Plerocercoids of the cestode Diphyllobothrium ditremum in brown trout Salmo trutta: substantial increase in infection after establishment of European minnow Phoxinus phoxinus.

This study focuses on plerocercoids of the cestode Diphyllobothrium ditremum in brown trout Salmo trutta from the subalpine lake Øvre Heimdalsvatn in south-central Norway. Salmo trutta was the only fish species in this lake until European minnow Phoxinus phoxinus was registered in 1969. The P. phoxinus population increased substantially in the following years. In contrast with the 1969-1972 period, when plerocercoids of D. ditremum were practically absent in S. trutta, there was a high prevalence and intensity of infection in the 2013 S. trutta samples. Because the life cycle of D. ditremum involves two larval stages, in copepods and salmonids and mature worms in piscivorous birds, such as mergansers and loons, a change in feeding ecology of S. trutta or changes in population densities of copepods, fish or birds might have influenced the infection pattern. No relationships between D. ditremum infection and muscle-tissue δ15 N signature or Hg concentration were found, indicating that infection is not a result of piscivory or cannibalism. Furthermore, consumption of copepods by S. trutta during summer and autumn was low. On the other hand, the number of piscivorous birds has increased, probably due to the presence of P. phoxinus as a new and numerous prey. An increased number of final D. ditremum hosts may have produced a higher output of cestode eggs, resulting in more infected copepods that in turn are consumed by S. trutta. Indirectly, P. phoxinus may therefore have caused the observed increased infection in S. trutta and thereby imposed further negative effects on S. trutta in high mountain areas.

Genetic variation for tolerance to acidic water in salmonids.

Water pH is an important factor affecting the general water quality as well as quality traits in fishes, and the magnitude of the effect varies among species. The massive and negative effect of acidification of rivers and lakes became evident during the 1960s and 1970s and caused the depletion of fish stocks in several countries in the northern hemisphere. Significant variation in tolerance to acidic water has been documented among salmonid species, and large genetic variation has been identified among strains of brown trout Salmo trutta, brook trout Salvelinus fontinalis and Atlantic salmon Salmo salar. For S. trutta, S. fontinalis and S. salar, there is considerable additive genetic variation in tolerance to acidic water, with heritabilities (h(2)) ranging from 0·09 to 0·27 for dead eyed-eggs (the period most sensitive to low pH). The main reasons for depletion of freshwater fish stocks are discussed.

Effect of nutritional status and sampling intensity on recovery after dorsal aorta cannulation in free-swimming Atlantic salmon (Salmo salar L.).

Recovery from implantation of a cannula in the dorsal aorta (DA) of Atlantic salmon (Salmo salar) was studied in relation to nutritional status and sampling intensity. The incentive for the study was the inconsistency between published reports and our own experience of recovery and longevity of fish exposed to this protocol. In two studies using starved fish, blood (0.3 ml) was sampled 0, 1 and 24 h after DA-cannulation, and thereafter at 48 and 72 h and thereafter once weekly for four weeks. In a third study using fed fish, four consecutive samples (0, 3, 6 and 12 h after a meal) were obtained twice a week over a four-week period. All fish displayed a sharp increase in pCO(2) and haematocrit (Hct) during surgery, followed by a marked raise in cortisol, glucose, sodium and potassium (1 h). pCO(2), pH and Hct approached baseline levels as early as the 1 h post-surgery sample, while this was not the case for cortisol and electrolytes before the 24 h post-surgery sample. Glucose did not display any significant changes post surgery. From then on, all variables displayed minor but non-significant (P > 0.05) changes indicating a steady state close to baseline values for unstressed fish. This pattern was independent of sampling procedure, i.e. repeated single or multiple samples and thus volume of blood removed. Nutritional status (fed vs. starved) did not affect post-surgical recovery pattern. Only K(+) and Hct displayed consistent and significant post-prandial patterns. We found marked differences between baseline level of cannulated fish and uncannulated control fish, in pH, K(+) and Hct indicating that cannulation may be the preferred method to obtain representative resting values in fish.

Effects of production intensity and production strategies in commercial Atlantic salmon smolt (Salmo salar L.) production on subsequent performance in the early sea stage.

A data set from commercial Atlantic salmon (Salmo salar L.) producers on production intensity and production strategies in smolt tanks (N = 63-94) was obtained during 1999-2006. The effects of production intensity on subsequent fish mortality and growth during the early sea phase (90 days) were examined by principal component analysis and subsequent generalized linear model analysis. Levels of accumulated metabolites (CO(2), total ammonia nitrogen and NH(3)), and information provided by producers (production density (kg fish m(3-1)), specific water use (l kg fish(-1) min(-1)) and oxygen drop (mg l(-1)) from tank inlet to tank outlet), were used as predictor variables. In addition, several other welfare relevant variables such as disease history, temperature during freshwater and sea stage; season (S1) or off-season (S0) smolt production; and the use of seawater addition during the freshwater stage were analyzed. No strong intensity effects on mortality or growth were found. CO(2) levels alone (P < 0.001, R(2) = 0.16), and in combination with specific water use (R(2) = 0.20), had the strongest effect on mortality. In both cases, mortality decreased with increasing density. For growth, the intensity model with the most support (R(2) = 0.17) was O(2) drop, density and their interaction effects, resulting in the best growth at low and high intensity, and poorer growth at intermediate levels. Documented viral disease outbreaks (infectious pancreatic necrosis and two cases of pancreas disease) in the sea phase resulted in significantly higher mortalities at 90 days compared with undiagnosed smolt groups, although mortalities were highly variable in both categories. The temperature difference between the freshwater stage and seawater had a small, but significant, effect on growth with the best growth in groups stocked to warmer seawater (P = 0.04, R(2) = 0.06). S0 and S1 smolt groups did not differ significantly in growth, but the mortality was significantly (P = 0.02) higher in S1 groups. Seawater addition as a categorical variable had no significant effects, but when analyzed within the seawater addition group, intermediate salinities (15-25 ppt) gave the best results on growth (p = 0.04, R(2) = 0.15). Production intensity had small explanatory power on subsequent seawater performance in the analyzed smolt groups. If anything, the analysis shows a beneficial effect of intensive production strategies on subsequent performance. Analysis of the various production strategies indicates better survival of S0 compared with S1 smolt groups, improved growth when stocked in seawater warmer than freshwater, and a negative effect of viral disease outbreaks on survival. The results clearly demonstrate the difficulty of extrapolating results from experimental work on fish welfare and production intensity variables to commercial production. On the other hand, the presented results may simply demonstrate that the traditional fish welfare criteria growth and mortality may not suffice to evaluate welfare consequences of suboptimal water quality or production strategies in the aquaculture industry.

Changes in arterial oxygen tension and physiological status in resting, unrestrained Arctic charr Salvelinus alpinus (L.) exposed to mild hypoxia and hyperoxia.

In arctic charr Salvelinus alpinus, arterial blood partial pressures of oxygen (PaO2) and carbon dioxide increased with increasing water oxygen tension (PwO2), while the water to arterial PO2 difference (PwO2-PaO2) did not change in relation to PwO2.

Lack of arterial PO2 downregulation in Atlantic salmon (Salmo salar L.) during long-term normoxia and hyperoxia.

Regulation of arterial partial pressure of O2 (PaO2) in Atlantic salmon (Salmo salar) was investigated during resting conditions in normoxic and hyperoxic water. Dorsal aorta cannulated adult Atlantic salmon (1.2-1.6 kg, n=8) were exposed to 2 week sequential periods of normoxia [16.7±1.1 kPa (mean±SD)] and hyperoxia (34.1±4.9 kPa) in individual tanks containing seawater (33.7±0.2 ppt) at stable temperature conditions (8.7±0.7°C) and a light regime of L:D=12:12. Tank design and sampling procedures were optimized to provide suitable shelter and current for the fish, and to allow repeated, undisturbed sampling of blood from free-swimming fish. Fish were sampled regularly through the experimental period. PwO2, PaO2, blood ion composition (Na+, K+, Cl-), acid-base status (pH, PCO2, HCO3-), haematocrit and glucose were measured. The most frequently observed PaO2 values were in the range of 60-80% of PwO2, both during normoxia and hyperoxia, and PaO2 values were significantly lower during normoxia than during hyperoxia. Blood pH, PCO2 and HCO3- were significantly elevated during hyperoxia, while, Na+, Cl- and Hct were significantly lower. K+ and glucose showed no significant differences. This study demonstrates a lack PaO2 regulation in Atlantic salmon to low partial pressures, in contrast to previous reports for many aquatic gill breathing animals. Both during normoxia and hyperoxia, PaO2 reflects PwO2, and alterations in external PO2 consequently result in proportional arterial PO2 changes. Physiological adaptation to hyperoxia, as illustrated by changes in several blood parameters, does not include down-regulation of PaO2 in Atlantic salmon. The lack of PaO2 regulation may make Atlantic salmon vulnerable to the oxidative stress caused by increased free radical formation in hyperoxic conditions.

Multiple stressor effects of radiation and metals in salmon (Salmo salar).

These experiments were designed to look at the cellular effects in key organs in Atlantic salmon (Salmo salar) after exposure in vivo to radiation and subtoxic levels of aluminum (Al) and cadmium (Cd), alone or in combination. Salmon (25g) were exposed to a single 0.5Gy dose of gamma-irradiation in water containing Cd, Al or Cd+Al. Three fish per group were sacrificed after 1h and the liver, pronephros, fin and gill of each was dissected. Small explants of each tissue were set up. After 2 days, the culture medium was harvested and filtered then placed on a reporter cell line for determination of stress signal activity (bystander effects). Radiation in combination with Cd and/or Al, caused bystander effects in tissues harvested from in vivo exposed salmon. The effects vary between different organs and are not consistently additive or synergistic for a given treatment. Tissue type appears to be critical. Liver cultures produce a toxic factor which is lethal to reporter cells, and therefore no liver data could be obtained. It is hoped that this stress signal response will prove to be a useful indicator of environmental stress in species inhabiting aquatic ecosystems.

Age dependence of the accumulation of organochlorine pollutants in brown trout (Salmo trutta) from a remote high mountain lake (Redó, Pyrenees).

Polychlorobiphenyls (PCBs), hexachlorocyclohexanes (HCHs), hexachlorobenzene (HCB) and DDT were examined in the muscle of brown trout (Salmo trutta) from a high mountain lake located in the Pyrenees (Catalonia, Spain) that was used as a model of these lacustrine environments. Results indicate that fish age is the main factor of variability among specimens in this population that is subjected to atmospheric inputs of the organochlorine compounds (OC). Increases of 2- and 20-fold between fish aged 1 year and 15 years old are found. The observed pattern cannot be explained in terms of fish size, condition factor, or muscle lipid content. Higher molecular weight compounds (higher lipophilicity) are better correlated with fish age than low molecular weight compounds. A transformation from 4,4'-DDT to 4,4'-DDE occurs in fish after ingestion; this results in amplified age-dependent signals, especially in male specimens. In contrast, PCB congener #180 has lower age dependence than the general OC group, which could be due to its high hydrophobicity (log K(ow) > 7). In any case, selective accumulation of hydrophobic compounds is already observed among younger fish (age, 1 year). Due to this effect, the relative OC composition does not reflect the main OC pollutants in the lake waters.

The mixing zone between limed and acidic river waters: complex aluminium chemistry and extreme toxicity for salmonids.

When liming running waters, dosers must compensate for different flow and water qualities and for the downstream inflow from acid tributaries which creates mixing zones. At a certain point in the mixing zone, a constant or fluctuating chemical disequilibrium will appear due to transformation processes. In laboratory assays, over-saturated solutions of aluminium with ongoing active precipitation of aluminium have been found to be especially toxic to fish. Recent experiments in a mixing zone in the limed River Audna, Norway, have confirmed this phenomenon. Atlantic salmon (Salmo salar L.) and sea trout (Salmo trutta L.) smolts were exposed to acid and limed waters and mixtures of the two waters downstream from the point of connection. In the acid tributary (mean values: pH=4.8, Ca=1.3 mg litre (-1)), Ali 236 microg litre(-1)=), LT5) was 22 and 40 h for Atlantic salmon and sea trout, respectively. In the mixing zone (pH=4.8-6.5, Ca=1.2-3.2 mg litre(-1), Ali=50-240 microg litre(-1)), LT50 was 7 h for both species, masking the normal species difference in tolerance. Osmoregulatory failure and rapid gill lesions occurred in the mixing zone as an effect of the transformation of Al into high molecular weight precipitating species. This is the first documentation of the existence of such highly toxic mixing zones in nature, and the results clearly show that the mixing zone is even more toxic to fish than acid aluminium-rich waters.

Po-210 and Pb-210 in water and fish from Taboshar uranium mining Pit Lake, Tajikistan.

Polonium-210 in water and (210)Pb and (210)Po in different fish organs from 3 different fish species in Taboshar Pit Lake (n = 13), located in the uranium mining area in Tajikistan, and in Kairakkum Reservoir (reference lake, n = 3), have been determined as part of a Joint project between Norway, Kazakhstan, Kyrgyzstan and Tajikistan. The average activity concentration of (210)Pb and (210)Po in liver, muscle and bone of Carassius auratus was higher than the concentration in similar tissues of C. carpio and Sander lucioperca from the reference site. The accumulation of (210)Po was higher than for (210)Pb, and the accumulation of (210)Po was highest in the liver of C. auratus (3673 ± 434 Bq kg(-1) ww). Although the average activity concentration of (210)Pb in liver and bones of C. auratus from Pit Lake were fairly similar, a huge variation in the liver activity concentrations (25-327 Bq kg(-1) ww) was found. The results confirm direct uptake of unsupported (210)Po into the liver, and that the distributions of (210)Po and (210)Pb in fish organs were different. The BCF (L/kg) for (210)Po in bone, liver and muscle clearly demonstrates high accumulation of (210)Po in C. auratus, especially in the liver. The average BCFs of liver, bone and muscle were >1.4 × 10(5), >2.5 × 10(4) and >1.4 × 10(4), respectively. All fish in the Pit Lake were found to be in the same trophic level, however, a linear correlation between log (210)Po in liver and δ(15)N could indicate biomagnification of (210)Po in liver of C. auratus. In regards to the recommended Annual Limit of Intake (ALI) for (210)Po, the concentration of (210)Po in muscle tissues of C. auratus is alarming, as there is a high probability for the local population at risk to exceed the recommended ALI through consumption of fish from Taboshar Pit Lake.

Environmental impact assessment of radionuclides and trace elements at the Kurday U mining site, Kazakhstan.

The Kurday uranium mining site in Kazakhstan operated from 1954 to 1965 as part of the USSR nuclear weapon programme. To assess the environmental impact of radionuclides and trace elements associated with the Kurday mining site, field expeditions were performed in 2006. In addition to in situ gamma and (220)Rn dose rate measurements, sampling included at site fractionation of water as well as sampling of water, fish, sediment, soils and vegetation. The concentrations of U and associated trace metals were enriched in the Pit Lake and in the artesian water (U exceeding the WHO guideline value for drinking water), and decreased downstream from the mining area. Uranium, As, Mo and Ni were predominantly present as mobile low molecular mass species in waters, while a significant proportion of Cr, Mn and Fe were associated with colloids and particles. Due to oxidation of divalent iron in the artesian ground water upon contact with air, Fe served as scavenger for other elements, and peak concentrations of U-, Ra-isotopes, As and Mn were seen. Most radionuclides and trace elements were contained in minerals in soils and sediments, and good correlations were obtained between U and As, Cd, Mo and (226)Ra. Based on sequential extractions, a significant fraction of U, Pb and Cd could be considered mobile. Radioactive particles carrying significant amount of trace metals may represent a hazard during strong wind events. The transfer of radionuclides and metals from soils or sediments to water was in general low. The Kd levels varied with the element in question, ranging from 0.5 to 3 × 10(2) L/kg d.w. for (238)U being relatively mobile, 10(3) for (226)Ra, As, Cd, Ni, to 10(4) L/kg d.w. for Cu, Cr and Pb being rather inert The transfer of radionuclides and metals from soils to vegetation (TF) was low, while higher if the transfer to vegetation, especially underwater mosses, occurred via water (e.g., BCF 37 L/kg w.w. for (238)U and 3 × 10(3) L/kg w.w. for (226)Ra). The transfer of Cd, Pb and As from water to fish liver (BCF) was rather high, showing BCFs in the range of 10(2)-10(3) L/kg w.w., and may, if eaten, represent a health risk. Furthermore, the high Hg level in fish filet reaching 0.3 mg/kg w.w. muscle and the tendency of biomagnification call for dietary restrictions. Total gamma and Rn dose rate to man amounted to about 6 mSv/y, while the highest calculated dose rate for non-human species based on the ERICA Assessment Tool were obtained in aquatic plants, with calculated mean doses of 700 µGy/hr, mostly due to the U exposure. Overall, it is concluded that measures such as restricted access to the Pit Lake as well as dietary restrictions with respect to drinking water and intake of fish should be taken to reduce the environmental risk to man and biota.

Environmental impact assessment of radionuclide and metal contamination at the former U sites Taboshar and Digmai, Tajikistan.

Uranium (U) ore mining and processing were initiated in the former Soviet Republics of Tajikistan after the Second World War as part of the USSR nuclear weapon programme. The U mine in Taboshar was opened in 1936, and mining took place from 1945 to 1965, while the Digmai tailings dump was exploited during 1963-1993. The mining, milling and extraction activities have resulted in large amounts of waste rock deposits and U tailing materials placed in the vicinity of inhabited areas. To assess the environmental impact of radionuclides and trace metals in the Taboshar and Digmai mining and tailing sites in Tajikistan, field expeditions were performed in 2006 and 2008. In addition to in situ gamma and radon dose rate measurements, sampling of water, fish, sediments, soils and vegetation including in situ fractionation of water were performed. The U concentrations in water from Taboshar Pit Lake (2.0 mg U/L) were higher than in waters collected in the Digmai area. The Pit Lake and the stream water from the tailing mountain were also characterised by elevated concentrations of As, Mo, Mn and Fe, exceeding the WHO recommended values for drinking water. Uranium, As, Mo and Ni were present as low molecular mass species in the waters, and are therefore considered mobile and potentially bioavailable. The (238)U concentrations in sediments and soils varied between the sites; with peak concentrations (6 kBq/kg dw) in sediments from the Pit Lake, while the soil concentrations were significantly lower (296-590 Bq/kg dw). In contrast, high levels of the radium isotopes ((226)Ra and (228)Ra) were found in the Digmai soil (17-32 kBq/kg dw). Based on sequential extraction results, both U and Pb were found to be quite mobile at the Pit Lake site, showing that these elements were associated with the pH sensitive and redox sensitive amorphous fractions. In tailings, U was found to be quite mobile, but here Pb was rather inert. The transfer of radionuclides and metals from sediments to waters was in general low. In the Pit Lake, U was quite mobile (Kd = 90 L/kg), followed by Ni (1.5 × 10(3) L/kg) and As (6 × 10(3) L/kg), Cu and Cd (1.5 × 10(4) L/kg), while Pb (3 × 10(5) L/kg) was rather inert. The transfer from soil to plant, TFs (kg/kg dw), was in general low, while the bioconcentration factor for water living Poaceae and for fish from water was relatively high (Pb 1.8 × 10(5) and Cd 1 × 10(4)). These legacy sites, containing enhanced levels of natural radioactive material (TENORM) as well as heavy metals, may represent a hazard having a potential radiological and chemical impact on man and the environment, and measures should be taken to reduce the environmental risk to man and biota.

Uranium activity ratio in water and fish from pit lakes in Kurday, Kazakhstan and Taboshar, Tajikistan.

Kurday in Kazhakstan and Taboshar in Tajikistan were U mining sites operated during the 1950s and 1960s as part of the USSR nuclear weapon program. Today, they represent sources of potential U contamination of the environment. Within both mining sites, open pits from which U ore was extracted have been filled with water due to ground water inflow and precipitation. These artificial pit lakes contain fish consumed occasionally by the local people, and wild and domestic animals are using the water for drinking purposes. To assess the potential impact from U in these pit lakes, field work was performed in 2006 in Kurday and 2006 and 2008 in Taboshar. Results show that the U concentration in the lake waters were relatively high, about 1 mg/L in Kurday Pit Lake and about 3 mg/L in Taboshar Pit Lake. The influence of U-bearing materials on the lakes and downstream waters were investigated by measuring the U concentration and the (234)U/(238)U activity ratios. In both Kurday and Taboshar, the ratios increased distinctively from about 1 at the pit lakes to >1.5 far downstream the lakes. The concentrations of (238)U in gill, liver, muscle and bones in fish from the pit lakes were much higher than in the reference fish. Peak concentration of U was seen in bones (13 mg/kg w.w.), kidney (9.1 mg/kg w.w.) and gills (8.9 mg/kg w.w.) from Cyprinus auratus caught in the Taboshar Pit Lake. Bioconcentration factors (BCF) calculated for organs from fish caught in the Taboshar Pit Lake, with the same tendency seen in the Kurday Pit Lake, showed that U accumulates most in bone (BCF = 4.8 L/kg w.w.), gills (BCF = 3.6 L/kg w.w.), kidney (BCF = 3.6 L/kg w.w.), and liver (BCF = 2.5 L/kg w.w.), while least was accumulated in the muscle (BCF = 0.12 L/kg w.w.).

Environmental impact assessment of radionuclide and metal contamination at the former U site at Kadji Sai, Kyrgyzstan.

During 1949-1967, a U mine, a coal-fired thermal power plant and a processing plant for the extraction of U from the produced ash were operated at the Kadji Sai U mining site in Tonsk district, Issyk-Kul County, Kyrgyzstan. The Kadji Sai U legacy site represents a source of contamination of the local environment by naturally occurring radionuclides and associated trace elements. To assess the environmental impact of radionuclides and trace metals at the site, field expeditions were performed in 2007 and 2008 by the Joint collaboration between Norway, Kazakhstan, Kyrgyzstan, Tajikistan (JNKKT) project and the NATO SfP RESCA project. In addition to in situ gamma and Rn dose rate measurements, sampling included at site fractionation of water and sampling of water, fish, sediment, soils and vegetation. The concentrations of radionuclides and trace metals in water from Issyk-Kul Lake were in general low, but surprisingly high for As. Uptake of U and As was also observed in fish from the lake with maximum bioconcentration factors for liver of 1.6 and 75, respectively. The concentrations of U in water within the Kadji Sai area varied from 0.01 to 0.05 mg/L, except for downstream from the mining area where U reached a factor of 10 higher, 0.2 mg/L. Uranium concentrations in the drinking water of Kadji Sai village were about the level recommended by the WHO for drinking water. The (234)U/(238)U activity ratio reflected equilibrium conditions in the mining pond, but far from equilibrium outside this area (reaching 2.3 for an artesian well). Uranium, As and Ni were mainly present as low molecular mass (LMM, less than 10 kDa) species in all samples, indicating that these elemental species are mobile and potentially bioavailable. The soils from the mining sites were enriched in U, As and trace metals. Hot spots with elevated radioactivity levels were easily detected in Kadji Sai and radioactive particles were observed. The presence of particles carrying significant amount of radioactivity and toxic trace elements may represent a hazard during strong wind events (wind erosion). Based on sequential extractions, most of the elements were strongly associated with mineral matter, except for U and As having a relatively high remobilization potential. Low Kd was obtained for U (3.5 × 10(2) L/kg d.w.), intermediate Kds (~3 × 10(3) L/kg d.w.) were obtained for (226)Ra, As and Ni, while a high Kd (2.2 × 10(5) L/kg d.w.) were obtained for Pb. The accumulation of metals in fish gills reflected the LMM species in the Issyk-Kul water, and did not show any bioaccumulation. The muscle Hg concentrations in all fish species were low and did not represent any health risk even for groups at risk. Total gamma and Rn dose rate to man amounted to about 12 mSv/y, while the highest calculated dose rate for non-human species based on the ERICA Assessment Tool were obtained in terrestrial plants (164 µGy/h) due to the Ra exposure. The results obtained showed that radiation doses to resident public at all of the investigated sites in the Kadji Sai area were in general relatively low. Low radiological risk and no detrimental health impact on resident public can be expected at these sites. However, exposure to Rn and Tn in the living environment can be further reduced by implementing simple countermeasures such as ventilation of dwelling cellars. More focus in the Kadji Sai area should probably be put on trace elements, especially the As uptake in fish in Lake Issyk-Kul.

Acute and sub-lethal effects in juvenile Atlantic salmon exposed to low µg/L concentrations of Ag nanoparticles.

Silver nanoparticles (Ag-NP) are components in numerous commercial products and are discharged into the environment in quantities that are largely unknown. In the present study, juvenile Atlantic salmon were exposed to 1, 20, and 100 µg/L (48 h, static renewal) of a commercially available Ag-NP colloidal suspension in natural (soft) lake water. A solution of AgNO(3) containing 20 µg/L Ag(I) ions was also included to discriminate the effect of NPs from that of ionic silver. Furthermore, the commercial Ag-NP suspension was compared to an in-house synthesised colloidal NP suspension prepared from AgNO(3) and NaBH(4) in citrate buffer. The size distribution of Ag in all exposure solutions was characterised by 0.22 µm filtration and 10 kDa hollow fibre cross-flow ultrafiltration in combination with ICP-MS. All exposures were characterised by a relatively high proportion of Ag-NP in the colloidal size fraction 3-220 nm. For assessment of biological effects, acute toxicity, gill histopathology, blood plasma parameters (Na, Cl, glucose, haemoglobin), and gene expression of a selection of gill biomarkers were measured. Results showed that the gills accumulated Ag in all exposure groups apart from the fish exposed to 1 µg/L Ag-NP. Accumulated Ag caused concentration-dependent response increases in general stress markers such as plasma glucose and gill gene expression of heat shock protein 70. Furthermore, induction of the metallothionein A gene indicated that Ag had been internalized in the gills, whereas a concentration-dependant inhibition of Na/K ATPase expression indicated impaired osmoregulation at as low as 20 µg/L concentrations of Ag-NP. The commercial Ag-NP suspension caused acute gill lamellae necrosis at high concentrations (100 µg/L), potentially giving rise to the substantial (73%) fish mortality at this concentration. The two different Ag-NP preparations gave comparable results for several endpoints measured, but differed in MT-A induction and mortality, thus emphasising the variation in effects that may arise from different Ag-NP preparations.

Effects of combined gamma-irradiation and metal (Al+Cd) exposures in Atlantic salmon (Salmo salar L.).

These experiments were designed to investigate transcriptional effects in Atlantic salmon (Salmo salar) after exposure in vivo to ionizing gamma radiation combined with subtoxic levels of aluminum (Al) and cadmium (Cd). Juvenile fish (35 g) in freshwater with or without Al and Cd (255 microg Al/L + 6 microg Cd/L) were exposed to a 75 mGy dose of gamma-irradiation, and induced responses were compared to those of controls. The transcriptional levels of eight genes encoding proteins known to respond to stress in fish were quantified in liver of fish exposed for 5 h to gamma radiation, to Al and Cd or to the combination of Al, Cd and gamma radiation. The studied genes were caspase 3B, caspase 6A, caspase 7, p53 (apoptosis), glutathione reductase (GR), phospholipid hydroperoxide glutathione peroxidase (GSH-Px), (oxidative stress), metallothionein (MT-A) (metal stress) and ubiquitin (Ubi) (protein degradation). The results showed that gamma-irradiation alone induced significant upregulation of caspase 6A, GR, GSH-Px, MT-A and Ubi compared to the control group, while 5 h exposure to Al+Cd alone did not induce any of the studied genes compared to the control. No significant upregulation of the series of investigated genes could be observed in fish exposed to gamma-irradiation in combination with Al+Cl. In conclusion, the results suggest that the presence of Al+Cd in the water counteracted the gamma-irradiation effect by modifying the transcription of genes encoding proteins involved in the defense mechanisms against free radicals in the cells.

Environmentally relevant mixed exposures to radiation and heavy metals induce measurable stress responses in Atlantic salmon.

These experiments were designed to identify stress effects in 3 key organs in Atlantic Salmon (Salmo salar, L.) after exposure in vivo to very low doses of radiation, and subtoxic levels of aluminum (Al) and cadmium (Cd) alone or in combination. Six fish per group were sacrificed after exposure and the anterior kidney, fin, and gill were dissected and sentfor assay of bystander signal production as a stress response end point. Radiation doses as low as 4 mGy delivered over 5 h, alone or in combination with Cd and/or Al, caused bystander signals to be produced in tissues harvested from in vivo exposed salmon. The effects vary among different organs and are not consistently additive or synergistic for a given treatment although gill cells do show high degrees of synergism between radiation and metal exposure. Data for individual fish did not suggest any systemic sensitivity to the stressors. Interestingly, the data for Cd suggest that lower toxicity is found when the metal is used in combination with radiation exposure. Expression of two proteins associated with survival responses (Bcl-2) or death responses (cmyc) after radiation was measured in the tissue cultures and showed a highly significant correlation with response outcome. The results, although complex, indicate that these stress signal responses may aid in the mechanistic investigation of mixed contaminant effects in fish exposed to metals and radiation.

Effects of hypo- and hyperoxia on transcription levels of five stress genes and the glutathione system in liver of Atlantic cod Gadus morhua.

The transcript levels of three genes coding for antioxidants, Cu/Zn superoxide dismutase (SOD), catalase and phospholipid hydroperoxide glutathione peroxidase (GSH-Px), and those of two stress proteins, metallothionein (MT) and CYP1A, were examined with real-time quantitative (q) RT-PCR in hepatic tissue of Atlantic cod exposed to 46% (hypoxia), 76% (normoxia) and 145% (hyperoxia) O(2) saturation (tank outlet). To evaluate the oxidative stress state, the levels of total glutathione (tGSH), reduced glutathione (GSH) and oxidized glutathione (GSSG) and subsequently the oxidative stress index (OSI), were determined in the same tissue samples. The transcript level of GSH-Px was significantly upregulated in fish exposed to hyperoxia, and significantly downregulated in fish exposed to hypoxia, compared to the normoxia group. Significant downregulation was also found for SOD and CYP1A transcriptional levels in fish exposed to hypoxia. The transcript levels of catalase and MT did not change in liver of cod exposed to suboptimal oxygen levels. No significant differences were seen between the groups for tGSH, GSH, GSSG or OSI. Prolonged exposure to unfavourable oxygen saturation levels did not alter the OSI, indicating that the antioxidant glutathione system is maintained at an unchanged level in liver of the examined cod.

mRNA expression of antioxidant enzymes (SOD, CAT and GSH-Px) and lipid peroxidative stress in liver of Atlantic salmon (Salmo salar) exposed to hyperoxic water during smoltification.

The mRNA levels of three antioxidant genes, Cu/Zn superoxide dismutase (SOD), catalase (CAT) and phospholipid hydroperoxide glutathione peroxidase (GSH-Px), were quantified with real-time qRT-PCR in liver of Atlantic salmon Salmo salar exposed to 80% (normoxia), 105% and 130% O2 saturation for 54 days. The salmon were then translocated and exposed to 90% and 130% O2 saturation for additional 72 days during smoltification. TBARS and vitamin E levels in liver and the levels of oxidized glutathione (GSSG), total glutathione (GSH) and the resulting oxidative stress index (OSI) in blood were quantified as traditional oxidative stress markers. No significant mean normalized expression (MNE) differences of SOD, CAT or GSH-Px were found in liver after hyperoxia exposure at the two sampling times. Significantly decreased OSI was found in smolt exposed to 130% O2 saturation after 126 days (n = 18, P < 0.0001), indicating hyperoxia-induced oxidative stress. No effects were seen on growth, or on the levels of thiobarbituric reactive substances (TBARS) and vitamin E in liver after the exposure experiment. Overall, the mRNA expression of SOD, CAT and GSH-Px in liver related poorly with the hyperoxic exposure regimes, and more knowledge are needed before the expressed levels of these antioxidant genes can be applied as biomarkers of hyperoxia in Atlantic salmon.

Environmental effects of aluminium.

Aluminium (Al), when present in high concentrations, has for long been recognised as a toxic agent to aquatic freshwater organisms,i.e. downstream industrial point sources of Al-rich process water. Today the environmental effects of aluminium are mainly a result of acidic precipitation; acidification of catchments leads to increased Al- concentrations in soil solution and freshwaters. Large parts of both the aquatic and terrestrial ecosystems are affected.In the aquatic environment, aluminium acts as a toxic agent on gill-breathing animals such as fish and invertebrates, by causing loss of plasma- and haemolymph ions leading to osmoregulatory failure. In fish, the inorganic (labile) monomeric species of aluminium reduce the activities of gill enzymes important in the active uptake of ions. Aluminium seems also to accumulate in freshwater invertebrates. Dietary organically complexed aluminium, maybe in synergistic effects with other contaminants, may easily be absorbed and interfere with important metabolic processes in mammals and birds.The mycorrhiza and fine root systems of terrestrial plants are adversely affected by high levels of inorganic monomeric aluminium. As in the animals, aluminium seems to have its primary effect on enzyme systems important for the uptake of nutrients. Aluminium can accumulate in plants. Aluminium contaminated invertebrates and plants might thus be a link for aluminium to enter into terrestrial food chains.