Sub-lethal effects induced by a mixture of different pharmaceutical drugs in predicted environmentally relevant concentrations on Lithobates catesbeianus (Shaw, 1802) (Anura, ranidae) tadpoles.

The increasing consumption of medications by humans has negative effects such as the increased disposal of these compounds in the environment. Little is known about how the disposal of a "drug mix" (DM) in aquatic ecosystems can affect their biota. Thus, we evaluated whether the exposure of Lithobates casteibeianus tadpoles to a DM composed of different medication classes (antibiotic, anti-inflammatory, antidepressant, anxiolytic, analgesic, and antacid drugs)-at environmentally relevant concentrations-may change their oral morphology, trigger behavioral disorders, and have mutagenic effects on erythrocyte cells. Based on our data, animals exposed to the DM showed changes in mandibular sheath pigmentation, dentition, and swimming activity, as well as atypical behavior in the social aggregation test [with co-specific and interspecific (Physalaemus cuvieri) individuals] and antipredatory defensive response deficit (chemical stimulus from Odonata larvae), after 15 exposure days. The mutagenic analysis revealed higher frequency of nuclear abnormalities in the erythrocytes of tadpoles exposed to the DM (e.g., multilobulated, blebbed, kidney-shaped, notched nucleus, binuclear, and micronucleated erythrocytes). Given the chemical complexity of the DM, we assumed that several organic functions may have been affected, either by the isolated, synergistic, antagonistic, or additive action of DM compounds. Finally, our study confirms the toxicological potential of DM in L. catesbeianus tadpoles, with emphasis to impacts that can affect the fitness of individuals and their natural populations. Thus, we suggest that more attention should be given to the disposal of medications in the environment and reinforce the need of improving water and sewage treatment systems.

The potential reproductive toxicity of tannery effluent to the estrous cycle and ovarian follicular dynamics of female Swiss mice.

Although the toxic effects of tannery effluent (TE) on tanning-industry workers have been reported in many studies, its effects on females' reproductive system are unknown. We aimed at evaluating the effects of direct contact with TE on the "emotional" status, estrous cycle (during 15 consecutive exposure days), and ovarian follicular dynamics of female Swiss mice at the end of the experiment to broaden the knowledge about the toxicity of this pollutant. The herein adopted exposure protocol simulated tanning-industry workers' exposure to TE. The test animals were subjected to 45 exposure days, for 1 h a day, 5 days a week (from Monday to Friday). Based on the collected data, female mice exposed to TE recorded high anxiety index in the elevated plus maze test, although we did not observe changes in their estrous cycle. The smaller total and specific number of ovarian follicles (types 1 to 6) and the higher frequency of degenerating follicles (atresic) in female mice exposed to TE marked the folliculogenesis reduction in them. Therefore, our study was the first to provide evidences that the exposure to TE can cause reproduction issues in female mice, as well as the first experimental insight about the impact of unhealthy work activities in tanning industries on women's reproductive system.

Mice exposure to haloxyfop-p-methyl ester at predicted environmentally relevant concentrations leads to anti-predatory response deficit.

Although the efficiency of haloxyfop-p-methyl ester (HPME) as selective herbicide is acknowledged, its impact on non-target organisms is poorly known. It is not known whether the short exposure of mammals to low HPME concentrations (consistent with a realistic contamination scenario) poses risks to these animals. Thus, the aim of the current study is to evaluate the effects of HPME on the anti-predatory behavior of female Swiss mice exposed to it. The animals were divided in groups: non-exposed (control) and exposed (route: i.p., for 2 days) to different herbicide concentrations (2.7 × 10-4 g/kg and 2.7 × 10-2 g/kg of body weight), which were considered environmentally relevant predicted concentrations. The animals were subjected to the open field and elevated plus-maze tests; results showed that the HPME did not lead to anxiolytic or anxiety behavior, or to locomotive changes in the tested animals, fact that was confirmed through the Basso Mouse Scale for locomotion scores. On the other hand, animals exposed to the herbicide were incapable of recognizing the snake as potential predator. Animals in the control group, exposed to a real snake (Pantherophis guttatus) remained longer in the safety zone of the test device, presented lower frequency of self-grooming behaviors for a shorter period-of-time, besides showing longer freezing time, which was not observed in animals exposed to HPME. Therefore, our study indicates the ecotoxicological potential of the herbicide, since anti-predatory behavior disorders may affect preys' responses and population dynamics.

Behavioral response and dynamics of Eisenia fetida hemocytes exposed to environmentally relevant concentration of sulfentrazone.

Although the toxicity of the pesticide sulfentrazone in some aquatic organisms is known, its effects on edaphic organisms such as earthworms remain completely unknown. Thus, we aimed at evaluating the behavior and immune response of Eisenia fetida exposed to sulfentrazone at environmentally relevant concentrations (EC). E. fetida representatives exposed to this contaminant (for 48 h) were divided in the following groups: environmental concentration (EC1x: 318 ng sulfentrazone/g of dry weight soil) and EC100x (concentration 100 times higher than in EC1x). Based on the avoidance test results, earthworms responded to this pesticide and proved the toxicity of sulfentrazone. The observed immune response induction was expressed by increased granulocytes presenting phagocytic vacuoles and agglomerations/encapsulations, mainly in animals belonging to groups EC1x and EC100x. However, the reduced frequency of plasmocytes in these animals' hemolymphs suggested that the phagocytic immune response was not efficient to assure 100% survival. Our study is the first to report sulfentrazone toxicity in an edaphic organism, at environmental concentration.

Mutagenic assessment of Lithobates catesbeianus tadpoles exposed to the 2,4-D herbicide in a simulated realistic scenario.

The aim of the current study is to assess possible erythrocyte mutagenic effects on Lithobates catesbeianus tadpoles exposed to water contaminated with 2,4-D. In order to do so, tadpoles were exposed to a predictive and environmentally relevant herbicide concentration (1.97 mg/L), which is likely to be found in lentic environments formed by superficial water runoffs in pasture areas where the herbicide was applied. The micronucleus test, as well as tests for other nuclear abnormalities, was conducted after 3, 5, and 9 days of exposure (d.e.). Changes in the biomass and mouth-cloaca length or interference in the larval development of the animals (in the three evaluated times) were not recorded. However, tadpoles exposed to 2,4-D showed the highest total number of nuclear abnormalities, as well as the highest frequency of binucleated erythrocytes and kidney-shaped nuclei (shortly after 3 d.e.). The micronucleus frequency was also higher in animals exposed to 2,4-D (in the 3rd, 5th, and 9th d.e.), as well as the frequency of binucleated cells (3rd, 5th, and 9th d.e.) presenting notched (9th d.e.) and blebbled (9th d.e.) nuclei in comparison to those of the control, after 5 and 9 days of exposure. Therefore, the current study is a pioneer in showing that 2,4-D has a mutagenic effect on L. catesbeianus tadpoles, even at low concentrations (environmentally relevant) and for a short period of time, a fact that may lead to direct losses in anuran populations living in areas adjacent to those subjected to 2,4-D herbicide application.

Correction to: Histological liver chances in Swiss mice caused by tannery effluent.

There is a problem in the original publication of this paper (Figure 2 Graphs are in Portuguese). Shown in this paper is the correct version.

The intake of water containing a mix of pollutants at environmentally relevant concentrations leads to defensive response deficit in male C57Bl/6J mice.

Previous studies have individually confirmed the toxic effects from different pollutants on mammals. However, effects resulting from the exposure of these animals to multi-pollutant mixes have not been studied so far. Thus, the aim of the current study is to assess the effect from the chronic exposure (105days) of C57Bl/6J mice to a mix of pollutants on their response to potential predators. In order to do so, the following groups were formed: "control", "Mix 1× [compounds from 15 pollutants identified in surface waters at environmentally relevant concentration (ERC)]", "Mix 10×" and "Mix 25×" (concentrations 10 and 25 times higher than the ERC). From the 100th experimental day on, the animals were subjected to tests in order to investigate whether they showed locomotor, visual, olfactory and auditory changes, since these abilities are essential to their anti-predatory behavior. Next, the animals' behavior towards potential predators (Felis catus and Pantherophis guttatus) was assessed. The herein collected data did not show defensive response from any of the experimental groups to the predatory stimulus provided by P. guttatus. However, the control animals, only, presented anti-predatory behavior when F. catus was introduced in the apparatus, fact that suggests defensive response deficit resulting from the treatments. Thus, the current study is pioneer in showing that the chronic intake of water containing a mix of pollutants (even at low concentrations) leads to behavioral disorders able to affect the survival and population dynamics of mammalian species at ecological level.

Behavioral and mutagenic biomarkers in tadpoles exposed to different abamectin concentrations.

It is known that pesticides such as abamectin (ABA) present cytotoxic effects on target organisms; however, the effects from ABA on non-target organisms such as amphibians are poorly understood. The aim of the current study is to investigate whether the exposure of Lithobates catesbeianus tadpoles to different abamectin concentrations [12.5, 25, and 50% of the median lethal concentration (LC50)] leads to behavioral and morphological changes and/or generates possible cytotoxic effects. The aggregation test showed that tadpoles exposed to the highest ABA concentrations did not respond to the stimulus from non-familial and unrelated co-specific species. On the other hand, there was no difference in the total number of crossings in the central line of the herein adopted apparatus between groups; it suggests that ABA did not affect animal locomotion in the aforementioned test, although changes in the normal swimming pattern of tadpoles exposed to the pesticide were recorded in the swimming activity test. In addition, the herein exposed animals did not respond to the predatory stimulus in the antipredator response test; this result suggests defensive response deficit caused by the pesticide. With respect to their oral morphology, tadpoles exposed to ABA presented the lowest scores for mandibular pigmentation and structures, as well as for dentition condition. Finally, it was possible seeing that the exposure to ABA, even at the lowest concentration (12.5% of the LC50), resulted in nuclear changes in the erythrocytes of the animals; these changes became evident in the increased number of micronuclei and in other nuclear abnormalities. Thus, besides confirming the cytotoxic potential of ABA in amphibians, the current study corroborates the hypothesis that the exposure to the herein investigated pesticide leads to behavioral and morphological changes in tadpoles, fact that may negatively reflect on the survival, as well as on natural populations of these individuals.

Analysis of various effects of abamectin on erythrocyte morphology in Japanese quails (Coturnix japonica).

Although previous studies have already confirmed the toxicological potential of abamectin (ABA) in different experimental models (from invertebrates to vertebrates), almost nothing is known about the impacts the exposure to this pesticide can cause on birds. Thus, the aim of our study is to investigate the cytotoxic effects on the erythrocytes of female Japanese quails (Coturnix japonica) exposed to low abamectin concentrations. In order to do so, three experimental groups were proposed: "control," composed of quails exposed to abamectin-free drinking water; "ABA 1% median lethal dose (LD50)," comprising birds exposed to water containing 15.5 mg a.i./L of abamectin (via commercial formulation Kraft® 36EC), and "ABA 10% LD50," composed by birds exposed to water containing 155.0 mg a.i./L of abamectin. The micronucleus test and the tests applied to other nuclear abnormalities in the peripheral blood of birds were conducted 40 days after exposure. Our study revealed significant physical abnormalities in nuclear shapes (erythrocytes with asymmetric constriction nuclei, notched nuclei, indented and moved nucleus) of those birds exposed to higher abamectin levels. When all nuclear abnormalities were tallied, a significant dose-dependent trend was noted. Therefore, our study presents initial imprints on determination of abamectin-mediated cellular toxicity in avifauna which can be instrumental in checking polluted ecosystems.

Histological liver chances in Swiss mice caused by tannery effluent.

Although tannery effluents are known for being highly toxic to organisms, reports about the effects of the intake of these xenobiotics on experimental mammal models are recent. Studies about the damages the chronic intake of these effluents can cause in the liver of outbred mice remain an unexplored field. Thus, the aim of the present study is to assess (histological) the hepatic condition of Swiss mice (outbred strain) chronically exposed to the intake of different raw tannery effluent concentrations diluted in water for 150 days. Accordingly, the mice (males and females) were divided in the following groups: control group-animals treated with drinking water, only; and groups 5 and 10%-treated with raw tannery effluent diluted in water. After exposure, the animals were subjected to euthanasia for liver fragment sample collection and histological analysis, respectively. Moderate hydropic degeneration was observed in the centrilobular regions of the liver of mice exposed to 5 and 10% tannery effluent, as well as greater amounts of hepatocytes presenting karyomegaly and necrotic hepatocytes, and a smaller amount of Kuffer cells in the liver of mice exposed to the xenobiotic. Finally, animals exposed to 10% tannery effluent showed mild hyperplasia of the bile ducts in the portal areas and fibroblast proliferation around the bile ducts, thus suggesting a fibrous process. Except for the frequency of hepatocytes presenting karyomegaly (lower in females), the herein observed hepatic changes were similar in male and female Swiss mice. Accordingly, the present data support the hypothesis that the chronic intake of tannery effluent by outbred mice (Swiss) causes damages in the liver, a fact that broadens the knowledge about the toxic potential of this pollutant, which goes beyond that of C57Bl/6J male mice (inbred strain).

Impacts of tannery effluent on development and morphological characters in a neotropical tadpole.

Previous studies have shown the contamination of rivers and streams caused by tannery effluents (TE). However, the effects of TE on amphibians are still unknown. Thus, we evaluated whether the TE, even at low concentrations (0.2%), could cause asymmetric development (inferred through the fluctuating asymmetry indices (FA)) and changes in morphological characteristics of Physalaemus cuvieri larvae. Accordingly, the tadpoles were distributed in groups non-exposed (n=160) and exposed (n=160) to this TE, for 15days. The TE used presented a complex chemical composition (especially high concentrations of Cr, Cd and Ni and a diverse array of toxic organic compounds). We used three individual FA indices: FA1:=|R-L|, ii) FA2=|R-L|÷[(R+L)÷2] and FA3=(R-L)2, where "R" are "L" are the right and left sides, respectively. The larvae exposed to the TE had higher FA indexes (FA1, FA2 and FA3) for nostril-snout distance, relative to the control group larvae. Regarding eye-snout distance, asymmetry values were higher for the FA1 and FA2 indices (with no difference for the FA3 index), which were similar to those observed for the eye-width character. For eye length, FA2 was higher in animals of the effluent group. Tadpoles exposed to TE had significantly lower body masses than those in the control group, when controlling for body length and larval development stage. Furthermore, tadpoles in the effluent group had smaller body size and lower interocular distance. The frequency of tadpoles that showed any alteration in tail morphology as well as tail attachment position (dorsal and ventral) was significantly higher in the effluent group than in animals not exposed to the TE. Finally, our data show that exposure to TE delayed larval development of tadpoles. Thus, our pioneering study confirms the hypothesis that exposure of P. cuvieri to TE, at low concentrations, impacts development and morphological characters.

Inbred mice strain shows neurobehavioral changes when exposed to tannery effluent.

The bovine leather processing (tanning industries) stands as a generating activity of potentially toxic waste. The emission of untreated effluents into the environment may cause serious harm to human and environmental health. Nevertheless, few studies have investigated the possible effects of intake of these effluents in experimental mammalian models. Thus, this study aimed to evaluate the neurobehavioral effects of chronic intake of different tannery effluent concentrations diluted with water (0.1, 1, and 5%) in male C57BL/6J mice. After 120 days of exposure, the animals were subjected to different behavioral tests, predictive of anxiety (elevated plus maze (EPM), open-field (OF), and neophobia test), depression (forced swim), and memory deficits (object recognition test). From the EPM test, it was observed that the mice exposed to 0.1, 1, and 5% of tannery effluents showed higher anxiety scores compared to the animals in the control group. However, the results of this study revealed no differences among the experimental groups in the proportion (percentage) of locomotion in the central quarters/total locomotion calculated (by OF), considered an indirect measure for anxiety. At neophobia test, all the animals exposed to chronic intake of tannery effluents showed higher latency time to start eating, which corresponds to an anxiogenic behavior. Regarding the forced swim test, it was observed that the animals exposed to tannery effluents had longer time in immobility behavior, suggesting a predictive behavior to depression. Finally, the object recognition test showed that the treatments did not cause damage to the animals' memory. The recognition rate of the new object did not differ among the experimental groups. Thus, it is concluded that male C57BL/6J mice (inbred strain) exposed to tannery effluents have predictive neurobehavioral changes of anxiety and depression, without memory deficit.

Memory deficit in Swiss mice exposed to tannery effluent.

Although it is known that tannery effluents constitute highly toxic pollutants whose effects in humans represent public health problems in several countries, studies involving experimental mammalian models are rare. In this context, the objective of the present study was to assess the effect of the exposure to tannery effluent on the memory of male and female Swiss mice. Animals of each sex were distributed into two experimental groups: the control group, in which the animals received only drinking water and the effluent group, in which the mice received 1% of gross tannery effluent diluted in water. The animals were exposed to the effluent by gavage, oral dosing, for 15days, ensuring the administration of 0.1mL of liquid (water or effluent)/10g of body weight/day. On the 14th and 15th experimental days the animals were submitted to the object recognition test. It was observed that the new object recognition indices calculated for the animals exposed to the effluent (males and females) were significantly lower than those obtained with the control group. The exposure to tannery effluent caused memory deficit in Swiss mice in a similar way for both sexes, reinforcing previous findings that these pollutants affect the central nervous system. It contributes to the knowledge in the area by attesting harmful effects to the cognition of such animals.

Predictive behaviors for anxiety and depression in female Wistar rats subjected to cafeteria diet and stress.

Obesity and chronic stress have been considered important public health problems that affect millions of people worldwide. Our aim was to analyze the effect of obesity associated with chronic stress on neurobehavioral parameters in female rats, considering that the association of these syndromes can enhance the negative effects on homeostasis. The animals were distributed into standard diet (Std), standard diet+stress (Std+stress), cafeteria diet (Cafe), and cafeteria diet+stress (Cafe+stress) groups. The animals of groups Std and Std+stress were fed with rodent standard feed. Groups Cafe and Cafe+stress, additionally to the standard feed, were offered palatable and calorie-rich processed food and cola-type soft drink ad libitum. From the eighth experimental week, groups Std+stress and Cafe+stress were subjected to restraint chronic stress model (50 days). After the stress protocol, predictive anxiety (open-field and elevated plus-maze tests) and depression (forced swim) were applied. The cafeteria diet was effective in inducing obesity. The ratio locomotion in the central quadrants/total locomotion evaluated during the open field test was not indicative of anxiogenic or anxiolytic effect in the animal's behavior. However, the elevated plus maze test showed that obese and stressed animals were prone to higher anxiety levels. In addition, the obese and stressed animals display less climbing behavior than all the other groups, which can be considered an indicator of depression-like behavior. Nevertheless, it is suggested that the mechanisms involved in effects of obesity associated with chronic stress be better investigated in female rats, considering the organic complexity related to these modern illnesses.