Solid-State Fermentation as Green Technology to Improve the Use of Plant Feedstuffs as Ingredients in Diets for European Sea Bass (Dicentrarchus labrax) Juveniles.

This study aimed to evaluate the utilization by juvenile European sea bass of a SSFed PF mixture with Aspergillus niger CECT 2088. A 22-day digestibility and a 50-day growth trial were performed testing four diets, including 20 or 40% of an unfermented or SSFed PF mixture (rapeseed, soybean, rice bran, and sunflower seed meals, 25% each). SSF of the PF added cellulase and ß-glucosidase activity to the diets. Mycotoxin contamination was not detected in any of the experimental diets except for residual levels of zearalenone and deoxynivalenol (100 and 600 times lower than that established by the European Commission Recommendation-2006/576/EC). In diets including 20% PF, SSF did not affect growth but increased apparent digestibility coefficients of protein and energy, feed efficiency, and protein efficiency ratio. On the contrary, in diets including 40% PF, SSF decreased growth performance, feed intake, feed and protein efficiency, and diet digestibility. SSF decreased the intestinal amylase activity in the 40% SSFed diet, while total alkaline proteases decreased in the 20% and 40% SSFed diets. Hepatic amino acid catabolic enzyme activity was not modulated by SSF, and plasma total protein, cholesterol, and triglyceride levels were similar among dietary treatments. In conclusion, dietary inclusion of moderate levels of the SSFed PF, up to 20%, improves the overall feed utilization efficiency without negatively impacting European sea bass growth performance. The replacement of PF with the SSFed PF mixture may contribute to reducing the environmental footprint of aquaculture production.

Solid-State Fermentation of Plant Feedstuff Mixture Affected the Physiological Responses of European Seabass (Dicentrarchus labrax) Reared at Different Temperatures and Subjected to Salinity Oscillation.

This study aimed to evaluate the effects of dietary inclusion of plant feedstuff mixture (PFM) pre-treated by solid-state fermentation (SSF) on the physiological responses of European seabass. For that purpose, two diets were formulated to contain: 20% inclusion level of non-fermented plant ingredients mixture (20Mix) and 20Mix fermented by A. niger in SSF conditions (20Mix-SSF). Seabass juveniles (initial body weight: 20.9 ± 3.3 g) were fed the experimental diets, reared at two different temperatures (21 and 26 °C) and subjected to weekly salinity oscillations for six weeks. Growth performance, digestive enzyme activities, humoral immune parameters, and oxidative stress indicators were evaluated. A reduction in weight gain, feed intake, and thermal growth coefficient was observed in fish fed the fermented diet (20Mix-SSF). Salinity oscillation led to an increase in weight gain, feed efficiency, daily growth index, and thermal growth coefficient, regardless of dietary treatment. Higher rearing temperatures also increased daily growth index. No dietary effect was observed on digestive enzymes activities, whereas rearing temperature and salinity oscillation modulated digestive enzyme activities. Oxidative stress responses were significantly affected by experimental diets, temperature, and salinity conditions. Catalase and glutathione peroxidase activities showed an interactive effect. Fish reared at 21 °C showed higher enzymatic activity when fed the 20Mix-SSF. Conversely, fish reared at 26 °C showed higher GPx activity when fed the 20Mix diet. Fish reared at 26 °C showed reduced peroxidase and lysozyme activities, while salinity fluctuation led to increased lysozyme activity and decreased ACH50 activity. ACH50 activity increased in fish fed the 20Mix-SSF. Overall, the dietary inclusion of PFM fermented by A. niger was unable to mitigate the impact of environmental stress on physiological performance in European seabass. In fact, fermented feed caused an inhibition of growth performances and an alteration of some physiological stress indicators.

Assessment of multiple biomarkers in Lithobates catesbeianus (Anura: Ranidae) tadpoles exposed to zinc oxide nanoparticles and zinc chloride: integrating morphological and behavioral approaches to ecotoxicology.

The ecotoxicological risk to vertebrates posed by zinc oxide nanoparticles (ZnO NPs) is still poorly understood, especially in animals with a biphasic life cycle, which have aquatic and terrestrial phases, such as amphibians. In the present study, we investigated whether acute exposure (7 days) to ZnO NPs and zinc chloride (ZnCl2) at three environmentally relevant concentrations (0.1, 1.0, and 10 mg L-1) induces changes in the morphology, chondrocranium, and behavior of the tadpoles of Lithobates catesbeianus (Anura: Ranidae). Tadpoles exposed to both forms of Zn did not undergo any morphological or behavioral changes at the lowest concentrations (0.1 and 1.0 mg L-1). However, the animals exposed to the highest concentration (10 mg L-1) lacked oral disc structures, were smaller in size, had a longer tail, and presented changes in the position and coiling of the intestine and malformations of the chondrocranium in comparison with the control group. This indicates that ZnO NPs and ZnCl2 altered the development of the tadpoles, causing delays in their metamorphosis and even reducing individual fitness. The tadpoles exposed to both forms of Zn at 10 mg L-1 also had reduced mobility, especially in the presence of conspecifics. Based on these findings, we emphasize the importance of studying morphological, skeletal, and behavioral biomarkers to evaluate the toxic effects of metal-based nanoparticles in amphibians.

Environmental heterogeneity drives the distribution of copepods (Crustacea: Copepoda) in the Amazon, Araguaia, Pantanal, and Upper Paraná floodplains.

Environmental heterogeneity influences the physiology and behavior of organisms, leading to alterations in populations, communities, and ecosystems. This study was designed to evaluate the effect of the spatial and temporal limnological heterogeneity on the distribution of copepods in the Amazon River floodplain, Araguaia River floodplain, Pantanal floodplain, and Upper Paraná River floodplain. We performed dispersion homogeneity tests (PERMIDISP) to analyze the limnological heterogeneity, a Generalized Linear Mixed Model (GLMM) to test the relationship between limnological heterogeneity and species distribution, and a Canonical Correspondence Analysis (CCA) to analyze which limnological variables explain the variation in the community composition. In our study, we observed higher environmental heterogeneity during dry periods, especially in the Amazon floodplain. The Amazon showed the highest species richness (the total number of species that occurred in each floodplain), with endemic species, also the highest abundance of individuals, especially during dry periods, which was possibly due to the biogeography, the size of the basin, and the heterogeneity of the environments. The Pantanal presented the second highest species richness and showed the highest species richness and abundance of individuals during rainy periods. The distribution of copepods was highly correlated with environmental heterogeneity. The species were mainly related to temperature, nutrients, and depth.

Titanium dioxide nanoparticles as a risk factor for the health of Neotropical tadpoles: a case study of Dendropsophus minutus (Anura: Hylidae).

The production and use of titanium dioxide (TiO2) nanoparticles are increasing worldwide. The release of this substance into the environment can induce toxic effects in aquatic invertebrates and vertebrates, although the exact nature of its impacts on Neotropical amphibians is still poorly understood. In this context, the present study evaluated the toxicity of TiO2 nanoparticles and their counterpart-dissolved titanium dioxide (TiO2)-in the tadpoles of Dendropsophus minutus. The biometric parameters, DNA damage, and behavioral changes were verified in tadpoles exposed to three different concentrations (0.1 mg·L-1, 1.0 mg·L-1, and 10 mg·L-1) of TiO2 nanoparticles and dissolved TiO2 for 7 days. We verified significant DNA damage in the D. minutus tadpoles exposed to both forms of Ti, in comparison with the control group. We also identified a reduction in total size, body length, and width, and the height of the musculature of the tail of the tadpoles exposed to all concentrations of both substances in comparison with the control. In the behavioral test, the tadpoles exposed to nanoparticles and dissolved TiO2 presented reduced mobility and a tendency to be less aggregated than normal. Here, the simultaneous use of multiple biomarkers was fundamental for the reliable assessment of the adverse effects of nanomaterials on anuran amphibians and the establishment of a systematic approach to the biomonitoring of aquatic ecosystems. The present study expands our understanding of the genotoxic, morphological, and behavioral effects of TiO2 nanoparticles and dissolved TiO2 on anuran amphibians, and contributes to the establishment of further research for the more systematic assessment of the environmental risk of nanomaterials.

Predation by an omnivorous fish and food availability alter zooplankton functional diversity: a microcosm approach.

Studies considering the functional traits of organisms, populations, and communities functional indices increase the understanding of many factors on ecosystem functioning. Here, we analyze the predation effect (by fish) on zooplankton functional diversity and the effects of biomass and density of periphytic algae on zooplankton feeding type trait and body size. We expect that intense predation by fish on zooplankton leads to higher values of zooplankton functional diversity and that food resource will be positively related to the abundance of zooplankton trait and body size. For that, microcosms were established (T1- fish-absence, and T2- fish-presence, both with periphytic algae as food). We observed that fish presence decreased zooplankton functional diversity through modifications in the availability of nutrients and algae, through the middle-out effect. We also observed that body size had a negative relationship with the food resource, reaffirming that high food availability in subtropical lakes is linked to small-bodied zooplankton. The raptorial copepods covariate positively with the periphytic algae, which was an alternative food resource and, in this case, the main form of carbon input into the system. In this study, omnivorous fish reduced zooplankton functional traits, which can alter the energy stock and energy flow in aquatic ecosystems.

Ecotoxicity of nanomaterials in amphibians: A critical review.

Nanomaterials (NMs) have been used in a growing number of commercial products, and their rapid expansion could lead to their release into the aquatic environments. However, there is limited knowledge about the impact of NMs in the biota, especially the amphibians. The present study revised the historical use of amphibian species as a model system for nanoecotoxicological studies and summarized the data available in the scientific literature about the genotoxic, mutagenic, histopathological, embryotoxic and reproductive effects of NMs in different groups of amphibians. The interaction, bioaccumulation, mode of action (MoA) and ecotoxicity of NMs on amphibians were also revised. The nanoecotoxicological studies were conducted with 11 amphibian species, being eight species of the order Anura and three species of the order Caudata. Xenopus laevis was the most studied species. The studies were conducted mainly with inorganic NMs (72%) compared to organic ones. The nanoecotoxicity depends on NM behavior and transformation in the environment, as well as the developmental stages of amphibians. The known effects of NMs in amphibians were mainly reported with reactive oxygen species (ROS) production, oxidative stress, and genotoxic effects. Results emphasize the need for further studies testing the ecotoxicity of different NMs, concentrations and exposure periods at environmentally relevant approaches. Furthermore, standard protocols for nanoecotoxicological tests using amphibians are required. Revised data showed that amphibians are suitable organisms to assess the environmental impact of NMs and indicated significant research gaps concerning the ecotoxicity of NMs on freshwater ecosystems and recommendations for future researches.

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.

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.

Insights about the toxic effects of tannery effluent on Lithobates catesbeianus tadpoles.

Tannery industries are considered highly polluting due to the large production of polluted wastewater [untreated tannery effluent (UTE)]. Although previous studies have already shown the consequences from fish, birds and mammals' exposure to this pollutant, little is known about its toxicological effect on representatives of class amphibian. Thus, we aim at assessing whether short-term UTE exposure, even at low concentrations (0.2% UTE diluted in water), would be able to induce behavioral, morphological and cyto-genotoxic changes in L. catesbeianus tadpoles. In order to do so, two experimental groups were set (control and tannery effluent) and exposed, or not, to UTE for seven days. A positive control group (cyclophosphamide) was included in the experimental design in order to assess cyto-genotoxicity. Our behavioral results showed that tadpoles exposed to the contaminant presented abnormal responses in the predator-response test; therefore, it evidenced losses in their capacity to recognize chemical olfactory cues of a potential predator. We also searched for changes in mouth length, in dentition, in body length before and after the eyes of animals exposed to UTE. Besides, we observed higher nuclear abnormality frequency in the circulating erythrocytes of tadpoles exposed to the contaminant, as well as in animals belonging to the positive control group. Some of the observed abnormalities were micronuclei, binucleated, notched, kidney-shaped and blebbed cells, multilobulated nuclei, as well as lower mitotic index. Therefore, our data confirm the hypothesis that UTE causes behavioral, morphological and cyto-genotoxic changes in L. catesbeianus tadpoles, fact that opens new perspectives to other investigations about how and which UTE constituents were responsible for the observed effects.

The advertisement and aggressive calls of Rhinella abei (Baldissera, Caramaschi, and Haddad, 2004) (Anura: Bufonidae) from Campo Largo, Paraná, Brazil.

Rhinella abei is a medium-sized species (snout-to-vent length 57.0-76.4 mm in males; 60.4-83.9 mm in females-Baldissera et al. 2004) of the Rhinella crucifer species group, distributed in the Brazilian Atlantic Rainforest, from the State of Paraná to northern Rio Grande do Sul (Frost 2016). It is recorded in forested areas (Conte & Rossa-Feres 2007), or while breeding along forest edges and open habitats. Information on advertisement calls of species in the R. crucifer group is available for R. casconi, R. crucifer, R. inopina and R. ornata (Andrade et al. 2015; Heyer et al. 1990; Oliveira et al. 2014; Roberto et al. 2014). Here we describe the advertisement and aggressive calls of R. abei recorded in the municipality of Campo Largo (25.507472° S, 49.376632° W, datum "WGS84"), southeast State of Paraná, Brazil.