Cytoprotective and anti-apoptotic action of HSP70 stress protein in Oreochromis niloticus exposed to residual dilutions of insecticides with fipronil and ethiprole.

The objective of this research was to investigate the potential damage caused by the residual concentrations of the insecticides Regent® WS 800 and Curbix® SC 200, containing fipronil and ethiprole, respectively as active ingredients, on the liver of Oreochromis niloticus. The analyses of HSP70 shock protein labelling and cell death process by TUNEL method were performed in order to measure the effects of the exposure of cell repair system of fish to both insecticides. Statistical analyses showed no significant molecular damage to the hepatic tissue of animals. Nevertheless, variations in HSP70 and DNA fragmentation levels, endpoint of cell repair system response and cellular death, respectively, were observed in several groups. These results indicate that the cell repair machinery was efficient when in contact with residual concentrations of insecticides. However, the DNA fragmentation detected by the TUNEL method suggests that even in face of the cytoprotective action of the HSP70 protein, there are damages that become irreparable. To finish, it is worth mentioning that given the results obtained from residual concentrations, use in the field should be with caution.

Toxicity evaluation of leached of sugarcane vinasse: Histopathology and immunostaining of cellular stress protein.

Sugarcane vinasse is a residue generated at a rate fifteen times greater than the ethanol production. Because of its high organic and micronutrient content, this residue is used as a fertilizer on sugarcane crops. However, when used in large quantities, vinasse can saturate the soil and contaminate nearby water resources by percolation and leaching. Given the proven toxic potential of in natura vinasse, the present study aimed to evaluate the toxic potential of leached sugarcane vinasse using Nile tilapia (Oreochromis niloticus) as a test organism. A bioassay was performed after vinasse percolation in laboratory soil columns. The bioassay included one control group containing fresh water and two treatment groups, the first exposed to a 2,5% dilution of leached of vinasse and the second to a 2,5% dilution of in natura vinasse. After exposure, histopathological analysis was performed in gills and livers, and the latter were labelled for HSP70 proteins. No significant changes were detected in the gills of the exposed fish. However, in the liver, both in natura and leached vinasse induced statistically significant histopathological changes. These changes include hydropic degeneration, cell boundary losses, pyknotic nuclei and cellular disorganization. HSP70 expression significant increase in liver of both treatment groups were observed, being higher for the in natura vinasse exposed group. Results suggested that both leached vinasse and in natura vinasse were toxic, its still able to provoke histological changes and induce the cytoprotective response in exposed fish liver, evidenced by a immunostaining of cellular stress proteins. Thus, in order to reduce its environmental impact, appropriated effluent disposal is essential.

Avaliação da eficiência do tratamento de esgoto doméstico pelo teste do micronúcleo em Oreochromis niloticus (Cichlidae) / Evaluation of the domestic wastewater treatment efficiency by micronucleus test on Oreochromis niloticus (Cichlidae)

RESUMO Este estudo teve por objetivo avaliar a eficiência no tratamento do esgoto doméstico de uma cidade do interior paulista, pela análise de micronúcleos e anormalidades nucleares nos eritrócitos de tilápias expostas ao efluente bruto e ao tratado. Duas coletas foram realizadas: uma na estação chuvosa e outra na estação seca. Foram utilizados três aquários de 20 L nos experimentos, com cinco peixes cada. No primeiro, os animais foram expostos à água de poço artesiano (controle negativo); no segundo, ao esgoto bruto diluído; no terceiro, ao esgoto após tratamento. Decorrido o tempo de exposição (72 h), coletou-se o sangue desses animais para obtenção de lâminas de esfregaço. Os resultados obtidos mostraram efeitos genotóxicos e mutagênicos do efluente bruto, em ambas as coletas; porém, esses efeitos não foram significativos após o tratamento do esgoto, o que mostra que o processo utilizado no tratamento foi eficiente para minimizar tais efeitos.

ABSTRACT This study aimed at evaluating the efficiency in the domestic wastewater treatment from a city of Sao Paulo State (Brazil), by micronuclei and nuclear abnormalities analysis in erythrocytes of tilapias exposed to raw and treated wastewater. Two samples were collected. Three aquariums of 20 L, with five fish each, were used in the bioassays. In the first, the animals were exposed to the artesian water (negative control); in the second, to the diluted raw wastewater; and in the third, to after wastewater treatment. After the exposure time (72 h), the blood was collected for preparing the smear slides. The results showed genotoxic and mutagenic effects of the raw wastewater in both samples, showing that the process used was efficient to minimize these effects.

Toxicity evaluation of vinasse and biosolid samples in diplopod midgut: heat shock protein in situ localization.

Large amounts of residues generated by agricultural, urban and industrial activities are dumped daily on the soil. This practice deserves special attention because it causes serious environmental problems. This study evaluated the toxic potential of the sugarcane vinasse, a by-product of the sugar-alcohol industry, and the biosolid, a residue produced by wastewater treatment plants, both widely used as fertilizers. The evaluation was performed through bioassays using a typical soil bioindicator, the diplopod Rhinocricus padbergi. The specimens were exposed to soils containing these residues in concentrations that are compatible with the Brazilian regulation for agricultural use. Semi-quantitative immunolabelling analyses of the stress protein HSP70 were performed on the midgut of the studied diplopods. There was a significant increase in the immunolabelling of HSP70 proteins as a response to xenobiotics from both residues, particularly in regions where the function of the cells is the detoxification of the organ (e.g. the hepatic cell layer and specific regions of the epithelium). Higher immunolabelling was observed in the specimens exposed to vinasse in comparison with the biosolid exposure. This demonstrates that the substances in the tested residues had proteotoxic action in the exposed animals and induced a cytoprotective response, which led to higher stress protein immunolabelling. Therefore, caution is needed for the use of such residues in agriculture.

Genotoxicity evaluation of two metallic-insecticides using Allium cepa and Tradescantia pallida: A new alternative against leaf-cutting ants.

In order to combat leaf-cutting ants, the pesticide sulfluramid used to be the most widely utilized active ingredient. However, its use was banned in 2009 by the Stockholm Convention, although some countries were allowed to continue using it. As an effective alternative to its replacement, researchers developed a metallic-insecticide system, which is a natural product linked to metal complexes. Thus, the aim of this study was to evaluate the ability of these new metallic-insecticides in change the genetic material of non-target organisms. The tests were performed utilizing chromosomal aberrations and micronucleus tests in the Allium cepa test system and the Trad-MCN test in Tradescantia pallida. To better understand the results, one of the components of the formula, 5-methyl-phenanthroline, was also analyzed according to the same parameters. To A. cepa, the results showed that one of the metallic insecticides induced cytotoxicity and genotoxicity at different concentrations, while the other metallic-insecticide showed chromosomal instability only at the highest concentration. The analysis of 5-methyl-phenanthroline revealed that it can be related with the positive results, since genotoxic effects were induced. In the Trad-MCN test, none of the metallic-insecticides showed genotoxic activity, although one of them induced more micronucleus formation.

Evaluation of herbicides action on plant bioindicators by genetic biomarkers: a review.

The use of pesticides has increased worldwide, owing to the demand for products of good quality and to satisfy a growing population. Herbicides represent almost half of the total amount of pesticides used. Although important to the reduction of costs and an increase of productivity, their indiscriminate use, as well as that of the other pesticides, is a global environmental problem, since they affect the living organisms. To evaluate the damage caused by herbicides to the environment, different organisms have been used as bioindicators, especially higher plants, due to several advantages. This is a literature review on herbicidal actions in plant bioindicators, as assessed by genetic biomarkers. Also, the present manuscript aimed to characterize the main organisms (Allium cepa, Vicia faba and Tradescantia spp.) and the most used biomarkers (mitotic index, chromosome aberrations, micronuclei, sister chromatid exchange and mutations). We concluded that herbicides induce cytotoxicity and genotoxicity in the assessed bioindicators. The data corroborate the existing warnings of the risks that the indiscriminate and increasing use of pesticides poses to the environment and its biodiversity.

Histopathological changes in the perivisceral fat body of Rhinocricus padbergi (Diplopoda, Spirobolida) triggered by biosolids.

Human activities generate a great amount of sewage daily, which is dumped into the sewer system. After sewage-treatment processes, sewage sludge is generated. Such byproduct can be treated by different methods; the result of treatment is a stabilized compost of reduced pathogenicity that has a similar inorganic chemical composition to the raw sewage sludge. After such pretreatment, sewage sludge is called a biosolids, and it can be used in agriculture. In this contest, the present study evaluated the effects of a sample of biosolids on the perivisceral fat body of a diplopod. These invertebrates are soil organisms that play an important role in the dynamics of terrestrial ecosystems, and as a consequence, they are in contact with xenobiotics present in this environmental compartment. Special emphasis is given on the interpretation of the effects of complex mixtures in target organs of diplopods. A semiquantitative analysis for the evaluation of histopathological changes in the perivisceral fat body was proposed. The sample-induced histopathological and ultrastructural changes in individuals exposed to it, and the severity of the effects was positively related to the exposure time, resulting in the deaths of exposed individuals after 90 days. Thus, the results indicate the need for caution in the use of biosolids as well as the need for improving waste management techniques, so they will produce environmentally innocuous final products.

Sugar cane vinasse in water bodies: impact assessed by liver histopathology in tilapia.

Aquatic ecosystems are the main receptors of toxic substances from human activities. With the increase in sugar cane production, vinasse - the main residue of ethanol production - is a potential contaminant of water resources, due to its high organic matter content. This study was aimed at evaluating the toxicity of vinasse by examining the liver of the fish Oreochromis niloticus exposed to different dilutions of sugar cane vinasse (1%, 2%, 5%, 5% and 10%) in laboratory bioassays. Portions of liver were collected and fixed for histological and histochemical techniques to detect total proteins, polysaccharides and lipids. In the histological analysis, the groups treated with vinasse exhibited significant alterations, such as loss of cytoplasmic integrity, loss of cell limit and tissue disorganization. Protein and lipid profiles were not altered. Higher accumulation of polysaccharides was detected in fish exposed to lower concentrations of vinasse, with a gradual decrease in animals treated with vinasse in higher concentrations. We concluded that vinasse has a dose-dependent toxic and cytotoxic potential in water bodies and that the liver is strongly affected when acutely exposed to this contaminant.

The use of diplopods in soil ecotoxicology - a review.

Diplopods play an important role in the dynamics of terrestrial ecosystems, actively participating in the decomposition of organic matter and soil aeration. They have gained increased attention from ecotoxicology research because they are continuously exposed to soil contaminants and biological effects of chemical stressors can be measurable at various levels of biological organization. This paper is the first review on the use of diplopods as soil bioindicators and compiles the effects of the different toxic chemical agents on these animals. Special emphasis is given on the interpretation of the effects of heavy metals and complex mixtures in target organs of diplopods.

Sugarcane vinasse: environmental implications of its use.

The inadequate and indiscriminate disposal of sugarcane vinasse in soils and water bodies has received much attention since decades ago, due to environmental problems associated to this practice. Vinasse is the final by-product of the biomass distillation, mainly for the production of ethanol, from sugar crops (beet and sugarcane), starch crops (corn, wheat, rice, and cassava), or cellulosic material (harvesting crop residues, sugarcane bagasse, and wood). Because of the large quantities of vinasse produced, alternative treatments and uses have been developed, such as recycling of vinasse in fermentation, fertirrigation, concentration by evaporation, and yeast and energy production. This review was aimed at examining the available data on the subject as a contribution to update the information on sugarcane vinasse, from its characteristics and chemical composition to alternatives uses in Brazil: fertirrigation, concentration by evaporation, energy production; the effects on soil physical, chemical and biological properties; its influence on seed germination, its use as biostimulant and environmental contaminant. The low pH, electric conductivity, and chemical elements present in sugarcane vinasse may cause changes in the chemical and physical-chemical properties of soils, rivers, and lakes with frequent discharges over a long period of time, and also have adverse effects on agricultural soils and biota in general. Thus, new studies and green methods need to be developed aiming at sugarcane vinasse recycling and disposal.

Clastogenicity of landfarming soil treated with sugar cane vinasse.

The addition of nutrients and/or soil bulking agents is used in bioremediation to increase microbial activity in contaminated soils. For this purpose, some studies have assessed the effectiveness of vinasse in the bioremediation of soils contaminated with petroleum waste. The present study was aimed at investigating the clastogenic/aneugenic potential of landfarming soil from a petroleum refinery before and after addition of sugar cane vinasse using the Allium cepa bioassay. Our results show that the addition of sugar cane vinasse to landfarming soil potentiates the clastogenic effects of the latter probably due the release of metals that were previously adsorbed into the organic matter. These metals may have interacted synergistically with petroleum hydrocarbons present in the landfarming soil treated with sugar cane vinasse. We recommend further tests to monitor the effects of sugar cane vinasse on soils contaminated with organic wastes.

Morphological and histochemical characterization of gill filaments of the Brazilian endemic bivalve Diplodon expansus (Küster, 1856) (Mollusca, Bivalvia, Hyriidae).

This study presents the morphological description and histochemical characterization of gill filaments of the Brazilian endemic bivalve Diplodon expansus, aiming to broaden the morphological knowledge of this species and establish the structure of the gills that will serve as control in histopathological studies applied to biomonitoring. The gill filaments are divided into three zones: frontal, intermediate, and abfrontal. In the center of the filament, haemocytes circulate through the haemolymph vessel, which is internally lined by endothelium. The frontal surface of the filament is covered with cilia, the lateral surface exhibits aquifer ducts, and the abfrontal surface presents ciliated and nonciliated cells. The epithelium of the filaments is composed of ciliated cells, nonciliated absorptive cells, and mucocytes. The support of the filaments is made by two specialized structures called skeletal rod and skeletal loop. Based on the obtained information, the gill filaments of the studied species present some peculiar characteristics that are not yet reported in detail in the literature such as the simultaneous presence of skeletal rod and skeletal loop. On the other hand, the general constitution of the filament is similar to that described for both marine and limnic bivalves and seems to be suitable for ecotoxicological studies.

Toxicity of trifluralin herbicide in a representative of the edaphic fauna: histopathology of the midgut of Rhinocricus padbergi (Diplopoda).

The aim of this study was to use the diplopod Rhinocricus padbergi as test organism to verify the possible toxic effects of the herbicide trifluralin in a representative of the edaphic fauna. Histological and histochemical techniques were applied in the midgut of the animals after exposure to different concentrations of trifluralin for 7 and 90 days (acute and subchronic exposure, respectively). The individuals from the control group did not present alterations to any of the exposures. In the acute exposure, the group exposed to a quarter and half of the field dose presented an accumulation of cytoplasmic granules in the hepatic cells; the field dose group presented an increase in the rate of epithelial renewal and the group exposed to double the field concentration presented an increase in the release of secretory vesicles. The subchronic response was obtained with the field dose and double field dose because the animals from a quarter and half groups did not survive until the end of the experiment. In both groups, the animals presented disruption of the epithelium and higher occurrence of hemocytes among the hepatic cells. The group exposed to double the field dose presented sites of cytoplasmic vacuolization, dilatation of the intercellular space, increase in the epithelial renewal, and release of secretory vesicles. The results alert for a careful use of this herbicide, since it promoted alterations in the midgut cells of the test organism used.

Effectiveness of estradiol valerate on sex reversion in Astyanax altiparanae (Characiformes, Characidae)

The objective of this study was to evaluate the effectiveness of estradiol valerate in an attempt to feminize the male Astyanax altiparanae. One thousand newly-hatched larvae were randomly distributed in four incubators. During a period of 30 days, estradiol valerate was administered in different dosages (20, 40, and 80 mg/kg of diet); the control group did not receive hormone in the diet. After 161 days of hatching, biometry was performed on 50 fishes per treatment group, and the gonads were analyzed. The hormone treatments were effective in feminizing A. altiparanae, achieving 70-76% of the desired sex, while the control treatment had 44% females. The hormone did not affect the growth of fish.

Alterations in the fat body cells of Rhinocricus padbergi (Diplopoda) resulting from exposure to substrate containing sewage sludge.

The final disposal of residues generated at sewage treatment plants (STPs) has become a major problem for cities, due to the increase in the amount of treated sewage. One of the alternatives for the residue, labeled "sewage sludge," is its reuse in agriculture and in degraded soil. However, not all pathogens and metals present in it are eliminated during treatment. Diplopods have been used as bioindicators in ecotoxicological tests as they are constantly in close contact with the soil. Owing to this fact, the purpose of this study was to expose specimens of the diplopod Rhinocricus padbergi to substrate containing sewage sludge collected at STPs to analyze morphological alterations in their parietal and perivisceral fat body, where substances are stored. The exposures were held for 7, 15, or 90 days at different concentrations of sewage sludge (control, 1%, 10%, and 50%). The parietal fat body showed no alterations in any of the three exposure periods or concentrations. Alterations in the perivisceral fat body were observed for all exposure periods. According to the results, we suggest that the sludge used has toxic agents responsible for changing the animal's perivisceral fat body.

Sewage sludge toxicity in edaphic organism: analysis of midgut responses in the diplopod Rhinocricus padbergi.

Diplopods are agile saprophagous invertebrates with the ability to colonize several soil layers. They feed on waste and decaying organic matter, making the soil in which they are present better aerated and mineral-enriched. Because of their habits, diplopods have been suggested to be used in studies to analyze possible toxic effects of impacted soils in edafic fauna. In that respect, this study aimed at checking the feasibility of using morphological analysis from millipede's midgut as a biomarker for soils exposed to sewage sludge from sewage treatment plants. Histological and histochemical techniques, applied to the species Rhinocricus padbergi's midgut, were used. Two sewage sludge samples were used; they were collected in two small towns in the countryside of São Paulo State. Both cities are part of the Piracicaba-Capivari-Jundiaí basin (PCJ-1 and PCJ-3). For 7 and 15-day periods, the animals were exposed to 10% concentrations and crude sludge for the PCJ-1sample and to 1, 10, and 50% concentrations and sludge for the PCJ-3 sample. The material exposed to the PCJ-1 sample showed an increase in the presence of cytoplasmic granules in both concentrations in all periods and a slight increase in the rate of epithelial renewal. The material of PCJ-3 sample presented the same alterations observed in PCJ-1 sample in higher frequence and over that high quantity of neutral polysaccharides in the hepatic cells layer. We can conclude that with the increase of sewage sludge concentrations tested, there is an increase in morphophysiologicals alterations frequencies found in the diplopods' midgut.

Surface morphology of Diplodon expansus (Küster, 1856; Mollusca, Bivalvia, Hyriidae) gill filaments after exposure to environmentally relevant concentrations of atrazine herbicide.

Brazilian endemic species Diplodon expansus (Küster, 1856) is found in freshwater bodies in the country's southeast, in large anthropogenic influence regions especially with an extensive agriculture emphasis. One of the main pesticides used in the species occurrence region is the atrazine herbicide, which has a great contamination potential in the aquatic environment. Therefore, several studies into its toxicity in aquatic systems have been developed. However, the tested concentrations are usually very high and rarely found in the environment and the short-term exposure responses in other aquatic organisms such as native bivalves are still scarce. Thus, this study sought to consider the potential effects of environmentally realistic concentrations of atrazine herbicide on the surface morphology of gill filaments of the bivalve D. expansus under laboratory-controlled conditions after short-term exposure. None of the animals died before the end of the experiment. The main alterations were observed on the frontal surface of filaments, which include mucus accumulation, cilia loss, and disruption. Mucus increased secretion and accumulation in the frontal filaments region preceded as a protective mechanism. Cilia loss and disruption on the frontal surface of the gill filament indicated that ciliated frontal cells were more sensitive to atrazine exposure and these alterations may cause gills functional damages, compromising the uptake of food particles and respiration. Therefore, higher sublethal concentrations of atrazine may compromise the survival and consequently the population of D. expansus in agriculture areas after a longer period of continuous exposure.

Morphological biomarkers in the Rhinocricus padbergi midgut exposed to contaminated soil.

Morphological studies have been performed in invertebrates to elucidate cellular and/or tissue damage caused by environmental contamination. In present work, the exposure to industrial soil contaminated with polycyclic aromatic hydrocarbons and metals led to histological alterations in the midgut of Rhinocricus padbergi (Diplopoda). The following alterations were observed: increase of cytoplasmic granules in the fat body; increase in the number of regions of the epithelium in the process of renewal; increase in the number of hemocytes present among the cells of the fat body and degenerative changes in the midgut epithelium. Based on the results, we concluded that R. padbergi proved to be an excellent bioindicator for the evaluation of soil quality. The observed changes could be used as biomarkers for assessing sublethal effects of contaminated soils.

Hemocitical responses to environmental stress in invertebrates: a review.

Although invertebrates are recognized by the great facility to accumulate pollutants present in their environment and many of them are used as sentinel species in biomonitoring studies, little is known about the impact of toxicants on the immune system of these animals. In this regard, hemocytes play a fundamental role: these cells circulate freely through the hemolymph of invertebrates and act on the recognition of foreign material to the organism, mediating and effecting the cellular defense, such as phagocytosis, nodulation, and encapsulation. Different morphological types can be recognized but still there is controversy among the researchers about the exact classification of the hemocytes due to the diversity of techniques for the preservation and observation of these cells. In the present study, a review on the main hemocyte responses to environmental stress in different invertebrate organisms is presented, emphasizing the contamination by heavy metals. It is discussed parameters such as: alteration in the number of cells involved in the defense reaction, phagocytic activity, lysosomal responses, and production of reactive oxygen species.

Acute and subchronic exposure of diplopods to substrate containing sewage mud: Tissular responses of the midgut.

Several discussions regarding the agricultural use of sewage mud have occurred; however, its use has been questioned due to the risks of soil contamination mainly by metals. Diplopods are saprophagous invertebrates, agile in colonizing several layers of soil. Due to the habit of this group, several researchers have proposed their use in ecotoxicological analyses. This study aimed to expose these invertebrates to substrate containing sewage mud of a STS (Sewage Treatment Station) from São Paulo State, Brazil and analyzed its toxic potential by morphological analysis of the midgut of the diplopod Rhinocricus padbergi, region of the digestive tube where digestion and nutrient absorption occur. The animals were exposed in mud concentrations at 1%, 10% and 50% mixed with soil from the collection site for periods of 7 (acute exposure), 15 (intermediary exposure) and 90 (subchronic exposure) days in each treatment. The animals from the control group did not present alterations at all the exposition times. As an acute response (7 days), it was observed in the animals exposed to mud at 1% and 10% an increase in the epithelial renovation and in the liberation of secretory vesicles of glycoprotein content. In the animals exposed to mud at 50%, the main alteration observed was the increase in the number of cytoplasmatic granules in the fat body cells; the epithelium seemed to be in reorganization process in all the observed individuals, suggesting an intense epithelial renovation. In the intermediary period of exposure (15 days), in the animals exposed to mud at 1% it was observed an increase in the number of haemocytes among the cells of the fat body, distributed both separately and grouped; the animals exposed to mud at 10% presented the same evidence of epithelial renovation observed in the animals exposed for 7 days at a concentration of 50% of mud; it was also observed an increase in the number of haemocytes, which reacted strongly to the PAS technique. Cytoplasmatic granules in the hepatic cells, fat body cells and haemocytes were also observed in great number, being that great part presented calcium in their constitution. In the animals exposed to mud at 50%, the alteration observed was the presence of numerous secretory vesicles of the apocrine type through all the epithelia of vacuolated aspect; hepatic cells with cytoplasmatic granules were also observed. As subchronic response (90 days), it was observed that all the fat body cells were completely taken by cytoplasmatic granules, into which it was, detected the presence of calcium; such granules were also observed in the apical region of the principal epithelial cells. These results suggest the existence of toxic agents in the analyzed mud sample, whose different concentrations trigger responses that occur in different manners, intensities and velocities that might prevent the entrance and action of toxic agents in the organism of the studied invertebrate.