Genotoxic effects of Roundup on the fish Prochilodus lineatus

Glyphosate-based herbicides, such as Roundup, represent the most extensively used herbicides worldwide, including Brazil. Despite its extensive use, the genotoxic effects of this herbicide are not completely understood and studies with Roundup show conflicting results with regard to the effects of this product on the genetic material. Thus, the aim of this study was to evaluate the genotoxic effects of acute exposures (6, 24 and 96 h) to 10 mg L(-1) of Roundup on the neotropical fish Prochilodus lineatus. Accordingly, fish erythrocytes were used in the comet assay, micronucleus test and for the analysis of the occurrence of nuclear abnormalities and the comet assay was adjusted for branchial cells. The results showed that Roundup produces genotoxic damage in erythrocytes and gill cells of P. lineatus. The comet scores obtained for P. lineatus erythrocytes after 6 and 96 h of exposure to Roundup were significantly higher than respective negative controls. For branchial cells comet scores were significantly higher than negative controls after 6 and 24 h exposures. The frequencies of micronucleus and other erythrocyte nuclear abnormalities (ENAs) were not significantly different between Roundup exposed fish and their respective negative controls, for all exposure periods. In conclusion, the results of this work showed that Roundup produced genotoxic effects on the fish species P. lineatus. The comet assay with gill cells showed to be an important complementary tool for detecting genotoxicity, given that it revealed DNA damage in periods of exposure that erythrocytes did not. ENAs frequency was not a good indicator of genotoxicity, but further studies are needed to better understand the origin of these abnormalities.     

Acute effects of glyphosate herbicide on metabolic and enzymatic parameters of silver catfish (Rhamdia quelen)

Silver catfish (Rhamdia quelen; Teleostei) were exposed to commercial formulation Roundup, a glyphosate herbicide: 0 (control), 0.2 or 0.4 mg/L for 96 h. Fish exposed to glyphosate showed an increase in hepatic glycogen, but a reduction in muscle glycogen at both concentrations tested. Glucose decreased in liver and increased in muscle of fish at both herbicide concentrations. Glyphosate exposure increased lactate levels in liver and white muscle at both concentrations. Protein levels increased in liver and decreased in white muscle while levels of ammonia in both tissues increased in fish at both glyphosate concentrations. Specific AChE activity was reduced in brain after treatments, no changes were observed in muscle tissue. Catalase activity in liver did not change during of exposure. Fish exposed to glyphosate demonstrated increased TBARS production in muscle tissue at both concentrations tested. For both glyphosate concentrations tested brain showed a reduction of TBARS after 96 h of exposure. The present results showed that in 96 h, glyphosate changed AChE activity, metabolic parameters and TBARS production. The parameters measured can be used as herbicide toxicity indicators considering environmentally relevant concentration.     

Acute lethal and sublethal effects of neem leaf extract on the neotropical freshwater fish Prochilodus lineatus

The aim of this study was to determine the toxicity of the aqueous extract of neem leaves, a product extensively used in fish-farms as alternative for the control of fish parasites and fish fry predators, for the neotropical fish Prochilodus lineatus. The 24 h LC(50) of neem leaf extract for juveniles P. lineatus was estimated as 4.8 g L(-1); the fish were then exposed for 24 h to 2.5, 5.0 and 7.5 g L(-1) or only clean water (control). Plasma glucose levels were higher in fish exposed to 2.5 g L(-1) and 5.0 g L(-1) neem extract, relative to control, indicating a typical stress response. Neem extract did not interfere with the osmoregulating capacity of the fish, as their plasma sodium, chloride, total protein and osmolarity did not change. The presence of the biopesticide interfered with the antioxidant defense system of P. lineatus, as there was a decrease in liver catalase activity at all neem concentrations and the detoxifying enzyme glutathione-S-transferase was activated in fish exposed to 5.0 g L(-1). Fish exposed to all neem extract concentrations exhibited damaged gill and kidney tissue. These results indicate that although neem extract is less toxic to P. lineatus than other synthetic insecticides used in fish-farming it does cause functional and morphological changes in this fish species.     

Acute morphological and physiological effects of lead in the neotropical fish Prochilodus lineatus.

The present study investigated lead effects on gill morphology, hematocrit, blood sodium, glucose, lipids, protein, and cholesterol of Prochilodus lineatus exposed to two sublethal lead concentrations for 96 h. Preliminary series of short-term static toxicity tests were run to determine LC50 (96 h) of lead in P. lineatus, which was 95 mg Pb.L-1. Therefore, lead concentrations tested in the sublethal experiments were 24 and 71 mg Pb.L-1, which correspond to 25% and 75% of the LC50 (96 h), respectively. Gills of P. lineatus exposed to both lead concentrations during 96 h presented a higher occurrence of histopathological lesions such as epithelial lifting, hyperplasia, and lamellar aneurism. P. lineatus did not show significant alterations in hematocrit during exposure to both lead concentrations. Fish exposed to the highest lead concentration showed a significant decrease in Na+ plasma concentration after 48 h, possibly reflecting a sodium influx rate decrease. P. lineatus exposed to both lead concentrations presented a "classical general adaptation syndrome to stress", as hyperglycemia associated with lowered lipids and proteins was reported. Stress-response magnitude was dose-dependent. While the response to the lowest lead concentration might represent adaptation, the highest concentration seems to characterize exhaustion.     

Acute effects of nitrite on ion regulation in two neotropical fish species

To broaden the understanding of physiological responses of tropical fish to environmental stressors, the effects of nitrite on haematological parameters and plasma and red blood cell ion regulation were studied in two neotropical fish species, Astyanax altiparanae and Prochilodus lineatus. Both fish species were exposed to NaNO2 (30 mg l(-1)) over a 96-h period and blood samples were taken for ion and haematological analyses. The results revealed that nitrite leads to a decrease in P. lineatus blood haematocrit and haemoglobin content and an increase in blood methaemoglobin. A. altiparanae did not exhibit any significant difference in these haematological parameters. During the exposure to NO2- both fish species had significantly reduced plasma Na+ concentration and red blood cell (RBC) K+ concentration, but only P. lineatus showed an increase in extracellular K+ concentration. When RBC volume was analyzed in vitro, after 2 min of exposure to NaNO2, a 36% shrinkage was observed in P. lineatus cells, while only a 10% shrinkage was observed in A. altiparanae cells. These results suggest that for P. lineatus, nitrite entrance into the cell leads to methaemoglobin formation and K+ efflux, causing red cell shrinkage and increased plasma K+. However, A. altiparanae proved to be a species more resistant to nitrite, exhibiting fewer responses to this compound.     

Acute effects of diflubenzuron on the freshwater fish Prochilodus lineatus

The effects of diflubenzuron (DFB), an insecticide to control ectoparasites in fish farms, on muscle acetylcholinesterase (AChE), detoxifying and antioxidant enzymes, hematological and physiological parameters, and liver histopathology were evaluated in Prochilodus lineatus after 6, 24 and 96 h of exposure to 25 mg L(-1) of DFB. The insecticide caused a reduction in the number of erythrocytes and hemoglobin content after 96 h exposure, probably due to hemolysis. Hyperglycemic response indicated energy mobilization, and may have contributed to the increase in osmolarity after 96 h exposure to DFB. The induction of glutathione-S-transferase (GST) and catalase activities in liver pointed to the activation of xenobiotic metabolic pathways and antioxidant defenses. The decrease in muscle AChE at all experimental times showed that DFB is an AChE inhibitor. In addition, DFB induced hepatic alterations that might impair normal liver functions. These results show that DFB can cause health disorders in fish and further studies are required to better define its safe use in aquaculture.     

Effect of the herbicide Roundup on the activity of Glycosidases of invertebrates and juvenile fish

The effects of in vitro exposure to the herbicide Roundup at concentrations of 0.1–50 μg/L on the activity of maltase and sucrase and the total amylolytic activity in the organism of invertebrates and fish fry have been investigated. Glycosidases in invertebrates are less sensitive to the herbicide than those in juvenile fish. Roundup has a greater inhibitory effect on glycosidase activity in the tissues of actual prey (roach recovered from pike stomach) than in potential prey (roach captured in the pond). The magnitude and direction of the effects depend on the animal species and the concentration of the toxicant.     

DNA damage in fish (Anguilla anguilla) exposed to a glyphosate-based herbicide -- elucidation of organ-specificity and the role of oxidative stress

Organophosphate herbicides are among the most dangerous agrochemicals for the aquatic environment. In this context, Roundup(?), a glyphosate-based herbicide, has been widely detected in natural water bodies, representing a potential threat to non-target organisms, namely fish. Thus, the main goal of the present study was to evaluate the genotoxic potential of Roundup(?) in the teleost fish Anguilla anguilla, addressing the possible causative involvement of oxidative stress. Fish were exposed to environmentally realistic concentrations of this herbicide (58 and 116 μgL(-1)) during one or three days. The standard procedure of the comet assay was applied to gill and liver cells in order to determine organ-specific genetic damage. Since liver is a central organ in xenobiotic metabolism, nucleoids of hepatic cells were also incubated with a lesion-specific repair enzyme (formamidopyrimidine DNA glycosylase - FPG), in order to recognise oxidised purines. Antioxidants were determined in both organs as indicators of pro-oxidant state. In general, both organs displayed an increase in DNA damage for the two Roundup(?) concentrations and exposure times, although liver showed to be less susceptible to the lower concentration. The enzyme-modified comet assay showed the occurrence of FPG-sensitive sites in liver only after a 3-day exposure to the higher Roundup(?) concentration. The antioxidant defences were in general unresponsive, despite a single increment of catalase activity in gills (116 μgL(-1), 3-day) and a decrease of superoxide dismutase activity in liver (58 μgL(-1), 3-day). Overall, the mechanisms involved in Roundup(?)-induced DNA strand-breaks showed to be similar in both organs. Nevertheless, it was demonstrated that the type of DNA damage varies with the concentration and exposure duration. Hence, after 1-day exposure, an increase on pro-oxidant state is not a necessary condition for the induction of DNA-damaging effects of Roundup(?). By increasing the duration of exposure to three days, ROS-dependent processes gained preponderance as a mechanism of DNA-damage induction in the higher concentration.     

Genotoxic and mutagenic effects of diesel oil water soluble fraction on a neotropical fish species

Numerous spills and leakages involving petroleum and its derivatives have recently occurred in Brazilian rivers. Considering the lack of information regarding the genotoxic response of neotropical fish to these events and the predominance of information regarding saltwater fish, which offers no genuine comparisons, the present work aimed to evaluate the genotoxicity and mutagenicity of the diesel water soluble fraction (DWSF) on the neotropical fish Prochilodus lineatus under acute (6, 24 and 96h) and subchronic (15 days) exposures, using the comet (SCGE) and micronucleus assays. The results indicated genotoxic and mutagenic damage in erythrocytes of P. lineatus exposed to DWSF. Comet scores for fish exposed to DWSF in all experimental periods were significantly higher than the respective negative control groups (fish exposed to clean water for the same period). The relative frequencies of micronucleated erythrocytes for P. lineatus exposed to DWSF under acute and subchronic treatment were also significantly higher than their respective negative controls. Taken together these results showed that acute and subchronic exposures to DWSF produce mutagenic and genotoxic effects on the blood cells of P. lineatus and that the combination of comet and micronucleus assays proved to be both suitable and useful in the evaluation of the genotoxicity of diesel oil due to their complementary action.     

Goldfish can recover after short-term exposure to 2,4-dichlorophenoxyacetate: Use of blood parameters as vital biomarkers

This study investigated the effects of 2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, on the metabolism of goldfish, Carassius auratus, using only vital (non-lethal) approaches. After 96 h exposure to 1, 10 or 100 mg/L of 2,4-D selected hematological (total hemoglobin and hematocrit) and biochemical (glucose content, aspartate transaminase and acetylcholinesterase activities) parameters were unchanged in blood of exposed fish. At 100 mg/L of 2,4-D lymphocyte numbers decreased by 8%, whereas promyelocyte and metamyelocyte numbers increased by 7- and 2-fold, respectively. Exposure to 100 mg/L of 2,4-D also elevated carbonyl protein levels (by 2-fold), triglyceride content (by 43%) and alanine transaminase activity (by 46%) in goldfish plasma. All of these hematological and biochemical parameters reverted to control values after a 96 h recovery period. These data indicate that 2,4-D has toxicological effects on goldfish that can be monitored with multiple diagnostic tests using non-lethal blood testing.     

四氯乙烯和镉对草鱼的单一与联合毒性效应

以静水生物测试法研究了四氯乙烯和重金属镉对草鱼的单一与联合毒性,同时采用Marking相加指数法对二者的联合毒性进行了评价.单一毒性试验表明:四氯乙烯对草鱼24、48、72和96 h的半数致死浓度(LC₅₀)分别为49.12、41.68、36.37和34.30 mg·L^-1;镉对草鱼24、48、72和96h的LC₅₀分别为45.58、34.81、28.63和24.05mg·L^-1;二者对草鱼均有毒性,而且为高毒,镉的毒性大于四氯乙烯.联合毒性试验表明:二者毒性比为1∶1,暴露时间为24、48、72和96 h时四氯乙烯和镉的LC₅₀分别为24.63、12.54、9.88和7.08 mg·L^-1以及17.11、8.71、6.87和4.92 mg·L^-1,相加指数AI(additive index)分别为0.14、0.81、0.95和1.43,联合作用结果为协同效应,并且随着时间的增加,协同作用增强;二者浓度比为1∶1, 暴露时间为24、48 、72和96 h时四氯乙烯和镉的LC₅₀分别为17.00、11.18、10.61和9.19 mg·L^-1,AI分别为0.39、0.70、0.51和0.54,联合作用为协同作用.     

除草剂乐草隆对红鲫的遗传毒性研究

目的　探讨除草剂乐草隆对红鲫的遗传毒性。方法　用单细胞凝胶电泳检测不同浓度的乐草隆对红鲫外周血淋巴细胞DNA的损伤作用。结果　乐草隆致毒红鲫的淋巴细胞DNA的迁移度均较阴性对照组高 (P<0 0 5 ) ,在一定浓度范围内 (0～ 7 0 0mg L)DNA损伤程度与浓度呈正相关 (r=0 982 ,P <0 0 1)。在 12h、2 4h、4 8h、96h、10d实验组DNA损伤程度均有增强的趋势。结论　乐草隆对红鲫具有一定的遗传毒性     

Acute and sublethal effects in an Indian major carp Cirrhinus mrigala exposed to silver nitrate: Gill Na+/K+-ATPase, plasma electrolytes and biochemical alterations

Due to prolonged use of silver in many applications, it enters into the freshwater and affects the aquatic organisms. Fingerlings of Cirrhinus mrigala were exposed to acute and sublethal concentrations of silver nitrate and the alterations of gill Na(+)/K(+)-ATPase, plasma electrolytes and biochemical parameters were assessed. The median lethal concentration of silver nitrate to the fish C. mrigala for 96 h was found to be 0.107 mg/l (with 95% confidence limits). 1/10th of LC 50 96 h value (0.0107 mg/l) was selected for sublethal study. During acute treatment branchial Na(+)/K(+)-ATPase activity was inhibited approximately 44.34% after 96 h of exposure. In sublethal treatment, silver nitrate could not produce a significant change in the activity of the enzyme at the end of 7th day. However, after 14th day, significant (p < 0.05) decrease was noted showing 22.52%-49.11% in rest of the study period. Silver intoxication resulted hyponatremia, hypokalemia, hypochloremia, and hypoproteinemia in both the treatments. Despite the decrease in these parameters, plasma glucose level was found to be increased in both the treatments to endure the silver toxicity. We suggest that the alterations in branchial Na(+)/K(+)-ATPase activity, plasma electrolytes, and biochemical parameters of fish may be useful in environmental biomonitoring and to assess the health of fish in freshwater habitat contaminated with silver.     

双酚A和对硝基酚对泥鳅的急性毒性效应

采用单因子急性毒性试验法及组织切片法,检测了双酚A(bisphenol A,BPA)和对硝基酚(p-nitrophenol,P-NP)对泥鳅(Misgurnus anguillicadatus)的急性毒性效应以及对鳃组织结构的破坏,统计了半数致死浓度(median lethal concentration,LC_(50))和安全浓度(safe concentration,SC).结果表明:BPA对泥鳅24 h、48 h、72 h、96 h LC_(50)分别为8.32、8.02、6.97、6.43 mg·L~(-1),SC为2.24 mg·L~(-1);P-NP对泥鳅24 h、48 h、72 h、96 h LC_(50)分别为19.14、17.58、16.98、14.69 mg·L~(-1),SC为4.40 mg·L~(-1);HE染色结果显示,两种化学物质高浓度处理均使鳃小叶细胞脱落,鳃小叶变细;BPA与p-NP均为高毒性污染物,BPA比P-NP毒性更大.对泥鳅的毒性作用是随着体内积累浓度的增加、作用时间的延长而增强,并可造成鳃组织的严重受损.

Abstract：

By the methods of single factor toxicity test and histological observation, this paper studied the acute toxicity of bisphenol A (BPA) and p-nitrophenol (p-NP) to Misgurnus anguilli-cadatus and the histological changes of M. Anguillicadatus gill. The median lethal concentration (LC_(50)) and safe concentration (SC) were calculated. The LC_(50) of BPA for 24, 48, 72, and 96hours were 8. 32, .8. 02, 6. 97 and 6. 43 mg·L~(-1), respectively, and the SC was 2. 24 mg·L~(-1);while the mean LCw values of p-NP for 24, 48, 72, and 96 hours were 19. 14, 17. 58, 16. 98, and14. 69 mg·L~(-1), respectively, and the SC was 4. 40 mg·L~(-1). After exposed to BPA and p-NP,the ceils of branchial leaflets fell off, and the branchial leaflets became thin. Both BPA and p-Nphad high toxicity to M. Anguillicadatus, and BPA had higher toxicity than p-NP. With the increas-ing concentration and exposure time of BPA and p-NP, their toxicity effects to M. Anguillicadatus increased, and the damage on gill of M. Anguillicadatus was more severe.     

Acute toxicity of copper, zinc and manganese in single and mixed salt solutions to juvenile longfin dace, Agosia chrysogaster

The acute toxicity of copper, zinc and manganese and copper-zinc and copper-manganese mixtures were determined for juvenile longfin dace, Agosia chrysogaster in hard water bioassays (mean=218 mg 1⁻¹ CaCO₃). Copper-zinc was the most lethal toxicant (96-h L.c.₅₀= 0.21 mg 1⁻¹ copper and 0.28 mg 1⁻¹ zinc) and exhibited a more than additive toxicity which was in contrast to the additive toxicity of copper-manganese mixtures (96-h L.c.₅₀= 0.45 mg 1⁻¹ copper and 64.0 mg 1⁻¹ manganese). The toxicity of copper (96-h L.c.₅₀= 0.86 mg 1⁻¹) and zinc (96-h L.c.₅₀= 0.79 mg 1⁻¹) to the fish was similar but both were considerably more lethal than manganese (96-h L.c.₅₀= 130 mg 1⁻¹).     

Biomarkers of toxicity in Clarias gariepinus exposed to sublethal concentrations of polycyclic aromatic hydrocarbons

Physiological, biochemical and histological indices in Clarias gariepinus broodstock, and teratogenic indices in embryos exposed to sublethal concentrations of naphthalene, phenanthrene and pyrene were investigated in 2014 using a static-renewal bioassay protocol. Phenanthrene (1.41 mg l^?1) was the most toxic, followed by pyrene (1.53 mg l^?1) and naphthalene (7.21 mg l^?1), based on 96 h LC50 values. Hepatosomatic indices were significantly higher in naphthalene- and pyrene-treated males compared with solvent controls, whereas fecundity in females was significantly lower by factors of 2.4 (naphthalene), 2.8 (phenanthrene) and 2.4 (pyrene), compared with controls. Catalase levels were lower in female phenanthrene-treated fish compared with controls. Histological alterations observed in PAH-treated fish include oedema, inflammatory cells, epithelial lifting and hyperplasia in the gills, vacuolation, haemosiderin pigments and sinusoidal congestion in the liver, and degenerated zona radiata in the ovary. Teratogenic effects were not observed, as evidenced by the lack of histological alterations in embryos spawned from pre-exposed broodstock. Sex-specific responses and the utility of biomarkers at cellular and individual levels of organisation are therefore demonstrated for holistic evaluations of polycyclic aromatic hydrocarbons in ecotoxicological studies.     

A note on copper bioaccumulation in Mozambique tilapia,Oreochromis mossambicus (Osteichthyes: Cichlidae)

Copper (Cu) is one of the most commonly reported metal pollutants in African water bodies, but there are few studies on African freshwater fish species of copper accumulation and copper toxicity. Adult O. mossambicus were exposed to 0mg l^?1 (control) and 0.75mg l^?1 Cu for 96h and 0 (control), 0.11, 0.29 and 0.47mg l^?1 copper for 64 days. Samples of liver and gills were collected after 96h, and after 1, 32 and 64 days, respectively. There were significant differences in the mean Cu accumulation values in the liver and gills between the control and the Cu-exposed fish after the 96-h exposure. In fish exposed to 0.11 and 0.29mg l^?1 Cu for 64 days there was an increase in copper level in the tissues. In fish exposed to 0.47mg l^?1 Cu the concentration in the gill and liver tissue did not increase between Day 1 and Day 32. At this time, Cu accumulation in the liver was higher than for fish exposed to 0.11 and 0.29mg l^?1 Cu for 64 days. Exposure to approximately 0.47mg l^?1 Cu for more than 32 days induced mortality.     

Acute effects of sediments taken from an urban stream on physiological and biochemical parameters of the neotropical fish Prochilodus lineatus

Juveniles of Prochilodus lineatus were exposed to sediments collected from one of five sites along an urban stream into which various types of contaminants are discharged. After 24 or 96 h fish were examined and the results compared with those from control groups (fish exposed only to water, for the same period). Plasma ion levels varied significantly and fish exposed to site 5 sediment showed a transient increase in both sodium and chloride concentrations. Plasma glucose was significantly higher in fish exposed to sediment from sites 2 and 5. The higher liver glutathione-S-transferase activity registered in fish exposed to sediment from sites 1, 4 and 5 suggests the presence of organic contaminants at these sites and the enhancement of liver catalase activity in fish exposed to sediment from sites 3 and 4 may be due to contaminant-mediated oxyradical production. The overall results revealed that sites 4 and 5 are more severely contaminated, probably due to organic contaminants from agricultural sources and municipal landfill.