Metal X-ray microanalysis in the olfactory system of rainbow trout exposed to low level of copper

Summary— It has recently been shown that a chronic copper exposure induces specific degeneration of olfactory receptor cells in rainbow trout; however, the exact mechanism of action of the metal is not yet known. Using X-ray microanalysis in transmission electron microscopy, we have studied the distribution of metal in the olfactory system of fish exposed for 15, 30 and 60 days to 20 μg/l of copper. This was done in order to determine if it was accumulated in receptor cells and transported into the central nervous system via the olfactory nerve. No copper accumulation was detected either in the olfactory epithelium, in the olfactory nerve or in the olfactory bulb. The heavy metal was exclusively found within melanosomes of melanophores located in the lamina propria. After 60 days of exposure, the copper content in melanosomes was about two-fold higher than that in controls. As far as some morphological recovery took place in the olfactory organ during the metal exposure, which lets us suppose that some detoxication mechanism occurs, it could be suggested that melanophores might be somehow involved in such a mechanism.     

Hormonal and ion regulatory response in three freshwater fish species following waterborne copper exposure

We evaluated effects of sublethal copper exposure in 3 different freshwater fish: rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio). In a first experiment we exposed these fishes to an equally toxic Cu dose, a Cu level 10 times lower than their 96 h LC₅₀ value: 20, 65, and 150 µg/L Cu. In a second series we exposed them to the same Cu concentration (50 µg/L). Na⁺/K⁺-ATPase activity in gill tissue was disturbed differently in rainbow trout then in common and gibel carp. Rainbow trout showed a thorough disruption of plasma ion levels at the beginning of both exposures, whereas common carp and gibel carp displayed effects only after 3 days. Rainbow trout and common carp thyroid hormones experienced adverse effects in the beginning of the exposure. The involvement of prolactin in handling metal stress was reflected in changes of mRNA prolactin receptor concentrations in gill tissue, with an up regulation of this mRNA in rainbow trout and a down regulation in gibel carp, which was more pronounced in the latter. Overall, rainbow trout appeared more sensitive in the beginning of the exposure, however, when it overcame this first challenge, it handled copper exposure in a better manner then common and gibel carp as they showed more long term impacts of Cu exposure.     

Influence of available dietary carbohydrate content on tolerance of waterborne copper by rainbow trout,Salmo gairdneri Richardson*

The effect of increased available dietary carbohydrate on the tolerance of waterborne copper by rainbow trout was investigated. Trout were reared for six to eight weeks on one of four isocaloric and isonitrogenous diets which varied in available carbohydrate content due to either processing (extrusion v. steam-pelleting) or supplementation with cerelose (0 or 21%). The incipient lethal level (ILL) of copper for trout reared on the low-available-carbohydrate steam-pelleted diet was 350 μg I^?1, significantly higher than the 276 μg I^?1 shown for trout reared on the high-available-carbohydrate extruded diet. The ILL of copper for the low-carbohydrate group (0% cerelose) was 408 μg I^?1, significantly higher than the ILL of 246 μg I^?1 of trout reared on the high-carbohydrate diet (21% cerelose). Increased available dietary carbohydrate resulted in increased liver glycogen content and liver-body weight ratios and decreased liver protein content. Significant correlations were evident between waterborne copper tolerance and liver glycogen, liver-body weight ratios and liver protein. Relative to those on high carbohydrate diets, livers of fish on low-carbohydrate diets contained higher concentrations of copper after seven days of exposure to 0 or 196 μg I^?1 of copper. The elevated liver glycogen levels, resulting from increased dietary carbohydrate, are thought to have impaired liver function, thus reducing copper tolerance.     

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 μg L⁻¹), propineb (3, 6 and 24 mg L⁻¹), and benomyl (2, 5 and 20 mg L⁻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.     

The effects of early chronic exposure to sublethal copper on the olfactory discrimination of rainbow trout,Oncorhynchus mykiss

Synopsis The impact of a long-term sublethal copper exposure on the olfactory discrimination performance was examined in rainbow trout,Oncorhynchus mykiss. Two fish groups, one from the 14th day post-fertilization and the other from hatching, were exposed continuously to 22 g Cu²⁺·1^–1 for 41 and 37 weeks, respectively. Preference/avoidance responses to different odor conditions (own rearing water/well water; own rearing water/heterospecific water) were evaluated during the 8th month after hatching. The 2 odor conditions appear reliable criteria to evaluate the fish discrimination ability. When controls were given a choice between own rearing water against either well water or heterospecifc water, they significantly preferred their own rearing water, whereas both copper-exposed groups showed no preference. The behavioral response of exposed fish indicates impairment of their olfactory discrimination ability; however, it cannot be determined if this reflects a loss of olfactory sensitivity or an olfactory hyposensitivity. Behavioral tests performed 2 and 10 weeks after removal of copper, showed some functional recovery of the olfactory discrimination ability which could be related to the renewal property of olfactory receptor cells. Results demonstrate that a long-term sublethal exposure to copper, as it commonly occurs in the natural condition, may result in olfactory dysfunction with potential impacts on fish survival and reproduction.     

Menthol as an alternative anaesthetic and sedative for rainbow trout,Oncorhynchus mykiss

Menthol is known for its analgesic properties, but relatively little information is available on its potential as an anaesthetic on fish. The purpose of this study was to assess anaesthetic and sedative effectiveness of menthol and its safety in rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout 180 ± 28 g (mean ± SD) within a range of 152–208 g fish^?1 were individually exposed to menthol concentrations between 10 and 150 mg l^?1 and observed for behavioural responses, induction time to anaesthesia and recovery time. Menthol concentrations of 40–150 mg l^?1 induced anaesthesia with varying exposure times. There was an exponential relationship (p < 0.001) between induction time and menthol concentration. Menthol concentrations of 80–150 mg l^?1 induced anaesthesia within three minutes of exposure and fish recovered within three minutes. Induction and recovery data showed that 80 mg l^?1 was most suitable for anaesthesia in juveniles of this species. Concentrations of 10–20 mg l^?1 had sedative effects. Menthol stock solutions prepared using ethanol and acetone and storage time of stock solutions at room temperature for up to 48 h showed no significant differences in anaesthetic efficiency. When exposure time to menthol was kept constant at three minutes, 22% of fish had temporary cessation of gill ventilation. These fish had longer recovery times than those that did not show that response. Menthol was an effective anaesthetic and could be tested as a sedative for trout.     

The distribution of zinc and copper in plasma, erythrocytes and erythrocyte membranes of rainbow trout (Salmo gairdneri)

1. The zinc and copper concentration of plasma was determined in rainbow trout, lake trout, walleye and whitefish. 2. These fish had mean plasma zinc concentrations ranging from 9.3 to 15.1 ppm and copper concentrations from 0.6 to 1.3 ppm. 3. In rainbow trout, the concentration of zinc and copper is greater in the erythrocyte membrane than in the total erythrocyte. 4. Ultrafilterable plasma zinc and copper concentration in rainbow trout was determined to be 0.03 and 0.019 ppm, respectively. 5. Dialysis of rainbow trout plasma against 20 mM EDTA results in removal of 99% of the zinc and 88% of the copper from plasma proteins.     

Accumulation of chemostimulatory amino acids by a sedimentable fraction isolated from olfactory rosettes of rainbow trout (Salmo gairdneri)

The accumulation of amino acids by a sedimentable fraction isolated from olfactory rosettes of rainbow trout (Salmo gairdneri) was examined to determine the correspondence with established criteria for receptors. Physical parameters such as saturability, reversibility, affinity and quantity of sites were reasonably consistent with receptor criteria. The kinetics of association were slow and not in accord with values expected of sensory receptors. Accumulation correlated with electrophysiological effectiveness for most amino acids. The correlation was tissue specific, not being evident in brain or gill. Denervation experiments showed that high-affinity accumulation depended upon the presence of neuronal elements in the olfactory mucosa. Inhibition studies with amino acids and analogues generally indicated good correspondence with previous electrophysiological findings. Experiments with transport inhibitors, lack of Na⁺, counter-transport and increasing osmotic strength showed some involvement of aminoc acid transport. Inconsistencies between the various aspects of amino acid accumulation by the sedimentable fraction and electrophysiological response preclude unequivocal classificatin of the accumulation as representing olfactory receptors.     

The effect of sub-lethal ammonia exposure on fed and unfed rainbow trout: the role of glutamine in regulation of ammonia

Many species of fishes have evolved mechanisms for coping with ammonia caused by either high ammonia environments or an inability to excrete nitrogenous wastes. Rainbow trout (Oncorhynchus mykiss), have not been known to have such a mechanism. The present study investigated whether rainbow trout can use amino acid synthesis and storage to cope with ammonia. Experiments were performed on fed and unfed rainbow trout under both control and elevated ammonia conditions (0 and 10 mgN/l (total ammonia nitrogen), pH 7.2). The results indicate that both feeding and ammonia exposure increased plasma ammonia significantly 6 h postprandial and post ammonia exposure. After 48 h the fed/ammonia exposed fish had plasma ammonia levels that were not significantly different than the fed/control fish. Plasma ammonia was reduced by more than 50%, attributable to ammonia being converted to glutamine in brain, liver and muscle tissue. Feeding alone also increased glutamine levels in brain tissue. Activity of glutamine synthetase in brain and liver was increased corresponding to an increase in glutamine concentrations when fish were exposed to ammonia. This is the first report showing that rainbow trout can detoxify endogenous and exogenous ammonia.     

Comparison of dietary and waterborne exposure to benzo a pyrene: bioavailability,tissue disposition and CYP1A1 induction in rainbow trout Oncorhynchus mykiss

Absorption and tissue distribution of benzo\[ a ]pyrene (BaP)-derived radioactivity were studied in juvenile rainbow trout following dietary or waterborne exposure. In order to compare the bioavailability of BaP, the fish were exposed to 1.5 mCi 3H-BaP kg-1 fish, either in the diet or in the water as a 2 days static exposure. Furthermore, tissue levels of BaP-derived radioactivity bound to macromolecules in different tissues were studied in non-induced fish, and in fish induced by additional treatment with unlabelled BaP (corresponding to 5 mg kg-1 fish) in the water. Absorption and tissue distribution of 3H BaP were studied by liquid scintillation counting and whole-body autoradiography. BaPderived radioactivity bound to macromolecules in different tissues was studied by autoradiography of solvent-extracted whole-body sections. The hepatic CYP1A induction was measured as EROD activity. Exposure to unlabelled BaP resulted in a marked induction of hepatic EROD activity in rainbow trout 2 days after the start of the exposure. Significant higher concentrations of radiolabelled compound were observed in waterborne-exposed fish, in contrast to dietary-exposed fish. High concentrations of radiolabelling were observed in the gills, liver, bile, intestines, olfactory organ, kidney and the skin of the waterborne-exposed fish. In the dietary-exposed fish, high levels of radioactivity were observed in the intestines and the bile, whereas lower concentrations were present in the liver. Only traces of radioactive compound were observed in the gills. In contrast to waterborne-exposed fish, no radioactivity was detected in the olfactory organ or skin. In autoradiograms of sections extracted with a series of polar and non-polar solvents, a large fraction of radioactivity was still present in the gills, olfactory organ, liver, kidney, skin and intestinal mucosa of the waterborne-exposed fish, indicating that reactive BaP intermediates formed by CYP1A-mediated metabolism were bound to macromolecules in these tissues.     

The acute lethal toxicity to rainbow trout of mixtures of copper,phenol, zinc and nickel†

The acute lethal toxicities to rainbow trout, Salmo gairdneri Richardson, of mixtures of copper and phenol, of copper, zinc and phenol, and of copper, zinc and nickel, over an exposure period of 48 h were determined. The concentrations selected for testing were such that each poison theoretically contributed equally to the toxicity of the mixture. It was found that the toxicities of these mixtures could be adequately predicted by summation of the fractional toxicities of the particular poisons which were present.     

Thermal Tolerance of Diploid Versus Triploid Rainbow Trout and Brook Trout Assessed by Time to Chronic Lethal Maximum

Synopsis An effect of ploidy on thermal tolerance in juvenile trout was assessed in a series of tests comparing time to chronic lethal maximum (CLMax). Diploid and triploid fish were produced from a common spawn for three different groups each of brook trout Salvelinus fontinalis and of rainbow trout Oncorhynchus mykiss. One or two CLMax tests were performed per group, on between 15 and 50 individuals per ploidy within groups. The tests involved exposure of fish to a progressive 2°C day⁻¹ water temperature increase and recording of the time at which each individual fish reached loss of equilibrium (LE). The time to LE data were rank transformed and analyzed as a randomized complete block design. Although relative performance varied among trials, the analysis indicated overall differences due to ploidy were small and nonsignificant among both brook trout and rainbow trout. Size proved to be significantly correlated with time to LE in the brook trout trials, but not in the rainbow trout trials. Two of the six groups included a large proportion of fish which had received a heat shock following fertilization, but were not successfully triploidized. In both cases, thermal tolerance of the heat-shocked diploids was similar to that of the non-heat shocked control diploids, indicating no persistent effect of the heat shock on thermal tolerance.     

Rainbow trout metallothionein

Two forms of hepatic metallothionein were isolated and purified from rainbow trout injected intraperitoneally with cadmium chloride. Both forms showed similarities with mammalian metallothioneins, had a high cystein content (30 mol%), and were void of aromatic amino acids and histidine. The molecular weight was estimated to be about 6000 dalton for the apothioneins, and the thiol groups of the cysteine residues complexed with the heavy metals (Cd, Cu, Zn) in a SH/Me⁺⁺ ratio of about 2.4. The amount of copper in metallothionein from rainbow trout was very high, greater than the amount of cadmium and zinc after injections of 3 mg cadmium/kg body weight. The total metal content of cadmium, copper and zinc in metallothionein 1 and 2 were about 7 and 8 atoms per molecule respectively.     

Trace Metal Levels in Rainbow Trout (Oncorhynchus mykiss) Cultured in Net Cages in a Reservoir and Evaluation of Human Health Risks from Consumption

Although fish consumption has positive health effects, metals accumulated in fish can cause human health risks. In this study, the levels of ten metals in rainbow trout (Oncorhynchus mykiss) farmed in the Keban Dam Reservoir, which has the biggest rainbow trout production capacity in Turkey, were determined and compared with the maximum permissible levels (MPLs). Also, human health risks associated with rainbow trout consumption were assessed. The metal concentrations in rainbow trout were found below the MPLs. The estimated daily intake of each metal was much lower than the respective tolerable daily intake. The target hazard quotient (THQ) for individual metal and total THQ for combined metals did not exceed 1, indicating no health risk for consumers. The cancer risk (CR) value for inorganic arsenic was within the acceptable lifetime risk range of 10⁻⁶ and 10⁻⁴. For carcinogenic and non-carcinogenic effects, the maximum allowable fish consumption rates were high enough to ensure the human health. According to these results, the consumption of rainbow trout farmed in the Keban Dam Reservoir does not pose a risk on human health.

     

Effect of short‐term (0–72 h) fasting on serum biochemical characteristics in rainbow trout Oncorhynchus mykiss

An experiment was carried out to investigate the effects of short‐term fasting periods on the serum biochemical characteristics of rainbow trout, Oncorhynchus mykiss. For this purpose, fish were fasted 0, 6, 12, 24, 48 and 72 h before blood sampling. Thereafter the serum levels of thyroxine (T₄), 3,5,3′‐triiodothyronine (T₃), cortisol, glucose, lactate, triglyceride, cholesterol, total protein, albumin, globulin and albumin : globulin ration (A : G) were determined. Results show that serum levels of T₄ (4.60–8.77 ng ml^?1), T₃ (7.50–13.3 ng ml^?1), cortisol (7.91–24.5 ng ml^?1), glucose (18.5–80.1 mg dl^?1), lactate (12.7–29.6 mg dl^?1), triglyceride (171–500 mg dl^?1), and cholesterol (321–535 mg dl^?1) were significantly affected by the fasting period. However, there were no significant changes in serum total protein (3.03–3.68 g dl^?1), albumin (1.78–2.01 g dl^?1), globulin (1.15–1.70 g dl^?1) or A : G (1.13–1.93) among the fish fasted 0–72 h. Results clearly show the importance of a fasting period on the serum biochemical properties of rainbow trout. According to the results, 24 h fasting is suggested as a pre‐sampling fasting period in rainbow trout to measure serum levels of T₄, T₃, cortisol, glucose, lactate, triglyceride, and cholesterol. Potential mechanisms related to the changes in biochemical properties are discussed.     

Viral load of various tissues of rainbow trout challenged with viral haemorrhagic septicaemia virus at various stages of disease

Market-sized rainbow trout Oncorhynchus mykiss were challenged by waterborne exposure to viral haemorrhagic septicaemia virus (VHSV isolate of genogroup Ia). Fish were sampled at 4 stages of infection (before onset of clinical signs, clinically affected fish, mortalities and survivors) and the viral load determined in (1) internal organs, (2) muscle tissue and (3) brain and gill tissue. Virus levels were determined by virus titration and real-time RT-PCR. VHSV was detected by either method in the majority of fish before onset of clinical signs and in the survivor group as well as in all fish in the clinically affected fish and mortality groups. Mean virus amounts per mg of tissue determined by virus titration (TCID50) or real-time RT-PCR (copy number) were > 10(4) in preclinical fish, > 10(3.8) in clinically affected fish, > 10(3.9) in mortalities and > 10(1.2) in survivors. Virus levels tended to be highest in the internal organs of subclinical and clinically affected fish and in brain and gill tissue of survivors. The results demonstrate that significant levels of VHSV can be found in tissues of rainbow trout that may be marketed for human consumption, which may have relevance for the biosecurity of VHS-free areas.     

A note on copper bioaccumulation in Mozambique tilapia,Oreochromis mossambicus (Osteichthyes: Cichlidae)

Copper (Cu) is one of the most commonly reported metal pollutants in African water bodies, but there are few studies on African freshwater fish species of copper accumulation and copper toxicity. Adult O. mossambicus were exposed to 0mg l^?1 (control) and 0.75mg l^?1 Cu for 96h and 0 (control), 0.11, 0.29 and 0.47mg l^?1 copper for 64 days. Samples of liver and gills were collected after 96h, and after 1, 32 and 64 days, respectively. There were significant differences in the mean Cu accumulation values in the liver and gills between the control and the Cu-exposed fish after the 96-h exposure. In fish exposed to 0.11 and 0.29mg l^?1 Cu for 64 days there was an increase in copper level in the tissues. In fish exposed to 0.47mg l^?1 Cu the concentration in the gill and liver tissue did not increase between Day 1 and Day 32. At this time, Cu accumulation in the liver was higher than for fish exposed to 0.11 and 0.29mg l^?1 Cu for 64 days. Exposure to approximately 0.47mg l^?1 Cu for more than 32 days induced mortality.