Geochemical and morphological characterization of particles originating from tunnel construction.

Rock particles from drilling and blasting during tunnel construction (DB particles) are released to the aquatic environment where they may cause negative toxicological and ecological effects. However, there exists little research on the difference in morphology and structure of these particles. Despite this DB particles are assumed to be sharper and more angular than naturally eroded particles (NE particles), and in consequence cause greater mechanical abrasion to biota. Moreover, morphology of DB particles is assumed to depend on geology, thus depending on where construction takes place different morphologies may be emitted. The objectives in the current study were to investigate the morphological differences between DB and NE particles, and the influence of mineral and elemental content on DB particles. Particle geochemistry and morphology were characterized by inductively coupled plasma mass spectrometry, micro-X-ray fluorescence, X-ray diffraction, environmental scanning electron microscope interfaced with energy dispersive X-ray, stereo microscope, dynamic image analysis and coulter counter. DB particles (61-91% < 63 µm) collected from five different tunnel construction locations in Norway were 8-15% more elongated (lower aspect ratio) than NE particles from river water and sediments, although their angularity was similar (solidity; diff 0.3-0.8%). Despite distinct mineral and elemental characteristics between tunnel construction locations, DB morphology was not explained by geochemical content since only 2-2.1% of the variance was explained. This suggests that particle formation mechanisms during drilling and blasting are more influential of morphology than mineralogy, when working in granite-gneiss terrain. When tunnelling in granite-gneiss terrain, particles with greater elongation than natural particles may enter aquatic systems.

Accumulation of lead (Pb) in brown trout (Salmo trutta) from a lake downstream a former shooting range.

An environmental survey was performed in Lake Kyrtjønn, a small lake within an abandoned shooting range in the south of Norway. In Lake Kyrtjønn the total water concentrations of Pb (14µg/L), Cu (6.1µg/L) and Sb (1.3µg/L) were elevated compared to the nearby reference Lake Stitjønn, where the total concentrations of Pb, Cu and Sb were 0.76, 1.8 and 0.12µg/L, respectively. Brown trout (Salmo trutta) from Lake Kyrtjønn had very high levels of Pb in bone (104mg/kg w.w.), kidney (161mg/kg w.w.) and the gills (137mg/kg d.w), and a strong inhibition of the ALA-D enzyme activity were observed in the blood (24% of control). Dry fertilized brown trout eggs were placed in the small outlet streams from Lake Kyrtjønn and the reference lake for 6 months, and the concentrations of Pb and Cu in eggs from the Lake Kyrtjønn stream were significantly higher than in eggs from the reference. More than 90% of Pb accumulated in the egg shell, whereas more than 80% of the Cu and Zn accumulated in the egg interior. Pb in the lake sediments was elevated in the upper 2-5cm layer (410-2700mg/kg d.w), and was predominantly associated with redox sensitive fractions (e.g., organic materials, hydroxides) indicating low potential mobility and bioavailability of the deposited Pb. Only minor amounts of Cu and Sb were deposited in the sediments. The present work showed that the adult brown trout, as well as fertilized eggs and alevins, may be subjected to increased stress due to chronic exposure to Pb, whereas exposure to Cu, Zn and Sb were of less importance.

Radiation-induced bystander effects in the Atlantic salmon (salmo salar L.) following mixed exposure to copper and aluminum combined with low-dose gamma radiation.

Very little is known about the combined effects of low doses of heavy metals and radiation. However, such "multiple stressor" exposure is the reality in the environment. In the work reported in this paper, fish were exposed to cobalt 60 gamma irradiation with or without copper or aluminum in the water. Doses of radiation ranged from 4 to 75 mGy delivered over 48 or 6 h. Copper doses ranged from 10 to 80 µg/L for the same time period. The aluminum dose was 250 µg/L. Gills and skin were removed from the fish after exposure and explanted in tissue culture flasks for investigation of bystander effects of the exposures using a stress signal reporter assay, which has been demonstrated to be a sensitive indicator of homeostatic perturbations in cells. The results show complex synergistic interactions of radiation and copper. Gills on the whole produce more toxic bystander signals than skin, but the additivity scores show highly variable results which depend on dose and time of exposure. The impacts of low doses of copper and low doses of radiation are greater than additive, medium levels of copper alone have a similar level of effect of bystander signal toxicity to the low dose. The addition of radiation stress, however, produces clear protective effects in the reporters treated with skin-derived medium. Gill-derived medium from the same fish did not show protective effects. Radiation exposure in the presence of 80 µg/L led to highly variable results, which due to animal variation were not significantly different from the effect of copper alone. The results are stressor type, stressor concentration and time dependent. Clearly co-exposure to radiation and heavy metals does not always lead to simple additive effects.

Changes in arterial PO2, physiological blood parameters and intracellular antioxidants in free-swimming Atlantic cod (Gadus morhua) exposed to varying levels of hyperoxia.

Free-swimming Atlantic cod (Gadus morhua) were exposed to water oxygen pressures (P(w)O2) ranging from 18.1 to 41.5 kPa and sampled for blood using an indwelling caudal artery cannula. Arterial blood oxygen pressure (P(a)O2) increased with increasing P(w)O2, from 12.0 kPa in normoxia (18.1 kPa) to 34.2 kPa in the highest hyperoxic level tested (41.5 kPa). Blood CO2 pressure and plasma bicarbonate concentration increased with P(w)O2, indicating reduced ventilation with increased P(w)O2. Plasma glucose, sodium and potassium were not affected by water oxygen level. Blood oxidative stress biomarkers, reduced glutathione, oxidized glutathione and the oxidative stress index (ratio between oxidized and total glutathione) differed intermittently between normoxia and hyperoxia. The oxidative stress index was higher in the blood of exposed compared to unexposed control cod. Together with elevated P(a)O2, these findings suggest increased production of reactive oxygen species and increased oxidative stress in Atlantic cod exposed to hyperoxia.

Uptake and effects of 2, 4, 6 - trinitrotoluene (TNT) in juvenile Atlantic salmon (Salmo salar).

Organ specific uptake and depuration, and biological effects in Atlantic salmon (Salmo salar) exposed to 2, 4, 6-trinitrotoluene (TNT) were studied. Two experiments were conducted, the first using radiolabeled TNT (14C-TNT, 0.16mg/L) to study uptake (48h) and depuration (48h), while the second experiment focused on physiological effects in fish exposed to increasing concentrations of unlabeled TNT (1µg-1mg/L) for 48h. The uptake of 14C-TNT in the gills and most of the organs increased rapidly during the first 6h of exposure (12h in the brain) followed by a rapid decrease even though the fish were still exposed to TNT in the water. The radioactivity in the gall bladder reached a maximum after 55h, 7h after the transfer to the clean water. A high concentration of 14C-TNT in the gall bladder indicates that TNT is excreted through the gall bladder. Mortality (2 out of 14) was observed at a concentration of 1mg/L, and the surviving fish had hemorrhages in the dorsal muscle tissue near the spine. Analysis of the physiological parameters in blood from the high exposure group revealed severe effects, with an increase in the levels of glucose, urea and HCO3, and a decrease in hematocrit and the levels of Cl and hemoglobin. No effects on blood physiology were observed in fish exposed to the lower concentrations of TNT (1-100µg/L). TNT and the metabolites 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT) were found in the muscle tissue, whereas only 2-ADNT and 4-ADNT were found in the bile. The rapid excretion and estimated bioconcentration factors (range of 2-18 after 48h in gills, blood, liver, kidney, muscle and brain) indicated a low potential for bioaccumulation of TNT.

Differences in copper bioaccumulation and biological responses in three Mytilus species.

Mytilus species are important organisms in marine systems being highly abundant and widely distributed along the coast of Europe and worldwide. They are typically used in biological effects studies and have a suite of biological effects endpoints that are frequently measured and evaluated for stress effects in laboratory experiments and field monitoring programmes. Differences in bioaccumulation and biological responses of the three Mytilus species following exposure to copper (Cu) were investigated. A laboratory controlled exposure study was performed with three genetically confirmed Mytilus species; M. galloprovincialis, M. edulis and M. trossulus. Chemical bioaccumulation and biomarkers were assessed in all three Mytilus species following a 4 day and a 21 day exposure to waterborne copper concentrations (0, 10, 100 and 500µg/L). Differences in copper bioaccumulation were measured after both 4 and 21 days, which suggests some physiological differences between the species. Furthermore, differences in response for some of the biological effects endpoints were also found to occur following exposure. These differences were discussed in relation to either real physiological differences between the species or merely confounding factors relating to the species natural habitat and seasonal cycles. Overall the study demonstrated that differences in chemical bioaccumulation and biomarker responses between the Mytilus spp. occur with potential consequences for mussel exposure studies and biological effects monitoring programmes. Consequently, the study highlights the importance of identifying the correct species when using Mytilus in biological effects studies.

Dose-dependent hepatic transcriptional responses in Atlantic salmon (Salmo salar) exposed to sublethal doses of gamma radiation.

Due to the production of free radicals, gamma radiation may pose a hazard to living organisms. The high-dose radiation effects have been extensively studied, whereas the ecotoxicity data on low-dose gamma radiation is still limited. The present study was therefore performed using Atlantic salmon (Salmo salar) to characterize effects of low-dose (15, 70 and 280 mGy) gamma radiation after short-term (48h) exposure. Global transcriptional changes were studied using a combination of high-density oligonucleotide microarrays and quantitative real-time reverse transcription polymerase chain reaction (qPCR). Differentially expressed genes (DEGs; in this article the phrase gene expression is taken as a synonym of gene transcription, although it is acknowledged that gene expression can also be regulated, e.g., at protein stability and translational level) were determined and linked to their biological meanings predicted using both Gene Ontology (GO) and mammalian ortholog-based functional analyses. The plasma glucose level was also measured as a general stress biomarker at the organism level. Results from the microarray analysis revealed a dose-dependent pattern of global transcriptional responses, with 222, 495 and 909 DEGs regulated by 15, 70 and 280 mGy gamma radiation, respectively. Among these DEGs, only 34 were commonly regulated by all radiation doses, whereas the majority of differences were dose-specific. No GO functions were identified at low or medium doses, but repression of DEGs associated with GO functions such as DNA replication, cell cycle regulation and response to reactive oxygen species (ROS) were observed after 280mGy gamma exposure. Ortholog-based toxicity pathway analysis further showed that 15mGy radiation affected DEGs associated with cellular signaling and immune response; 70mGy radiation affected cell cycle regulation and DNA damage repair, cellular energy production; and 280mGy radiation affected pathways related to cell cycle regulation and DNA repair, mitochondrial dysfunction and immune functions. Twelve genes representative of key pathways found in this study were verified by qPCR. Potential common MoAs of low-dose gamma radiation may include induction of oxidative stress, DNA damage and disturbance of oxidative phosphorylation (OXPHOS). Although common MoAs were proposed, a number of DEGs and pathways were still found to be dose-specific, potentially indicating multiple mechanisms of action (MOAs) of low-dose gamma radiation in fish. In addition, plasma glucose displayed an apparent increase with increasing radiation doses, although the results were not significantly different from the control. These findings suggested that sublethal doses of gamma radiation may cause dose-dependent transcriptional changes in the liver of Atlantic salmon after short-term exposure. The current study predicted multiple MoA for gamma radiation and may aid future impact assessment of environmental radioactivity in fish.

Global transcriptional analysis of short-term hepatic stress responses in Atlantic salmon (Salmo salar) exposed to depleted uranium.

Potential environmental hazards of radionuclides are often studied at the individual level. Sufficient toxicogenomics data at the molecular/cellular level for understanding the effects and modes of toxic action (MoAs) of radionuclide is still lacking. The current article introduces transcriptomic data generated from a recent ecotoxicological study, with the aims to characterize the MoAs of a metallic radionuclide, deplete uranium (DU) in an ecologically and commercially important fish species, Atlantic salmon (Salmo salar). Salmon were exposed to three concentrations (0.25, 0.5 and 1.0 mg/L) of DU for 48 h. Short-term global transcriptional responses were studied using Agilent custom-designed high density 60,000-feature (60 k) salmonid oligonucleotide microarrays (oligoarray). The microarray datasets deposited at Gene Expression Omnibus (GEO ID: GSE58824) were associated with a recently published study by Song et al. (2014) in BMC Genomics. The authors describe the experimental data herein to build a platform for better understanding the toxic mechanisms and ecological hazard of radionuclides such as DU in fish.

Sublethal effects in Atlantic salmon (Salmo salar) exposed to mixtures of copper, aluminium and gamma radiation.

The present study was designed to investigate the effects in presmolt of Atlantic salmon (Salmo salar) exposed to copper (Cu), aluminium (Al) and gamma radiation, individually or in combination. Fish were exposed for 48 h to metals added to lake water; 10, 40 and 80 µg Cu/L, 250 µg Al/L and a combination of 40 µg Cu/L and 250 µg Al/L. In addition, gamma radiation (4-70 mGy delivered over 48 h) was added as an additional exposure stressor. Selected endpoints were chosen to reveal different toxic mechanisms and included Cu and Al accumulation on gills, blood chemistry and haematological variables (plasma sodium and chloride, haematocrit, glucose), hepatic levels of reduced and oxidised glutathione (GSH and GSSG) and hepatic transcriptional response of glutathione peroxidase (GPx), gamma-glutamylcysteine synthetase (GCS), metallothionein (MT) and ubiquitin. Exposure to Cu alone resulted in gill accumulation of Cu, reduction of plasma ions and increased transcriptional response of GPx, MT and ubiquitin. Exposure to Al alone reduced plasma ion levels but did not affect any of the hepatic biomarkers except for ubiquitin. The combined metal exposure (Cu + Al) altered the GSH levels, however GPx and MT were not affected suggesting a different mode of detoxification in the combined exposure. Gamma radiation appeared to influence GSH and ubiquitin levels. The observed effects seemed to be both stressor and concentration dependent.

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.

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.