Toxic risk evaluation of effluents from a swine biodigester in the plant models Lactuca sativa and Allium cepa.

Pig farming is recognized as an activity with great polluting potential. The aim was to investigate possible environmental risks of effluents from the stabilization pond (SP) and the raw effluent (RE) from the biodigestion process of swine residues, in different concentrations in the models Lactuca sativa and Allium cepa. Seeds were germinated in different dilutions, 100% (C1), 50% (C2), 25% (C3), 12.5% (C4), 6.25% (C5), 3.12% (C6), 0.78% (C7), and 0.39% (C8). Distilled water was used as the negative control (CN) and trifluralin (0.84 g/L-1) as the positive control. Germination (GR), root growth (RG), cell cycle, and oxidative stress (OS) were analyzed. To assess OS, the activity of the enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) and the quantification of glutathione (GSH) and lipid peroxidation (LPO) were analyzed. Data were submitted to ANOVA (one way), followed by the Kruskal-Wallis mean test (P ≤ 0.05). Chemical analysis showed high values of Cu, Fe, Mn, and Zn. Dilutions (C1, C2, C3 RE) and (C1 and C2 SP) inhibited GR and RG of L. sativa and A. cepa than other concentrations. The mitotic index showed a reduction in C5 (RE), C6, and C7 (SP) of L. sativa and C3 and C4 (SP) of A. cepa in relation to CN and higher frequencies of chromosomal alterations. Regarding the OS, only the concentrations of SP treatment showed statistical difference in relation to the NC: in L. sativa model, GSH at (C5 and C8) concentrations and LPO (C7); in A. cepa model, SOD (C3 and C4), GST (C4, C5 and C6), GSH (C5 and C8), and CAT (C3 and C7). The alterations in metabolism are possibly related to the metals, such as zinc and copper, observed in high amounts in the raw waste. The results allowed us to conclude that the raw and stabilization pond effluents offer environmental risks, requiring caution and monitoring in the use of these effluents.

Toxicogenetic, biochemical, and anatomical effects of the herbicide Clethodim on Allium cepa L.

Pesticides are compounds with several chemical or biological agents developed to potentiate the biocide action. Their use is associated with increased economic and agricultural productivity worldwide but can harm health and the environment, damaging existing biota. Clethodim is a systemic post-emergent herbicide for grasses, highly selective for cotton, coffee, onions, carrots, soybeans, etc. Therefore, this work aimed to evaluate the harmful effect of the herbicide Clethodim with the model plant Allium cepa. A series of tests were conducted to evaluate the effects of the herbicide under study. Germination tests, root growth, cell, and nucleolar cycle analysis, as well as oxidative stress assessment and histological analysis of the roots, were performed. The results indicated that the herbicide demonstrated phytotoxicity, inhibiting germination at C1 (1.92 g/L) and C3 (0.84 g/L), and root growth at all concentrations, presenting mutagenicity at C1 (1.92 g/L) and C4 (0.24 g/L), evidenced by the increased frequency of micronuclei. In addition, changes were observed in the enzymatic activity of the enzymes catalase at concentrations C1 (1.92 g/L) and C2 (0.96 g/L) and ascorbate peroxidase at concentrations C1 (1.92 g/L), C2 (0. 96 g/L), and C3 (0.48 g/L) and in cell elongation at concentrations C1 (1.92 g/L) and C3 (0.48 g/L), demonstrated in histological analyses of the root apex.

Distribution and behavior of lipid droplets in hepatic cells analyzed by variations of citochemical technique and scanning electron microscopy.

Toxicity evaluations involve the analysis of multiple biomarkers. In this study, the liver, target organ analyzed by treatments with iron concentrations, indicated the accumulation of lipids as a response. Considering that the distribution of lipids in an organ is directly related to the induction of inflammatory processes by aquatic contaminants, this study proposes to carry out an integrative investigation of the behavior and the distribution of lipids in the liver tissue. Techniques of light and electron microscopy were performed in order to propose a new way of assessing and quantifying the distribution of lipid droplets, also presenting methodological alternatives that can be chosen by the reader according to the interests and resources available. Thus, it is assumed that the method begins with the fixation of the liver with Glutaraldehyde 2,5% in PBS 0,1 M and continues with post fixation with osmium tretoxide 1%, which marks lipids. For this proposition, two inclusion methodologies were performed to histological analyses in Historesin and ultrastructural analyses in EMBeed 812. For light microscopy (LM) analyses, cuts were obtained with 2,5 micrometers thickness, which were stained with (1) Mayers hematoxylin and (2) toluidine blue. The images obtained were processed in software Image J Fiji to evidence the lipid distribution in liver.•Cytological reactions with osmium tetroxide constitute low complexity methods that allow the optimization of the localization, identification and quantification of lipid droplets in the liver tissue when analyzed under the conventional light microscope.•Samples included in EMBeed 812 resin commonly used in Transmission Electron Microscopy can be analyzed by SEM-BEC, as complementary analyses for the detection of lipids.•Using SEM-BEC and conventional light microscopy, it is possible to quantify the area occupied by lipid droplets using Image J Fiji software, as these are contrasted due to the reaction with osmium tetroxide.

Morphological and histopathological changes in seahorse (Hippocampus reidi) gills after exposure to the water-accommodated fraction of diesel oil.

Industrial activities and urbanization are the main sources of pollutants in estuarine environments. Diesel, which is widely used in urban and port activities, is an important source of hydrocarbons in the aquatic environment, and its water-accommodated fraction (WAF) is toxic to the local biota. This study was performed to analyze the effects of diesel oil WAF on fish. Specifically, we characterized the gill morphology of the seahorse Hippocampus reidi and analyzed the histopathological changes in the gills after exposure to 50% diesel oil WAF. Acute (12, 24, 48, and 96 h) and subchronic (168 and 336 h) toxicity tests were performed. Furthermore, a recovery protocol was conducted: after exposure to 50% WAF for 168 h, the fish were transferred and kept in seawater without contaminants for 336 h, for a total experimental period of 504 h. The seahorse branchial apparatus was found to be tufted with short filaments; the apical surfaces of the pavement cells in the filament and lamellar epithelia formed "crests" and had microridges. Mitochondria-rich cells were distributed exclusively in the lamellar epithelium, while mucous cells were distributed in the filament epithelium. All pathologies observed after acute and subchronic exposure featured progressive time-dependent alterations of lamellar structure that might disrupt gill physiological and metabolic functions. During the recovery period, the gill alterations were gradually repaired.

Water-soluble fraction of petroleum induces genotoxicity and morphological effects in fat snook (Centropomus parallelus).

Petroleum hydrocarbons are one of the primary organic chemicals found in water bodies, and the water-soluble fraction of petroleum (WSFP) may be responsible for much of the toxic effects. In the present study, genotoxicity assays and histopathological analysis of the gills were analyzed for two experimental protocols: 1) Juvenile Centropomus parallelus were exposed to different concentrations of WFSP (0%, 25%, 50% and 75%) for 96h; 2) A second fish group was exposed to 50% WFSP for 168h followed by a post-exposure period for 168h in clean water (recovery). The total benzene, toluene, ethyl benzene and xylene (BTEX) and polycyclic aromatic hydrocarbon (PAH) concentrations at time 0 were 254µgL-1 and 4.72µgL-1 in 25%; 552.9µgL-1 and 9.36µgL-1 in 50%; and 842.4µgL-1 and 9.97µgL-1 in 75% WSFP, respectively. Based on the alkaline comet assay, the damage index (DI) values of fish exposed to 25% WSFP for 96h were significantly higher than those in the control group, and in the micronucleus test, the higher damage values were found in fish exposed to 75% WSFP. Furthermore, this last genotoxic test showed recovery after 168h. At all concentrations of WSFP, several histopathological changes were observed, and overall, most of these changes observed in the gills were classified as proliferative changes and represented a protective mechanism against pollutant uptake. Based on the recovery experiment, the damage was also significantly reduced after recovery. Our results showed that short-term exposure to WSFP compounds triggered cellular alterations in C. parallelus, but total recovery did not occur with time. Additionally, the different periods of exposure were not sufficient to induce severe gill damage in C. parallelus. Moreover, this fish demonstrated its usefulness as a sentinel species.

Ecotoxicity and hematological effects of a natural insecticide based on tobacco (Nicotiana tabacum) extract on Nile tilapia (Oreochromis niloticus) / Ecotoxicidade e efeitos hematológicos de um inseticida natural de extrato de tabaco (Nicotiana tabacum) em tilápia do Nilo (Oreochromis niloticus)

Natural insecticides from plant extracts represent an alternative to the highly toxic synthetic products in order to reduce environmental contamination; however some might also be toxic for non-target organisms. The present study determined the 50% lethal concentration (48h; LC50) for adults Nile tilapia (Oreochromis niloticus) exposed to the natural insecticide Fumydro®, based on the tobacco plant (Nicotiana tabacum), and evaluated its effect on hematological variables. After preliminary tests, adult specimens of O. niloticus were exposed to four Fumydro® concentrations (200, 300, 400 and 500 µL L-1). The 48h; LC50 of Fumydro® was determined at 370 ± 50 µL L-1. The surviving fish after exposure to Fumydro® showed an increase in the number of red blood cells, hemoglobin concentration, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. The number of thrombocytes and leukocytes has not changed, unlike the differential leukocyte count that presented an increased percentage of neutrophils. The results indicated that the insecticide Fumydro® is highly toxic to Nile tilapia and changes in erythrocyte variables suggested the induction to hypoxemia with low effect on the immune system.

Inseticidas naturais produzidos a partir de extratos de plantas pode ser uma alternativa para diminuir a contaminação do ambiente por agrotóxicos sintéticos e altamente tóxicos, embora alguns deles possam também ser tóxicos para organismos não alvos. O presente estudo determinou a concentração letal 50% (CL50; 48h) para tilápia do Nilo (Oreochromis niloticus) expostas ao inseticida natural Fumydro®, produzido a base de planta de tabaco (Nicotiana tabacum) e analisou o seu efeito nas variáveis hematológicas. Após testes preliminares, exemplares adultos de O. niloticus foram expostos a quatro concentrações de Fumydro® (200, 300, 400 e 500 µL L-1). A CL50; 48h foi estimada em 370 ± 50 µL L-1. Os peixes sobreviventes a exposição ao Fumydro® apresentaram aumento no número de eritrócitos, concentração de hemoglobina, hemoglobina corpuscular média e concentração de hemoglobina corpuscular média. O número de trombócitos e de leucócitos não foi alterado, mas ocorreu alteração na contagem diferencial de leucócitos ocorrendo um aumento na porcentagem de neutrófilo nos peixes expostos a 200, 300 µL L-1 de Fumydro®. Estes resultados indicaram que o inseticida Fumydro® é extremamente tóxico para tilápia do Nilo e as alterações nas variáveis eritrocitárias sugerem que causa hipoxemia, mas tem pouca ação sobre o sistema imune.

Opercular epithelial cells: a simple approach for in vitro studies of cellular responses in fish.

This study evaluated the efficacy of fish opercular external (skin) and inner (opercular membrane) epithelium as an in vitro model for toxic and other substances studies. The rainbow trout (Oncorhynchus mykiss) operculum was cultured in 12-well dishes containing sterile Leibovitz 15 (L-15) supplemented with glutamine medium during 24h at 9 degrees C, and the effect of copper, a toxic agent, and/or cortisol, an endogenous agent, on the epithelial cells was analyzed using light microscopy techniques. The opercula were submitted to four treatments: (i) control (Cont), L-15 medium only, (ii) 0.28 microM cortisol (Cort), (iii) 100 microM CuSO(4) (Cu), and (iv) 0.28 microM cortisol+100 microM CuSO(4) (Cort-Cu). The tissue condition after 24h incubation was analyzed by staining the mucous cells for neutral and acid mucosubstances. Cellular necrosis was evaluated by measuring the lactate dehydrogenase (LDH) leakage at 12 and 24h incubation. Cellular proliferation, apoptosis, metallothionein (MT) and glucocorticoid receptor (GR) expression were evaluated by immunohistochemistry. The LDH leakage was higher and the proliferating cell nuclear antigen (PCNA) positive-stained cells were lower in Cu treatment in both, epidermis and opercular membrane. Apoptotic cells in the opercular membrane were higher in the Cort and Cort-Cu treatments while, in the epidermis, they were higher in Cu and Cort-Cu treatments. GR-positive stained cells decreased significantly in all treatments in both epithelia and the MT-positive cells increased in the Cu and Cort-Cu treated groups. Copper showed to be a potent toxic stressor killing the cells via necrosis, decreasing the number of PCNA-positive cells and inducing MT synthesis while cortisol did not affect the MT synthesis, although it might stimulate apoptosis. The results are evidence that the opercular epithelia serve as a suitable model for studying in vitro effects of toxic agents, as well as endogenous factors on the cellular responses without interference of the physiological state of fish being useful to predict in vivo toxicity.