Jack F. Klaverkamp

Accumulation and distribution of dietary uranium in lake whitefish (Coregonus clupeaformis).

We investigated the accumulation and distribution of uranium (U) in adult lake whitefish (Coregonus clupeaformis) fed a commercial diet containing 100, 1000 and 10000 µg U/g for 10, 30, and 100 days. No food avoidance or refusal occurred. The major sites of U accumulation were in the mineralized tissues, bone and scales, and in intestine, liver, kidney, and, in the highest treatment, gonads. Significant accumulation in fish fed 100 µg U/g was observed only in scales. Duration-dependent accumulation was observed in bone, scales, liver, and kidney of fish fed 10000 µg U/g and in scales of fish fed 1000 µg U/g. Dose-dependent accumulation was observed in scales of fish exposed for 100 days. U accumulation in gonads and gill peaked on day 30 when fish gonads were in the most advanced stage of maturation, of the sampling days evaluated. To evaluate the biological availability of U to fish inhabiting contaminated aquatic systems, analyses of U in scales, bone, intestine, kidney, and liver are recommended for biomonitoring programmes. The toxicology of U in these fish is described in the following manuscript (Cooley, H.M., Evans, R.E. Klaverkamp, J.F., 2000. Toxicology of dietary uranium in lake whitefish (coregonus clupeaformis) Aquat. Toxicol., 48, 393-413).

Toxicology of dietary nickel in lake whitefish (Coregonus clupeaformis)

The sublethal toxicity associated with exposure of adult lake whitefish (Coregonus clupeaformis) to diets containing 0, 10, 100, and 1000 microg Ni per g for 10, 31, and 104 days was assessed through the measurement of responses, through a range of levels of biological organization. The accumulation and distribution of Ni in these fish are described in the previous manuscript (Ptashynski and Klaverkamp, 2001. Aquat. Toxicol. in press). Hematological parameters, including concentrations of glucose and hemoglobin and hematocrit, were not different between control and treated fish. Organ and whole organism parameters, including LSI, growth, and condition factor, were also unaffected. Histopathological lesions in kidney and liver proved to be the most sensitive and reliable indicators of Ni exposure. In livers of treated fish, areas of focal necrosis and altered bile ducts were observed. Histological alterations were observed throughout the posterior kidneys, in glomeruli, tubules, collecting ducts, and hematopoietic tissue, in fish fed medium and high dose diets. In whitefish kidneys, the frequency (%) of altered distal tubules and fields of views with alterations increased with the dose and duration of exposure. Significant increases in metallothionein concentrations were observed in intestine of whitefish fed the high dose diet on day 10, but these increases were not sustained. Significant increases in lipid peroxide concentrations were also observed in plasma of whitefish fed the high dose diet on day 31, but were not observed on day 104. These biochemical responses to dietary Ni require further evaluation. To evaluate exposure of natural populations of fish to Ni, analysis of Ni in kidney and liver and assessment of renal and hepatic histopathology are recommended for use in field bio-monitoring programs.

Toxicology of dietary uranium in lake whitefish (Coregonus clupeaformis)

Using responses ranging from whole animal to the molecular level, the toxicological significance of the accumulation and distribution of uranium (U) in adult lake whitefish fed a commercial diet contaminated with three concentrations of U, 100, 1000 and 10000 µg U/g, for 10, 30, and 100 days, as described in the previous manuscript (Cooley, H.M., Klaverkamp, J.F., 2000. Accumulation and distribution of dietary uranium in Lake whitefish (Coregonus clupeaformis), Aquat. Toxicol., 48, 375-392), was evaluated. Whole organism morphometrics were unaltered by U exposure. Haematological variables were either unchanged or only transiently affected. Liver and kidney metallothionein concentrations were largely unaltered. Concentrations of serum lipid peroxides were significantly elevated in all treatment groups on days 30 and 100, indicating that U may damage cellular and sub-cellular membranes. Dose- and duration-dependent histopathologies were present in liver and posterior kidney of treated lake whitefish. The most consistent and pronounced lesions in liver were focal hepatocyte necrosis and alterations of bile ductule epithelium. Dose- and duration-dependent renal lesions were most evident in proximal tubules. However, we observed a range of histopathologies in this organ, including tubular necrosis, inflammation, haemorrhaging, depletion of haematopoietic tissues, alterations of distal tubules and collecting ducts, tubule dilation, pigmented macrophage proliferation, and glomerular lesions. Pathologies were observed in lake whitefish at all concentrations of dietary U following prolonged exposure. When used in conjunction with U tissue residue analyses, histopathological lesions and indices of lipid peroxidation may prove useful indicators of U toxicity when assessing fish health in U biomonitoring programmes.

Oxidative stress in Lake Sturgeon (Acipenser fulvescens) orally exposed to 2,3,7,8-tetrachlorodibenzofuran

Juvenile lake sturgeon were orally dosed with gelatin containing nominal concentrations of 0, 0.16 or 1.6 ng [3H]-2,3,7,8-tetrachlorodibenzofuran (TCDF) kg−1 fish weight. Liver, kidney and blood were collected 10 and 27 days after exposure. Phase I mixed-function oxidase (MFO) and Phase II (glucuronyltransferase) enzyme activities were determined in liver. Concentrations of non-enzymatic (tocopherol, retinoids) and enzymatic (superoxide dismutase, catalase, glutathione peroxidase) antioxidant parameters were also quantified in liver and kidney. TCDF concentrations were elevated in liver and kidney of both dose groups at 10 and 27 days. Lower tissue concentrations of TCDF at 27 days, compared with 10 day exposures, as well as the presence of polar metabolites in bile, may indicate rapid metabolism and clearance of the contaminant. MFO enzyme activity, measured as ethoxyresorufin-O-deethylase (EROD), was induced in liver with activity at the two sample periods reflecting the TCDF concentrations. Greater concentrations of hepatic lipid peroxides in both dose groups, indicate that oxidative stress was produced by contaminant exposure and/or induced metabolism enzyme activity.

Toxicological effects of dietary arsenic exposure in lake whitefish (Coregonus clupeaformis)

Adult lake whitefish were fed As contaminated diets at nominal concentrations of 0, 1, 10, and 100 microg As/g food (dry weight) for 10, 30, and 64 days. Reduced feed consumption was observed in lake whitefish fed the 100 microg As/g food, beginning on day 45 of exposure. The accumulation and distribution of As in these fish are described in the previous manuscript [Pedlar, R.M., Klaverkamp, J.F., 2001. The accumulation and distribution of dietary arsenic in lake whitefish (Coregonus clupeaformis). Aquat. Toxicol., in press]. At the molecular level of organization, metallothionein (MT) induction occurred in lake whitefish fed the 100 microg As/g food after 10 and 30 days, and in fish fed the 1 and 10 microg As/g diets for 64 days. Dietary As exposure did not have a significant effect on plasma lipid peroxide (LPO) concentrations. At the tissue and organ level, mean liver somatic index decreased significantly in lake whitefish fed the 100 microg As/g food for 64 days. Blood parameters (hematocrit, hemoglobin concentration, red blood cell count, mean cell volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration) were not affected by exposure to As contaminated diets. Liver and gallbladder histopathologies were observed in lake whitefish fed all As contaminated diets after each duration of exposure. Histopathology observed in liver included nuclear, architectural and structural alterations, areas of inflammation, and focal necrosis. Sloughing of the epithelium, dilation of vascular elements, inflammation, edema, fibrosis, and increased width of the submucosa were some of the alterations observed in gallbladders of lake whitefish fed As contaminated diets. Both organs were sensitive to As exposure, as damage occurred with exposure to dietary concentrations of As as low as 1 microg/g. Whole organism parameters were unaltered by dietary As exposure. Based on the results of this study, histopathological alterations in liver and gallbladder, and hepatic MT induction may be useful indicators of As toxicity in environmental monitoring programs that also measure As concentrations in those tissues.

The accumulation, distribution, and toxicological effects of dietary arsenic exposure in lake whitefish (Coregonus clupeaformis) and lake trout (Salvelinus namaycush).

A 20-day experiment was conducted to compare the accumulation, distribution, and toxicological effects of dietary As, as arsenate, in lake whitefish (LWF, Coregonus clupeaformis) and lake trout (LT, Salvelinus namaycush). Results of this experiment were used to design an experiment of longer duration in which one of the fish species was selected and exposed three times per week to lower dietary As doses. In the present study each treatment group was exposed to a combination of one of three doses of As (0, 100, or 1000 microg As/g) and one of two types of diet, no brine shrimp (NS) or with brine shrimp (WS) for a total of eight dosing events. Brine shrimp were added to determine whether their presence enhanced consumption of As-contaminated food. Modified feeding behavior occurred in both fish species fed As contaminated diets, with the exception of the 100 microg As/g NS food. Brine shrimp addition did not affect feed consumption of the As contaminated diets. Significant As accumulation occurred in stomach, pyloric caeca, intestine, liver, kidney, and gallbladder, but not in bile or muscle. As exposure did not have a significant effect on hepatic and renal metallothionein concentrations. Concentrations of lipid peroxides were only significantly elevated in the plasma of LT fed the 1000 microg As/g WS food. Liver somatic indices decreased significantly in both species, whereas hematological parameters were not affected in either species. Histological lesions occurred in gallbladder, liver, kidney, pyloric caeca and intestine from LWF. These lesions were not observed in LT; however, gallbladders were not examined in this species. Weight gain was lower in both species fed As contaminated diets, however, condition factors were not affected.

Accumulation, distribution and toxicology of dietary nickel in lake whitefish (Coregonus clupeaformis) and lake trout (Salvelinus namaycush)

An 18-day experiment was conducted to investigate the uptake and sublethal toxicity of dietary Ni in adult lake whitefish (LWF, Coregonus clupeaformis) and lake trout (LT, Salvelinus namaycush) fed diets containing 0, 1000 and 10000 microg Ni/g, prepared with and without brine shrimp. The results of this experiment were used to design an experiment of longer duration in which one of the fish species was selected and exposed to lower dietary Ni doses. In the present study feed refusal was observed in LT and LWF fed 10000 microg Ni/g, after three and 4-5 feedings, respectively. LT fed Ni-contaminated diets exhibited different patterns of Ni accumulation than LWF. Increased Ni concentrations in all LWF tissues, except the intestine, were associated with increased doses of Ni. Copper and Zn concentrations in kidney and liver of LWF were altered. Metallothionein concentrations in kidneys of LT fed 1000 microg Ni/g and 10000 microg Ni/g and LWF fed 10000 microg Ni/g and in livers of LWF fed 10000 microg Ni/g (diet without shrimp only) increased significantly. Increased lipid peroxide production in the plasma of LT fed 10000 microg Ni/g was observed. Blood glucose and electrolytes were affected by Ni exposure. Histopathological alterations were observed in kidneys of LWF fed low and high dose diets, livers of whitefish fed high dose diets, and intestines of LWF fed high dose diets and LT fed low and high dose diets. LT fed high dose diets exhibited significant decreases in weight.

An assessment of Ah-inducible phase I and phase II enzymatic activities and oxidative stress indices in adult lake trout (Salvelinus namaycush) from Lake Ontario and Lake Superior

Abstract Relationships between phase I/mixed function oxidase (MFO) and phase II (UGT) enzymatic activities and the appearance of oxidative stress were investigated in the livers of lake trout collected during 1992–1994 from Lake Ontario and Lake Superior. These lakes represent the most and least contaminated of the Great Lakes, respectively. Despite historically different contaminant profiles, lake trout from Lake Ontario had only marginally greater hepatic MFO activity and equivalent UGT activity when compared to lake trout from Lake Superior. Renal MFO activity was significantly greater in lake trout from Lake Ontario. UGT activity in the kidney was similar in lake trout from the two lakes except for those from Black Bay of Lake Superior, which had significantly lower activity. The Lake Superior population had greater hepatic activites of the enzymatic antioxidants catalase in 2 of the 3 years sampled, greater glutathione peroxidase in 1 of the 3 years sampled, and had consistently greater concentrations of the non-enzymatic antioxidant tocopherol. Tocopherol concentrations and glutathione peroxidase activity were also greater in the kidney of lake trout from Lake Superior. Hepatic and renal ascorbic acid concentrations were not different between the two populations. The predominant vitamin A compounds in lake trout from Lake Superior were retinol based, whereas didehydroretinoids were more predominant in lake trout from Lake Ontario. Total alcohol and esterified concentrations of retinoids were significantly higher in the kidneys of lake trout from Lake Ontario. This suggests a compensatory mechanism, whereby filtered retinol is reabsorbed by cells of the kidney. Lake Ontario lake trout had significantly greater hepatic concentrations of lipid hydroperoxides, a measure of oxidative stress.

An evaluation of the relationships among oxidative stress, antioxidant vitamins and early mortality syndrome (EMS) of lake trout (Salvelinus namaycush) from Lake Ontario

Mortality of the offspring in up to 50% of female lake trout from Lake Ontario appears just before swimup, and is known as early mortality syndrome (EMS). Adult lake trout from Lake Ontario also have high tissue concentrations of organic contaminants and elevated activities of hepatic MFO (mixed-function oxidase) biotransformation enzymes. Previous examinations of lake trout exposed to organochlorines in the laboratory and with elevated MFO activity have also identified increased indices of oxidative stress. We assayed MFO activity, as ethoxyresorufin-O-deethylase (EROD), and Ah-induced phase II activity, as glucuronyltransferase (UGT), in livers of adult female lake trout from Lake Ontario and from a reference population in Lake Manitou in order to examine relationships to oxidative stress. Enzymatic (superoxide dismutase, catalase, glutathione peroxidase) and non-enzymatic antioxidants (vitamins A, C and E), and oxidative membrane breakdown products (lipid hydroperoxides) were also quantified in livers of adult females. Eggs from each of the females were reared in the laboratory and analyzed at six time periods before the swimup stage for content of the antioxidant vitamins C and E and for vitamin A and its provitamin forms. MFO activity of the fry was assessed only at the postswimup stage. Adults whose offspring had EMS (>10% mortality) had lower content of vitamin E compared to the group from Lake Ontario without EMS (<10% mortality). Hepatic lipid hydroperoxides were elevated in both groups of adult lake trout from Lake Ontario. Except for total carotenoid concentrations, which were lower in eggs of lake trout from the EMS group, concentrations of antioxidant vitamins were comparable in eggs from lake trout with and without EMS from Lake Ontario and reference lake trout. No correlation was found among MFO activity, vitamin concentrations and the prevalence of EMS in lake trout fry at any of the developmental stages that were examined.

Metabolism of 3H-retinol by lake trout (Salvelinus namaycush) pre-exposed to 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126)

To provide insight into retinoid metabolism in fish exposed to polychlorinated biphenyl (PCB), juvenile lake trout were orally exposed to 0, 3, 10 or 30 μg 3,3′,4,4′,5-pentachloro-biphenyl (PCB 126) kg−1 body weight for 12 weeks. Phase I (MFO) enzyme activity was induced by 60, 177 and 450 fold, while glucuronyltransferase enzyme activity was 2.3, 2.5 and 5 fold higher in the 3, 10 and 30 μg PCB kg−1 dose groups, respectively, compared with untreated fish. After 12 weeks, cannulae were inserted into the dorsal aorta. Two days later, 3H-retinol was injected as a bolus in charcoal stripped plasma through the cannula. Blood samples were obtained 15 and 30 min and 1, 3, 6, 15, 30, 50 and 75 h after the injection. The radioactivity recovered from plasma was compared for each PCB dose group and was not significantly different for any of the treatments at any of the sample times. However, greater radioactivity recovered from bile after 75 h indicates accelerated metabolism of 3H-retinol with increasing PCB dose. Tissue distributions of aqueous and lipophilic soluble radioactivity indicates that direct metabolism of retinol by MFO and conjugation enzymes is probably responsible for the depletion of retinoids in PCB 126 exposed lake trout.