Sequestration of environmental cadmium by metallothionein in the roach (Rutilus rutilus) and the stone loach (Noemacheilus barbatulus)

Two species of coarse fish that are relatively resistant to cadmium poisoning were exposed to sub-lethal concentrations of the metal in their aquarium water. Thus, roach were exposed to cadmium concentrations between 30 and 500 micrograms/l for periods of 14-70 days whereas stone loach were exposed to 1250 micrograms Cd/l for 21-77 days. Under all conditions of exposure, it was found upon analysis of the major organs of accumulation of cadmium in the two species that the toxic metal was sequestered by a single isoform of metallothionein. The amino acid compositions of roach and stone loach metallothionein were determined and found to be similar to those reported for other piscine metallothioneins. The two proteins were found to contain Cd:Zn:Cu in approximate ratios of 4:1:2 per mole of protein. The sequestration of Cd by metallothionein in the two resistant species of fish is contrasted with the situation observed previously in a cadmium-sensitive species, the rainbow trout.     

A comparison of cadmium-induced metallothionein gene expression and Me2+ distribution in the tissues of cadmiumsensitive (rainbow trout; Salmo gairdneri) and tolerant (stone loach; Noemacheilus barbatulus) species of freshwater fish

1. The accumulation of cadmium in the liver, kidney and gills of rainbow trout and stone loach was measured during exposure of the fish to the metal at 3 smg/l in their aquarium water. The pattern of accumulation of the toxic metal in the individual organs was different between the two species.2. The tissue concentrations of metallothionein-specific mRNA and metallothionein protein were also determined in these organs from the same fish. In rainbow trout, the induction of metallothionein gene expression resulted in a gradual increase in metallothionein concentration in gill over the course of the experiment whereas increases in metallothionein in the liver and kidney were detected only at the later time points of analysis (beyond 19 weeks). By contrast, in the same tissues from stone loach, relatively minor changes were quantified in specific mRNA and metallothionein concentrations.3. Throughout the experimental period, tissue concentrations of zinc and copper were determined in the liver, kidney and gills of the rainbow trout and stone loach. Subtle decreases were observed in the zinc concentration of gills in rainbow trout and substantial increases were observed in the hepatic copper concentrations in both species at the later time points of analysis.4. The ability of cadmium to induce metallothionein gene expression and its subsequent ability to compete for the sequestration sites on the newly-synthesized protein is discussed with regard to the relative levels of cadmium, zinc and copper in the organs studied and differing regimes of cadmium administration.     

Studies on the toxicity of zinc sulphate and of cadmium sulphate to stone loach Noemacheilus barbatulus (L.) in hard water

The toxicity of zinc sulphate and cadmium sulphate to stone loach Noemacheilus barbatulus was examined in an attempt to explain the distribution of the species in two polluted streams. Stone loach in hard water were more sensitive than rainbow trout to zinc, but much more resistant to cadmium. Their disappearance from the Willow Brook, Northants, in 1971–72 could be accounted for by zinc toxicity. A behavioural change, the loss of the instinct to hide during daylight, occurred in subsequently lethal concentrations of cadmium. The accumulation of cadmium in several tissues was also determined.     

Uptake and distribution of radioiodine, and the effect of ambient nitrate, in some fish species

125I uptake by the thyroid was most pronounced in the smelt (Osmerus eperlanus), perch (Perca fluviatilis), rainbow trout (Salmo gairdneri), roach (Rutilus rutilus) and bleak (Alburnus alburnus). Unlike other tissues, only the muscle in the species studied no relative accumulation of 125I as compared to the ambient water. The Crusian carp (Cyprinus carassius) indicated the lowest levels of accumulation of 125I among the species tested. Thyroid radioiodine concentration was independent of the length of the fish in the rainbow trout and perch. In the rainbow trout, however, the liver and muscle radioiodine concentrations decreased significantly with increasing length of the fish. Exposure of rainbow trout to water containing supranormal concentration of nitrate (1500 micrograms/l) resulted in lowered 125I uptake. The same phenomenon was observed for the perch and Crusian carp, with low nitrate concentrations, while with higher nitrate concentrations, the uptake levels were again increased. Thyroid function, as judged from the conversion ratio and T/S ratio, was not affected by nitrate.     

The survival and growth of six species of freshwater fish, in tapwater and diluted and undiluted effluent from sewage percolating filters

The survival and growth of rainbow trout, Salmo gairdneri Richardson, stone loach, Noe-macheilus barbatulus (L), bullhead, Cottus gobio L. , three-spined stickleback, Gasterosteus aculeatus L., minnow, Phoxinus phoxinus (L), and roach, Rutilus rutilus (L), have been studied in water of three qualities—Birmingham tap water, 50% and 100% settled effluent from filter beds. Predicted toxicities (the sum of the proportions of the 48-h LC50 of individual poisons to rainbow trout) were compared with the observed toxicity in 50% and 100% effluent. The behaviour of each species offish and the rates of mortality over an exposure period of 210 days are given together with cumulative growth rates (as a percentage of the initial weight). The Mean Percentage Change in Weight (MPW) and Mean Growth Rate (MGR) for each group offish was calculated from the initial mean wet weight and the mean weight of surviving fish at the end of the first and second intervals. The Mean Production Rate (MPR) over the experimental period was also calculated and was used as a holistic measure of the rate of total tissue elaboration or utilization, or loss in biomass due to mortality. The Condition Factor (K) also reported was calculated from the body weight and length and was used to examine (a) the effects of water quality, and (b) the timing and duration of maturation of the gonads. The latter was further used as a measure of the energy status of the fish.     

A comparison of the sequestration of cadmium and zinc in the tissues of rainbow trout (Salmo gairdneri) following exposure to the metals singly or in combination

Rainbow trout were exposed to either cadmium (9 micrograms/l) or zinc (100 micrograms/l) in their aquarium water. They were then transferred to water containing concentrations of cadmium (54 micrograms/l) that would have otherwise proved fatal to the majority of the fish without the pretreatment. Most of the fish survived under both sets of conditions. However, two different mechanisms seem to be involved in the protection of the animals against the toxic manifestations of cadmium. In both cases, more than 99% of the total body load of cadmium was found in the liver, kidney and gills of the animals. Analysis of the metal-binding proteins in these organs was carried out. In the fish exposed to the two concentrations of cadmium, the toxic metal was found only in association with two low mol. wt specific binding proteins despite the presence of zinc- (and copper)-containing isometallothioneins in all three organs. On the other hand, cadmium was distributed between these binding-proteins and metallothioneins in the liver, kidney and gill of the trout pretreated with zinc before their exposure to cadmium.     

Hepatic biotransformation enzymes of fish exposed to non-point source pollution in small streams

The response of hepatic phase I and phase IIbiotransformation enzymes in brown trout(Salmo trutta f. fario) and stone loach(Barbatula barbatula) to differentpollution exposure scenarios was investigatedduring a 5-year-study. Phase I enzymes wereassessed by means of7-ethoxyresorufin-O-deethylase (EROD) activityand cytochrome P4501A (CYP1A) protein level,and phase II enzymes were measured by thecatalytic activity of glutathione-S-transferase(GST). Fish were exposed in the laboratory totap water (controls) or under semi-fieldconditions in so-called bypass systems to waterfrom two streams, with one of the streams(Krähenbach) receiving contaminants mainlyfrom non-point sources (for instance, surfacerun-off), and the second stream (Körsch)receiving contaminants from both non-pointsources and point sources (sewage plants). Inaddition to the fish in the bypass systems,feral fish from the streams were sampled. Thequestions addressed by the study were: (1) isit possible to discriminate the study sites onthe basis of hepatic biotransformation indices?(2) are the metabolic enzyme responses of thetwo species, brown trout and stone loach,comparable? (3) are the metabolic enzymeresponses of fish from the bypass systemscomparable to those of feral fishes? HepaticEROD activities were elevated in both browntrout and stone loach exposed in the bypasssystems compared to the laboratory control. Adiscrimination of the two field sites relativeto pollution intensity was possible by hepaticEROD activities of brown trout only, whereasbiotransformation enzymes of stone loach showedno consistent differences between the stream.Hepatic GST levels of both speciesdid not discriminate between the streams orbetween streams and laboratory. The enzymeresponses measured in fishes from the bypasssystems were comparable to those of feralfishes living in the streams. The findings ofthis study point to critical factors for thebiological monitoring of low-level pollutionwith complex chemical mixtures, including theselection of appropriate test species,monitoring endpoints, and sampling strategies.     

Induction of metallothionein gene expression by cadmium and the retention of the toxic metal in the tissues of rainbow trout (Salmo gairdneri)

1. When rainbow trout were exposed to cadmium by intraperitoneal injection, there was a rapid (within 3hr) and significant (approx. 63%) loss of the metal from the whole bodies of the fish.2. Of the metal retained in the bodies of the fish (approx. 37% of the injected dose), more than 98% was accounted for collectively among the liver, kidney and gills.3. Subsequent maintenance of the rainbow trout in fresh water for up to 98 days post-metal administration, indicated that there was no further loss of the cadmium accumulated in the organs studied and that the distribution of the metal among the liver, kidney and gills remained unchanged over that period.4. During this 98-day period of maintenance of the fish, tissue concentrations of metallothionein-specific mRNA and metallothionein protein were quantified using riboprobe and ELISA systems respectively. Metallothionein-specific mRNA concentrations increased rapidly (within 24 hr) before falling back to levels similar to, or slightly greater than, those found in control animals. The concentration of metallothionein protein also increased significantly (within 3 days) then remained elevated thereafter.5. Throughout the experimental period, the concentrations of zinc and copper were also monitored in the liver, kidney and gills of the rainbow trout. The concentrations of each ion differed between each of the organs but did not change during the experiment.6. The induction of metallothionein gene expression by cadmium in the liver, kidney and gill of rainbow trout and the subsequent sequestration of the toxic metal is discussed with regard to the relative levels of these other essential metal ions.     

Temperature effects on anaesthesia with clove oil in six temperate‐zone fishes

The potential use of clove oil (eugenol) as an anaesthetic for Atlantic salmon Salmo salar , brown trout Salmo trutta , rainbow trout Oncorhynchus mykiss , whitefish Coregonus lavaretus , perch Perca fluviatilis and roach Rutilus rutilus was examined at 5, 10, 15 and 20° C using three anaesthetic concentrations (varying from 20 to 200 mg l⁻¹) at each temperature. Substantial species differences in sensitivity to clove oil were observed, even amongst congeners, and there may be some disadvantages (slow recovery and possibly mortality) with using clove oil for 0+ year whitefish and at low temperatures for perch and roach.     

Three species of fishes from an eutrophic, seasonally alkaline lake are not more tolerant to acute exposure to high pH in the laboratory

A number of different freshwater fish species (perch Perca fluviatilis , roach Rutilus rutilus and rudd Scardinius erythrophthalamus ) from either eutrophic (Slapton Ley, a seasonally alkaline lake) or non-eutrophic waters were compared with respect to their sodium uptake kinetics and tolerance to acute (1 h) exposure to pH 9·5. Further comparisons were made with rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta . The influence of fish size was also investigated in rainbow trout. Exposure to pH 9·5 was found to disrupt sodium balance and inhibit ammonia excretion in all species and sizes of fishes. The origin of fishes did not have a significant effect on the sodium uptake kinetics or the physiological responses to high pH water. The fishes from the eutrophic lake therefore did not appear to have any increased tolerance to acute exposure to alkaline water. In contrast to previous studies there was no inhibition of Na⁺ uptake during exposure to high pH. Indeed in some groups of fish Na⁺ uptake was actually stimulated, as was Na⁺ efflux. These differences are attributed to experimental water composition and interspecific differences in physiology. It was not always possible to size-match fishes of the different species, so rainbow trout were used to assess the effect of body mass (from 2 to 40 g), on Na⁺ uptake kinetics and Na⁺ or ammonia fluxes during alkaline water exposure in rainbow trout. Size had no significant effect on these measurements within this narrow range, which helps validate the comparison between species in this study.     

Development of the activities of oxidative, glycolytic and muscle enzymes during early larval life in three families of freshwater fish

The development of the activities of oxidative (COX, CS), glycolytic (PFK, PK, LDH) and muscle enzymes (CK, MK, Pase) was studied in representatives of the families Coregonidae, Salmonidae and Cyprinidae, from hatching to an age of approximately 100 days. In addition, the activities of two enzymes of amino acid metabolism (GOT, GPT) were followed in rainbow trout and in roach.
Water content of fresh body weight and protein content of dry body weight decrease during the early larval period. Specific activities of the two oxidative enzymes decline, whereas those of glycolytic and muscle enzymes increase in all species.
A family-specific event is the enormous increase in glycolytic and muscle enzymes from very low values in the early larva to very high levels in adult Coregonus sp. In rainbow trout, CS activity begins with a low-level period lasting throughout the yolk-sac period, whereas in the other species CS activity is high immediately after hatching.
Acclimation to either 15 or 20° C has no effect on the mass-specific activities of PFK, M K, CK and Pase in roach and chub, but the former three enzymes appear to be strongly dependent on rearing conditions during the early larval period, whereas Pase is not.     

Mass immigration of juvenile fishes into a small,once-dried tributary demonstrates the importance of remnant tributaries as wintering habitats

The winter ecology of stream fishes is still poorly understood, especially in non-salmonid species. A small tributary (<4 m in width, <1 km in length) of the Otofuke River (central Hokkaido, northern Japan) dewatered in the summer of 2013, which allowed us to evaluate immigration after the summer draught. More than 10000 juveniles of Siberian stone loach Barbatula toni, Japanese dace Tribolodon spp., and non-native rainbow trout Oncorhynchus mykiss immigrated to the tributary in the four months following the summer dewatering. This suggests that even remnant tributaries may be critical wintering habitats for some juvenile fishes.     

Effects of stress on growth, cortisol and glucose levels in non-domesticated Eurasian perch (Perca fluviatilis) and domesticated rainbow trout (Oncorhynchus mykiss)

Eurasian perch (Perca fluviatilis) is a promising aquaculture candidate, but the growth performance of this non-domesticated species may be negatively affected by its stress responsiveness to intensive culture conditions. To evaluate this potential problem, juvenile Eurasian perch were exposed to a standardized handling stressor twice a week for an 8-week period. A similar study was conducted on domesticated rainbow trout (Oncorhynchus mykiss) for comparison of intra- and inter-specific differences. The stressed fish of both species showed lower body growth than the non-stressed control fish, however, the final mean body mass was 35.4% lower in the stressed Eurasian perch than in the non-stressed controls, compared to 22.8% difference between the two groups in rainbow trout. The stress responsiveness was examined by comparing the post-stress cortisol and glucose levels in repeatedly stressed fish and fish exposed to the stressor only once. The cortisol stress response in both species strongly indicated a habituation to the repeated stressor. Thus, repeatedly stressed Eurasian perch reached maximum cortisol levels of 130 ng/mL after 0.5 h compared to 200 ng/mL in the fish stressed once, while considerably smaller differences in cortisol levels were shown between the repeatedly and single stressed rainbow trout. Rainbow trout also showed lower post-stress glucose levels in the repeatedly stressed fish compared to the single stressed fish. In contrast, the glucose levels in both groups of Eurasian perch increased abruptly after stress treatment and remained elevated at approximately 19 mM for 6 h; levels were three times as high as the peak levels 3 h post-stress in rainbow trout. Together, the habituation of the stress response shown in both species did not eliminate the growth difference found in the repeatedly stressed fish versus the control fish. Further, the lower growth performance of Eurasian perch compared to rainbow trout could partly be due to the increased energy consumption in the more stress responsive Eurasian perch.     

Effects of temperature, salinity, and feeding on aminotransferase activity in the liver and white muscle of rainbow trout (Salmo gairdneri Richardson).

1. The liver-somatic index of rainbow trout is governed by temperature and salinity, and by the interaction of these two factors. 2. The overall liver-alanine aminotransferase activity (in units/100 g body weight) increases slightly with increasing salinity of the surroundings in the case of rainbow trout. 3. The overall liver-aspartate aminotransferase activity (in units/100 g body weight) in rainbow trout depends on their food and the temperature at which they are kept. 4. Salinity adaptation leads to reductions in the specific alanine and aspartate aminotransferase activity in the liver of rainbow trout. 5. The specific alanine aminotransferase activity in the muscle of starving rainbow trout kept in diluted seawater (580 mOsm/l, 18 degrees C) is clearly higher than in control animals kept in tapwater.     

The effects of cadmium on ALA-D activity, growth and haemoglobin content in the water flea, Daphnia magna

The ALA-D activity, haemoglobin content and growth was studied in the water flea, Daphnia magna, exposed to 0, 0.1, 0.2, 0.4, 0.8 and 1.6 micrograms Cd/l. The ALA-D activity in water fleas exposed to 0.2-1.6 micrograms Cd/l fluctuated around the control value. The activity in animals exposed to 0.1 micrograms Cd/l decreased during the entire experiment. After 16 days exposure to cadmium the haemoglobin content in water fleas ranged between 80 and 31% of control value. In animals exposed to 0.8 and 1.6 micrograms Cd/l the haemoglobin content decreased progressively during the experiment. Growth was not affected by cadmium at these concentrations.     

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.     

Ultrastructural biomarkers as tools to characterize the health status of fish in contaminated streams

Ultrastructural biomarkers in gill, liver, andkidney of brown trout (Salmo truttaf. fario) and stone loach (Barbatulabarbatula) were investigated over afive year period. The cellular damage of theorgans was assessed semi-quantitatively basedon a three-step classification ofultrastructural responses. Data obtained forfish exposed under semi-field conditions in twodifferently polluted test streams and in thelaboratory demonstrated that the ultrastructureof the organs can be correlated with differentpollutant exposure conditions. Cellular damagewas generally more severe in fish exposed to acomplexly polluted stream than in those exposedto a moderately polluted stream or to tap waterin the laboratory. Histopathological effects inliver and gill of trout were more pronounced inferal fish than in transplanted fish, whereasresponses in the kidney in both species, and inliver and gill of loach, were similar forintroduced and feral fish. In a laboratoryexperiment where trout were exposed todifferent mixtures of pollutants includingpesticides, PAH, and ammonia, only theultrastructure of kidney and liver showedsignificant differences between the threeexperimental set-ups. In a recovery experiment,where trout were transferred from thesemi-field condition back to the laboratory,ultrastructural investigations showed adifferential capacity of the respective organsto recover from stress under field conditions.Kidney and liver fully recovered after threemonths under control condition whereas gillsdemonstrated only partial recovery.     

Recovery of Historically Contaminated Watercourse Polluted by the Chemical Wood Industry: EROD Activity in Fish as Biomarker

Despite outstanding process alterations over decades, pulp- and paper-mill-contaminated sediments and continuing exposure by the effluents may still have effects on biota. In this study, ecotoxicological impacts in the boreal watercourse were analyzed by measuring ethoxyresorufin-O-deethylase (EROD) induction from wild fish populations and from experimentally exposed fish. In order to assess the role of sediment-borne chemicals, juvenile rainbow trout (Oncorhynchus mykiss) were exposed in the laboratory to the surface sediments of Lake Vatianjärvi and Southern Lake Saimaa, both watercourses impacted by the chemical wood industry for approximately a century. Hepatic EROD activity was also measured from roach (Rutilus rutilus) and perch (Perca fluviatilis) caught in Lake Vatianjärvi. Increased EROD activity was not observed in wild fish caught in Lake Vatianjärvi nor in rainbow trout exposed to the sediment of Lake Vatianjärvi, but it was observed in rainbow trout exposed to the sediment of Southern Lake Saimaa, probably explained by critically high concentrations of retene. The results of both the laboratory and field study indicate the absence of EROD-inducing compounds of the previously heavily contaminated Lake Vatianjärvi.     

THE EFFECT OF TENSION OF DISSOLVED OXYGEN ON THE TOXICITY OF UN-IONIZED AMMONIA TO SEVERAL SPECIES OF FISH

The resistance to rapidly lethal concentrations of un-ionized ammonia ranging from about 2.0 to 8.8 p. p. m. N was determined for rainbow trout ( Salmo gairdnerii Richardson), perch ( Perca fluviatilis L.), roach ( Rutilus rutilus (L.)), and gudgeon ( Gobio gobio (L.)) in tensions of dissolved oxygen 53.4 and 96.7% of air-saturation value at 15.2°C.
The relation between concentration of un-ionized ammonia and period of survival from a few hours to 13 days was investigated for rainbow trout in two tensions of dissolved oxygen, 45.7 and 100.3% of air-saturation value, at 20.1°C.
Period of survival of all the species tested decreased with rise in concentration of un-ionized ammonia.
Decrease in tension of dissolved oxygen increased the toxicity of un-ionized ammonia to all species except gudgeon, for which there was no significant difference in period of survival at the two levels of oxygen.
The effect of oxygen tension on period of survival was greatest in the lowest concentrations of un-ionized ammonia.
Resistance of perch and roach to lack of oxygen was not significantly affected, but that of rainbow trout was reduced significantly, by the presence of a small concentration of ammonia (not toxic to trout within at least 13 days).