Effects of single and combined exposures of gold (nano versus ionic form) and gemfibrozil in a liver organ culture of Sparus aurata.

In vitro methods have gained rising importance in ecotoxicology due to ethical concerns. The aim of this study was to assess the single and combined in vitro effects of gold, as nanoparticle (AuNPs) and ionic (Au+) form, and the pharmaceutical gemfibrozil (GEM). Sparus aurata liver organ culture was exposed to gold (4 to 7200 µg·L-1), GEM (1.5 to 15,000 µg·L-1) and combination 80 µg·L-1 gold +150 µg·L-1 GEM for 24 h. Endpoints related with antioxidant status, peroxidative/genetic damage were assessed. AuNPs caused more effects than Au+, increasing catalase and glutathione reductase activities and damaging DNA and cellular membranes. Effects were dependent on AuNPs size, coating and concentration. GEM damaged DNA at an environmentally relevant concentration, 1.5 µg·L-1. Overall, the effects of the combined exposures were higher than the predicted, based on single exposures. This study showed that liver culture can be a useful model to study contaminants effects.

Effects of gold nanoparticles in gilthead seabream-A proteomic approach.

Despite the widespread use of nanoparticles (NPs), there are still major gaps of knowledge regarding the impact of nanomaterials in the environment and aquatic animals. The present work aimed to study the effects of 7 and 40 nm gold nanoparticles (AuNPs) - citrate and polyvinylpyrrolidone (PVP) coated - on the liver proteome of the estuarine/marine fish gilthead seabream (Sparus aurata). After 96 h, exposure to AuNP elicited alterations on the abundance of 26 proteins, when compared to the control group. AuNPs differentially affected several metabolic pathways in S. aurata liver cells. Among the affected proteins were those related to cytoskeleton and cell structure, gluconeogenesis, amino acids metabolism and several processes related to protein activity (protein synthesis, catabolism, folding and transport). The increased abundance of proteins associated with energy metabolism (ATP synthase subunit beta), stress response (94 kDa glucose-regulated protein) and cytoskeleton structure (actins and tubulins) may represent the first signs of cellular oxidative stress induced by AuNPs. Although higher gold accumulation was found in the liver of S. aurata exposed to 7 nm PVP-AuNPs, the 7 nm cAuNPs were more bioactive, inducing more effects in liver proteome. Gold accumulated more in the spleen than in the other assessed tissues of S. aurata exposed to AuNPs, highlighting its potential role on the elimination of these NPs.

Biological effects and bioaccumulation of gold in gilthead seabream (Sparus aurata) - Nano versus ionic form.

The question of whether gold (Au) is more toxic as nanoparticles or in its ionic form remains unclear and controversial. The present work aimed to clarify the effects of 96 h exposure to 4, 80 and 1600 µg·L-1 of 7 nm gold nanoparticles (AuNPs) - (citrate coated (cAuNPs) or polyvinylpyrrolidone coated (PVP-AuNPs)) - and ionic Au (iAu) on gilthead seabream (Sparus aurata). Effects at different levels of biological organization (behaviour, neurotransmission, biotransformation, oxidative stress/damage and genotoxicity) were assessed. cAuNPs induced oxidative stress and damage (lipid peroxidation increase), even at 4 µg·L-1, and reduced the ability of S. aurata to swim against a water flow at 1600 µg·L-1. Exposure to cAuNPs induced more adverse effects than exposure to PVP-AuNPs. All tested concentrations of Au (nano or ionic form) induced DNA breaks and cytogenetic damage in erythrocytes of S. aurata. Generally, iAu induced significantly more effects in fish than the nano form, probably associated with the significantly higher accumulation in the fish tissues. No fish mortality was observed following exposure to AuNPs, but mortality was observed in the group exposed to 1600 µg·L-1 of iAu.

Effects and bioaccumulation of gold nanoparticles in the gilthead seabream (Sparus aurata) - Single and combined exposures with gemfibrozil.

Gold nanoparticles (AuNPs) are found in a wide range of applications and therefore expected to present increasing levels in the environment. There is however limited knowledge concerning the potential toxicity of AuNPs as well as their combined effects with other pollutants. Hence, the present study aimed to investigate the effects of AuNPs alone and combined with the pharmaceutical gemfibrozil (GEM) on different biological responses (behaviour, neurotransmission, biotransformation and oxidative stress) in one of the most consumed fish in southern Europe, the seabream Sparus aurata. Fish were exposed for 96 h to waterborne 40 nm AuNPs with two coatings - citrate and polyvinylpyrrolidone (PVP), alone or combined with GEM. Antioxidant defences were induced in liver and gills upon both AuNPs exposure. Decreased swimming performance (1600 µg.L-1) and oxidative damage in gills (4 and 80 µg.L-1) were observed following exposure to polyvinylpyrrolidone coated gold nanoparticles (PVP-AuNPs). Generally, accumulation of gold in fish tissues and deleterious effects in S. aurata were higher for PVP-AuNPs than for cAuNPs exposures. Although AuNPs and GEM combined effects in gills were generally low, in liver, they were higher than the predicted. The accumulation and effects of AuNPs showed to be dependent on the size, coating, surface charge and aggregation/agglomeration state of nanoparticles. Additionally, it was tissue' specific and dependent on the presence of other contaminants. Although, gold intake by humans is expected to not exceed the estimated tolerable daily intake, it is highly recommended to keep it on track due to the increasing use of AuNPs.

Genotoxicity of gold nanoparticles in the gilthead seabream (Sparus aurata) after single exposure and combined with the pharmaceutical gemfibrozil.

Due to their diverse applications, gold nanoparticles (AuNPs) are expected to increase of in the environment, although few studies are available on their mode of action in aquatic organisms. The genotoxicity of AuNPs, alone or combined with the human pharmaceutical gemfibrozil (GEM), an environmental contaminant frequently detected in aquatic systems, including in marine ecosystems, was examined using gilthead seabream erythrocytes as a model system. Fish were exposed for 96 h to 4, 80 and 1600 µg L-1 of 40 nm AuNPs with two coatings - citrate or polyvinylpyrrolidone; GEM (150 µg L-1); and a combination of AuNPs and GEM (80 µg L-1 AuNPs + 150 µg L-1 GEM). AuNPs induced DNA damage and increased nuclear abnormalities levels, with coating showing an important role in the toxicity of AuNPs to fish. The combined exposures of AuNPs and GEM produced an antagonistic response, with observed toxic effects in the mixtures being lower than the predicted. The results raise concern about the safety of AuNPs and demonstrate interactions between them and other contaminants.

A multibiomarker approach highlights effects induced by the human pharmaceutical gemfibrozil to gilthead seabream Sparus aurata.

Lipid regulators are among the most prescribed human pharmaceuticals worldwide. Gemfibrozil, which belongs to this class of pharmaceuticals, is one of the most frequently encountered in the aquatic environment. However, there is limited information concerning the mechanisms involved in gemfibrozil effects to aquatic organisms, particularly to marine organisms. Based on this knowledge gap, the current study aimed to assess biochemical and behavioral effects following a sublethal exposure to gemfibrozil (1.5, 15, 150, 1500 and 15,000 µg L-1) in the estuarine/marine fish Sparus aurata. After the exposure to 1.5 µg L-1 of gemfibrozil, fish had reduced ability to swim against a water flow and increased lipid peroxidation in the liver. At concentrations between 15-15,000 µg L-1, the activities of some enzymes involved in antioxidant defense were induced, appearing to be sufficient to prevent oxidative damage. Depending on the organ, different responses to gemfibrozil were displayed, with enzymes like catalase being more stimulated in gills, whereas glutathione peroxidase was more activated in liver. Although there were no obvious concentration-response relationships, the integrated biomarker response version 2 (IBRv2) analysis revealed that the highest concentrations of gemfibrozil (between 150-15,000 µg L-1) caused more alterations. All the tested concentrations of gemfibrozil induced effects in S. aurata, in terms of behavior and/or oxidative stress responses. Oxidative damage was found at a concentration that is considered environmentally relevant, suggesting a potential of this pharmaceutical to impact fish populations.

Genotoxicity of gemfibrozil in the gilthead seabream (Sparus aurata).

Widespread use of pharmaceuticals and suboptimal wastewater treatment have led to increased levels of these substances in aquatic ecosystems. Lipid-lowering drugs such as gemfibrozil, which are among the most abundant human pharmaceuticals in the environment, may have deleterious effects on aquatic organisms. We examined the genotoxicity of gemfibrozil in a fish species, the gilthead seabream (Sparus aurata), which is commercially important in southern Europe. Following 96-h waterborne exposure, molecular (erythrocyte DNA strand breaks) and cytogenetic (micronuclei and other nuclear abnormalities in cells) endpoints were measured. Gemfibrozil was positive in both endpoints, at environmentally relevant concentrations, a result that raises concerns about the potential genotoxic effects of the drug in recipient waters.

PAH metabolites in fish bile: From the Seine estuary to Iceland.

Polycyclic aromatic hydrocarbons (PAH) are environmental contaminants that pose significant risk to health of fish. The International Workshop on Integrated Assessment of Contaminant Impacts on the North Sea (ICON) provided the framework to investigate biomarker responses as well as contaminant concentrations side by side in marine ecosystems. Concentrations of the main PAH metabolites 1-hydroxypyrene, 1-hydroxyphenanthren and 3-hydroxybenzo(a)pyrene were determined in bile by HPLC with fluorescence detection. Fish species under investigation were dab (Limanda limanda), flounder (Platichthys flesus) and haddock (Melanogrammus aeglefinus). A contamination gradient was demonstrated from the low contaminated waters of Iceland and off-shore regions of the North Sea towards higher concentrations in coastal areas. Concentrations of PAH metabolites differed primarily according to sampling region and secondarily to species.

Effects of water accommodated fractions of crude oils and diesel on a suite of biomarkers in Atlantic cod (Gadus morhua).

The aim of this study was to characterize concentration- and time-dependent responses in juvenile Atlantic cod (Gadus morhua) following exposure for one and three weeks to the water-soluble fraction (WAF) of three weathered oils: Arabian Light crude oil (ALC), North Sea crude oil (NSC) and ship-diesel. The sum of polycyclic aromatic hydrocarbons (PAH) in water was highest after one week of exposure and within environmentally relevant concentrations. PAH metabolites in bile confirmed exposure to and uptake of PAHs. Hepatic cytochrome P450 1A (CYP1A) gene expression (mRNA quantification) increased dramatically following exposure to all three oil types (fold-change up to 165) and there was a time lag between gene and protein expression. Hepatic CYP1A protein concentration and ethoxyresorufin-O-deethylase (EROD) activity were more variable among individuals and treatments than gene expression. EROD activity in liver and gills increased in fish exposed to WAF from the two crude oils, but not in fish exposed to WAF from diesel. Exposure to diesel appeared to induce oxidative stress to a greater extent than exposure to crude oils. Other biomarkers (glutathione S-transferases, acetylcholine esterase, vitellogenin) did not appear to respond to the exposure and hence did not discriminate among oils. Biomarker responses in cod after exposure to weathered crude oils and diesel suggested that the CYP1A system and oxidative stress markers have the highest potential for discriminating among different oil types and to monitor the environmental consequences of spills.

Toxicity screening of produced water extracts in a zebrafish embryo assay.

Produced water is the largest effluent discharge from oil and gas/condensate production facilities in the North Sea. There is concern that contaminants originating from the reservoir and chemicals used in the production process may affect marine organisms. Developmental toxicity of extractable organic compounds in produced water effluents from oil and gas/condensate production platforms in the Norwegian sector of the North Sea was assessed in a temporal and spatial manner using zebrafish (Danio rerio) embryos. Large-scale solid-phase extraction (SPE) and on-column fractionation of water-soluble fraction (WSF) and an oil/particulate fraction was used in a rapid screening bioassay for embryotoxicity. Exposure to produced water extracts increased rate of mortality and reduced pigmentation and heart rate, as well as delaying time to hatch. The oil/particulate fraction was 10-fold less toxic than WSF, indicating that toxicity was predominantly produced by moderately polar and bioavailable compounds. Large spatial and temporal variation in produced water toxicity was observed, displaying considerable variability in the reservoir, oil well, and effluent composition over time. The noted toxicity did not correlate well with either reported produced water composition or parameters such as total hydrocarbons, thus challenging chemical measurements as a reliable source of information for predicting complex effects. Although embryotoxicity was observed following exposure to the extracts, dilution and transformation of produced water in the recipient are expected to rapidly reduce the concentrations of compounds in the effluents to levels below the thresholds of observed effects.

Assessment of lysosomal membrane stability and peroxisome proliferation in the head kidney of Atlantic cod (Gadus morhua) following long-term exposure to produced water components.

There is a need for sensitive biological effect methods by which to detect impacts of chronic exposure to low concentrations of contaminants. Two methods shown to be potentially useful for monitoring purposes in fish include lysosomal membrane stability and peroxisome proliferation. These biological endpoints were assessed in Atlantic cod (Gadus morhua) head kidney following exposure to a mixture of produced water components including polycyclic aromatic hydrocarbons, phenol, and alkylphenols. Lysosomal damage of head kidney cells occurred within the first two weeks and did not recover during the entire exposure period (32 weeks). Lysosomal membrane stability was not affected by gender and was responsive at low concentrations of contamination, indicating that lysosomal membrane stability measured in the head kidney could be a useful biomarker for effects of offshore pollution. Peroxisome proliferation, measured as acyl-CoA oxidase activity in the head kidney, appeared to be a potential biomarker in male cod exposed less than 16 weeks.

Endocrine modulation in Atlantic cod (Gadus morhua L.) exposed to alkylphenols, polyaromatic hydrocarbons, produced water, and dispersed oil.

Effluent from oil production activities contains chemicals that are suspected of inducing endocrine disruption in fish. In this study, Atlantic cod (Gadus morhua L.) were exposed to mixtures of low- and medium-molecular-weight alkylphenols (AP) (methyl- to heptylphenol), polycyclic aromatic hydrocarbons (PAH), diluted produced water, and dispersed oil for 15 d in a flow-through exposure system. Condition index (CI), hepatosomatic index (HSI), gonadosomatic index (GSI), concentration of the estrogenic biomarker vitellogenin (Vtg), and modulation of the total sex steroid-binding capacity in plasma were determined to assess whether these mixtures were capable of interfering with endocrine-regulated physiological processes in Atlantic cod. No marked differences in plasma Vtg levels were found between control and exposed groups of either males or females, possibly due to high intergroup variances and low sample numbers. An apparent numerical increase in the number of male and female fish with high plasma Vtg levels was, however, observed in some exposure groups compared to control. This purported weak estrogenic effect was several orders of magnitude lower than that observed for potent estrogens and suggested that the levels of estrogen receptor (ER) agonists were low. Exposure of female fish to a mixture of dispersed oil and a mixture of AP, PAH, and dispersed oil led to upregulation of the plasma total sex steroid-binding capacity, indicating interference with the normal blood steroid transport. No significant effects were seen for CI, HSI, and GSI, suggesting that the endocrine-disrupting potential was not sufficient to elicit effects on general physiological conditions and gonad development during this short exposure period.

Repeated sampling of Atlantic cod (Gadus morhua) for monitoring of nondestructive parameters during exposure to a synthetic produced water.

The past decades of monitoring discharges from oil and gas industry have revealed that although there are indications of adverse effects in tissues of aquatic organisms, little is known about their temporal development. Furthermore, observations in wild-caught individuals have not been clearly reproduced in laboratory studies or caging studies, and vice versa, and the results are therefore not easily interpretable. There is clearly a need for exposure studies designed for monitoring the development of effect markers in individual fish over chronic periods to low contaminant levels. Through repetitive nondestructive sampling, the progression of effects may be monitored in individuals, significantly reducing the number of fish needed in exposure studies. A laboratory exposure study was designed to be able to monitor selected parameters in individual Atlantic cod (Gadus morhua). Passive integrated transponders in combination with visible implant elastomers were used to study individual fish during the exposure period (44 wk). Fish were measured (weight and length) and a blood sample was taken for analysis of hematocrit, DNA damage (micronucleus), and oxidative stress (total oxyradical scavenging capacity) at up to seven time points. There were no apparent adverse effects of treatments on the health of experimental fish, frequency of micronucleated erythrocytes, or oxidative stress in whole blood. It is possible that the time scale was not sufficient for development and detection of parameters included here or that red blood cells may not be a suitable matrix for the selected analyses. Future studies need to include other parameters in blood to investigate their sensitivity to low-concentration exposures.

Using biological effects tools to define Good Environmental Status under the European Union Marine Strategy Framework Directive.

The use of biological effects tools offer enormous potential to meet the challenges outlined by the European Union Marine Strategy Framework Directive (MSFD) whereby Member States are required to develop a robust set of tools for defining 11 qualitative descriptors of Good Environmental Status (GES), such as demonstrating that "Concentrations of contaminants are at levels not giving rise to pollution effects" (GES Descriptor 8). This paper discusses the combined approach of monitoring chemical contaminant levels, along side biological effect measurements relating to the effect of pollutants, for undertaking assessments of GES across European marine regions. We outline the minimum standards that biological effects tools should meet if they are to be used for defining GES in relation to Descriptor 8 and describe the current international initiatives underway to develop assessment criteria for these biological effects techniques.

Oxidative stress responses in rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to pro-oxidants and a complex environmental sample.

A wide range of pollutants in the aquatic environment have the capacity to induce toxic effects expressed as cellular oxidative stress. In the current study, the potential of an in vitro toxicity testing system was therefore investigated using rainbow trout (Oncorhynchus mykiss) hepatocytes to assess different endpoints of oxidative stress. The pro-oxidants CuSO(4) and paraquat were used as models for comparison to a complex environmental sample. Results following 6, 24, 48 and 96h exposure to different concentrations of these substances show cellular effects on intracellular ROS formation, glutathione levels and redox status, expression of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, gamma-glutamyl-cysteine synthetase (GCS) and thioredoxin, as well as cytotoxicity parameters. The most consistent effects (maximum values within brackets), observed in dose and time parameters for both model compounds and environmental sample, were the depletion of total glutathione (9.4% of control), induced levels of oxidized glutathione (695% of control), and gene expression regulation depicted relative to the control gene beta-actin of GCS mRNA (239% of control) and catalase (29% of control). In conclusion, the responses on several antioxidant defence system parameters demonstrated the validity of the in vitro toxicity testing system. Not only could multiple effects be detected at sub-lethal exposure concentrations, but these effects also gave valuable insight to the toxic mechanisms at the molecular level.

Produced water extracts from North Sea oil production platforms result in cellular oxidative stress in a rainbow trout in vitro bioassay.

Produced water (PW) discharged from offshore oil industry contains chemicals known to contribute to different mechanisms of toxicity. The present study aimed to investigate oxidative stress and cytotoxicity in rainbow trout primary hepatocytes exposed to the water soluble and particulate organic fraction of PW from 10 different North Sea oil production platforms. The PW fractions caused a concentration-dependent increase in reactive oxygen species (ROS) after 1h exposure, as well as changes in levels of total glutathione (tGSH) and cytotoxicity after 96 h. Interestingly, the water soluble organic compounds of PW were major contributors to oxidative stress and cytotoxicity, and effects was not correlated to the content of total oil in PW. Bioassay effects were only observed at high PW concentrations (3-fold concentrated), indicating that bioaccumulation needs to occur to cause similar short term toxic effects in wild fish.

Differential gene expression and biomarkers in zebrafish (Danio rerio) following exposure to produced water components.

The main effluent from oil and gas production is produced water (PW), a waste that contains low to moderate concentrations of oil-derived substances such as polycyclic aromatic hydrocarbons (PAHs) and alkylphenols (APs). PW components may be present in seawater at low concentrations over large areas in the vicinity of oil and gas production facilities. In this study, zebrafish (Danio rerio) were exposed to control and three treatments (high-, pulsed-, low-dose) of a synthetic PW mixture for 1, 7 and 13 weeks. The aim was to investigate the development of transcriptome and biomarker responses as well as relationships between early responses and population-relevant effects. The synthetic PW contained a mixture of low-molecular-weight PAHs (<5 ring) and short-chain APs (C1-C4). The water-borne exposure levels (sum PAH) ranged from 0.54 ppb (low dose) to 5.4 ppb (high dose). Bile pyrene metabolites ranged from 17-133 ng g(-1) bile in the control group to 23-1081 ng g(-1) bile in the high exposure group. Similar levels have been observed in wild fish, confirming an environmentally relevant exposure. The expression of mRNAs of hepatic genes was investigated in the high exposure group using the Zebrafish OligoLibrary from Compugen. Functional clustering analysis revealed effects in the reproductive system, the nervous system, the respiratory system, the immune system, lipid metabolism, connective tissue and in a range of functional categories related to cell cycle and cancer. The majority of differentially expressed mRNAs of genes were down-regulated, suggesting reduction in gene transcription to be as relevant as up-regulation or induction when assessing biological responses to PW exposure. Biomarkers for effects of PAHs (cytochrome P450 1A) and environmental estrogens (vitellogenin) did not appear to be affected by the chronic exposure to low concentration of PW components. Effects at the population level included a reduction in condition factor in male fish from all exposed groups and spinal column deformations in the F1 generation of exposed groups. The different exposure regimes did not produce any significant differences in reproduction or recruitment. The results from this study demonstrate that environmentally relevant concentrations of PW affect gene expression and population-relevant endpoints in zebrafish, although links between the two were not obvious.

The partial pressure of oxygen affects biomarkers of oxidative stress in cultured rainbow trout (Oncorhynchus mykiss) hepatocytes.

Oxidative stress, the imbalance between production of reactive oxygen species and the cellular detoxification of these reactive compounds, is believed to be involved in the pathology of various diseases. Several biomarkers for oxidative stress have been proposed to serve as tools in toxicological and ecotoxicological research. Not only may exposure to various pro-oxidants create conditions of cellular oxidative stress, but hyperoxic conditions may also increase the production of reactive oxygen species. The objective of the current study was to determine the extent to which differences in oxygen partial pressure would affect biomarkers of oxidative stress in a primary culture of hepatocytes from rainbow trout (Oncorhynchus mykiss). Membrane integrity, metabolic activity, levels of total and oxidized glutathione (tGSH/GSSG) was determined, as well as mRNA expression levels of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), gamma-glutamyl-cystein synthetase (GCS) and thioredoxin (TRX). The results show that different biomarkers of oxidative stress are affected when the cell culture is exposed to atmospheric oxygen, and that changes such as increased GSSG content and induction of GSSG-R and GSH-Px can be reduced by culturing the cells under lower oxygen tension. Oxygen tension may thus influence results of in vitro based cell research and is particularly important when assessing parameters in the antioxidant defence system. Further research is needed to establish the magnitude of this effect in different cellular systems.

Immunolocalization of metallothioneins in different tissues of turbot (Scophthalmus maximus) exposed to Cd.

Metallothioneins (MT) were localized by immunochemistry in different organs and cell compartments of turbot exposed to sublethal concentrations (100 ppb) of Cd for 7 days. The polyclonal rabbit anti-cod MT antibody (NIVA, Norway) applied herein exhibited positive cross-reactivity with turbot MTs. Immunoreactive MTs were localized in the branchial epithelium, in the liver and in the kidney of turbot. In Cd exposed fishes MTs were demonstrated mainly in branchial chloride cells (CC) and to a lesser extend in the area where progenitor cells are located and in the cells of the respiratory epithelium (secondary lamellae). A higher staining intensity for MTs was observed in CC of the interlamellar space of the main branchial epithelium in comparison with control CC. MT-staining was also observed in the chondroblasts of the cartilage and in the erythrocytes within blood vessels both in control and Cd-exposed specimens. MT immunoreaction was high in the liver hepatocytes and weak in the epithelium of the proximal portion of the kidney in exposed turbot. The tegument, spleen and muscle were devoid of any immunolabelling in both treatments. Ultrastructural studies at the transmission electron microscope revealed that Cd-induced MTs were mainly located in the cytoplasm of gill CC, the lysosomes and the cytoplasm of hepatocytes and in the basal labyrinth of kidney proximal nephrocytes. The differential localization/induction of MTs in different cell types described hereby suggests that the quantification of the specific expression of MT may be used in biomonitoring programs as a biomarker of Cd exposure in aquatic environments.

Toxicogenomic responses in rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to model chemicals and a synthetic mixture.

As more salmon gene expression data has become available, the cDNA microarray platform has emerged as an appealing alternative in ecotoxicological screening of single chemicals and environmental samples relevant to the aquatic environment. This study was performed to validate biomarker gene responses of in vitro cultured rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to model chemicals, and to investigate effects of mixture toxicity in a synthetic mixture. Chemicals used for 24h single chemical- and mixture exposures were 10 nM 17alpha-ethinylestradiol (EE2), 0.75 nM 2,3,7,8-tetrachloro-di-benzodioxin (TCDD), 100 microM paraquat (PQ) and 0.75 microM 4-nitroquinoline-1-oxide (NQO). RNA was isolated from exposed cells, DNAse treated and quality controlled before cDNA synthesis, fluorescent labelling and hybridisation to a 16k salmonid microarray. The salmonid 16k cDNA array identified differential gene expression predictive of exposure, which could be verified by quantitative real time PCR. More precisely, the responses of biomarker genes such as cytochrome p4501A and UDP-glucuronosyl transferase to TCDD exposure, glutathione reductase and gammaglutamyl cysteine synthetase to paraquat exposure, as well as vitellogenin and vitelline envelope protein to EE2 exposure validated the use of microarray applied to RNA extracted from in vitro exposed hepatocytes. The mutagenic compound NQO did not result in any change in gene expression. Results from exposure to a synthetic mixture of the same four chemicals, using identical concentrations as for single chemical exposures, revealed combined effects that were not predicted by results for individual chemicals alone. In general, the response of exposure to this mixture led to an average loss of approximately 60% of the transcriptomic signature found for single chemical exposure. The present findings show that microarray analyses may contribute to our mechanistic understanding of single contaminant mode of action as well as mixture effects, but that its use in screening of complex environmental samples will need to be further evaluated.