Long-term exposure to polycyclic musk tonalide - A potential threat to juvenile zebrafish (Danio rerio)?

Polycyclic musk compounds are commonly used in personal care products to replace expensive natural fragrances. Due to their huge consumption, they have become a part of the aquatic environment. In the present study, a two-month exposure effect of tonalide on juvenile zebrafish (Danio rerio) was investigated. We determined the vitellogenin concentration to define the potential endocrine-disrupting effect of tonalide and also analysed selected indices to evaluate the induction of oxidative stress. The environmentally relevant concentration of tonalide (i.e., 500 ng/l) caused a significant decrease in the catalase activity (P < 0.05) and a significant increase (P < 0.05) in the lipid peroxidation. An increasing lipid peroxidation was also recorded for the highest concentration group tested (i.e., 50 000 ng/l). On the other hand, no significant changes were recorded in vitellogenin in all the exposed groups. Thus, based on these results, we have not demonstrated the endocrine-disrupting activity of tonalide in zebrafish. The results of the oxidative stress indices showed a significant impairment of the antioxidant defence after the two-month tonalide exposure, which could indicate part of the adaptive response to the tonalide toxicity.

Efficacy and safety of higher oral doses of azaperone to achieve sedation in pigs.

The aim of this study is to evaluate the possibility of achieving more effective and prolonged sedation in pigs by the oral administration of increased doses of azaperone and to evaluate its safety. This was performed through a prospective randomised and double blinded study. A total of 32 weaned piglets were divided into 4 groups (8 in each group). Group A was given 1 ml of saline orally and served as the control group. Group B received azaperone orally at a dose of 4 mg/kg b.w. Group C received azaperone orally at a dose of 8 mg/kg b.w. Group D was given azaperone orally at a dose of 12 mg/kg b.w. The response to the defined stimulus, movement level, degree of salivation, body temperature, respiratory frequency, blood plasma azaperone concentration and biochemical variables were included in the trial. We found that by increasing the dose of the orally administered azaperone, the onset of the sedation is faster, the end of the sedation starts later and the sedation time is longer. However, the use of higher doses of oral azaperone is not suitable for piglets because the doses negatively affect the respiratory rate, body temperature, some biochemical parameters and cause the immobility of the piglets.

Use of IHF-QD Microscopic Analysis for the Detection of Food Allergenic Components: Peanuts and Wheat Protein.

The aim of the study was to analytically evaluate quantum dots in immunohistofluorescence (IHF-QD) microscopic imaging as detectors of food allergens-peanut and wheat. The experiment was designed as two in silico experiments or simulations: (a) models of pastry samples were prepared with the addition of allergenic components (peanut and wheat protein components) and without the addition of allergenic components, and (b) positive and negative commercial samples underwent food allergen detection. The samples from both simulations were tested by the ELISA and IHF-QD microscopic methods. The primary antibodies (secondary antibodies to a rabbit Fc fragment with labeled CdSe/ZnS QD) were labelled at 525, 585, and 655 nm emissions. The use of quantum dots (QDs) has expanded to many science areas and they are also finding use in food allergen detection, as shown in the study. The study indicated that differences between the ELISA and IHF-QD microscopic methods were not observable among experimentally produced pastry samples with and without allergenic components, although differences were observed among commercial samples. The important value of the study is certainly the differences found in the application of different QD conjugates (525, 585, and 655). The highest contrast was found in the application of 585 QD conjugates that can serve for the possible quantification of present food allergens-peanuts and wheat. The study clearly emphasized that QD can be used for the qualitative detection of food allergens and can represent a reliable analytical method for food allergen detection in different food matrixes.

Comprehensive fitness evaluation of common carp (Cyprinus carpio L.) after twelve weeks of atrazine exposure.

Atrazine is frequently detected in surface waters and negatively affect physiological function in aquatic organisms. Even though numerous authors have intensively studied its toxicity, only limited information concerning the long-term fish exposure is available. The aim of this study was evaluation of chronic exposure in common carp. Fish were exposed to a range of atrazine concentrations (0.3 - environmentally relevant concentration; 300; 1000; and 3000 µg/l) for twelve weeks. The potential impact of atrazine exposure was studied using various markers (behaviour; biometrical characteristics; haematological, biochemical and oxidative stress indices and histopathological changes). Most alterations were recorded at the highest concentration (3000 µg/l) which is probably due to a combined effect of both the herbicide exposure and intensive parasite infection development during second week. This group was cancelled after three weeks due to adverse health status, which partly confirmed atrazine immunotoxicity. Chronic exposure resulted in long-term reduction in feed intake followed by a significant decrease in body weight and morphological changes in gill at 1000 µg/l. At the same concentration, significant alterations in haematological (e.g. increase in erythrocyte, leukocyte, lymphocytes and neutrophil counts as well as decrease in monocyte counts) and biochemical (e.g. changes in enzyme activities; increase in glucose; decrease in ammonia) indices were documented, especially during first three weeks. Similar trend, but not so intensive, was observed at 300 µg/l. Moreover, significant changes were observed in various indicators of oxidative stress. These alterations were highly variable with both increasing and decreasing trends depending on dose and analysed tissue. Significant changes, especially in white blood profile, enzyme activities and oxidative stress indices, were proven even at 0.3 µg/l. Obtained results indicate that chronic atrazine exposure of common carp can negatively influence many indicators of health status such as behaviour, immune response, haematological and biochemical profile, oxidative stress indices and organ histopathology.

Herpesvirus associated dermal papillomatosis in Williams' mud turtle Pelusios williamsi with effects of autogenous vaccine therapy.

An adult female of Williams' mud turtle, Pelusios williamsi long-term captive, that was allegedly caught wild in Kenya was found to have developed papilloma-like skin lesions. Excised tumors were examined histologically after routine processing with hematoxylin and eosin (H & E) stained slides, examined for the presence of viral particles by electron microscopy employing negative staining, and examined for the presence of viral DNA by PCR. Microscopic features in pre-treatment biopsies were fully diagnostic and consistent with multifocal squamous cell papilloma. Viral-type inclusion bodies were not identified. Turtle was found to be infected by reptilian herpesvirus. Association with herpesvirus and vast multiplicity of tumors thwarted surgical solution. An autogenous vaccine was prepared using 5 g of excised fresh tissue, aseptically ground, treated with diluted formalin, centrifuged to obtain a supernatant, and subsequently exposed to UV light. Autogenous vaccine induced substantial areas of necrosis of the papillomatous lesions noted by the loss of cytological architecture, nuclear loss, and by edema. The outer edges of the healing biopsies appeared to be regenerating. Therefore, our vaccine application could be considered as effective. It is difficult to treat and eliminate herpesvirus infection because of its cryptic presence and sudden onset of disease. Successful application of autogenous vaccine could be a potentially promising strategy, which deserves further testing.

Multibiomarker Responses of Juvenile Stages of Zebrafish (Danio rerio) to Subchronic Exposure to Polycyclic Musk Tonalide.

Synthetic polycyclic musks, widely used as additives in personal care products, are present in both biotic and abiotic matrices of the aquatic environment at concentrations of ng/l to µg/l. Although they are determined at comparatively low concentrations, these levels are biologically relevant and pose a significant growing risk as stressors to aquatic organisms. The purpose of our study was to evaluate the effects of 28-day-long exposure to polycyclic musk tonalide in zebrafish juvenile stages (Danio rerio) using selected biomarkers. Environmentally relevant concentrations of tonalide caused significant changes in selected enzyme activities in the experimental groups exposed to the highest concentrations. The activity of glutathione S-transferase and lipid peroxidation increased significantly (p < 0.05) after exposure to the highest concentration (50,000 ng/l) compared with the control. A similar trend was observed in catalase activity; there was a significant increase (p < 0.05) after exposure to two highest concentrations of tonalide (5000 and 50,000 ng/l). In addition, a statistically significant decrease (p < 0.05) in glutathione reductase activity was found in the lowest test concentration of tonalide (50 ng/l). None of the tested concentrations resulted in histopathological changes in liver, kidney, skin, or gill. Furthermore, no effects on body weight, body length, specific growth rate, and behavior were observed. Our results showed that tonalide exposure induced profound changes in the activities of antioxidant and detoxifying enzymes, such changes representing an adaptive response of the fish organism to tonalide toxicity.

Effect of T-2 toxin-contaminated diet on common carp (Cyprinus carpio L.).

The T-2 toxin, a fungal metabolite produced by Fusarium molds, occurs in a range of agriculture products. Reduced availability of fish meal has led to increasing use of cereals as a source of protein in commercial aquaculture feeds, which has increased the potential for mycotoxin contamination. The purpose of this study was to investigate toxicity of T-2 toxin intake in common carp (Cyprinus carpio L.) using haematological, biochemical and immunological parameters and oxidative stress indices. In a four-week feeding trial, fish were fed a commercial diet with 5.3 mg/kg T-2 toxin added. Ingestion of contaminated diet did not lead to mortality of fish, probably due to lower feed intake. On the other hand, it significantly affected haematological variables such as haematocrit, haemoglobin, red blood cell counts leading to anemia and white blood cell counts leading to leukopenia due to lymphopenia. Plasma glucose concentration and alanine amino transferase activity showed a significant increase while triglycerides concentration decreased. Activity of ceruloplasmin was significantly decreased in plasma. Further, liver glutathione S-transferase activity was significantly increased and catalase activity decreased, in parallel with a significant increase in caudal kidney catalase activity and a decrease in glutathione peroxidase activity. Finally, lipid peroxidation (detected as malondialdehyde) was significantly increased in the liver and caudal kidney. Changes in non-specific immune response and cytokine levels in head kidney indicated immune system sensitivity to T-2 toxin. Overall, the results demonstrate that this feed-borne mycotoxin is able to induce anaemia and oxidative stress and cause changes in the immune response of common carp.

Haemogregarines of freshwater turtles from Southeast Asia with a description of Haemogregarina sacaliae sp. n. and a redescription of Haemogregarina pellegrini Laveran and Pettit, 1910.

The uniform morphology of the developmental stages of Haemogregarina species and the insufficient information supplied by the simplistic descriptions of previous authors complicates their differential diagnosis and proper species identification. In this study, we detected Haemogregarina spp. in 6 out of 22 (27.2%) examined turtles originating from Southeast Asia, Malayemys subtrijuga (n = 4), Sacalia quadriocellata (n = 1) and Platysternon megacephalum (n = 1), and compared them with the available literature data. Microscopic analysis of our isolates distinguished 2 morphological species, Haemogregarina pellegrini and one new species, being described in this paper as Haemogregarina sacaliae sp. n. Phylogenetic analyses based on 1210 bp long fragment of 18S rDNA sequences placed both haemogregarines firmly within the monophyletic Haemogregarina clade. Isolates of H. pellegrini from 2 distantly related turtle hosts, M. subtrijuga and P. megacephalum, were genetically identical. Despite the fact that numerous Haemogregarina species of turtles have been described, the incompleteness of the morphological data and relatively low host specificity provides the space for large synonymy within this taxon. Therefore, a complex approach combining microscopic analyses together with molecular-genetic methods should represent the basic standard for all taxonomic studies.

Oxidative stress indices and histopathological effects of the nonsteroidal antiinflammatory drug naproxen in adult zebrafish (Danio rerio).

OBJECTIVES: The aim of this study was to investigate the effects of the nonsteroidal antiinflammatory drug naproxen on adult zebrafish (Danio rerio). METHODS: Three months old zebrafish (Danio rerio) were exposed to naproxen at concentrations of 0.001, 0.1 and 5 mg.L(-1). We focused on the changes in oxidative stress indices during and at the end of the experiment and histopathological examination of tissues after a two week long exposure period. RESULTS: We found that a 3 day long exposure to naproxen causes mild oxidative stress and affects detoxification in zebrafish, which is demonstrated by the increased activity of glutathione peroxidase and glutathione S-transferase at 0.001 and 0.1 mg.L(-1) of naproxen, respectively. After a 7 day long exposure to 0.1 and 5 mg.L(-1), more potent effects on enzymes occur. However, these effects are only short lasting. At the end of the experiment, the activities of the target enzymes recover back to homeostatic baseline levels. Except catalase, which is induced only after a two week long exposure to the environmental concentration of naproxen. Despite the fact that naproxen causes mild oxidative stress in zebrafish, exposure to this drug does not result in lipid peroxidation. Histopathological examination revealed obvious changes to the gills and liver even at exposure to the environmental concentration of naproxen. CONCLUSION: This study demonstrates that the environmental concentration of naproxen can slightly influence both the antioxidant defense system and histopathology of non-target fish.

Effects of subchronic exposure to N,N-diethyl-m-toluamide on selected biomarkers in common carp (Cyprinus carpio L.).

DEET (N,N-diethyl-m-toluamide) is the most common active ingredient in the insect repellents commonly detected in European groundwater. The aim of this study was to investigate the effect of subchronic DEET exposure on biochemical and haematological parameters, antioxidant enzymes, including catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase, and the amount of thiobarbituric acid reactive substances (TBARS) in common carp (Cyprinus carpio L.). Two specific proinflammatory and anti-inflammatory cytokine genes were selected to assess an immunological status of the fish. Fish were exposed for 28 days to three concentrations of DEET (1.0 µg/L, 0.1 mg/L, and 1.0 mg/L) where 1 µg/L is corresponding to the concentration found in the environment. DEET had a significant (P < 0.05) effect on increased RBC, decreased mean corpuscular volume (MCV), and mean corpuscular haemoglobin value (MCH) compared to control groups in the concentration of 1 mg/L. A significant decline (P < 0.05) in triacylglycerols (TAG) in plasma was found in the concentration of 1 mg/L compared to the control groups. The parameters of oxidative stress in tissues of common carp were weekly affected and immunological parameters were not affected.

Effects of subchronic exposure of diclofenac on growth, histopathological changes, and oxidative stress in zebrafish (Danio rerio).

The aim of this study was to investigate effects of subchronic exposure to sublethal levels of diclofenac on growth, oxidative stress, and histopathological changes in Danio rerio. The juvenile growth tests were performed on Danio rerio according to OECD method number 215. Fish at the age of 20 days were exposed to the diclofenac environmental concentration commonly detected in the Czech rivers (0.02 mg L(-1)) and the range of sublethal concentrations of diclofenac (5, 15, 30, and 60 mg L(-1)) for 28 days. A significant decrease (P < 0.01) in the fish growth caused by diclofenac was observed in the concentrations of 30 and 60 mg L(-1). The identified value of LOEC (lowest observed effect concentration) was 15 mg L(-1) of diclofenac and NOEC (no observed effect concentration) value was 5 mg L(-1) of diclofenac. We did not find histopathological changes and changes of selected parameters of oxidative stress (glutathione S-transferase, glutathione reductase) in tested fish. The environmental concentration of diclofenac in Czech rivers did not have any effect on growth, selected oxidative stress parameters (glutathione S-transferase, glutathione reductase), or histopathological changes in Danio rerio but it could have an influence on lipid peroxidation.

Effects of the exposure of fish to triazine herbicides.

Triazines are an important group of herbicides, which have been used extensively or selectively both in agricultural and non-agricultural areas worldwide for more than fifty years to control broadleaf and some grassy weeds. As a consequence of their massive application, they have become and remain significant environmental pollutants, especially in aquatic ecosystems. Fish are an integral part of the aquatic environment and are, therefore, suitable models for the study of the behavioral, biological, and biochemical effects of triazine exposure. We have summarized and evaluated the effects of triazine herbicides on fish in order to provide an overview of current information on triazines. The overall effects of triazine herbicide exposure on the physiology of fish were evaluated by considering a variety of parameters in a number of reports. Haematological and biochemical profiles of blood provide important information about the internal environment of the organism and the general physiology and health status of triazine exposed fish. According to studies using biotransformation and bioaccumulation indices to estimate the effects of triazines on fish, changes in fish metabolism reflect the pollution of the environment by these herbicides. The responses of antioxidant defence systems in fish to triazine exposure could be an adaptive mechanism to protect the fish from triazine-induced oxidative stress. Acute exposure to triazines affects reproduction or reproductive development in fish; however, some triazines did not affect fish behavior during long-term exposure to low concentrations. Nevertheless, an impact on the overall behavioral response of fish cannot be excluded.

Biochemical markers for the assessment of pollution of selected small streams in the Czech Republic.

OBJECTIVES: The objective of the presented study was to investigate levels of the most important biochemical markers in fish which were caught upstream and downstream from sites near waste water treatment plants situated at 3 selected small streams of the Czech Republic. Organic pollutants and mercury were determined in muscle tissue of fish for complex assessment. DESIGN: Levels of biochemical markers (cytochrome P450 (CYP450), ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), vitellogenin (VTG)) were measured in selected tissues (liver, plasma, brain, gill and kidney) samples of brown trout. The concentrations of persistent organic pollutants (POPs) and mercury were determined in muscle samples. RESULTS: Significantly higher levels of biochemical markers (EROD, GST, VTG) were measured downstream from waste water treatment plant (WWTP) in Vlachovo Brezí (the small Libotýnský brook). Values of biochemical markers at the other localities Cerný brook and Moravice River were measured downstream from WWTP consistently or slightly lower with values of upstream. The values of POPs and mercury were in all monitored sites higher upstream compared with downstream from WWTP. The highest values of POPs and mercury were found on Cerný brook in Bruntál (more industrial and agricultural activity). CONCLUSION: The highest occurrence of selected POPs and mercury was generally upstream suggesting that small WWTP are able to partially remove these substances in those locations. But finding higher values of biochemical markers in downstream shows that there are many other substances endocrine disrupting chemicals (EDCs) that WWTP are unable to remove.

Oxidative stress responses in zebrafish Danio rerio after subchronic exposure to atrazine.

Atrazine is one of the most used pesticides all over the world and it is frequently detected in surface water. The aim of this study was to investigate if zebrafish exposure to atrazine could induce oxidative stress and changes in detoxifying system. Juvenile fish were exposed to sublethal concentrations of 0.3, 3, 30, or 90 µg L(-1) for 28 days. The level of oxidized lipids increased in experimental groups exposed to atrazine at 30 and 90 µg L(-1) compared to control. Activity of glutathione S-transferase decreased in group with the highest concentration compared to control. A significant decline was observed in catalase activity in all experimental groups compared to control. Activity of superoxide dismutase increased only in experimental group exposed to atrazine at 30 µg L(-1) compared to control. Activity of glutathione peroxidase and reductase (GR) increased in experimental groups exposed to atrazine at 0.3 (only for GR activity) and 90 µg L(-1) compared to control. Our results showed that atrazine exposure had profound influence on the oxidative stress markers and detoxifying enzyme of the exposed zebrafish. The changes in antioxidant enzyme activities could be an adaptive response to protect the fish from the atrazine-induced toxicity.

Decrease in the antioxidant capacity in beverages containing tea extracts during storage.

The aim of this work was to determine antioxidant capacity of beverages containing black, white, and green tea extracts using the photochemiluminescence method, and to monitor its changes based on the storage temperature and time. Samples were stored at two different temperatures (refrigerated at 4°C and laboratory temperature 22°C), analyzed after opening of the original package, and consequently after 4 and 7 days. Results of the antioxidant capacity are expressed as the standard equivalents, that is, ascorbic acid in mmol/L. The highest mean value of the antioxidant capacity was found after opening of the original package in fruit-juice-enriched samples and totaled 9.793 mmol/L. This group revealed significant dependence (P < 0.05) not only on the storage time, but also temperature. In samples without added fruit juices containing preservatives the value was 0.428 mmol/L. This group showed significant dependence (P < 0.05) on the decrease of antioxidant capacity only when based on the storage time. Samples without fruit juices or preservatives showed significant decrease in the antioxidant capacity (P < 0.05) after 4 days of storage based on the storage time. The dependence on temperature was revealed only after 7 days of storage.

The effects of subchronic exposure to terbuthylazine on zebrafish.

OBJECTIVES: Terbuthylazine belongs to the group of symmetrical triazine herbicides used extensively in agriculture and non-agricultural sites, primarily to control broadleaf and some grassy weeds that have become ubiquitous contaminants of the environment. The aim of this study was to assess the impact of terbuthylazine in surface waters on fish under experimental conditions. The effects of subchronic exposure to terbuthylazine on fish growth and the development of histopathological changes in selected organs (gill, kidney, liver), and on the activity of some biochemical parameters - glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) and on the concentration of thiobarbituric acid reactive substances (TBARS) in Danio rerio were investigated during a 28 day toxicity test. METHODS: Juvenile growth tests were performed on Danio rerio according to OECD guideline No. 215. Fish at the age of 30 days were exposed for 28 days to a range of sublethal concentrations of terbuthylazine (0.55 - environmental concentration, 150, 400, 700 and 1000 µg/L). RESULTS: There were no significant differences (p<0.05) between the specific growth rates (r) of the test groups and control group. Histopathological examination revealed pathological changes only in the liver in fish exposed to terbuthylazine at concentrations of 700 and 1000 µg/L. Significant differences (p<0.05) in activities of biochemical markers were found in GST (400, 700 and 1000 µg/L), GR (700 and 1000 µg/L), significant differences (p<0.05) of TBARS concentration were found at 1000 µg/L. CONCLUSIONS: Based on the results of growth rate, the histopathological examination of selected organs, and the results of biochemical parameters, it was found that the environmental concentration of terbuthylazine did not have any effects on juvenile D. rerio. The values of NOEC and LOEC for terbuthylazine for juvenile D. rerio were 150 µg/L and 400 µg/L.

Oxidative stress parameters in early developmental stages of common carp (Cyprinus carpio L.) after subchronic exposure to terbuthylazine and metribuzin.

OBJECTIVES: This work assessed the subchronic effects of the triazine compounds terbuthylazine and metribuzin on embryo-larval stages of common carp (Cyprinus carpio L.) using biotransformation enzyme and selected oxidative stress parameters. DESIGN: Early developmental stages of fish, from embryo to larvae, were exposed to terbuthylazine at concentrations of 0.9, 160, 520, and 820 µg/L, and metribuzin at concentrations of 0.9, 4, 14, and 32 mg/L for 30 days and compared to the non-treated control group. For assessment of free radical defence the activities of glutathione S-transferase (GST) and glutathione reductase (GR) were measured; the lipid peroxidation (TBARS) level was determined as a marker of free radical damage. RESULTS: Terbuthylazine had no significant effect (p>0.05) at any tested concentrations on GST activity. Terbuthylazine caused an increase (p<0.05) in GR activity at the concentration of 520 µg/L. We found a non-significant (p>0.05) effect of terbuthylazine exposure on TBARS level. In metribuzin treated groups, the activity of GST was increased (p<0.05) in all experimental groups, with the highest at the concentration of 32 mg/L. GR activity was increased in the 0.9, 4, and 14 mg/L groups and the TBARS levels were the highest in the 0.9 mg/L group. CONCLUSION: Our results suggest that terbuthylazine and metribuzin induced oxidative stress in embryo-larval stages of common carp. Increased activities of biotransformation enzymes or antioxidant defence enzymes were markers of free radical attack. There was low lipid peroxidation in early developed fish after triazine exposure.