Lene Sørlie Heier

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.

Speciation of lead, copper, zinc and antimony in water draining a shooting range—Time dependant metal accumulation and biomarker responses in brown trout (Salmo trutta L.)

The speciation of Pb, Cu, Zn and Sb in a shooting range run-off stream were studied during a period of 23 days. In addition, metal accumulation in gills and liver, red blood cell ALA-D activity, hepatic metallothionine (Cd/Zn-MT) and oxidative stress index (GSSG/ tGSH levels) in brown trout (Salmo trutta L.) exposed to the stream were investigated. Fish, contained in cages, were exposed and sampled after 0, 2, 4, 7, 9, 11 and 23 days of exposure. Trace metals in the water were fractionated in situ according to size (nominal molecular mass) and charge properties. During the experimental period an episode with higher runoff occurred resulting in increased levels of metals in the stream. Pb and Cu were mainly found as high molecular mass species, while Zn and Sb were mostly present as low molecular mass species. Pb, Cu and Sb accumulated on gills, in addition to Al origination from natural sources in the catchment. Pb, Cu and Sb were also detected at elevated concentration in the liver. Blood glucose and plasma Na and Cl levels were significantly altered during the exposure period, and are attributed to elevated concentrations of Pb, Cu and Al. A significant suppression of ALA-D was detected after 11 days. Significant differences were detected in Cd/Zn-MT and oxidative stress (tGSH/GSSG) responses at Day 4. For Pb the results show a clear link between the HMM (high molecular mass) positively charged Pb species, followed by accumulation on gills and liver and a suppression in ALA-D. Thus, high flow episodes can remobilise metals from the catchment, inducing stress to aquatic organisms.

Effects of combined γ-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.

Early stress responses in Atlantic salmon (Salmo salar) exposed to environmentally relevant concentrations of uranium

Uranium (U) is a naturally occurring heavy metal widely used in many military and civil applications. Uranium contamination and the associated potential adverse effects of U on the aquatic environment have been debated during recent years. In order to understand the effect and mode of action (MoA) of U in vivo, juvenile Atlantic salmon (Salmo salar) were exposed to 0.25 mg/L, 0.5 mg/L and 1.0mg/L waterborne depleted uranyl acetate, respectively, in a static system for 48 h. The U concentrations in the gill and liver were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) and the resulting biological effects were determined by a combination of analysis of gene expression and micronuclei formation. The hepatic transcriptional level of 12 biomarker genes from four stress-response categories, including oxidative stress (γ-glutamyl cysteine synthetase (GCS), glutathione reductase (GR), glutathione peroxidase (GPx)), DNA damage and repair (P53, cyclin-dependent kinase inhibitor 1 (P21), growth arrest and DNA damage-inducible gene gamma (Gadd45G), proliferating cell nuclear antigen (PCNA), Rad51), apoptosis (Bcl2-associated X protein (BAX), Bcl-x, Caspase 6A,) and protein degradation (Ubiquitin) were evaluated by quantitative real-time polymerase chain reaction (q-rtPCR). The results clearly showed accumulation of U in the gill and liver with increasing concentrations of U in the exposure water. The effects of U on differential hepatic gene expression also occurred in a concentration-dependent manner, although deviations from ideal concentration-response relationships were observed at the highest U concentration (1.0 mg/L). All the genes tested were found to be up-regulated by U while no significant micronuclei formation was identified. The results suggest that U may cause oxidative stress in fish liver at concentrations greater than 0.25 mg/L, giving rise to clear induction of several toxicologically relevant biomarker genes, although no significant adverse effects were observed after the relatively short exposure period.

Short-term temporal variations in speciation of Pb, Cu, Zn and Sb in a shooting range runoff stream.

This study was designed to explore the changes in physico-chemical forms of Pb, Cu, Zn and Sb in a stream draining a contaminated shooting range, located at Steinsjøen in the South-Eastern part of Norway, during a period of 21days. To obtain information on the element species distribution, an interphased size and charge fractionation system was applied, where membrane filtration (0.45microm) and ultrafiltration using hollow fibre (nominal cut off 10kDa) were performed prior to charge fractionation using chromatography (cationic and anionic exchange resins). The results show that Pb mainly was present as particulate and colloidal high molecular mass (HMM) species, Cu as colloidal (HMM) and low molecular mass (LMM) species, while Sb and Zn were mainly present as LMM species. The total element concentrations of Pb, Cu, Zn and Sb were positively correlated to water flow and dissolved organic carbon (DOC), suggesting these are important factors in controlling the run-off of the investigated elements in this catchment. During episodes of higher water flow, the increase in element concentration was mainly in the colloidal fraction. Partial redundancy analysis (pRDA) revealed that variations in pH, HMM organic carbon (HMM OC) and LMM organic carbon (LMM OC) explained 47% of the variation in size distribution of the elements, while variations in precipitation and water flow explained 48% of the variation in the charge distribution of the elements. The variation in concentrations during the period varied by a factor of 4, also stressing the importance of frequent sampling opposed to spot sampling in environmental surveys and risk assessments.

Chemical and ecological effects of contaminated tunnel wash water runoff to a small Norwegian stream

Cleaning and washing of road tunnels are routinely performed and large volumes of contaminated wash water are often discharged into nearby recipients. In the present study, traffic related contaminants were quantified in tunnel wash water (the Nordby tunnel, Norway) discharged from a sedimentation pond to a nearby small stream, Arungselva. In situ size and charge fractionation techniques were applied to quantify traffic related metal species, while PAHs were quantified in total samples. All metals and several PAHs appeared at elevated concentrations in the discharged wash water compared with concentrations measured in Arungselva upstream the pond outlet, and to concentrations measured in the pond outlet before the tunnel wash event. In addition, several contaminants (e.g. Cu, Pb, Zn, fluoranthene, pyrene) exceeded their corresponding EQS. PAH and metals like Al, Cd, Cr, Cu, Fe and Pb were associated with particles and colloids, while As, Ca, K, Mg, Mo, Ni, Sb and Zn were more associated with low molecular mass species (<10kDa). Calculated enrichment factors revealed that many of the metals were derived from anthropogenic sources, originating most likely from wear of tires (Zn), brakes (Cu and Sb), and from road salt (Na and Cl). The enrichment factors for Al, Ba, Ca, Cr, Fe, K, Mg and Ni were low, suggesting a crustal origin, e.g. asphalt wear. Based on calculated PAH ratios, PAH seemed to originate from a mixture of sources such as wear from tires, asphalt and combustion. Finally, historical fish length measurement data indicates that the fish population in the receiving stream Arungselva may have been adversely influenced by the chemical perturbations in runoffs originating from the nearby roads and tunnels during the years, as the growth in summer old sea trout (Salmo trutta L.) in downstream sections of the stream is significantly reduced compared to the upstream sections.

Exposure of brown trout (Salmo trutta L.) to tunnel wash water runoff — Chemical characterisation and biological impact

Washing and cleaning of road tunnels are a routinely performed maintenance task, which generate significant amount of polluted wash-water runoff that normally is discharged to the nearest recipient. The present study was designed to quantify chemical contaminants (trace metals, hydrocarbons, PAH and detergents) in such wash water and assess the short term impact on brown trout (Salmo trutta L.) based on in situ experiments. Selected endpoints were accumulation of trace metals in gills, haematological variables and hepatic mRNA transcription of five biomarkers reflecting defence against free radicals, trace metals, planar aromatic hydrocarbons and endocrine disruptions which were measured prior (-3h), during (1 and 3h) and after the tunnel wash (14, 38 and 86h). Our findings showed that the runoff water was highly polluted, but most of the contaminants were associated with particles which are normally considered biologically inert. In addition, high concentrations of calcium and dissolved organic carbon were identified in the wash water, thus reducing metal toxicity. However, compared to the control fish, a rapid accumulation of trace metals in gills was observed. This was immediately followed by a modest change in blood ions and glucose in exposed fish shortly after the exposure start. However, after 38-86h post wash, gill metal concentrations, plasma ions and glucose levels recovered back to control levels. In contrast, the mRNA transcription of the CYP1A and the oxidative stress related biomarkers TRX and GCS did not increase until 14h after the exposure start and this increase was still apparent when the experiment was terminated 86h after the beginning of the tunnel wash. The triggering of the defence systems seemed to have successfully restored homeostasis of the physiological variables measured, but the fish still used energy for detoxification four days after the episode, measured as increased biomarker synthesis.

Hepatic transcriptomic profiling reveals early toxicological mechanisms of uranium in Atlantic salmon (Salmo salar)

BackgroundUranium (U) is a naturally occurring radionuclide that has been found in the aquatic environment due to anthropogenic activities. Exposure to U may pose risk to aquatic organisms due to its radiological and chemical toxicity. The present study aimed to characterize the chemical toxicity of U in Atlantic salmon (Salmo salar) using depleted uranium (DU) as a test model. The fish were exposed to three environmentally relevant concentrations of DU (0.25, 0.5 and 1.0 mg U/L) for 48 h. Hepatic transcriptional responses were studied using microarrays in combination with quantitative real-time reverse transcription polymerase chain reaction (qPCR). Plasma variables and chromosomal damages were also studied to link transcriptional responses to potential physiological changes at higher levels.ResultsThe microarray gene expression analysis identified 847, 891 and 766 differentially expressed genes (DEGs) in the liver of salmon after 48 h exposure to 0.25, 0.5 and 1.0 mg/L DU, respectively. These DEGs were associated with known gene ontology functions such as generation of precursor metabolites and energy, carbohydrate metabolic process and cellular homeostasis. The salmon DEGs were then mapped to mammalian orthologs and subjected to protein-protein network and pathway analysis. The results showed that various toxicity pathways involved in mitochondrial functions, oxidative stress, nuclear receptor signaling, organ damage were commonly affected by all DU concentrations. Eight genes representative of several key pathways were further verified using qPCR No significant formation of micronuclei in the red blood cells or alterations of plasma stress variables were identified.ConclusionThe current study suggested that the mitochondrion may be a key target of U chemical toxicity in salmon. The induction of oxidative stress and uncoupling of oxidative phosphorylation may be two potential modes of action (MoA) of DU. These MoAs may subsequently lead to downstream events such as apoptosis, DNA repair, hypoxia signaling and immune response. The early toxicological mechanisms of U chemical toxicity in salmon has for the first time been systematically profiled. However, no other physiological changes were observed. Future efforts to link transcriptional responses to adverse effects have been outlined as important for understanding of potential risk to aquatic organisms.

Fish mortality during sea salt episodes—catchment liming as a countermeasure

Aluminium (Al) toxicity is usually associated with acid rain and acidified freshwater systems. The present work demonstrates that acute fish mortality (50%) also occurs in moderate acidified salmon rivers during sea salt episodes. Furthermore, catchment liming was proved to be an efficient measure to counteract the fish toxicity. The impact of sea salt episodes on river water qualities and on Atlantic Salmon (Salmo salar L.) was studied in two rivers situated at the west coast of Norway. During February-May 2002, fish were kept in tanks and continually exposed to the changing water qualities. Changes in Al-species were followed using in situ fractionation techniques. During storm events and high sea salt deposition, the sea salt concentration increased (190 to 580 microM Cl), pH decreased (pH 5.3 to 4.6) and the concentration of low molecular mass (LMM) cationic Al-species (Al(i)) increased (0.7 to 3.0 microM) in the river. Subsequently, Al accumulated in fish gills (6 to 19 micromol g(-1) dw) causing ionoregulatory and respiratory failures as well as mortality. In water the concentration of LMM Al(i) stayed enhanced during four weeks, while the physiological stress responses in surviving fish remained high for a longer time (>eight weeks). To counteract Al toxicity, one of the tributary catchments had been limed four years earlier. Due to catchment liming (1000 kg ha(-1)) the water concentration of LMM Al(i)(<0.7 microM) and the Al accumulation in gills remained relatively low (<7 micromol g(-1) dw) during the storm and no fish mortality occurred.

Hepatic gene expression profile in brown trout (Salmo trutta) exposed to traffic related contaminants

In recent decades there has been growing concern about highway runoff as a potential threat and a significant source of diffuse pollution to the aquatic environment. However, identifying ecotoxicological effects might be challenging, especially at sites where the traffic density is modest to low. Hence, there is a need for alternatives e.g. small-scale toxicity tests using conventional endpoints such as mortality and growth. The present paper presents result from a transcriptional (microarray) screening performed on liver from brown trout (Salmo trutta) acutely exposed (4h) to traffic-related contaminants during washing of a highway tunnel outside the city of Oslo, Norway. The results demonstrated that traffic-related contaminants caused a plethora of molecular changes that persisted several hours after the exposure (i.e. during recovery). Beside an evident transcriptional up-regulation of e.g. cytochrome P450 1A1 (CYP1A1), cytochrome P450 1B1 (CYP1B1), and cytosolic sulfotransferase (SULT) involved in xenobiotic biotransformation, the observed responses were predominantly associated with immunosuppression, oxidative damage, and endocrine modulation. The observed responses were likely caused by an interaction of several contaminants including trace metals and organic micro-pollutants such as PAHs.