Erol Capkin

Histopathology of Rainbow Trout Exposed to Sublethal Concentrations of Methiocarb or Endosulfan

Liver, spleen, trunk kidney, gills, and brain of rainbow trout (Oncorhynchus mykiss) were examined histologically after exposure to different concentrations of methiocarb (2.5 and 3.75 mg/L) or endosulfan (0.6 and 1.3 μg/L) for 21 days. Histological recovery was also studied by maintaining the pesticide-exposed fish in a freshwater system for an additional 30 d. Lesions were not evident in liver, kidney, spleen, or brain of fish exposed to either concentration of methiocarb for 21 d. Lesions were observed in gills, liver, spleen, and trunk kidney (but not brain) of rainbow trout exposed to either concentration of endosulfan. There was no concentration-related effect observed on the histopathological lesions. After 30 days of recovery, fish had no histological lesions in gills, kidney, spleen, liver, or brain. Therefore all the changes observed during exposure were reversible.

Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens.

A multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of the five major fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida, Flavobacterium columnare, Renibacterium salmoninarum, and Yersinia ruckeri. Each of the five pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The detection limits of the multiplex PCR was in the range of 2, 1, 1, 3, and 1CFU for A. hydrophila, A. salmonicida, F. columnare, R. salmoninarum, and Y. ruckeri, respectively. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria. The multiplex PCR assay was useful for the detection of the bacteria in naturally infected fish. This assay is a sensitive and specific and reproducible diagnostic tool for the simultaneous detection of five pathogenic bacteria that cause disease in fish. Therefore, it could be a useful alternative to the conventional culture based method.

Assessment of acute toxicity and histopathology of the fungicide captan in rainbow trout.

Acute toxicity of the fungicide, captan, to juvenile rainbow trout was evaluated under static-renewal test condition. Actual concentrations of captan ranged from 0.05 to 1.00 mg/L. The concentrations of captan that killed 50% of the rainbow trout (3.11±0.8 g) within 24 (24 h; LC(50)), 48, 72 and 96 h were 0.57±0.09, 0.49±0.10, 0.44±0.11 and 0.38±0.13 mg/L (95% confidence limits), respectively. None of the unexposed control fish died and the first fish died 6 h after exposure to captan (≥0.65 mg/L). Hypertrophy, separation of epithelium from lamellae, lamellar fusion, and epithelial cell necrosis were observed on captan exposed fish. Gills also had scattered areas of focal lamellar hyperplasia. Fish exposed to fungicide had inflammation and necrosis in liver, trunk kidney and spleen. In order, the most affected organs were gill, trunk kidney and liver.

Effects of water quality and fish size on toxicity of methiocarb, a carbamate pesticide, to rainbow trout

The acute toxicity of methiocarb in juvenile rainbow trout (Oncorhynchus mykiss, 3.25±0.79g) was evaluated in glass aquaria under static conditions. Nominal concentrations of methiocarb in the toxicity test ranged from 1.25 to 7.50mgL(-1). The concentrations of methiocarb that killed 50% of the rainbow trout within 24-h (24-h LC(50)), 48-h LC(50), 72-h LC(50), and 96-h LC(50) were 5.43±0.19, 5.04±0.18, 4.95±0.19, and 4.82±0.21mgL(-1) (95% confidence limits), respectively. Mortality of fish increased with increasing water temperature. Increasing alkalinity from 19mgL(-1) as CaCO(3) to 40, 60, or 90mgL(-1) as CaCO(3) significantly decreased mortality of fish. Total hardness ranging from 50mgL(-1) as CaCO(3) to 147mgL(-1) as CaCO(3) did not affect mortality of fish exposed to methiocarb. Fish exposed to methiocarb had histological alterations such as lamellar edema, separation of epidermis from lamellae, and lamellar fusion. Methiocarb exposed fish had necrosis between molecular and granular layer of cerebellum where Purkinje cells present. Results indicate that alkalinity, temperature, and fish size affect methiocarb toxicity of rainbow trout.

Histopathological changes induced by maneb and carbaryl on some tissues of rainbow trout, Oncorhynchus mykiss

Acute toxicity of the pesticides, maneb and carbaryl, to juvenile rainbow trout were evaluated under static-renewal test conditions. Actual concentrations of maneb ranged from 0.10mg/L to 2.00mg/L and carbaryl ranged from 0.20mg/L to 3.90mg/L. The concentrations of maneb that killed 50% of the rainbow trout (3.27+/-0.9g) within 24-h (24-h; LC(50)), 48-h, 72-h and 96-h were 1.19+/-0.12, 1.04+/-0.11, 0.92+/-0.12 and 0.81+/-0.14mg/L (95% confidence limits), respectively. LC(50) values of carbaryl for 24-h, 48-h, 72-h and 96-h were 2.52+/-0.71, 2.16+/-0.63, 1.71+/-0.46 and 1.39+/-0.15mg/L, respectively. None of the unexposed control fish died and the first fish died 6h after exposure to maneb (>or=1.30mg/L), and carbaryl (>or=2.60mg/L). Lamellar edema, separation of epithelium from lamellae, lamellar fusion, swelling of the epithelial cells and epithelial cell necrosis were observed on maneb and carbaryl exposed fish. Gills also had scattered areas of focal lamellar hyperplasia. Fish exposed to pesticides had inflammation and focal necrosis in liver, trunk kidney and spleen. Maneb and carbaryl had similar histopathological lesions. In order, the most affected organs were gill, trunk kidney and liver.

Histopathological changes in rainbow trout (Oncorhynchus mykiss) after exposure to sublethal composite nitrogen fertilizers.

Subchronic toxicity of composite inorganic fertilizers such as ammonium sulfate [fertilizer A; (NH(4))(2)SO(4); 21% NH(4)-N)], composite fertilizer 15-15-15 (fertilizer B; commercial formulation: 15% NH(4)-N, 15% phosphorus, and 15% potassium oxide), and composite fertilizer 25-5-10 (fertilizer C; commercial formulation: 25% NH(4)-N, 5% phosphorus, and 10% potassium oxide) on the skin, liver, kidney, pancreas, and gills of the juvenile rainbow trout (Oncorhynchus mykiss) was studied in two-week toxicity tests under static-renewal test conditions. Fish exposed to sublethal concentrations of fertilizers did not show any behavioral abnormality compared to control groups. Histological lesions were observed in skin, gills, liver, pancreas, and trunk kidney of the fish. In the epidermis, degenerated/vacuolated epithelial cells, microcystic dilatations, and intracellular edema of mucus cell were observed. Liver had swollen and degenerated hepatocytes without losing adenoid structure. Hematopoietic tissues had necrosis and vacuolar degeneration on proximal tubules of the kidney. In order, the most affected organs were skin, liver, and kidney.

Effects of some pesticides on the vital organs of juvenile rainbow trout (Oncorhynchus mykiss)

Gill, trunk kidney, spleen, and liver of rainbow trout (Oncorhynchus mykiss) were examined after exposure to different sublethal concentrations of carbosulfan (25, 50 and 200 μgL(-1)), propineb (3, 6 and 24 mgL(-1)), and benomyl (2, 5 and 20 mgL(-1)) for 14 days. Lesions were observed in gill, trunk kidney, spleen, and liver of rainbow trout exposed to either concentration of pesticides. The most important lesions were determined in the highest concentrations of pesticides. Lamellar fusion, lamellar hyperplasia, epithelial lifting, vacuolization of epithelial tissue, epithelial necrosis, hypertrophy and sloughing of epithelium were observed on fish exposed to carbosulfan, propineb and benomyl. Fish had cell necrosis, degeneration and oedemas in liver, trunk kidney and spleen. None of these lesions were seen in control fish.

Effects of chronic carbosulfan exposure on liver antioxidant enzyme activities in rainbow trout.

Catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and selenium-dependent glutathione peroxidase (Se-GPx) activities in liver of rainbow trout (Oncorhynchus mykiss; 116.88±21.69g) were evaluated after exposing fish to sublethal concentrations (25μg/L) of carbosulfan in flow-through tanks for 60 days. During the experiment activities of CAT, SOD, GST, and Se-GPx and histopathological effects were determined once a week and once at the end of the 21 days of recovery period. All enzymes were affected by carbosulfan when compared to control fish. Fish had intracellular oedema, cell necrosis, pycnotic nucleus, and increase of sinusoidal space in the liver. After 21 days of the recovery period, all enzyme activities had returned to control levels and fish had no histological lesions in liver. Therefore all the changes observed during exposure were reversible. Results indicate that the liver CAT, SOD and GST enzymes are highly sensitive to carbosulfan as their activities altered significantly, suggesting they could be useful in predicting sublethal pesticide toxicity and useful as an indicator for assessment of pesticides in contaminated water.

Antimicrobial agents, triclosan, chloroxylenol, methylisothiazolinone and borax, used in cleaning had genotoxic and histopathologic effects on rainbow trout

Triclosan (TRC), chloroxylenol (PCMX) and methylisothiazolinone (MIT) have been commonly used as an antimicrobial in soaps while borax (BRX) is used in household cleaning. After using these chemicals, they are washed down drains and getting into the aquatic ecosystem in which they may affect aquatic living organisms. In the present study, the chronic effects of TRC, PCMX, MIT and BRX on genotoxicity, gene expression and histopathology of rainbow trout (Oncorhynchus mykiss) were evaluated for 40 days under semi static condition. The comet assay results indicated that MIT, TRC and PCMX caused significant DNA damage to erythrocytes of the fish. Transcription of SOD, GPX1, GPX2, GSTA, HSP90BB, HSP90BA, CAT, and HSC70A genes were significantly regulated as a result of TRC, PCMX, MIT, and BRX exposure except PCMX exposed GSTA gene. Histological lesions were detected in gills, spleen liver, and trunk kidney of the fish. Lamellar fusion, hyperplasia and epithelial necrosis in gills, melanomacrophage centers and splenic necrosis in spleen, pyknotic nucleus, fat vacuoles, necrotic hepatocytes in liver, cloudy swelling in the tubules, renal tubule epithelial cells degeneration, glomerular capillaries dilation and glomerulus degeneration in kidney, were observed. Our study demonstrates the chronic toxic effect of TRC, PCMX, MIT, and BRX is high in rainbow trout. Therefore, we should be more careful when using these chemicals for cleaning in order to protect aquatic environment.

Occurrence of antibiotic resistance genes in culturable bacteria isolated from Turkish trout farms and their local aquatic environment

Antibiotic resistance and presence of the resistance genes were investigated in the bacteria isolated from water, sediment, and fish in trout farms. A total of 9 bacterial species, particularly Escherichia coli, were isolated from the water and sediment samples, and 12 species were isolated from fish. The antimicrobial test indicated the highest resistance against sulfamethoxazole and ampicillin in coliform bacteria, and against sulfamethoxazole, imipenem, and aztreonam in known pathogenic bacteria isolated from fish. The most effective antibiotics were rifampicin, chloramphenicol, and tetracycline. The multiple antibiotic resistance index was above the critical limit for almost all of the bacteria isolated. The most common antibiotic resistance gene was ampC, followed by tetA, sul2, blaCTX-M1, and blaTEM in the coliform bacteria. At least one resistance gene was found in 70.8% of the bacteria, and 66.6% of the bacteria had 2 or more resistance genes. Approximately 36.54% of the bacteria that contain plasmids were able to transfer them to other bacteria. The plasmid-mediated transferable resistance genes were ampC, blaCTX-M1, tetA, sul2, and blaTEM. These results indicate that the aquatic environment could play an important role in the development of antibiotic resistance and the dissemination of resistance genes among bacteria.