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The present study was aimed at evaluating the in vivo effects of microplastics (MP), in terms of oxidative stress and histopathological effects, in two crustacean species: Procambarus clarkii and Leptuca pugilator. In addition, MP accumulation in the hepatopancreas (HP) of both species was also determined. Adults of both crayfish and crabs were exposed for one month to fluorescent polystyrene beads (size: 1 µm) at nominal concentrations of 1000 or 5000 particles/mL. During the exposure, animals were maintained under controlled feeding, aeration, temperature, and photoperiod conditions. At the end of the exposure, HP and hemolymph (HL) samples were harvested for analysis of oxidative damage and total antioxidant levels. Additionally, the presence of MPs in both tissues was confirmed. Significant differences with the control groups were observed in lipid peroxidation levels in HP in animals exposed to the lowest concentration in P. clarkii and to the highest concentration in L. pugilator. A marked increase in antioxidant levels was also observed in the HL at both concentrations in P. clarkii, and at the highest MPs concentration in L. pugilator. Moreover, several histopathological changes were detected in both gills and HP, including hypertrophied lamellae, lifting or collapse of gill epithelia, loss of normal shape of hepatopancreatic tubules, and epithelial atrophy in the HP tissue. We conclude that exposure to MP beads at selected concentrations results in oxidative damage, induces histopathological changes in gills and HP, and triggers an antioxidant response in two crustacean species.

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Atrazine, a herbicide that is intensively used in Argentina, was assayed to evaluate the alteration of reproduction in a wild species of crustaceans. Adult females of the estuarine crab Neohelice granulata were exposed to formulated atrazine during the 3-month pre-reproductive period. Three atrazine concentrations (0.03, 0.3 and 3mg/L) were assayed, together with a water dilution control. At the end of the exposure period, several variables concerning the energetic status of animals were measured, such as weight gain, glycemia, and energy reserves in both muscle and hepatopancreas. The gonadosomatic index (GSI) was also determined, as well as the proportion and relative area of each oocyte type in histological sections. Besides, the total content of vitellogenin proteins (Vg) in both ovary and hepatopancreas was measured. A significant (p<0.05) decrease of glycogen content was observed in muscle, while a significant (p<0.05) lower area of both previtellogenic and vitellogenic oocytes was verified in the ovary by effect of atrazine, in correspondence with a Vg content significantly (p<0.05) diminished in the ovary and augmented in the hepatopancreas. Besides, a higher proportion of previtellogenic oocytes were seen by effect of atrazine. Taken together, these results indicate a clear reduction and delay in the ovarian growth of the studied species, during the period at which the ovary normally grows up prior to spawning. On the other hand, a decrease of Vg content was observed at 0.3 and 3mg/L of atrazine after 24-h in vitro assays carried out with ovarian explants, providing additional evidence about the inhibitory role of this herbicide on the ovarian growth. These results encourage future research on deleterious effects of atrazine on crustacean reproduction.

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The present study analyzes a battery of biomarkers in the water bug Belostoma elegans from a stream polluted with organic matter (OMS), and another one considered as reference site (RS) during spring-summer season (December to March). Biochemical parameters of glucidic, lipidic and oxidative metabolic pathways were analyzed in males and females of this insect. In general, no significant differences were observed in all biomarkers assayed between both sexes, except lactate concentration which was higher in males than in females (p < 0.0006) in the first three months. About carbohydrate metabolism parameters, only pyruvate-kinase showed significant differences between insects collected in both streams (p < 0.05) during December. However, the total lipid content, saturated fatty acid, and mainly triacylglycerol were higher in insects from RS compared to those from OMS (p < 0.002) in all sampled months. Levels of lipoperoxidation, protein oxidation, reduced glutathione and glutathione-S-transferase activity showed no differences between insects collected from both streams. Nevertheless, the significant increase observed in superoxide dismutase and catalase activities (p < 0.004) could be due to the elevated oxidative metabolism in insects from RS compared to those from OMS with lower dissolved oxygen. Regarding those responding parameters, males accounted for the differences between the two sites during the study period. In conclusion, our results support that lipidic energetic reserves and antioxidant enzyme activities in B. elegans could be used as biomarkers of environmental pollution by organic matter.

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Early juveniles of the crayfish Cherax quadricarinatus were exposed for 60 days to 10 and 40 mg/L of pure glyphosate (acid form) in freshwater. Mortality was 33 % at the highest concentration, while no differences in molting were noted among treatments. After the first month of exposure, weight gain was significantly (p < 0.05) reduced in the 40 mg/L group. At the end of the assay, lipid levels in muscle, as well as protein level in both hepatopancreas and muscle were significantly (p < 0.05) reduced. These results suggest long-term utilization of both lipid and protein as main energetic reserves, likely in response to the chronic stress associated with herbicide exposure. Besides, the lower pyruvate kinase activity in muscle suggests a possible metabolic depression in this tissue. The hemolymphatic ASAT:ALAT ratio showed higher levels than the control at the highest glyphosate concentration, indicating possible damage to several tissues.

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The toxicity of cadmium and copper were assayed in the estuarine crab Chasmagnathus granulata. Acute assays were made on the first larvae stage, and both acute and chronic assays were made on juvenile crabs. The acute lethal toxicity of the assayed heavy metals was three orders of magnitude higher in larvae than in juveniles. Cadmium proved to be more toxic than copper in most cases; this difference was more evident in the chronic assays on juveniles, according to the nonphysiological feature of cadmium and its persistent accumulation in organisms. During these chronic assays, cadmium produced both a significant mortality and a clear retardation of molting, in accordance with its inhibitory effect on the molting process as previously reported. Copper only caused a molting acceleration during chronic assays, perhaps as a detoxifying mechanism. Heavy metal concentrations having effects in the chronic assays have been reported in some sectors of the estuary where the assayed species lives.

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Whole body oxygen consumption and some hemolymph parameters such as pH, partial pressure of gases, level of ions and lactate were measured in the estuarine crab Chasmagnathus granulata after both acute (96 h) and chronic (2 weeks) exposure to cadmium at concentrations ranging from 0.4 to 6.3 mg/l. In all instances, the crabs developed hemolymph acidosis, but no respiratory (increased PCO2) or lactate increases were evident. Hemolymph levels of sodium and calcium were always increased by cadmium exposure. The chronic toxicity of cadmium was enhanced at 12 per mil salinity, even causing a significantly higher mortality in comparison with the higher salinity (30 per mil ) used. A general metabolic arrest took place at 12 per mil salinity in the crabs chronically exposed to cadmium, as indicated by decreases of oxygen consumption and PCO2, an increase of PO2, along with no changes in lactate levels. These imbalances were associated with severe necrosis and telangiectasia in the respiratory gills, probably leading to respiratory impairment and finally histotoxic hypoxia and death of the animals.

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Whole body oxygen consumption and some hemolymph parameters such as pH, partial pressure of gases, level of ions and lactate were measured in the estuarine crab Chasmagnathus granulata after both acute (96 h) and chronic (2 weeks) exposure to cadmium at concentrations ranging from 0.4 to 6.3 mg/l. In all instances, the crabs developed hemolymph acidosis, but no respiratory (increased PCO2) or lactate increases were evident. Hemolymph levels of sodium and calcium were always increased by cadmium exposure. The chronic toxicity of cadmium was enhanced at 12 0/00 salinity, even causing a significantly higher mortality in comparison with the higher salinity (30 0/00) used. A general metabolic arrest took place at 12 0/00 salinity in the crabs chronically exposed to cadmium, as indicated by decreases of oxygen consumption and PCO2, an increase of PO2, along with no changes in lactate levels. These imbalances were associated with severe necrosis and telangiectasia in the respiratory gills, probably leading to respiratory impairment and finally histotoxic hypoxia and death of the animals

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