Long-term effects of propolis on serum biochemical parameters of rainbow trout (Oncorhynchus mykiss)

Long-term effects of propolis administration on serum biochemical parameters of rainbow trout (Oncorhynchus mykiss) were investigated. To determine the possible toxicity and side effects of propolis, fish were fed on diets containing 0, 0.5, 1.5, 4.5 and 9 g propolis/kg diet for 8 weeks. At the end of the experiment, various seric biochemical parameters were determined. Our results showed that all dosages induced no significant alterations in growth parameters and the seric levels of total protein, albumin, globulin, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides and activities of glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, alkaline phosphatase and lactate dehydrogenase, when compared to the control group. On the basis of our findings, propolis is a non-toxic substance for rainbow trout and its long-term administration might not have any side effects.     

Effects of sublethal concentrations of triphenyltinacetate on rainbow trout (Oncorhynchus mykiss)

To evaluate the toxic effects of sublethal concentrations of the fungicide triphenyltinacetate (TPTAc), a prolonged toxicity study was made on rainbow trout (Oncorhynchus mykiss). Fish were exposed to TPTAc concentrations ranging from 1 to 6 g TPTAc/L for 28 and 18 days, respectively, using a flow-through exposure system. Hematological findings included an increase of the total number of erythrocytes and an elevated incidence of erythrocyte degradation stages at 4 g TPTAc/L and higher. The hemoglobin content and the packed-cell volume increased as well at 4 and 1 g TPTAc/L. Whereas the total number of leucocytes increased in fish exposed to 1 g TPTAc/L, the number of leucocytes tended to decrease at higher concentrations. The percentage of lymphocytes within the differential blood cell count decreased. The histopathological examination of TPTAc-exposed fish showed a dose-related lymphocytic depletion of the spleen, accompanied by a proliferation of reticuloendothelial cells and an increased erythrophagia even at the lowest TPTAc concentration. In severe cases, cell necrosis was evident. In liver tissue, a depletion of the glycogen deposits within the hepatocytes could be detected in fish exposed to 4 and 6 g TPTAc/L. The analysis of the phenyltin compounds within various organs of fish by HRGC-FPD revealed remarkable concentrations of triphenyltin of up to 16.1 g/g with the following order of residue levels: liver > kidney > spleen > gills > muscle. Di- and monophenyltin were found only in traces of 1–109 ng/g in these organs. The present study indicates that TPTAc negatively affects rainbow trout in a concentration range that might be present in aquatic environments.     

Effects of subchronic nitrite exposure on rainbow trout (Oncorhynchus mykiss)

Subchronic toxicity of nitrite in rainbow trout (Oncorhynchus mykiss; mean mass±S.D., 18.9±1.3 g) was assessed in a 28-day trial. The influence of nitrite on fish mortality, growth rate, haematology, blood biochemistry, and gill histology was observed. Survival was not affected by exposures up to 1 mg l⁻¹ NO₂⁻ (at 10 mg l⁻¹ Cl⁻). On the basis of growth rate inhibition data, the values of NOEC (28 d LC₀) and LOEC (28 d LC₁₀) were estimated at 0.01 and 0.2 mg l⁻¹ NO₂⁻, respectively. At 0.01 mg l⁻¹ NO₂⁻ (the lowest concentration tested), there was segmental hyperplasia of the respiratory epithelium of secondary lamellae and elevated glucose and decreased potassium. Elevated nitrite concentrations were found in blood plasma of fish exposed to concentrations of 1.0 mg l⁻¹ NO₂⁻ and higher, and in muscle tissue at the highest concentration 3.0 mg l⁻¹ NO₂⁻. Plasma and muscle nitrite levels were lower than those in the ambient water in all experimental groups.     

Acute toxicity of carbamazepine to juvenile rainbow trout (Oncorhynchus mykiss): effects on antioxidant responses, hematological parameters and hepatic EROD

Awareness of residual pharmaceutically active compounds (PhACs) in the aquatic environment is growing as investigations into these pollutants are increasing and analytical detection techniques are improving. However, the toxicological effects of PhACs have not been adequately researched. In this study, the toxic effects of carbamazepine (CBZ), an anticonvulsant drug commonly present in surface and groundwater, was studied in juvenile rainbow trout, Oncorhynchus mykiss, by acute semi-static bioassay. Bood parameters, liver xenobiotic-metabolizing response and tissue antioxidant status were evaluated. Compared to the control group, fish exposed to CBZ (96 h LC50) showed significantly higher Er, Hb, MCHC, monocytes, neutrophil granulocytes and plasma enzymes activity, and significantly lower MCV and lymphocytes. CF and HSI were not significantly different among groups such as hepatic EROD. SOD, CAT, GPx and GR activity was significantly higher in liver of experimental groups, but decreased significantly in brain and gill. In general, antioxidant enzyme activity in intestine and muscle was less evident than in liver. Oxidative stress indices (levels of LPO and CP) were significantly higher in gill and brain, despite a trend to increased values were manifested in the remaining tissues. In short, CBZ-induced stress responses in different tissues were reflected in the oxidant stress indices and hematological parameters. However, before those parameters are used as special biomarkers for monitoring residual pharmaceuticals in aquatic environment, more detailed experiments in laboratory need to be performed in the future.     

Effects of handling on heat shock protein expression in rainbow trout (Oncorhynchus mykiss)

As part of an effort to validate the use of heat shock proteins (HSPs) as biomarkers of exposure to and effects of contaminants, we evaluated the effect of two handling regimens on the induction of HSP 60 and 70 in rainbow trout (Oncorhynchus mykiss). Fish were acclimated to laboratory conditions for several weeks before the beginning of the experiment. Fish were then captured by net, placed in a cooler for 1 h while being transported in a truck, returned to their original tanks, then sacrificed 6 to 8 h later. Tricaine methane sulfonate (MS-222) was used during different phases of handling to reduce handling stress. Heat-stressed fish were included in the experiment as a positive control. Muscle, liver, gills, and heart were analyzed for HSP 60 and 70 by immunoblotting. We found no effect of any handling regimen on the induction of HSPs. These findings suggest that the capture and transport of fish for environmental monitoring purposes should not interfere with the use of stress proteins as biomarkers.     

Cytological and biochemical effects of a mixture of 20 pollutants on isolated rainbow trout (Oncorhynchus mykiss) hepatocytes

Isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) were exposed to different concentrations (1x, 10x, 50x) of a complex mixture of 20 environmentally relevant contaminants (PAHs, PCBs, pesticides) typical of the chemical burden of surface waters and sediments of small rivers in southwest Germany to investigate sublethal cytological and biochemical alterations. Results document that all concentrations clearly induced biochemical and morphological changes. The activities of enzymes such as lactate dehydrogenase and catalase as well as the rate of lipid peroxidation were significantly increased, whereas acid phosphatase activity decreased. Cytopathological effects included deformation of cellular shape, deformation and dilation of the nuclear envelope, increase in heterochromatin, nuclear inclusions, heterogeneity of mitochondria, vesiculation of RER cisternae, as well as increases in myelinated bodies, lysosomes, and peroxisomes. If compared with cytological alterations induced in isolated rainbow trout hepatocytes after exposure to sediment extracts from rivers of corresponding contamination, cytological effects of exposure to the complex chemical mixture displayed numerous similarities.     

Acute and chronic toxicity of nickel to rainbow trout (Oncorhynchus mykiss)

Of the fish species tested in chronic Ni exposures, rainbow trout (Oncorhynchus mykiss) is the most sensitive. To develop additional Ni toxicity data and to investigate the toxic mode of action for Ni, we conducted acute (96-h) and chronic (85-d early life-stage) flow-through studies using rainbow trout. In addition to standard toxicological endpoints, we investigated the effects of Ni on ionoregulatory physiology (Na, Ca, and Mg). The acute median lethal concentration for Ni was 20.8 mg/L, and the 24-h gill median lethal accumulation was 666 nmol/g wet weight. No effects on plasma Ca, Mg, or Na were observed during acute exposure. In the chronic study, no significant effects on embryo survival, swim-up, hatching, or fingerling survival or growth were observed at dissolved Ni concentrations up to 466 microg/L, the highest concentration tested. This concentration is considerably higher than the only other reported chronic no-observed-effect concentration (<33 microg/L) for rainbow trout. Accumulation of Ni in trout eggs indicates the chorion is only a partial barrier with 36%, 63%, and 1% of total accumulated Ni associated with the chorion, yolk, and embryo, respectively. Whole-egg ion concentrations were reduced by Ni exposure. However, most of this reduction occurred in the chorion rather than in the embryos, and no effects on hatching success or larval survival were observed as a result. Plasma ion concentrations measured in swim-up fingerlings at the end of the chronic-exposure period were not significantly reduced by exposure to Ni. Nickel accumulated on the gill in an exponential manner but plateaued in trout plasma at waterborne Ni concentrations of 118 microg/L or greater. Consistent with previous studies, Ni did not appear to disrupt ionoregulation in acute exposures of rainbow trout. Our results also suggest that Ni is not an ionoregulatory toxicant in long-term exposures, but the lack of effects in the highest Ni treatment precludes a definitive conclusion.     

Dietary accumulation of perfluorinated acids in juvenile rainbow trout (Oncorhynchus mykiss)

Perfluorinated acids (PFAs) recently have emerged as persistent global contaminants after their detection in wildlife and humans from various geographic locations. The highest concentrations of perfluorooctane sulfonate are characteristically observed in high trophic level organisms, indicating that PFAs may have a significant bioaccumulation potential. To examine this phenomenon quantitatively, we exposed juvenile rainbow trout (Oncorhynchus mykiss) simultaneously to a homologous series of perfluoroalkyl carboxylates and sulfonates for 34 d in the diet, followed by a 41-d depuration period. Carcass and liver concentrations were determined by using liquid chromatography-tandem mass spectrometry, and kinetic rates were calculated to determine compound-specific bioaccumulation parameters. Depuration rate constants ranged from 0.02 to 0.23/d, and decreased as the length of the fluorinated chain increased. Assimilation efficiency was greater than 50% for all test compounds, indicating efficient absorption from food. Bioaccumulation factors (BAFs) ranged from 0.038 to 1.0 and increased with length of the perfluorinated chain; however, BAFs were not statistically greater than 1 for any PFA. Sulfonates bioaccumulated to a greater extent than carboxylates of equivalent perfluoroalkyl chain length, indicating that hydrophobicity is not the sole determinant of PFA accumulation potential and that the acid function must be considered. Dietary exposure will not result in biomagnification of PFAs in juvenile trout, but extrapolation of these bioaccumulation parameters to larger fish and homeothermic organisms should not be performed.     

Bioconcentration and tissue distribution of perfluorinated acids in rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss) were exposed simultaneously to a homologous series of perfluoroalkyl carboxylates and sulfonates in a flow-through system to determine compound-specific tissue distribution and bioconcentration parameters for perfluorinated acids (PFAs). In general, PFAs accumulated to the greatest extent in blood > kidney > liver > gall bladder. Carboxylates and sulfonates with perfluoroalkyl chain lengths shorter than seven and six carbons, respectively, could not be detected in most tissues and were considered to have insignificant bioconcentration factors (BCFs). For detectable PFAs, carcass BCFs increased with increasing length of the perfluoroalkyl chain, ranging from 4.0 to 23,000, based on wet weight concentrations. Carboxylate carcass BCFs increased by a factor of eight for each additional carbon in the perfluoroalkyl chain between 8 and 12 carbons, but this relationship deviated from linearity for the longest PFA tested, possibly because of decreased gill permeability. In general, half-lives (3.9-28 d) and uptake rates (0.053-1.700 L/kg/d) also increased with increasing length of the perfluoroalkyl chain in all tissues. Sulfonates had greater BCFs, half-lives, and rates of uptake than the corresponding carboxylate of equal perfluoroalkyl chain length, indicating that hydrophobicity, as predicted by the critical micelle concentration, is not the only determinant of PFA bioaccumulation potential and that the acid function must be considered.     

Dietary plant-protein substitution affects hepatic metabolism in rainbow trout (Oncorhynchus mykiss)

The high dietary protein requirements of salmonid fish are met with fishmeal-based feed in commercial aquaculture. The sustainability of this practice is questionable and, therefore, the feasibility of substituting fishmeal with plant-based products needs to be investigated. We investigated growth and metabolism in rainbow trout (Oncorhynchus mykiss) fed a diet composed of a mixture of plant proteins compared with those fed a fishmeal-based diet. Using two-dimensional gel electrophoresis of liver protein extracts, we showed that the liver protein profile changed in response to the alteration in the diet. A number of metabolic pathways were identified as sensitive to the protein source substitution. These included pathways involved in primary energy generation, maintenance of reducing potential, bile acid synthesis, and transport and cellular protein degradation. Interestingly, the pathways shown to be affected in the present study were somewhat different from those identified in our previous work with soyabean-based-protein replacement of fishmeal, with the effects on the abundance of several stress response proteins notably absent. We conclude, therefore, that the metabolic effects of plant protein replacement in aquaculture feed varies with plant-protein source.     

Effects of pulp mill effluents and restricted diet on growth and physiology of rainbow trout (Oncorhynchus mykiss)

Juvenile female rainbow trout was exposed for 4.5 months (June to October) to two dilutions of untreated and activated sludge treated whole mill effluent from a pulp mill producing bleached ECF pulp. Two controls were used, on fed ad libitum and a second receiving 0.5% feed of the body weight. All effluent exposed groups were fed ad libitum. Mean weight of the fish was measured monthly. At the end of the experiment a number of physiological and biochemical parameters were analyzed in order to establish the physiological status of the exposed fish in comparison with unexposed fish that obtained ad libitum or restricted amount of feed. The fish exposed to treated effluent grew significantly more than ad libitum control fish until August, whereupon growth retarded in fish exposed to the lower effluent dilution (400 v/v). The growth of fish exposed to untreated effluent did not deviate significantly from the control fed ad libitum. The results from the hematological analysis clearly showed that fish fed restricted amount of feed deviated significantly in most parameters compared with the control fed ad libitum. Fish exposed to treated effluent showed a response pattern similar to that of the control fed restricted amount of feed, whereas the fish exposed to untreated effluent showed a response pattern that did not deviate from that of the ad libitum control. The metabolic parameters suggested that fish exposed to treated effluent had a higher metabolic demand than ad libitum control and that the energy allocation at the end of the experiment was directed to processes other than growth. The responses on hematology were mainly a consequence of the increased energy demand and were not primary effects. The implications of using feed related parameters at field studies are discussed.     

Toxicokinetics of three polychlorinated biphenyl technical mixtures in rainbow trout (Oncorhynchus mykiss)

Accumulation and depuration parameters of polychlorinated biphenyls (PCBs) in fish have been reported only for a few congeners. As well, there is little information on the ability of fish to biotransform PCBs. To address these issues, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to dietary concentrations of three Aroclor mixtures (1248, 1254, 1260) in food for 30 d followed by an additional 160 d of nonspiked food at 8 degrees C. Accumulation, depuration, and potential biotransformation of 92 PCB congeners were assessed. Half-lives (t1/2) of PCB congeners ranged from 79 to 182 d, assimilation efficiencies ranged from 40 to 50% and biomagnification factors (BMF) ranged from 2.9 to 6.9. No evidence of significant biotransformation of any PCB congeners was found. All 92 congeners fell on the same t1/2 to Kow relationship as 16 preselected PCB congeners previously shown to persist in fish and no hydroxylated PCB metabolites (OH-PCBs) were detected in the plasma after 30 d of exposure. These findings suggest that OH-PCBs observed in feral fish may be accumulated from sources other than internal metabolism of the parent congeners, at least for juvenile fish at cool temperatures. Because t1/2s in this experiment were slower than t1/2s reported in other work, water temperature also may be an important factor in determining the t1/2s of all PCB congeners in fish.     

Effects of water hardness and temperature on the acute toxicity of mercuric chloride on rainbow trout (Oncorhynchus mykiss)

In this study, the toxicity of mercuric chloride (HgCl(2)), an important pollutant threatening water resources for many years, and the effects of water temperature and hardness on the toxicity in cultured rainbow trout Oncorhynchus mykiss (4.79 ± 0.16 g; 7.38 ± 0.24 cm; mean ± SD) were investigated at different temperatures (12 and 17°C) and hardness concentrations (35, 70 and 120 mg l(-1) as calcium carbonate, CaCO(3)). For this purpose, the acute toxicity tests were performed by 96-h static tests in different water temperatures and water hardness concentrations. For acute toxicity tests, solutions ranging from 0.4 to 1.2 mg l(-1) were used at 12°C and solutions ranging from 0.4 to 1.0 mg l(-1) at 17°C. The LC(50) values of HgCl(2) that killed 50% of rainbow trout within 96 h in the hardness concentrations of 35, 70 and 120 mg l(-1) CaCO(3) were calculated using probit analysis, and were found to be 0.725, 0.788, 0.855 mg l(-1) at 12°C and 0.670, 0.741, 0.787 mg l(-1) at 17°C, respectively. Consequently, the toxicity of HgCl(2) on rainbow trout decreased when the temperature decreased from 17 to 12°C. Toxicity increased when the hardness decreased from 120 to 35 mg l(-1) CaCO(3). In contrast to temperature, water hardness presents a negative effect on the toxicity of HgCl(2).     

Relative sensitivity of bull trout (Salvelinus confluentus) and rainbow trout (Oncorhynchus mykiss) to acute copper toxicity

Bull trout (Salvelinus confluentus) were recently listed as threatened in the United States under the federal Endangered Species Act. Past and present habitat for this species includes waterways contaminated with heavy metals released from mining activities. Because the sensitivity of this species to copper was previously unknown, we conducted acute copper toxicity tests with bull and rainbow trout (Oncorhynchus mykiss) in side-by-side comparison tests. Bioassays were conducted using water at two temperatures (8 degrees C and 16 degrees C) and two hardness levels (100 and 220 mg/L as CaCO3). At a water hardness of 100 mg/L, both species were less sensitive to copper when tested at 16 degrees C compared to 8 degrees C. The two species had similar sensitivity to copper in 100-mg/ L hardness water, but bull trout were 2.5 to 4 times less sensitive than rainbow trout in 220-mg/L hardness water. However, when our results were viewed in the context of the broader literature on rainbow trout sensitivity to copper, the sensitivities of the two species appeared similar. This suggests that adoption of toxicity thresholds that are protective of rainbow trout would be protective of bull trout; however, an additional safety factor may be warranted because of the additional level of protection necessary for this federally threatened species.     

Short-term modulation of lipogenesis by macronutrients in rainbow trout (Oncorhynchus mykiss) hepatocytes

Rainbow trout (Oncorhynchus mykiss) hepatocytes were cultured under simulated conditions of varying nutritional status to explore the short-term modulation by dietary substrates of the main lipogenic enzymes: glucose-6-phosphate dehydrogenase (G6PD), malic enzyme (ME), ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACoAC) and fatty acid synthetase (FAS). Primary cultures were individually exposed to varying amounts of glucose, hydrolysed casein and long-chain polyunsaturated fatty acids (PUFA) for 12 h. A second set of experiments was designed to evaluate the effects of mixing different relative amounts of these macronutrients in the culture medium. Glucose concentrations of up to 20-25 mm showed a stimulatory effect on G6PD, ME, ACL and ACoAC activity while an earlier inhibitory effect on FAS was observed at 10-20 mm glucose The use of hydrolysed casein as a nutritional source of amino acids inhibited the activity of FAS and ME and stimulated G6PD, ACoAC and ACL activity Low levels of linolenic acid exerted a stimulatory effect on all the lipogenic enzymes assayed with the exception of FAS, and increased amounts showed some inhibition of lipogenic activities Eicosapentaenoic acid and docosahexaenoic acid showed a similar effect, although the former strongly inhibited FAS activity while the latter showed greater potential to inhibit ACoAC and G6PD. A complete change in the relative levels of glucose, hydrolysed casein and PUFA in turn led to changes in the enzyme activity patterns observed. The present study shows the feasibility of exploring the direct regulation of lipogenesis in isolated fish cells by varying the relative amounts of main macronutrients, mimicking in vivo dietary conditions. It is felt that such an approach may serve to investigate the macronutrient regulation of other metabolic pathways.     

Adding magnesium to the silver-gill binding model for rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss; 2-17 g) were exposed to approximately 0.1 microM silver as AgNO3 for 3 to 4 h in synthetic, ion-poor water (20 microM Ca, 100 microM Na, 150 microM Cl, pH 7) to which was added Mg, Ca, or thiosulfate (S2O3). Gills were extracted and assayed for Ag using graphite furnace atomic absorption spectrophotometry. Up to 210 mM Mg (fourfold the concentration of Mg in seawater) did not reduce accumulation of Ag by trout gills. The conditional equilibrium stability constant (K) for Mg at silver-binding sites on the gills was calculated to be log K(Mg-gillAg) = 3.0, or approximately half-as-strong binding as for Ca at these sites. The inclusion of the Mg-gill stability constant into the original Ag-gill binding model increases the flexibility of the model, although the competitive effects of Mg are only important in sodium-poor systems.     

Effects of sodium chloride on chronic silver toxicity to early life stages of rainbow trout (Oncorhynchus mykiss)

The chronic (early life stage) toxicity of silver to rainbow trout (Oncorhynchus mykiss) was determined in flow-through exposures. Rainbow trout embryos were exposed to silver (as AgNO3) from 48 h or less postfertilization to 30 d postswimup in soft water in the presence and absence of 49 mg/L of NaCl (30 mg/L of Cl). The studies determined effect levels for rainbow trout exposed throughout an extended development period and assessed possible protective effects of sodium chloride. Lowest-observed-effect concentrations were greater than 1.25 microg/L of dissolved silver for survival, mean day to hatch, mean day to swimup, and whole-body sodium content in both studies. Whole-body silver concentrations increased significantly at 0.13 microg/L of dissolved silver in unmodified water and at 1.09 microg/L of dissolved silver in amended water. The maximum-acceptable toxicant concentration for growth was greater than 1.25 microg/L of dissolved silver in unmodified water and 0.32 microg/L of dissolved silver in amended water. Whole-body silver concentrations were more sensitive than survival and growth end points in unmodified water. Interpretation of sodium chloride effects on chronic silver toxicity to rainbow trout was complicated by differences in measured effect levels that were potentially the result of strain differences between test organisms in the two studies.     

Morphological effects of acute and chronic atrazine exposure in rainbow trout (Oncorhynchus mykiss)

The effects of the herbicide atrazine (2-Chlor-4-ethyl-amino-6-isopropyl-amino-s-triazine) on the kidney of rainbow trout (Oncorhynchus mykiss) were studied by exposing them to sublethal concentrations of 1.4 and 2.8 mg atrazine per liter of water for 96 h (acute exposure) respectively to 5, 10, 20, 40, and 80 g/L for a period of 28 days (chronic exposure).Alterations of the different components of renal corpuscles and of renal tubules, as well as an increase in cells with mitotic figures in renal hemopoietic interstitium were constant features at lower chronic (5, 10, 20, 40 g/L) exposure; necrosis of endothelial cells and renal hemopoietic tissue were prominent at concentrations of 80 g/L, and 1.4 and 2.8 mg/L atrazine.