeiro. Assim, este trabalho visa investigar os aspectos químicos que compõem a camada calcária, bem como a espessura da casca do ovo de Podocnemis expansa no período pós-eclosão. A espessura média da casca dos ovos foi de 183 ± 1,405 µm. Esta espessura é condizente com ovos de outros Testudines com casca flexível. Para os constituintes químicos, foram encontrados os seguintes valores médios percentuais: nitrogênio 7,983 ± 0,054, proteína bruta 49,91 ± 0,324, gordura bruta 0,068 ± 0,002, matéria mineral 20,302 ± 0,807, cálcio 13,374 ± 0,647 e fósforo 0,176 ± 0,003. Este conhecimento sobre a composição química da casca poderá auxiliar na criação em escala comercial dessa espécie, uma vez que a P. expansa tem se apresentado como fonte alternativa e promissora de carne exótica.

Effects of the exposure to atrazine on bone development of Podocnemis expansa (Testudines, Podocnemididae).

The use of pesticides is a widely spread practice in Brazilian agriculture, and dispersion of these substances is an important factor for the fauna and flora. Atrazine is an endocrine disruptor in the xenoestrogen class that is used worldwide in agricultural practices. In Brazil, its use is permitted in several crops. Podocnemis expansa is a representative of the Testudines order that is the largest freshwater reptile of South America. Its distribution enables it to get in contact with molecules that are commonly used as pesticides, which may cause deleterious effects in target populations. In order to evaluate the possible effects of the exposure to atrazine on bone ontogeny of this species, eggs were artificially incubated in sand moistened with water contaminated with atrazine at concentrations equal to 0, 2, 20 or 200 µg/L. Embryos were collected throughout incubation and submitted to diaphanization of soft tissues with potassium hydroxide (KOH); bones were stained with Alizarin red S and cartilages by Alcian blue. Embryos were evaluated for the presence of abnormalities during the different stages of pre-natal development of skeletal elements. No effect of atrazine was observed on bone development during the embryonic phase in P. expansa individuals, in the conditions of this study.