Chemical and biochemical tools to assess pollution exposure in cultured fish

There is little information regarding pollutant levels in farmed fish, and the risks associated to consumption. This study was designed to assess levels of exposure to metals, organochlorinated compounds, polycyclic aromatic hydrocarbons (PAHs) and alkylphenols (APEs) in farmed sea bass Dicentrarchus labrax from five aquacultures located in Southern Europe. Additionally, several biochemical responses (metallothionein, 7-ethoxyresorufin O-deethylase, vitellogenin) were determined as complementary tools. The obtained data indicate that pollutants exposure in farmed fish is similar to the levels reported in wild specimens from the area. Nonetheless, some biochemical responses were observed in the studied organisms, viz. metallothionein induction in Cu exposed organisms, and 7-ethoxyresorufin O-deethylase (EROD) and vitellogenin induction in PAHs and APEs exposed ones. The study further supports the usefulness of the biomarker approach as a first screening method to discriminate between basal and high levels of exposure in cultured fish.     

Responses of juvenile sea bass, Dicentrarchus labrax, exposed to acute concentrations of crude oil, as assessed by molecular and physiological biomarkers

In the present study, juvenile sea bass were exposed for 48 and 96 h to an Arabian light crude oil and their responses were assessed at the molecular and physiological levels. The aim of the study was therefore to assess (i) the short term effects of crude oil exposure by the measurement of several molecular biomarkers, (ii) the consequences of this short term exposure on fish health by using growth and condition indices measured after a decontamination period of 28 and 26 d in seawater. Hydrocarbon petroleum concentrations was monitored during the 96 h experiments and an increase of PAH concentrations were found in fish following both exposure times. An 7-ethoxyresorufin-O-deethylase (EROD) induction was observed after 48 h of exposure, while a significant decrease in the sea bass specific growth rate in length and for the RNA:DNA ratio was observed 28 d after that exposure ceased. The EROD induction doubled after the 96 h exposure, and a significant increase in GST activities was observed. A significant decrease in the specific growth rates, the otolith recent growth, the RNA:DNA ratio and the Fulton’s K condition index were then observed in sea bass 26 d after the 96 h exposure to mechanically dispersed crude oil compared to the control. The present study shows that growth and condition indices can prove useful in assessing fish health status following an oil spill. Their complementary analysis with sensitive molecular biomarkers as EROD could improve the determination of oil spill impact on fish populations.     

Field validation of a battery of biomarkers to assess sediment quality in Spanish ports

Two marine invertebrates, the crab Carcinus maenas and the clam Ruditapes philippinarum, were used as bioindicator species to assess contamination when exposed in situ to sediment from different sites from four Spanish ports Cadiz (SW Spain), Huelva (SW Spain), Bilbao (NE Spain) and Pasajes (NE Spain). In an attempt to determine sediments toxicity, a combination of exposure biomarkers was analyzed in both species: metallothionein-like-proteins (MTLPs), ethoxyresorufin O-deethylase (EROD), glutathione S-transferase activity (GST), glutathione peroxidase (GPX) and glutathione reductase (GR). In parallel, physical and chemical characterization of the different sediments was performed and biological responses related to the contaminants. Significant induction of MTLPs was observed when organisms were exposed to metal contaminated sediments (port of Huelva), and EROD and GPX activities after exposure to sediments containing organic compounds (port of Bilbao and Pasajes). No significant interspecies differences were observed in biomarker responses except for the GST and GR.     

Comparing the response of biochemical indicators (biomarkers) and biological indices to diagnose the ecological impact of an oil spillage in a Mediterranean river (NE Catalunya, Spain)

Three biomarkers of hydrocarbon exposure, liver 7-ethoxyresourfin-O-deethylase activity (EROD), fluorescent hydrocarbon compounds (FACs) in bilis, and the liver antioxidant enzyme catalase (CAT) were examined in the autochthonous fish species Barbus meridionalis collected in the river Fluvià (NE Catalunya, Spain) after an oil spillage. Four different locations were sampled, including the impacted site, upstream and downstream sites and a reference site. Biomarker responses were compared with diatom and macroinvertebrate community assemblage metrics (Specific Pollution Sensitivity index - IPS, and Iberian Biological Monitoring Working Party - IBMWP, respectively). Chemical analyses denoted that polycyclic aromatic hydrocarbon levels in sediment were much higher at the impacted site than in downstream reaches. Four fold increase of EROD activity together with increased levels of biliary FACs in barbs collected at the spilled site indicated exposure of inhabiting fish to the oil. Additionally, CAT activity was significantly depressed (four fold) when compared to other stations, thus suggesting that fish collected from the impacted sites could be more susceptible to suffer oxidative stress. Biological indices (particularly that of the diatom community IPS) showed slight significant effects between control and impacted sites, indicating that more tolerant taxa were favoured because of the oil spillage. These results support the need to include biochemical responses measured in local species in monitoring programmes aimed to diagnose specific pollution effects in stressed river ecosystems.     

Assessment of typical pollutants in waterborne by combining active biomonitoring and integrated biomarkers response

Organic pollutants, heavy metals and pharmaceuticals are continuously dispersed into the environment and have become a relevant environmental emerging concern. In this study, a situ assay to assess ecotoxicity of mixed pollutants was carried out in three typical sites with different priority contaminations in Guangzhou, China. Chemical analysis of organic pollutants, metals and quinolones in three exposure sites were determined by GC-ECD/MS, ICP-AES and HPLC, as well as, a combination of biomarkers including: ethoxyresorufin O-deethylase (EROD); aminopyrine N-demethylase (APND); erythromycin N-demethylase (ERND); glutathione S-transferase (GST); malondialdehyde (MDA); CYP1A; and P-glycoprotein (P-gp) mRNA expressions were evaluated in Mugilogobius abei. Results of chemical analysis in sediment samples revealed that the dominant chemicals were organic pollutants and heavy metals in Huadi River while quinolones in the pond. Bioassays indicated that differences among sites were in relation to some specific biomarkers. EROD and GST activities significantly increased after 72 h in situ exposure, but no difference was observed among the exposure sites. APND, ERND and MDA exhibited dissimilar change patterns for different priority pollutants. CYP1A and P-gp mRNA expressions were significantly induced at all exposure sites, whilst P-gp activity was typical for S2 with the highest levels of quinolones. The molecular biomarkers seemed to be more susceptible than enzyme activities. These assays confirmed the usefulness of applying a large array of various combined biomarkers at different levels, in assessing the toxic effects of mixed pollutants in a natural aquatic environment.     

Multiple biomarkers of pollution effects in Solea solea fish on the Tunisia coastline

This field study investigates the morphological indices (condition index, hepatosomatic index) and biochemical (catalase (CAT), glutathione S-transferase (GST), acetylcholinesterase (AChE), metallothionein (MT), lipid peroxidation) parameters in liver, gills and kidney of common sole (Solea solea) originating from different sites of the Tunisian coast area impacted by different anthropogenic activities. Differences among sites and tissues for AChE, GST, CAT, MT and TBARS were found and possibly related to known sources of domestic and industrial discharges in the studied sites. Liver, gills and kidney CAT, liver and kidney MT and brain AChE were key biomarkers to discriminate fish of different sites. So, we suggest using these biomarkers in future biomonitoring.     

Multiparametric approach for assessing environmental quality variations in West African aquatic ecosystems using the black-chinned tilapia (Sarotherodon melanotheron) as a sentinel species

The study highlights the potential of the black-chinned tilapia to be used as a sentinel to assess environmental contaminants based on the use of a set of biomarkers. The usefulness of fish species as sentinels for assessing aquatic environment contamination was tested using a set of biomarkers in Senegalese environments characterized by multi-pollution sources. The black-chinned tilapia (Sarotherodon melanotheron) was selected as a sentinel because of its abundance, wide distribution in all coastal aquatic ecosystems and physiological properties. The potential influence of confounding factors such as salinity on biomarker in the tilapia has been examined. Individuals were sampled during two seasons (dry and wet) in eight sites characterized by various degrees of anthropogenic contamination and different salinities (from 0 to 102?psu). Biomarkers??including growth rate (GR), condition factor (CF), biotransformation enzymes such as 7-ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST), lipid peroxidation (TBARS) and acetylcholinesterase (AChE)??were measured. Chemical contaminant [polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs)] levels showed different sources of contamination with relatively high concentrations of PAHs in the Hann Bay and Foundiougne locations. The most sensitive biomarker present in different sites according to the principal component analysis is EROD. Few variations of the AChE activity and TBARS levels were found. No clear relationship was found between biomarker responses and salinity, but GR and CF were lower in hypersaline conditions. Tilapia is responsive to environmental contaminants such as PAHs, OCPs and PCBs. The S. melanotheron multiparametric approach showed a better discrimination of sites.     

Effect of prochloraz fungicide on biotransformation enzymes and oxidative stress parameters in three-spined stickleback (Gasterosteus aculeatus L.)

The aim of this study was to characterize biomarker responses in three-spined sticklebacks exposed to prochloraz (Pcz). For this purpose, adult sticklebacks were exposed for 2 weeks to prochloraz at 0, 10, 50, 100 and 500 μg/L prior to one week of depuration in clean water. At days 7, 14 and 21, several hepatic biomarkers were measured including 7-ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), glutathione peroxidase (GPx), catalase (CAT), total glutathione (GSH) content and thiobarbituric acid reactive substances (TBARS). Pcz induced a transient increase of antioxidant enzymes and a depletion of glutathione content during the first 7 days of exposure. This study showed that EROD activity and antioxidants were disrupted in a transient manner. GST was rapidly induced in a dose-dependent manner and this induction was persistent and observed also after depuration. GST appeared as a valuable biomarker to assess the exposure to Pcz.     

Effect of prochloraz fungicide on biotransformation enzymes and oxidative stress parameters in three-spined stickleback (Gasterosteus aculeatus L.)

The aim of this study was to characterize biomarker responses in three-spined sticklebacks exposed to prochloraz (Pcz). For this purpose, adult sticklebacks were exposed for 2 weeks to prochloraz at 0, 10, 50, 100 and 500 microg/L prior to one week of depuration in clean water. At days 7, 14 and 21, several hepatic biomarkers were measured including 7-ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), glutathione peroxidase (GPx), catalase (CAT), total glutathione (GSH) content and thiobarbituric acid reactive substances (TBARS). Pcz induced a transient increase of antioxidant enzymes and a depletion of glutathione content during the first 7 days of exposure. This study showed that EROD activity and antioxidants were disrupted in a transient manner. GST was rapidly induced in a dose-dependent manner and this induction was persistent and observed also after depuration. GST appeared as a valuable biomarker to assess the exposure to Pcz.     

Inhibition of ethoxyresorufin-O-deethylase (EROD) activity in mixtures of 2,3,7,8-tetrachlorodibenzo-p-dioxin and polychlorinated biphenyls

Induction of ethoxyresorufin-O-deethylase (EROD) activity and porphyrin accumulation shows different structure-activity relationships for different polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Interactions between the two responses can strongly influence the induction and activity of EROD. The results support the conclusion that there are nonadditive interactions between nondioxin-like PCBs and dioxin-like compounds. The interaction between EROD activity and the porphyrin biosynthesis makes the prerequisite of additivity in the toxic equivalency factor concept for environmental mixtures highly spurious. Inhibition of EROD activity caused by non-dioxin like compounds could have a significant impact on the value of EROD activity as a biomarker in the present methods of risk assessment for these compounds.     

Comparision of the waterborne and dietary routes of exposure on the effects of Benzo(a)pyrene on biotransformation pathways in Nile tilapia (Oreochromis niloticus)

BaP is one of the most studied PAH, due to its ubiquitous presence in aquatic environments and toxicity to aquatic organisms. The main goal of this study was to assess BaP effects in Nile Tilapia after waterborne and dietary exposures, through the evaluation of EROD and GST activities in liver, gills and intestine, and BaP metabolites in bile; and also to evaluate the usefulness of these commonly used biomarkers after two different routes of exposure. Waterborne exposure to BaP led to a significant induction of EROD in all tissues analyzed (644%, 1640% and 2880% in relation to solvent in liver, gill and intestine respectively) while in dietary exposures EROD was induced only in intestine (3143%) after exposure to high BaP concentrations. GST activities with CDNB were slightly induced in liver (40%) and in gill (66%) after water exposure to BaP, and in intestine after dietary exposure to low BaP concentrations (182%). BaP metabolites in bile increased after both exposure routes, and were highly correlated with EROD activity after water exposure. In summary, this work has shown that the effects of BaP on biotransformation pathways depend on the route of exposure. Moreover, barrier tissues like gills and intestine also have an important role in the first-pass metabolism of BaP, reducing the amount of parent compound that reaches the liver to be metabolized. For that reason, EROD activity as a biomarker of exposure should also be applied in extrahepatic organs, like gills and intestine, in monitoring studies. Biliary BaP type metabolites are good reflectors of contamination levels under both exposure routes, while GST activity with CDNB as substrate, as a phase II enzyme, does not seem a reliable biomarker of exposure to BaP regardless the route of exposure.     

Changes in cell-type composition in digestive gland of slugs and its influence in biomarkers following transplantation between a relatively unpolluted and a chronically metal-polluted site

Changes in cell-type composition (CCTC) is a general phenomenon that takes place in the digestive gland epithelium of stressed molluscs. The aim of the present work was to determine whether CCTC is a reversible process in the digestive gland of sentinel slugs chronically exposed to metal pollution and how CCTC affects metal accumulation parameters and different cell and tissue biomarkers of exposure and effect. Slugs (Arion ater) from an abandoned zinc mine were transferred to a relatively unpolluted site and the other way around for 3, 10 and 28 d. The volume density of black silver deposits (Vv_BSD) after autometallography, and metallothionein (MT) levels were used as biomarkers of exposure to metals and CCTC and lysosomal responses were selected as effect biomarkers. Results indicated that slugs were sensitive to recent metal pollution; however, slugs chronically exposed to metals presented some characteristic features and were less responsive to pollution cessation without signs of CCTC reversal.     

Hepatic and immune biological effect assays in C57BL/6 mice to measure polycyclic aromatic hydrocarbon bioavailability under laboratory exposures with increasing environmental relevance

Background Monitoring biological responses that are mediated via the aryl-hydrocarbon receptor (AhR) in animals exposed to environmental contaminants can indicate both the presence to chemicals that act through this biochemical pathway and whether these chemicals are bioavailable. Objectives The use of an ex-situ method that incorporated biological responsiveness monitoring in mice for determining the presence of ‘biologically active’ hydrocarbons in contaminated soils was investigated. Methods The use of C57BL/6 as a test organism was validated by determining hepatic and immune responsiveness to two polyaromatic hydrocarbons (PAHs): 3,4 benz[a]pyrene (B[a]P) and 1,2 benz (a)anthracene (BA) administered via intraperitoneal (i.p.) injection. The responsiveness of mice exposed to soils spiked with hydrocarbons or ex situ exposures to soil removed from two contaminated sites was also investigated. Results and Discussion Mice that were exposed to B[a]P via i.p. injections showed a 14-fold increase in liver microsomal ethoxyresorufin O-deethylase (EROD) activity compared to the control group. In contrast EROD activity following BA exposure at the same level was not significantly enhanced. Mouse immune response was significantly inhibited in a dose-dependent manner by i.p. injections of B[a]P. No significant inhibition occurred with the same doses of BA. Following i.p. exposure, the retention of B[a]P in mouse carcasses was greater than BA. Mice exposed to clean soils spiked with environmentally relevant concentrations of B[a]P and BA failed to show any significantly different hepatic or immune responses. Carcass residue data indicated a limited uptake of PAH from the soil. In contrast, EROD activity in mice exposed (ex situ) to hydrocarbon-contaminated soils removed from a fuel-loading depot and decommissioned gas works was significantly enhanced (4- and 2-fold respectively). However, this increase in EROD activity did not appear to correlate with either soil or carcass PAH concentrations. Conclusions and Outlook These results support the assumption that B[a]P has a higher affinity for the aryl hydrocarbon receptor (AhR) compared to BA. Soil parameters such as organic carbon content, structure and particle size distribution can modulate the bioavailability of contaminants to biological receptors. These factors are implicated in the lack of responsiveness demonstrated in the spiked soil experiments. However the responsiveness of EROD activity in mice exposed (ex situ) to soil contaminated with complex mixtures of hydrocarbon compounds confirms the potential usefulness of this model to determine the presence of ‘biologically active’ compounds in aged soils removed from contaminated sites.     

Biomarker responsiveness in different tissues of caged Ruditapes philippinarum and its use within an integrated sediment quality assessment

Biomarkers comprising activities of biotransformation enzymes (ethoxyresorufin-O-deethylase -EROD-, dibenzylfluorescein dealkylase -DBF-, glutathione S-transferase -GST), antioxidant enzymes (glutathione reductase -GR- and glutathione peroxidase -GPX), lipid peroxidation -LPO- and DNA strand breaks were analyzed in the clam Ruditapes philippinarum caged at Cádiz Bay, Santander Bay and Las Palmas de Gran Canaria (LPGC) Port (Spain). Sediments were characterized. Digestive gland was the most sensitive tissue to sediment contamination. In Cádiz Bay, changes in LPO regarding day 0 were related with metals. In LPGC Port, DBF, EROD, and GST activity responses suggested the presence of undetermined contaminants which might have led to DNA damage. In Santander Bay, PAHs were related with EROD activity, organic and metal contamination was found to be associated with GR and GST activities and DNA damage presented significant (p < 0.05) induction. R. philippinarum was sensitive to sediment contamination at biochemical level. Biomarkers allowed chemical exposure and sediment quality assessment.     

In vitro toxicity of selected pesticides on RTG-2 and RTL-W1 fish cell lines

The rainbow trout fish cell lines RTG-2 and RTL-W1 were used to determine the cytotoxic effects of the pesticides bifenthrin, cypermethrin, cyhalothrin, lambda-cyhalothrin, quinalphos and chlorpyrifos. Cytotoxicity was measured by EROD and beta-Gal enzymatic activities, the neutral red (NR) uptake assay, and the FRAME KB protein (KBP) assay. The beta-Gal activity was unaffected by the pesticide exposure. The EROD activity was induced by cyhalothrin and lambda-cyhalothrin (RTG-2 and RTL-W1) and by bifenthrin (RTL-W1). Dose dependent inhibition responses were observed for EROD activity in cells exposed to quinalphos (RTL-W1) and chlorpyrifos (RTG-2 and RTL-W1). RTL-W1 offered a better response for EROD induction. The EC50 values on EROD endpoint were more sensitive than NR and KBP. The acute fish toxicity of chlorpyrifos and quinalphos depends highly on the species; the species sensitivity distributions cover several orders of magnitude and the values obtained for EROS were within the lowest part of the reported ranges.     

New evidences for old biomarkers: effects of several xenobiotics on EROD and AChE activities in Zebra mussel (Dreissena polymorpha)

The biomarker approach is widely used both in vertebrates and invertebrates for environmental biomonitoring, because it can supply an integrated response for multi-xenobiotics contamination. However, the use of biomarkers requires the identification of every possible variation that can influence the biochemical response, because ecosystems are generally subject to a mixture of pollutants, which can create additive, opposite or competitive effects. In recent years, there has been considerable interest in the use of biomarkers within marine bivalves, while very few data are available for freshwater molluscs. The aim of this research was to investigate changes on EROD and AChE activities in the freshwater bivalve Zebra mussel (Dreissena polymorpha) exposed to different pollutants (Arochlor 1260, CB 153 and 126, pp'DDT, chlorpyrifos, carbaryl) at laboratory conditions, in order to standardize the analytical procedures and to highlight eventual interferences on enzyme activities. Chemical concentrations in the mussel soft tissues were analyzed by GC/MS-MS. Main results showed a significant induction of EROD activity when mussels were exposed to 100 ng/l of PCB mixture of Arochlor 1260 and dioxin-like CB 126, but this congener showed also a clear competitive inhibition after 48 h of exposure. Surprisingly, pp'DDT determined a significant decrease of basal EROD activity after only 24 h of exposure, even if it was not possible to discriminate between the effect of the parent compound and that of its metabolites (DDD, DDE). We also found an interaction between the organophosphate insecticide chlorpyrifos, which does not directly decrease the AChE activity, and terbutilazine. This herbicide increased the biotransformation of the organophosphate compound to its oxidized metabolite (oxon), a much stronger AChE inhibitor. The possible use of the oxime Pyridine-2-Aldoxime Methochloride (2-PAM) to bring back the catalytic activity to basal levels was also demonstrated.     

Biomarker responses as indication of contaminant effects in Oreochromis niloticus

The current study investigated oxidative stress parameters (enzymes activities, metallothionein content and lipid peroxidation) in freshwater fish, Oreochromis niloticus, tilapia exposure to Monjolinho River (in 4 months of year: January, April, July and November). One critical site in Monjolinho River (site B) was assessed in comparison to a reference site (site A). Water pH and oxygen concentration was lower than that recommended by CONAMA (Brazilian National Environmental Committee), resolution 357/2005 for protection of aquatic communities, and ammonium and the metals Cu, Zn, Mn and Fe (on all months) concentrations were higher than the maximum concentration recommended. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were significantly decreased in liver and muscle in tilapia from Monjolinho River, throughout the year, in relation to reference except in gills that SOD activity increased. Glutathione S-transferase (GST) activity was significantly increased in liver of the tilapia from Monjolinho River in all sites, in relation to reference except in gills that GST activity increased in July and decreased in November, suggesting that GST activity could be induced to neutralize the pollutants toxicity. On the other hand, GST activity was significantly decreased in white muscle indicating a toxic effect of pollutants, resulting in a decreased ability of tilapia to perform defense reactions associated to GSTs. The decrease of catalase (CAT) activity in gills of the O. niloticus together with the increase of SOD activity, could explain the increased lipid peroxidation (LPO) level in this organ. Metallothionein levels in liver and gills were significantly high in all sites. Results indicate that the exposure to metals caused severe damage to tissues; despite the consensually assumed antioxidant induction as a sign of exposure to contaminants the effects seem in part to be mediated by suppression of antioxidant system with SOD, CAT and GPx as potential candidates for tissues toxicity biomarkers of pollutants.     

Screening of POP pollution by AChE and EROD activities in Zebra mussels from the Italian Great Lakes

The increase of ethoxyresorufin dealkylation (EROD) and the inhibition of acetylcholinesterase (AChE) as biomarkers have been commonly used in vertebrates for the persistent organic pollutants (POPs) biomonitoring of aquatic environments, but very few studies have been performed for invertebrates. Previous researches demonstrated the interference due to some chemicals on EROD and AChE activities of the freshwater bivalve Zebra mussel (Dreissena polymorpha) in laboratory and field studies, showing its possible use for the screening of POP effects. We investigated the contamination of the Italian sub-alpine great lakes (Maggiore, Lugano, Como, Iseo, Garda) by the biomarker approach on Zebra mussel specimens collected at 17 sampling sites with different morphometric characteristics and anthropization levels. Results showed a homogeneous contamination of AChE inhibitors in Lake Garda, Maggiore, Como and Iseo with values ranging from 0.5 to 3 nmol/min/mg proteins and with an average inhibition of about 66% to controls. The planar compounds pollution, able to activate the EROD activity, seems higher in some sampling stations of Lake Garda, Como and Iseo (2-4 pmol/min/mg proteins) than that measured in Lake Lugano (1.5-3 pmol/min/mg proteins). On the contrary, the enzyme activity in Lake Maggiore showed an interesting opposite effect of AhR-binding compounds and trace metals. Finally, the possible use of Zebra mussel specimens maintained at laboratory conditions as controls against the selection of the less polluted sampling site is discussed.     

Monitoring contaminants from oil production at sea by measuring gill EROD activity in Atlantic cod (Gadus morhua)

An ex vivo gill EROD assay was applied in Atlantic cod (Gadus morhua) as a biomarker for waterborne CYP1A-inducing compounds derived from oil production at sea. Exposure to nominal concentrations of 1 ppm or 10 ppm North Sea crude oil in a static water system for 24 h caused a concentration-dependent gill EROD induction. Further, exposure of cod for 14 days to environmentally relevant concentrations of produced water (PW, diluted 1:200 or 1:1000) from a platform in the North Sea using a flow-through system resulted in a concentration-dependent induction of gill EROD. Crude oil (0.2 ppm) from the same oil field also proved to induce EROD. Finally, gill EROD activity in cod caged for 6 weeks at 500-10 000 m from two platforms outside Norway was measured. The activities in these fish were very low and did not differ from those in fish caged at reference sites.