The effects of ciprofloxacin on early life stages of common carp (Cyprinus carpio).

The authors performed a toxicity test with ciprofloxacin in fertilized eggs of common carp according to guideline 210 of the Organisation for Economic Co-operation and Development. The tested concentrations were 1 µg L(-1) , 100 µg L(-1) , 500 µg L(-1) , 1000 µg L(-1) , and 3000 µg L(-1) . Accelerated hatching was found in all groups exposed to ciprofloxacin, but significant growth reduction was found only in the group exposed to the highest concentration (3000 µg L(-1) ). Increased numbers of macroscopic morphological anomalies were observed on day 6 of the test (after hatching). The highest numbers of macroscopic morphological anomalies were observed in the groups of free embryos and larvae exposed to ciprofloxacin concentrations of 100 µg L(-1) , 500 µg L(-1) , 1000 µg L(-1) , and 3000 µg L(-1) (20-23% of tested samples). A gradual decrease in glutathione S-transferase activity was detected in all experimental groups exposed to ciprofloxacin, but significant differences (p < 0.01) were found only in groups treated with 500 µg L(-1) and 3000 µg L(-1) . Glutathione peroxidase and catalase exhibited increased activity in most of the tested concentrations (p < 0.01 and <0.05, respectively), whereas decreased glutathione reductase activity was found in the groups exposed to ciprofloxacin concentrations of 500 µg L(-1) and 3000 µg L(-1) (p < 0.05). The concentration of thiobarbituric acid-reactive substances was significantly lower (p < 0.01) in all experimental groups exposed to ciprofloxacin. The lowest-observed-effect concentration of ciprofloxacin was 1 µg L(-1) . These results suggest that hatching, early ontogeny, occurrence of morphological anomalies, antioxidant and biotransformation enzyme activity, and lipid peroxidation in fish can be affected by ciprofloxacin. Environ Toxicol Chem 2016;35:1733-1740. © 2015 SETAC.

Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene.

Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-µM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants at 21 days after fertilization. Moreover, the body cavities of MO-treated and nontreated fish were examined by histology and in situ hybridization, showing gonads which had no germ cells in morphants at various stages (60, 150, and 210 days after fertilization). Taken together, these results report the first known and functional method of sturgeon sterilization.

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.

The effects of atrazine exposure on early life stages of common carp (Cyprinus carpio).

OBJECTIVES: Atrazine and its chloro-s-triazine metabolites are found in surface water and groundwater of the Czech Republic, although their use has been banned since 2005. The objective of the study was to determine the toxic effects of atrazine at an environmentally relevant concentration of atrazine, 0.3 µg.L-1 and at concentrations of 30, 100, and 300 µg.L-1 on morphometric and condition characteristics, development of early life stages, and antioxidant defense enzymes of common carp. METHODS: The embryo-larval toxicity test was performed according to the OECD Guidelines 210 (Fish, Early-life Stage Toxicity Test). RESULTS: Atrazine exposure showed no effect on morphometric and condition characteristics or histology. Exposure at 0.3 µg.L-1 was associated with significantly increased activity of glutathione peroxidase, glutathione S-transferase, superoxide dismutase, and catalase compared to control. Activity of glutathione reductase was slightly higher at a concentration of 0.3 µg.L-1, with significantly lower (p<0.05) activity observed in groups exposed to 30, 100, and 300 µg.L-1 compared to the group exposed to 0.3 µg.L-1. The level of oxidized lipids was slightly higher in groups exposed to atrazine at 100 and 300 µg.L-1 compared to controls. CONCLUSIONS: Atrazine has a significant influence on the biotransformation enzyme and oxidative defense enzymes of early life stages of common carp. The lowest observed effect concentration (LOEC) was 0.3 µg.L-1.

Oxidative stress parameters in fish after subchronic exposure to acetylsalicylic acid.

OBJECTIVES: The aim of this study was to investigate the effects of subchronic exposure of juvenile development stages of zebrafish (Danio rerio) to acetylsalicylic acid using selected oxidative stress biomarkers. DESIGN: Toxicity test with acetylsalicylic acid was performed according to the OECD Guideline No. 215, fish D. rerio aged 30 days were used. The tested concentrations were 0.004, 0.4, 40, 120 and 250 mg.L-1, duration of the test was 28 days. Products of lipid peroxidation and antioxidant enzymes were determined as the markers of oxidative stress. RESULTS: Significantly increased glutathione S-transferase activity was found in fish exposed to acetylsalicylic acid concentrations 40, 120 and 250 mg.L-1. The highest values of glutathione reductase activity were found in the groups exposed to acetylsalicylic acid concentrations 0.4, 40 and 120 mg.L-1. In the group exposed to acetylsalicylic acid concentrations 40 mg.L-1, catalase activity was significantly higher compared to the control group. Significantly higher glutathione peroxidase activity was found in the groups exposed to acetylsalicylic acid concentrations 0.004 and 120 mg.L-1. The concentrations of TBARS were lower in fish exposed to acetylsalicylic acid at all tested concentrations compared to control. CONCLUSION: The subchronic exposure of zebrafish to acetylsalicylic acid causes an increase in activity of antioxidant and biotransformation enzymes and a decrease in lipid peroxidation.

The effect of oyster mushroom ß-1.3/1.6-D-glucan and oxytetracycline antibiotic on biometrical, haematological, biochemical, and immunological indices, and histopathological changes in common carp (Cyprinus carpio L.).

The aim of the study was to evaluate the effect of micronized ß-1.3/1.6-D-glucan (BG) derived from the oyster mushroom Pleurotus ostreatus Hiratake and tetracycline antibiotic oxytetracycline (OTC) on biometrical, haematological, biochemical, and immunological indices, and histopathological changes in tissues of one- to two-year-old common carp (Cyprinus carpio L.). The fish tested were divided into five experimental groups and one control. Carp in the control group were fed commercial carp feed pellets. Fish in the five experimental groups were fed the same pellets supplemented with either OTC, a combination of OTC and BG, or BG as follows: 75 mg oxytetracycline kg(-1) bw (OTC group), 75 mg oxytetracycline kg(-1) bw and 0.5% ß-glucan (OTC + 0.5% BG group), 75 mg oxytetracycline kg(-1) bw and 2.0% ß-glucan (OTC + 2.0% BG group), 0.5% ß-glucan (0.5% BG group), and 2.0% ß-glucan (2.0% BG group). OTC- and BG-supplemented diets and the control diet were administered to experimental and control carp for 50 days (i.e. samplings 1-3, the exposure period); for the following 14 days, fish were fed only control feed pellets with no OTC or BG supplementation (i.e. sampling 4, the recovery period). Blood and tissue samples were collected both during, and at the end of the study. No significant changes in biometrical indices (i.e. total length, standard length, total weight, hepatosomatic and spleen somatic index, and Fulton's condition factor) were found in experimental carp compared to control in any sampling. In haematological indices, significant changes were found only in sampling 2, in which shifts in PCV (P < 0.01), Hb (P < 0.01), and WBC (P < 0.01), and in the counts of lymphocytes (P < 0.01), monocytes (P < 0.01), and neutrophil granulocytes-segments (P < 0.05) were revealed. As for biochemical profiling, plasma concentrations of glucose, albumins, cholesterol, natrium, and chlorides (all P < 0.01), and total proteins, lactate, phosphorus, and potassium (all P < 0.05) as well as the catalytic activity of ALP (P < 0.05) were altered in common carp. A significant change in induced (opsonizedzymosan particles, OZP) chemiluminescence (P < 0.05) in sampling 3 and no shifts in serum immunoglobulins concentration were found in the immunological analysis. Histopathological examination of skin, gills, liver, spleen, and cranial and caudal kidneys revealed no obvious specific changes in any tissue analysed. The use of ß-glucans in clinically healthy aquaculture remains an issue. Nevertheless, their use in breeding endangered by stress stimuli, infectious disease, or adverse environmental factors is defensible.

The effects of diclofenac on early life stages of common carp (Cyprinus carpio).

Diclofenac residues have been found in surface water, and thus could present a potential risk to aquatic species. The aim of this study was to assess the impact of diclofenac on the mortality, growth, and development of fish, as well as the impact of the drug on histological changes and selected parameters of oxidative stress in the fish. Subchronic toxic effects of diclofenac at concentrations of 0.015, 0.03, 1, and 3mg/L on embryos and larvae of common carp (Cyprinus carpio) were investigated during a 30-day toxicity test under experimental conditions. Exposure to diclofenac at 3mg/L was associated with increased mortality, increased activity of glutathione S-transferase, and decreased activity of glutathione reductase. Decreases in the levels of thiobarbituric-acid-reactive substances were associated with concentrations ≥ 0.03 mg/L. Based on these results a no observed effect concentration (NOEC)=0.015 mg/L and lowest observed effect concentration (LOEC)=0.03 mg/L were generated.

The effects of subchronic exposure to metribuzin on Danio rerio.

The aim of this study was to assess the impact of metribuzin in surface waters on fish under experimental conditions. The effects of subchronic exposure to metribuzin on fish growth and the development of histopathological changes in selected organs (gill, kidney, liver) and on activity of some biochemical markers (CYP450, EROD) in Danio rerio were investigated during a 28-day toxicity test. Juvenile growth tests were performed on D. rerio according to OECD method number 215. Fish at an initial age of 30 days were exposed to a range of metribuzin concentrations (1.5, 5, 16, 33, and 53 mg L⁻¹). Exposure to metribuzin at 53 mg L⁻¹ was associated with increased mortality. Negative effects with regard to total body weight, length, and the inhibition of specific growth rate were induced at concentrations of 33 and 53 mg L⁻¹. Histopathological examination revealed pathological lesions in the liver in pesticide-exposed fish only at the highest concentration of 53 mg L⁻¹ of metribuzin. Based on the results of growth rate, biochemical markers (CYP450, EROD), and histopathological examination, the lowest observed effect concentration (LOEC) value was 33 mg L⁻¹ and the no observed effect concentration (NOEC) value was 16 mg L⁻¹.

Effect of four selected carrier solvents on embryonal stages of Danio rerio.

OBJECTIVES: The aim of this study was to determine the effects of 96 hour exposure to selected solvents on the embryonic stages of zebrafish (Danio rerio). We investigated mortality and various types of changes which appeared (oedema, tail and eye defects, weak pigmentation, and deformation of the body). Based on the results, values of NOEC and LOEC for embryos of D. rerio were determined. METHODS: Embryonal toxicity tests were conducted according to OECD guideline 212. Ethanol and methanol were tested at concentrations of 0.1, 0.5, 1, 1.5, 2%; acetone at concentrations of 0.1, 0.5, 1, 1.25, 1.5%; and dimethylsulfoxide at concentrations of 1, 1.5, 2, 2.5, 3%. RESULTS: The LOEC values of ethanol and methanol were detected in the 1% concentration. Statistically significant changes (oedema) were reported in 1% ethanol, and oedema, weak pigmentation and deformation of the body were observed in 1% methanol. After exposure to acetone, the most common occurrence of oedema was in the 0.5% concentration (LOEC = 0.5%). The solvent dimethylsulfoxide caused oedema and body deformation at the 2% concentration (LOEC = 2%). CONCLUSIONS: The NOEC concentrations of the individual solvents were as follows: ethanol and methanol, 0.5%; acetone, 0.1%; and dimethylsulfoxide, 1.5%. These concentrations of individual solvents were higher than the maximum recommended concentration for toxicity tests on fish. For this reason, it can be assumed that the concentration of solvent allowed by the norm does not affect the procedure or results of such tests.

Acute toxicity of acetylsalicylic acid to juvenile and embryonic stages of Danio rerio.

OBJECTIVES: The aim of this study was to compare the acute toxicity of acetylsalicylic acid to embryonic and juvenile stages of aquarium fish - zebrafish (Danio rerio), oxidative stress parameters and detoxifying enzyme. METHODS: Tests were performed according to OECD No. 203 (Fish, acute toxicity test) and OECD No. 212 (Fish, short-term toxicity test on embryo and sac-fry stages) methodology. RESULTS: The results showed the mean acetylsalicylic acid LC50 value to be 567.7 mg/L in juvenile zebrafish. The acute toxicity of acetylsalicylic acid for zebrafish embryos was 274.6 mg/L. Statistically significantly higher activity of GST was found in concentrations 340, 380 and 420 mg/L of acetylsalicylic acid. TBARS, GPx and GST didn't show statistically significant activity in tested concentrations of acetylsalicylic acid. CONCLUSIONS: The results revealed a statistically significantly higher degree of sensitivity in the embryonic stages of zebrafish compared to its juveniles. Acetylsalicylic acid did not cause statistically significantly higher antioxidative defence in zebrafish.

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.

Comparison of acute toxicity of ketoprofen to juvenile and embryonic stages of Danio rerio.

OBJECTIVES: Ketoprofen is a common human medicine from a class of non-steroidal anti-inflammatory drugs (NSAIDs), which is provably detected in surface waters in concentrations ordinarily in µg.L-1. The aim of this study was to compare the acute toxicity of ketoprofen to embryonic and juvenile stages of aquarium fish - zebrafish (Danio rerio). METHODS: Tests were performed according to the methods of the Organisation for Economic Co-operation and Development (OECD) No. 203 (Fish, acute toxicity test) and OECD No. 212 (Fish, short-term toxicity test on embryo and sac-fry stages). RESULTS: The results showed (mean ± SD) LC50 value of ketoprofen to be 632.30 ± 10.10 mg.L-1 in juvenile zebrafish and 6.44 ± 2.22 mg.L-1 in embryonic stages of zebrafish. The results revealed statistically significantly higher sensitivity (p<0.01) of the embryonic stages of zebrafish to ketoprofen compared to its juveniles. The susceptibility of embryos depends on many factors, especially yet improperly developed enzymatic system in embryos, different ways of the absorption of the substance into the organism or differences in metabolism pathways. CONCLUSIONS: The acute toxicity of ketoprofen for juvenile stages of zebrafish is low, but the substance seems to be toxic for embryonic stages.

Effects of subchronic exposure to simazine on zebrafish (Danio rerio).

OBJECTIVES: Simazine is a triazine herbicide which has been used for a long period in agriculture and in the aquatic environment for control of weeds and algae. The aim of this study was to investigate the effects of subchronic exposure to simazine on growth and the development of histopathological changes in selected organs (gills, kidney, liver) in Danio rerio. METHODS: Juvenile growth tests were performed on D. rerio according to the OECD method No. 215. Fish at the age of 20 days were exposed to the environmental simazine concentration commonly detected in Czech rivers (0.06 µg.L-1) and a range of sublethal concentrations of simazine (0.6, 6.0 and 60.0 µg.L-1) for 28 days. RESULTS: There were no significant differences (p<0.05) between the specific growth rates (r) of the test groups and those of both control groups. Histopathological examination revealed pathological changes in fish exposed to a simazine concentration of 60.0 µg.L-1. The values of NOEC and LOEC of simazine were 6.0 µg.L-1 and 60.0 µg.L-1. CONCLUSIONS: The environmental concentration of simazine in Czech rivers did not have any effects on the growth and development of histopathological changes in D. rerio.