Praskova E

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 effect of oyster mushroom β-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 β-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% β-glucan (OTC + 0.5% BG group), 75 mg oxytetracycline kg(-1) bw and 2.0% β-glucan (OTC + 2.0% BG group), 0.5% β-glucan (0.5% BG group), and 2.0% β-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 Fultons 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 β-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.

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 donors 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 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.

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.