An ecotoxicological assessment of a strigolactone mimic used as the active ingredient in a plant biostimulant formulation.

A risk assessment on the aquatic toxicity of the plant biostimulant strigolactone mimic (2-(4-methyl-5-oxo-2,5-dihydro-furan-2-yloxy)-benzo[de]isoquinoline-1,3-dione (SL-6) was performed using a suite of standardised bioassays representing different trophic groups and acute and chronic endpoints. In freshwater, three trophic groups of algae, crustacea and fish were used. Whilst in seawater, algae (unicellular and macroalgae), Crustacea and Mollusca were employed. In addition, the genotoxicity of SL-6 was determined with the comet assessment performed on unicellular marine algae, oysters, and fish embryos. This was the first time ecotoxicity tests have been performed on SL-6. In freshwater, the lowest LOEC was measured in the unicellular algae at 0.31 mg/L SL-6. Although, similar LOEC values were found for embryo malformations and impacts on hatching rate in zebrafish (LOEC 0.31-0.33 mg/L). Consistent malformations of pericardial and yolk sac oedemas were identified in the zebrafish embryos at 0.31 mg/L. In marine species, the lowest LOEC was found for both Tisbe battagliai mortality and microalgae growth at an SL-6 concentration of 1.0 mg/L. Significant genotoxicity was observed above control levels at 0.0031 mg/L SL-6 in the unicellular algae and 0.001 mg/L SL-6 in the oyster and zebrafish larvae. When applying the simple risk assessment, based on the lowest NOECs and appropriate assessment factors, the calculated predicted no effect concentration (PNEC), for the ecotoxicity and the genotoxicity tests were 1.0 µg/L and 0.01 µg/L respectively.

Do DOM quality and origin affect the uptake and accumulation of lipid-soluble contaminants in coastal filter feeders? An experimental simulation of teflubenzuron exposure to blue mussels.

The increased export of terrestrial dissolved organic matter (terrDOM) to coastal marine ecosystems may affect local filter feeders and the local food web via the altered uptake of organic material and associated contaminants. To compare terrDOM to marine DOM (marDOM) as contaminant vectors to coastal biota, we exposed blue mussels (Mytilus sp.) to the different DOM types in combination with teflubenzuron, a widely applied lipophilic aquaculture medicine targeting salmon lice (Lepeophtheirus salmonis). A 16-day exposure of the blue mussels to DOM and teflubenzuron was followed by a depuration phase of 20 days without teflubenzuron. We calculated teflubenzuron adsorption rates and bioaccumulation factors (BAF) using a Bayesian model, expecting teflubenzuron uptake to be greater with terrDOM than marDOM due to the higher prevalence of large amphipathic humic acids in terrDOM. Humic acids have strong absorption properties and are able to envelope lipophilic molecules. Thus, humic acids can function as an efficient contaminant vector when taken up by filter feeders. Although there were varying degrees of overlap, the mussels tended to accumulate higher amounts of teflubenzuron in the DOM treatments than in the seawater control (bioaccumulation factor [BAF] in seawater: median 106 L/kg; 2.5 %-97.5 % percentile: 69-160 L/kg). Contrary to expectations, mussels exposed to marDOM showed a trend toward more bioaccumulation of teflubenzuron than those exposed to terrDOM (BAF marine 144 L/kg; 102-221 L/kg versus BAF terrestrial: 121 L/kg; 82-186 L/kg). The highest teflubenzuron accumulation was observed with the 50:50 mixture of marDOM and terrDOM (BAF mix: 165 L/kg; 117-244 L/kg). The slight difference in DOM-type accumulation rates observed in this experiment-especially the accumulation rate of terrDOM compared to that of the seawater-only treatment type-was not considered environmentally relevant. Further studies are necessary to see if the observed trends transfer to complex environmental systems.

New insights into submarine tailing disposal for a reduced environmental footprint: Lessons learnt from Norwegian fjords.

Submarine tailing disposal (STD) in fjords from land-based mines is common practice in Norway and takes place in other regions worldwide. We synthesize the results of a multidisciplinary programme on environmental impacts of STDs in Norwegian fjords, providing new knowledge that can be applied to assess and mitigate impact of tailing disposal globally, both for submarine and deep-sea activities. Detailed geological seafloor mapping provided data on natural sedimentation to monitor depositional processes on the seafloor. Modelling and analytical techniques were used to assess the behaviour of tailing particles and process-chemicals in the environment, providing novel tools for monitoring. Toxicity tests showed biological impacts on test species due to particulate and chemical exposure. Hypersedimentation mesocosm and field experiments showed a varying response on the benthos, allowing to determine the transition zone in the STD impact area. Recolonisation studies indicate that full community recovery and normalisation of metal leakage rates may take several decades due to bioturbation and slow burial of sulfidic tailings. The results are synthesised to provide guidelines for the development of best available techniques for STDs.

Determining the risk of calcium oxide (CaO) particle exposure to marine organisms.

Calcium oxide (CaO) is being considered as a possible treatment for both the control of echinoderm populations and the treatment against sea lice infestation in Norwegian salmon farms. CaO particles produce an exothermal reaction when in contact with water, which can cause epidermal burns and lesions to certain target organisms leading to death. The aim of the present study was to determine the effects of fine (<0.8 mm) and coarse (<2.5 mm) CaO particles to a range of marine species from different taxonomic groups: two echinoderms (Asterias ruben and Strongylocentrotus droebachiensis); two crustaceans (Carcinus maenas and Tisbe battagliai); two molluscs (Mytilus edulis and Hinia reticulata); a polychaete (Nereis pelagica); a fish (Cyclopterus sp.); and seaweed germlings (Fucus vesiculosus). Overall, the fine CaO particles were more toxic to the selected marine species than the coarse particles. Coarse CaO particle effects were only observed in four of the nine species tested (A. rubens, S. droebachiensis, N. pelagica, T. battagliai) with similar LC50 values between 207 and 268 g/m2. For the fine CaO particles, the lowest LC50 was for the epibenthic copepod (T. battagliai) at 3.14 g/m2, followed by the sea urchin (20.1 g/m2), starfish (22.2 g/m2), ragworm (29.6 g/m2), and netted dog whelk (41.9 g/m2). Lump sucker fish exhibited significant mortalities only at the highest fine CaO concentration tested (320 g/m2) and recorded an LC50 of 226 g/m2. The toxicity data were used to generate species sensitivity distributions (SSDs) for both fine and coarse CaO particles. The hazard concentrations for 5% of the species (HC5) calculated from the SSDs, based on NOEC values, for the coarse and fine particles were 35.5 and 1.5 g/m2 respectively. Using a recommended assessment factor of 5, the Predicted No Effect Concentration (PNEC) was calculated as 7.1 and 0.3 g/m2 for coarse and fine CaO particles respectively.

An ecotoxicological assessment of mine tailings from three Norwegian mines.

The study assessed the environmental toxicity of three Norwegian mine tailings from Omya Hustadmarmor, Sydvaranger, and Sibelco, which are all released into a seawater recipient. Ecotoxicity assessments were performed on the overlying water extracted from the mine tailings, the transformation/dissolution waters obtained from the mine tailings, and whole sediment assessment using a suite of marine organisms including algae, Crustacea, and Mollusca. Overall, based on the toxicity evaluation of the transformation/dissolution data, Sibelco tailings resulted in the highest toxicity albeit at relatively high concentrations, followed by Sydvaranger and Hustadmarmor. Sibelco was the only mine where process chemicals were not used. In contrast, the Corophium sediment contact assay revealed a significantly higher toxicity exerted by Hustadmarmor tailings, which may indicate a physical impact of the fine tailings. The effects observed were discussed with respect to both the measured chemical concentrations of the tailings and the potential physical impact of the tailing particles on organism health.

Seasonal and spatial variations in biomarker baseline levels within Arctic populations of mussels (Mytilus spp.).

Climate change and pollution resulting from human activities in the Arctic require reliable monitoring systems in sentinel species. Mytilus spp. are used as sentinel species all around the world. The use of Mytilus spp. in environmental monitoring requires knowledge about natural variations in pollution biomarkers. Seasonal variations in baseline levels of biomarkers were studied over a year in the mussels from both upper and lower littoral zones in Rakkfjorden, Norway, as they underwent their annual reproductive cycle. Spatial variations of these biomarker baseline levels were measured in five mussel populations within a 60-km radius from Rakkfjorden to investigate universality of the results from the specific population of Rakkfjorden at a regional scale. Seasonal variations in biomarker baseline levels were revealed and seemed to be related to the reproductive state of the mussels and the tidal zone. The mussels appeared to be more sensitive to oxidative stress during gametogenesis in autumn and winter, when having lower lysosome membrane stability and lower baseline levels of antioxidant biomarkers. An increase in baseline levels of these biomarkers was reported during spawning in spring, however, it was not possible to reveal whether these changes were due to spawning, or to a higher metabolic activity in mussels in response to elevated water temperature and food abundance. The differences between the tidal zones were reflected in reduced size of the mussels from the upper littoral zone, their late spawning in the season and increased baseline levels of antioxidant biomarkers during the coldest month, indicating a more challenging environment in the upper littoral zone. The spatial study indicated that the biomarker baseline levels measured in Rakkfjorden were no different from the levels measured in the mussels from five other sites and thus, are representative for mussels on a regional scale.

Weathering impacts the uptake of polyethylene microparticles from toothpaste in Mediterranean mussels (M. galloprovincialis).

Mediterranean mussels (Mytilus galloprovincialis) were exposed over 21 days to polyethylene (PE) particles (0.01 mg ml-1; 50-570 µm) isolated from toothpaste. PE was deployed in the Outer Oslofjord (Norway) for 21 days, before exposing the mussels to both virgin (PE-V) and weathered PE (PE-W) particles. The mussels ingested both types of particles, but significantly more weathered particles were ingested than virgin (p = .0317), based on PE dosed by weight (mg ml-1) but not when considering particle number (PE-V: 1.18 ±â€¯0.16 particles ml-1; PE-W 1.86 ±â€¯0.66 particles ml-1;). PE particle ingestion resulted in structural changes to the gills and digestive gland, as well as necrosis in other tissues such as the mantle. No differences were found regarding the degree of tissue alteration between PE-virgin and PE-weathered exposures. This current study illustrates the importance of using weathered particles in microplastic exposure studies to reflect the behaviour of plastic particles after entering the marine environment. The observed tissue alterations demonstrate the potential adverse effects to mussels exposed to microplastic particles.

DNA adducts in marine fish as biological marker of genotoxicity in environmental monitoring: The way forward.

DNA adducts in fish represent a very important genotoxicity endpoint in environmental monitoring, being a pre-mutagenic lesion that plays an essential role in the initiation of carcinogenesis. The analysis of DNA adducts is a challenging task due to the low concentration of the analyte. Methods are available to determine the presence of DNA adducts, although further knowledge is required to fully understand the nature of the adducts and responsible xenobiotics (i.e. position of adduct in DNA, most active xenobiotic and metabolite forms, structural information). At present, 32P-postlabeling is the most used method that has the required sensitivity for DNA adduct analyses in both human health and environmental monitoring. Development of new mass spectrometry based methods for identifying DNA adducts in complex matrixes is now considered as a necessary mission in toxicology in order to gain the necessary information regarding adduct formation and facilitate tracking sources of contamination. Mass spectrometry therefore represents the future of DNA adduct detection, bringing along a series of challenges that the scientific community is facing at present.

Individual and molecular level effects of produced water contaminants on nauplii and adult females of Calanus finmarchicus.

In the Barents Sea region new petroleum fields are discovered yearly and extraction of petroleum products is expected to increase in the upcoming years. Despite enhanced technology and stricter governmental legislation, establishment of the petroleum industry in the Barents Sea may potentially introduce a new source of contamination to the area, as some discharges of produced water will be allowed. Whether the presence of produced water poses a risk to the Arctic marine life remains to be investigated. The aim of this study was to examine effects of exposure to several compounds found in produced water-a mixture of selected organic compounds (APW), radium-226 ((226)Ra), barium (Ba), and a scale inhibitor-on the copepod species Calanus finmarchicus. Experiments were performed using exposure concentrations at realistic levels based on those detected in the vicinity of known discharge points. The influence of lethal and sublethal effects on early life stages was determined and significantly lower survival in the APW exposure groups was found. In the Ba treatment the life stage development did not proceed to the same advanced stages as observed in the control (filtered sea water). The scale inhibitor and (226)Ra treatments showed no significant difference from control. In addition, adult females were exposed to APW, (226)Ra, and a mixture of the two. Both individual-level effects (egg production and feeding) and molecular-level effects (gene expression) were assessed. On the individual level endpoints, only treatments including APW produced an effect compared to control. However, on the molecular level the possibility that also (226)Ra induced toxicologically relevant effects cannot be ruled out.

Identification of petrogenic produced water components as acetylcholine esterase inhibitors.

Effect-directed analysis (EDA) was applied to identify acetylcholine esterase (AChE) inhibitors in produced water. Common produced water components from oil production activities, such as polycyclic aromatic hydrocarbons (PAHs), alkylphenols, and naphthenic acids were tested for AChE inhibition using a simple mixture of PAHs and naphthenic acids. Produced water samples collected from two offshore platforms in the Norwegian sector of the North Sea were extracted by solid phase extraction and fractionated by open-column liquid solid chromatography and high-performance liquid chromatography (HPLC) before being tested using a high-throughput and automated AChE assay. The HPLC fractions causing the strongest AChE inhibition were analysed by gas chromatography coupled to a high-resolution time-of-flight mass spectrometry (GC-HR-ToF-MS). Butylated hydroxytoluene and 4-phenyl-1,2-dihydronaphthalene were identified as two produced water components capable of inhibiting AChE at low concentrations. In order to assess the potential presence of such compounds discharged into aquatic ecosystems, AChE activity in fish tissues was measured. Saithe (Pollachius virens) caught near two offshore platforms showed lower enzymatic activity than those collected from a reference location. Target analysis of saithe did not detected the presence of these two putative AChE inhibitors and suggest that additional compounds such as PAHs, naphthenic acids and yet un-identified compounds may also contribute to the purported AChE inhibition observed in saithe.

Marine ecotoxicity of nitramines, transformation products of amine-based carbon capture technology.

In the context of reducing CO2 emissions to the atmosphere, chemical absorption with amines is emerging as the most advanced technology for post-combustion CO2 capture from exhaust gases of fossil fuel power plants. Despite amine solvent recycling during the capture process, degradation products are formed and released into the environment, among them aliphatic nitramines, for which the environmental impact is unknown. In this study, we determined the acute and chronic toxicity of two nitramines identified as important transformation products of amine-based carbon capture, dimethylnitramine and ethanolnitramine, using a multi-trophic suite of bioassays. The results were then used to produce the first environmental risk assessment for the marine ecosystem. In addition, the in vivo genotoxicity of nitramines was studied by adapting the comet assay to cells from experimentally exposed fish. Overall, based on the whole organism bioassays, the toxicity of both nitramines was considered to be low. The most sensitive response to both compounds was found in oysters, and dimethylnitramine was consistently more toxic than ethanolnitramine in all bioassays. The Predicted No Effect Concentrations for dimethylnitramine and ethanolnitramine were 0.08 and 0.18 mg/L, respectively. The genotoxicity assessment revealed contrasting results to the whole organism bioassays, with ethanolnitramine found to be more genotoxic than dimethylnitramine by three orders of magnitude. At the lowest ethanolnitramine concentration (1mg/L), 84% DNA damage was observed, whereas 100mg/L dimethylnitramine was required to cause 37% DNA damage. The mechanisms of genotoxicity were also shown to differ between the two compounds, with oxidation of the DNA bases responsible for over 90% of the genotoxicity of dimethylnitramine, whereas DNA strand breaks and alkali-labile sites were responsible for over 90% of the genotoxicity of ethanolnitramine. Fish exposed to >3mg/L ethanolnitramine had virtually no DNA left in their red blood cells.

Integrated biomarker assessment of the effects of tailing discharges from an iron ore mine using blue mussels (Mytilus spp.).

The blue mussel (Mytilus spp.) has been used to assess the potential biological effects of the discharge effluent from the Sydvaranger mine, which releases its tailings into Bøk fjord at Kirkenes in the north of Norway. Metal bioaccumulation and a suite of biomarkers were measured in mussels positioned for 6 weeks at varying distances from the discharge outlet. The biomarkers used included: stress on stress (SS); condition index (CI); cellular energy allocation (CEA); micronuclei formation (MN); lysosomal membrane stability (LMS), basophilic cell volume (VvBAS); and neutral lipid (NL) accumulation. The individual biomarkers were integrated using the integrated biological response (IBR/n) index. The accumulation of Fe was significantly higher in mussels located closer to the discharge outlet, indicating that these mussels had been exposed to the suspended mine effluent. The IBR/n results were in good agreement with the location of the mussels in relation to the distance from the discharge outlet and expected exposure to the mine effluent. Several biomarkers showed responses resulting in higher IBR/n values of analysed mussels within a 3 km distance from the tailing discharge.

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.

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.

Parasitic infection manipulates sodium regulation in the freshwater amphipod Gammarus pulex (L.).

The acanthocephalan parasite Polymorphus minutus induces both physiological and behavioural effects in its intermediate host, Gammarus pulex. The net effect of parasite infection is to increase the likelihood of transmission to the definitive host. Osmoregulation is an energetically expensive mechanism that allows G. pulex to survive in dilute media. Any factor influencing osmoregulation is thus likely to affect the allocation of resources to other areas. This study investigated whether P. minutus infection alters sodium regulation in G. pulex. Haemolymph sodium concentration, water permeability and sodium fluxes were measured over the salinity acclimation range of G. pulex. Water permeability was unaltered by either acclimation salinity or parasite infection. Acclimation to 12‰ significantly raised the haemolymph sodium concentration, reduced the sodium influx, and increased the sodium efflux, to the same extent in both uninfected and infected G. pulex. However, parasite infection induced a significant increase in haemolymph sodium concentration in G. pulex acclimated to 6‰, which was not observed in uninfected G. pulex acclimated to the same salinity. Also, both sodium influx and sodium efflux were significantly lower in parasitized G. pulex acclimated to 6‰, when compared to uninfected G. pulex acclimated to the same salinity. It was concluded that the parasite induced disturbances to sodium regulation in G. pulex acclimated to 6‰ were a functional consequence of the manipulative strategy employed to alter behaviour, rather than a primary target.

Dissolved organic carbon reduces the toxicity of copper to germlings of the macroalgae, Fucus vesiculosus.

This study investigates the effects of waterborne copper exposure on germling growth in chemically defined seawater. Germlings of the macroalgae, Fucus vesiculosus were exposed to a range of copper and dissolved organic carbon (DOC as humic acid) concentrations over 14 days. Germling growth was found to be a sensitive indicator of copper exposure with total copper (TCu) and labile copper (LCu) EC(50) values of approximately 40 and 20 microg/L, respectively, in the absence of added DOC. The addition of DOC into the exposure media provided germlings with protection against copper toxicity, with an increased TCu EC(50) value of 117.3 microg/L at a corrected DOC (cDOC from humic acid only) concentration of 2.03 mg/L. The LCu EC(50) was not affected by a cDOC concentration of 1.65 mg/L or less, suggesting that the LCu concentration not the TCu concentration was responsible for inhibiting germling growth. However, at a cDOC concentration of approximately 2mg/L an increase in the LCu EC(50) suggests that the LCu concentration may play a role in the overall toxicity to the germlings. This is contrary to current understanding of aquatic copper toxicity and possible explanations for this are discussed.

Effects of dissolved organic carbon on the toxicity of copper to the developing embryos of the Pacific oyster (Crassostrea gigas).

The effects of humic acid (HA) on copper speciation and its subsequent toxicity to the sensitive early life stages of the Pacific oyster (Crassostrea gigas) are presented. Differential pulse anodic stripping voltammetry with a hanging mercury drop electrode was used to measure the copper species as labile copper (LCu; free ion and inorganic copper complexes) and total copper (TCu) with respect to increasing HA concentration. The TCu and LCu 50% effect concentrations (EC50s) in the absence of HA were 20.77 microg/L (95% confidence interval [CI], 24.02-19.97 microg/L) and 8.05 microg/L (95% CI, 9.6-5.92 microg/L) respectively. A corrected dissolved organic carbon (DOC) concentration (HA only) of 1.02 mg/L was required to significantly increase the TCu EC50 to approximately 41.09 microg/L (95% CI, 44.27-37.52 microg/L; p < 0.05), almost doubling that recorded when DOC (as HA) was absent from the test media. In contrast, the LCu EC50 was unaffected by changes in DOC concentration and was stable throughout the corrected DOC concentration range. The absence of change in the LCu EC50, despite increased HA concentration, suggests that the LCu fraction, not TCu, was responsible for the observed toxicity to the oyster embryo. This corresponds with the current understanding of copper toxicity and supports the free-ion activity model for copper toxicity.

Gill Na(+), K(+)-ATPase in a series of hyper-regulating gammarid amphipods: enzyme characterisation and the effects of salinity acclimation.

The enzyme Na(+), K(+)-ATPase was investigated in the gills of selected hyper-regulating gammarid amphipods. Gill Na(+), K(+)-ATPase was characterised with respect to the main cation and co-factor concentrations for the freshwater amphipod Gammarus pulex. The optimum cation and co-factor concentrations for maximal gill Na(+), K(+)-ATPase activity in G. pulex were 100mM Na(+), 15mM K(+), 15mM Mg(2+) and 5mM ATP, at pH 7.2. The effects of salinity acclimation on gill Na(+), K(+)-ATPase activity and haemolymph sodium concentrations was investigated in selected gammarid amphipods from different salinity environments. Maximal enzyme activity occurred in all gammarids when acclimated to the most dilute media. This maximal activity coincided with the largest sodium gradient between the haemolymph and the external media. As the haemolymph/medium sodium gradient decreased, a concomitant reduction in gill Na(+), K(+)-ATPase activity occurred. This implicates the involvement of gill Na(+), K(+)-ATPase in the active uptake of sodium from dilute media in hyper-regulating gammarids.

The effect of copper on osmoregulation in the freshwater amphipod Gammarus pulex.

The influence of copper on osmoregulation in the freshwater amphipod Gammarus pulex was determined from the analysis of water permeability, haemolymph sodium concentration, sodium influx and gill Na(+)/K(+) ATPase and Mg(2+) ATPase activity. Exposure to nominal copper concentrations of 100 microg l(-1) or greater caused a significant reduction in both haemolymph sodium concentration and sodium influx within 4 h. Measurements of water permeability, expressed as the half-time of exchange of body water (t(1/2)), excluded structural gill damage as the cause of this fall in haemolymph sodium. Copper at 10 microg l(-1) or above in the assay solution significantly reduced gill Na(+)/K(+) ATPase activity. In contrast gill Mg(2+) ATPase activity was markedly less affected by copper. These differences in enzyme sensitivity were considered with respect to the potential mechanisms of copper toxicity.