Toxicity assessment of polyethylene microplastics in combination with a mix of emerging pollutants on Physalaemus cuvieri tadpoles.

Studies in recent years have shown that aquatic pollution by microplastics (MPs) can be considered to pose additional stress to amphibian populations. However, our knowledge of how MPs affect amphibians is very rudimentary, and even more limited is our understanding of their effects in combination with other emerging pollutants. Thus, we aimed to evaluate the possible toxicity of polyethylene MPs (PE-MPs) (alone or in combination with a mix of pollutants) on the health of Physalaemus cuvieri tadpoles. After 30 days of exposure, multiple biomarkers were measured, including morphological, biometric, and developmental indices, behavioral parameters, mutagenicity, cytotoxicity, antioxidant and cholinesterase responses, as well as the uptake and accumulation of PE-MPs in animals. Based on the results, there was no significant change in any of the parameters measured in tadpoles exposed to treatments, but induced stress was observed in tadpoles exposed to PE-MPs combined with the mixture of pollutants, reflecting significant changes in physiological and biochemical responses. Through principal component analysis (PCA) and integrated biomarker response (IBR) assessment, effects induced by pollutants in each test group were distinguished, confirming that the exposure of P. cuvieri tadpoles to the PE-MPs in combination with a mix of emerging pollutants induces an enhanced stress response, although the uptake and accumulation of PE-MPs in these animals was reduced. Thus, our study provides new insight into the danger to amphibians of MPs coexisting with other pollutants in aquatic environments.

DNA damage and physiological responses in an Indian major carp Labeo rohita exposed to an antimicrobial agent triclosan.

This study is aimed to evaluate the toxic effects of triclosan (TCS) in an Indian major carp Labeo rohita. The 96-h LC50 value of triclosan to L. rohita was found to be 0.39 mg L-1. Fish were exposed to two sublethal concentrations (0.039 mg L-1, treatment I and 0.078 mg L-1, treatment II) of TCS for 35 days, and certain hematobiochemical, antioxidant, histopathological responses were measured. Compared to the control group, there was a significant (p < 0.05) decrease in the values and genotoxicity of hematological parameters such as hemoglobin (Hb), hematocrit (Hct), and erythrocyte (RBC) in TCS-exposed fish, but the values of leucocyte count (WBC), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) were found to be increased. A biphasic response in mean corpuscular hemoglobin concentration (MCHC) value was observed during the study period (35 days). Significant (p < 0.05) alterations in plasma biochemical parameters (glucose and protein), electrolytes (Na+, K+, and Cl-), and transaminases (GOT and GPT) were observed in fish treated with TCS in both treatments. Gill Na+/K+-ATPase activity was found to be decreased in fish treated with TCS in both treatments. Enzymatic and nonenzymatic antioxidant index levels have also fluctuated in all the tissues (gill, liver, and kidney). The histological lesions were comparatively more severe in the gill than the liver and kidney. Comet assay showed DNA damage on exposure at two sublethal concentrations. The present results suggest that TCS is highly toxic to fish even at sublethal concentrations.

Sublethal toxicity of quinalphos on oxidative stress and antioxidant responses in a freshwater fish Cyprinus carpio.

Extensive use of quinalphos, an organophosphorus pesticide, is likely to reach the aquatic environment and thereby posing a health concern for aquatic organisms. Oxidative stress and antioxidant responses may be good indicators of pesticide contamination in aquatic organisms. The data on quinalphos induced oxidative stress and antioxidant responses in carps are scanty. This study is aimed to assess the two sublethal concentrations of quinalphos (1.09 and 2.18 µL L-1 ) on oxidative stress and antioxidant responses of Cyprinus carpio for a period of 20 days. In liver, the malondialdehyde level was found to be significantly increased in both the concentrations. The results of the antioxidant parameters obtained show a significant increase in superoxide dismutase, catalase, and glutathione-S-transferase activity in liver of fish. These results demonstrate that environmentally relevant levels of the insecticide quinalphos can cause oxidative damage and increase the antioxidant scavenging capacity in C. carpio. This may reflect the potential role of these parameters as useful biomarkers for the assessment of pesticide contamination. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1399-1406, 2016.

"Fishcide" effect of the fungicide difenoconazole in freshwater fish (Labeo rohita): A multi-endpoint approach.

Difenoconazole is widely used to protect crops, fruits, and vegetables. However, this fungicide can enter aquatic environments and cause harmful effects to non-target organisms and induce little-known biological disorders. Thus, aiming to expand our knowledge about the ecotoxicity of difenoconazole on freshwater ichthyofauna, we aimed to determine the median lethal concentration (LC50) of difenoconazole and evaluate its possible impacts from different toxicity biomarkers, using freshwater fish Labeo rohita as a model system. Using the probit analysis method, the 96 h LC50 value of difenoconazole in the fish was calculated as 4.5 mg L-1. Posteriorly, fish were exposed to two sublethal concentrations (0.45 mg L-1 1/10th and 0.9 mg L-1 1/5th LC50 value) for 21 days. A significant reduction of superoxide dismutase (SOD) and catalase (CAT) activity was noted in the gill, liver, and kidneys of fish compared to the control groups. The level of glutathione-S-transferase (GST) and lipid peroxidation (LPO) activity was higher in all vital tissues of difenoconazole-treated fish. Histological alterations in the gill include epithelial lifting, lamellar fusion, hypertrophy, and epithelial necrosis. At the same time, the liver showed pyknotic nucleus, vacuolation, cellular edema and tubular necrosis, shrinkage of glomeruli, vacuolation, and pyknotic nuclei in the kidney. DNA damage was increased significantly with tail formation based on the concentration and time-dependent manner. Therefore, our study confirms that the exposure of L. rohita to difenoconazole induces negative biological consequences and sheds light on the danger of this fungicide for freshwater fish species. We believe that studies like ours can support actions and strategies for the remediation/mitigation of aquatic pollution by difenoconazole and for the conservation of freshwater ichthyofauna.

Gene expression profiling in liver of zebrafish exposed to ethylhexyl methoxycinnamate and its photoproducts.

In recent decades, the ecotoxicological potential of organic ultraviolet filters (OU-VFs) has received growing attention. However, the toxicity of its photoproducts or transformation products on freshwater vertebrates has been little explored. Therefore, the aim of the present study is to evaluate the possible adverse effects of ethylhexyl methoxycinnamate (EHMC) and its photoproducts [2-ethylhexanol (2-EH) and 4-methoxybenzaldehyde (4-MBA)] on the expression of stress-responsive and antioxidant genes. For this, zebrafish (Danio rerio) adults were exposed to pollutants at an environmentally relevant concentration (3 µg/L) and evaluated after 7, 14, and 21 days of exposure. The results of the principal component analysis (PCA) and two-way repeated measures (RM) ANOVA revealed that EHMC, 2-EH, and 4-MBA exposure caused significant downregulation of the genes hsp70, nrf2, cyp1a, ahr, sod1, sod2, cat, gstp1, gpx1a, gss, and gsr (on all trial days) in the liver of the animals. On the other hand, taken together, our data did not show significant differences between the effects induced by EHMC and its photoproducts. The genes evaluated in the present study play a major role in regulating the defensive antioxidant response against EHMC and its photoproducts. Additionally, our study provides an insight into the mechanisms of those OU-VFs in freshwater fish.

Transcriptional, biochemical and histological alterations in adult zebrafish (Danio rerio) exposed to benzotriazole ultraviolet stabilizer-328.

The occurrence of Benzotriazole Ultraviolet Stabilizer-328 (BUV-328) in different environmental and biological matrices is of immediate environmental concern. In the present study, we evaluated the toxicity of BUV-328 in zebrafish liver tissues to understand the role of oxidative damage in hepatotoxicity. Adult zebrafish were exposed to 0.01, 0.1 and 1 mg/L of BUV-328. At the end of 14, 28 and 42 days, liver tissues were examined for the responses of antioxidant enzymes, gene expression and histopathological alterations. The results indicated that superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were elevated at concentrations of 0.1 and 1 mg/L on 14th and 28th day. Glutathione S-transferase (GST) activity and malondialdehyde (MDA) levels were elevated in all the treated groups. The transcriptional levels of genes encoding sod, cat, gpx and gst enzymes were increased at 14th day and then declined (except sod on 28th day). Moreover, transcription of cyp1a and hsp70 were up-regulated throughout the study period. Histopathological lesions such as hypertrophy, cellular and nuclear enlargement, cytoplasmic and nuclear degeneration, necrosis with pyknotic nuclei, lipid and cytoplasmic vacuolization and nuclear displacement to the periphery were found to be increased with the dose and exposure duration. In brief, our findings indicate that even a low dose of BUV-328 is toxic to induce oxidative stress and liver damage in zebrafish over a long period of exposure.

Comparative toxicity of UV-filter Octyl methoxycinnamate and its photoproducts on zebrafish development.

In the present study, we explored the adverse effects of Octyl methoxycinnamate (OMC), and its photoproducts, namely 2-ethylhexanol (2-EH) and 4-methoxybenzaldehyde (4-MBA) on the developmental stages of zebrafish using various biomarkers such as developmental toxicity, oxidative stress, antioxidant response, neurotoxicity and histopathological changes. The 96 h effective concentrations (EC50) of OMC, 2-EH and 4-MBA were found to be 64.0, 34.0 and 3.5 µg/mL, respectively in the embryo toxicity test. Embryos exposed to the EC50 of OMC, 2-EH and 4-MBA showed time-dependent increases in the malformation, heart rate and hatching delay. The lipid peroxidation (LPO) level was significantly (p < 0.05) increased and both induction and inhibition of SOD, CAT, GPx and GST activities were observed in the zebrafish embryos exposed to OMC, 2-EH and 4-MBA. GSH activity was significantly (p < 0.05) decreased in the highest exposure groups, when compared with the control. AChE activity was increased in lower concentrations of OMC, 2-EH and 4-MBA exposed embryos whereas, the activity was found to be decreased in highest concentration. Moreover, the histopathological studies showed severe damage to the muscle fibers and yolk sac regions of the larvae with 4-MBA treatment. The photoproduct 4-MBA has the highest toxic effect, followed by 2-EH and OMC. Our results provide useful insights into the impacts of OMC and its photoproducts on zebrafish development.

Pyriproxyfen induced impairment of reproductive endocrine homeostasis and gonadal histopathology in zebrafish (Danio rerio) by altered expression of hypothalamus-pituitary-gonadal (HPG) axis genes.

Pyriproxyfen (PPF), a broad-spectrum insecticide known to cause reproductive and endocrine disruption in invertebrates, while the data is scarce in aquatic vertebrates. The goal of this study is to investigate the impact of PPF on reproductive endocrine system of male and female zebrafish along hypothalamus-pituitary-gonadal (HPG) axis. In brain, PPF caused significant alteration in the transcripts of erα, lhß, and cyp19b genes in male and fshß, lhß, and cyp19b genes in female zebrafish. The downstream genes of steroidogenic pathway like, star, 3ßhsd, 17ßhsd, and cyp19a expression were significantly altered in gonad of both sexes. Subsequent changes in circulatory steroid hormone levels lead to imbalance in hormone homeostasis as revealed from estradiol/testosterone (E2/T) ratio. Further, the vitellogenin transcript level was enhanced in hepatic tissues and their blood plasma content was increased in male (16.21%) and declined in female (21.69%). PPF also induced histopathological changes in gonads such as, reduction of mature spermatocytes in male and vitellogenic oocytes in female zebrafish. The altered E2/T ratio and gonadal histopathology were supported by the altered transcript levels of HPG axis genes. Overall, these findings provide new insights of PPF in zebrafish reproductive system and highlights for further investigations on its potential risks in aquatic environment.

Developmental toxicity and biological responses of zebrafish (Danio rerio) exposed to anti-inflammatory drug ketoprofen.

Ketoprofen a nonsteroidal anti-inflammatory drug (NSAID) is widely used in over-the-counter to treat pain, swelling and inflammation. Due to extensive application these drugs has been detected in surface waters which may create a risk to aquatic organisms. The aim of the present study is to assess the ecotoxicity of ketoprofen at different concentrations (1, 10 and 100 µg/ml) on embryos and adult zebrafish (1, 10 and 100 µg L-1) under laboratory conditions. In embryos, concentration dependent developmental changes such as edema, spinal curvature, slow heartbeat, delayed hatching, and mortality rate were observed. In adult zebrafish, biochemical enzymes such as AST, ALT and LDH activities were significantly (P < 0.05) increased whereas a decrease in Na+/K+-ATPase activity was noticed in all the tested concentrations of the drug ketoprofen. Similarly, exposure of ketoprofen caused a significant decrease in antioxidant levels in liver tissue (SOD, CAT, GSH, GPx, and GST). However, lipid peroxidation (LPO) level in liver tissue was found to be increased. The histopathological studies further evidenced the impact of ketoprofen in the liver tissue of zebrafish. The present result concludes that ketoprofen could have an impact on the development and biological endpoints of the zebra fish at above concentrations. The malformation in the development of the embryo and changes in the biological end points may provide integrated evaluation of the toxic effect of ketoprofen on zebrafish in a new perspective.

Toxicity assessment of pyriproxyfen in vertebrate model zebrafish embryos (Danio rerio): A multi biomarker study.

Pyriproxyfen (2-[1-methyl-2-(4-phenoxyphenoxy) ethoxy] pyridine) (PPF), a pyridine-based pesticide widely used to control agricultural insect pests and mosquitoes in drinking water sources. However, its ecotoxicological data is limited in aquatic vertebrates particularly in fish. Hence, the present study aimed to evaluate the adverse effect of PPF in zebrafish embryo development (Danio rerio). In order to investigate the impact of PPF, embryos were exposed to 0.16, 0.33 and 1.66 µg/mL (0.52, 1.04 and 5.2 µM, respectively) for 96 hpf and various biomarker indices such as developmental toxicity (edema formation, hyperemia, heart size and scoliosis), oxidative stress (reactive oxygen species (ROS), lipid peroxidation (LPO) and nitric oxide (NO)), antioxidant responses (superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx) and reduced glutathione (GSH)), biochemical (lactate dehydrogenase (LDH) and acid phosphatase (AP)), neurotoxicity (acetylcholinesterase (AChE)), genotoxicity (apoptosis and DNA damage) and histopathological changes were determined. The results showed that severe developmental deformities and changes in heart rate were observed in embryos treated with highest (1.66 µg/mL) concentration than the control (P < 0.05). Heart size measurement showed that, significant change in heart size (P < 0.01) was observed in embryos of 96 hpf only at 1.66 µg/mL PPF exposure. The oxidative stress was apparent at highest test concentration (1.66 µg/mL) as reflected by the elevated ROS, LPO and NO and changes in antioxidant enzyme activities including SOD, CAT, GST and GPx (P < 0.05). Besides, GSH level and AChE activity were significantly lowered in 1.66 µg/mL PPF exposed group than the control. After 96 hpf of PPF exposure, no significant changes were found in AP activity whereas, a biphasic response was observed in the LDH activity. There was no genotoxic effect in embryos exposed to PPF at 0.16 and 0.33 µg/mL, while significant (P < 0.05) DNA damage and apoptosis were found in 1.66 µg/mL treated group. Histopathological analysis revealed that exposure to PPF at 1.66 µg/mL resulted in thinning of heart muscles, pericardial edema and hyperemia while there was no obvious changes were observed in other treatment groups. Hence, the results of the present study demonstrate that PPF could cause adverse effect on early developmental stages of zebrafish at higher concentration.