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.

Development of a screening system for the detection of chemically induced DNA methylation alterations in a zebrafish liver cell line.

Early molecular events with correlation to disease, such as aberrant DNA methylation, emphasize the importance of DNA methylation as a potential environmental biomarker. Currently, little is known regarding how various environmental contaminants and mixtures alter DNA methylation in aquatic organisms, and testing is both time- and labor-consuming. Therefore, the potential of an in vitro screening method was evaluated by exposing zebrafish liver cells (ZF-L) for 96 h to the nonmutagenic model substance 5'-azacytidine (AZA), as well as a selection of environmental pollutants such as sodium arsenite (NAS), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 17α-ethinylestradiol (EE2), and diethylstilbestrol (DES). Six single genes with reported and anticipated importance in cancer were selected for analysis. Methylation of gene promoter areas was monitored by bisulfite conversion and high-resolution melt (HRM) analysis after exposure to sublethal concentrations of the test compounds. Subsequently, results were validated with direct bisulfite sequencing. Exposure of ZF-L cells to 0.5 µM AZA for 96 h led to hypomethylation of genes with both low and high basal methylation indicating similarity to mechanism of action in mammals. Further, NAS, EE2, and DES were shown to induce significant alterations in methylation, whereas TCDD did not. It was concluded that cell line exposure in combination with HRM may provide an initial contaminant screening assay by quantifying DNA methylation alterations with high throughput capacity. In addition, the rapid determination of effects following contaminant exposure with this in vitro system points to the possibility for new in vivo applications to be useful for environmental monitoring.

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.

Effects of dispersed aggregates of carbon and titanium dioxide engineered nanoparticles on rainbow trout hepatocytes.

The purpose of this study was to investigate the cytotoxicity and oxidative stress responses of selected engineered carbon and titanium dioxide (TiO2) nanomaterials to rainbow trout (Oncorhynchus mykiss) primary hepatocytes. The engineered nanomaterials tested were C(60) fullerenes, multiwall nanotubes (MWNT), single-wall nanotubes (SWNT) (functionalized and nonfunctionalized), and TiO2 of 5 and 200 nm in size. Characterization of these materials showed that they were typically present in solution as agglomerates. The engineered nanoparticle agglomerates were cytotoxic at nominal concentrations of >3 mg/L, and certain MWNT and SWNT produced significant intracellular reactive oxygen species (ROS) production as well as cytotoxicity. Analyses of the MWNT responsible for ROS production and cytotoxicity for selected transition metals demonstrated the presence of residual cobalt (Co), which was not present in the nonreactive/non-bioactive MWNT. Cobalt alone was not able to induce the observed effects in hepatocyte cells; however, coexposure with MWNT resulted in an increase in cytotoxicity. Data suggest that trace metals often associated with commercial nanotubes are responsible for the observed biological effects. In addition, other mechanisms, such as the proposed facilitated transport (e.g., Trojan horse) type mechanism of uptake, may provoke an increased response compared to aqueous exposures of trace metals in the absence of carbon nanoparticles.

Water column monitoring of the biological effects of produced water from the Ekofisk offshore oil installation from 2006 to 2009.

The Norwegian water column monitoring program investigates the biological effects of offshore oil and gas activities in Norwegian waters. In three separate surveys in 2006, 2008, and 2009, bioaccumulation and biomarker responses were measured in mussels (Mytilus edulis) and Atlantic cod (Gadus morhua) held in cages at known distances from the produced water (PW) discharge at the Ekofisk oil field. Identical monitoring studies performed in all three years have allowed the biological effects and bioaccumulation data to be compared, and in addition, enabled the potential environmental benefits of a PW treatment system (CTour), implemented in 2008, to be evaluated. The results of the 2009 survey showed that caged animals were exposed to low levels of PW components, with highest tissue concentrations in mussels located closest to the PW discharge. Mussels located approximately 1-2 km away demonstrated only background concentrations of target compounds. Concentrations of polycyclic aromatic hydrocarbons (PAH) and alkyl phenol (AP) metabolites in the bile of caged cod were elevated at stations 200-250 m from the discharge. There was also a signal of exposure relative to discharge for the biomarkers CYP1A in fish and micronuclei in mussels. All other fish and mussel biomarkers showed no significant exposure effects in 2009. The mussel bioaccumulation data in 2009 indicated a lower exposure to the PW effluent than seen previously in 2008 and 2006, resulting in an associated general improvement in the health of the caged mussels. This was due to the reduction in overall discharge of PW components (measured as oil in water) into the area in 2009 compared to previous years as a result of the improved PW treatment system.

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.

Monitoring North Sea oil production discharges using passive sampling devices coupled with in vitro bioassay techniques.

Semipermeable membrane devices (SPMDs) and polar organic integrative chemical samplers (POCIS) were deployed in vicinity of an offshore oil production platform discharging production water (produced water) to the North Sea. Extracts from SPMDs and POCIS were subjected to chemical analysis for polycyclic aromatic hydrocarbons (PAHs) and alkylphenols (APs) respectively, and also assessed for acute toxicity (cytotoxicity), estrogen receptor (ER)-mediated production of vitellogenin (Vtg) and induction of 7-ethoxyresorufin-O-deethylase (EROD) activity in primary hepatocytes from rainbow trout (Oncorhynchus mykiss). Chemical analysis of the extracts revealed a gradient of exposure away from the platform for low molecular weight PAH and AP, whereas no exposure gradient was apparent for high molecular weight PAH, as expected. These data coupled with earlier work allowed a tentative general exposure scenario to be determined. The passive sampler extracts also caused modulation of the bioassay toxicity endpoints, although a clear gradient of response relative to the discharge point could not be identified.

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.