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Metal oxide nanoparticles are finding increasing application in various commercial products, leading to concerns for their environmental fate and potential toxicity. It is generally assumed that nanoparticles will persist as small particles in aquatic systems and that their bioavailability could be significantly greater than that of larger particles. The current study using nanoparticulate ZnO (ca. 30 nm) has shown that this is not always so. Particle characterization using transmission electron microscopy and dynamic light scattering techniques showed that particle aggregation is significant in a freshwater system, resulting in flocs ranging from several hundred nanometers to several microns. Chemical investigations using equilibrium dialysis demonstrated rapid dissolution of ZnO nanoparticles in a freshwater medium (pH 7.6), with a saturation solubility in the milligram per liter range, similar to that of bulk ZnO. Toxicity experiments using the freshwater alga Pseudokirchneriella subcapitata revealed comparable toxicity for nanoparticulate ZnO, bulk ZnO, and ZnCl2, with a 72-h IC50 value near 60 microg Zn/ L, attributable solely to dissolved zinc. Care therefore needs to be taken in toxicity testing in ascribing toxicity to nanoparticles per se when the effects may be related, at least in part, to simple solubility.

The physiological responses of juvenile rainbow trout (Oncorhynchus mykiss) to lithium (as LiCl) in moderately hard freshwater (CaCO(3) = 120-140 ppm, Na(+) = approximately 0.6 mM) were studied. The study employed a 15-day step-up exposure regime; 66 microg/L Li for the first 9 days and 528 microg/L for the next 6 days. The concentrations of plasma ions, apolipoprotein AI, total cholesterol, and fatty acids, as well as metabolic enzyme citrate synthase (CS) and Na(+),K(+)-ATPase activities in the gill were measured. Li affected fish by exacerbated diffusive Na(+) losses at the gills in the beginning of exposure and a decrease of branchial CS activity. Detrimental effects were shown in fish exposed to 528 microg Li/L. These included a reduction of gill Na(+),K(+)-ATPase activity, possibly related to observed lower concentrations of free fatty acids and cholesterol in gill tissue.

Recent studies have shown that dietary Ca(2+) supplementation strongly inhibits uptake of Ca(2+) and Cd at the fish gill. To better understand the influence of dietary Ca(2+) on branchial Ca(2+) transport, we examined the expression of two trout gill calcium transporters during waterborne and dietary Cd exposure, at two different levels of dietary Ca(2+). Quantitative polymerase chain reaction (PCR) was used to monitor epithelial calcium channel (ECaC) and sodium-calcium exchange (NCX) mRNA levels following 7-28 days of exposure to these treatments. In brief, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to control, 3 microg/L waterborne Cd, 500 mg/kg dietary Cd, or a combined 3 microg/L waterborne plus 500 mg/kg dietary Cd exposure, supplemented with either 20 mg/g or 60 mg/g dietary calcium (Ca(2+)). Two-way analysis of variance was used to discern the main effects of Cd exposure and dietary Ca(2+) supplementation on ECaC and NCX mRNA levels. We found that dietary Ca(2+) supplementation decreased significantly ECaC mRNA expression on days 14 and 21. In comparison, NCX mRNA levels were not influenced by dietary Ca(2+) supplementation, but rather were significantly inhibited in the combined waterborne and dietary Cd exposure on day 7 alone. Statistical analysis found no interactive effects between Cd exposure and dietary Ca(2+) exposure at any time point, except for day 28. This study provides evidence of the importance of nutritional status on the transcriptional regulation of ion transport at the fish gill. We discuss the importance of diet and nutritional status to the development of new regulatory approaches, such as the biotic ligand model, which currently do not account for the significance of diet on metal bioavailability in aquatic organisms.

Previous research showed that the spiny dogfish, Squalus acanthias, is much more sensitive to silver exposure than typical marine teleosts. The aim of the present study was to investigate if spiny dogfish were equally sensitive to copper exposure and whether the toxic mechanisms were the same. We exposed cannulated and non-cannulated spiny dogfish to measured concentrations of Cu (nominally 0, 500, 1000 and 1500 microg L(-1) Cu) for 72-96 h. All Cu exposures induced acidosis and lactate accumulation of either a temporary (500 microg L(-1)) or more persistent nature (1000 and 1500 microg L(-1)). At the two highest Cu concentrations, gill Na(+)/K(+)-ATPase activities were reduced by 45% (1000 microg L(-1)) and 62% (1500 microg L(-1)), and plasma Na(+) and Cl(-) concentrations increased by approximately 50 mM each. At the same time urea excretion doubled and plasma urea dropped by approximately 100 mM. Together with plasma urea, plasma TMAO levels dropped proportionally, indicating that the general impermeability of the gills was compromised. Overall plasma osmolarity did not change. Cu accumulation was limited with significant increases in plasma Cu and elevated gill and kidney Cu burdens at 1000 and 1500 microg L(-1). We conclude that Cu, like Ag, exerts toxic effect on Na(+)/K(+)-ATPase activities in the shark similar to those of teleosts, but there is an additional toxic action on elasmobranch urea retention capacities. With a 96 h LC(50) in the 800-1000 microg L(-1) range, overall sensitivity of spiny dogfish for Cu is, in contrast with its sensitivity to Ag, only slightly lower than in typical marine teleosts.

Copper and zinc toxicity to the freshwater alga Chlorella sp. was determined at a range of pH values (5.5-8.0) in a synthetic softwater (hardness 40-48 mg CaCO(3)/L). The effects of the metals on algal growth (cell division) rate were determined after 48-h exposure at pH 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. The toxicity of both metals was pH dependent. As pH decreased from 8.0 to 5.5, the copper concentration required to inhibit the algal growth rate by 50% (IC50) increased from 1.0 to 19 microg/L. For zinc, the IC50 increased from 52 to 2,700 microg/L over the same pH range. Changes in solution speciation alone did not explain the increased toxicity observed as the pH increased. Modelled Cu(2+) and Zn(2+) concentrations decreased with increasing pH, whereas toxicity was observed to increase. Measurements of extracellular (cell-bound) metal concentrations support the biotic ligand model (BLM) theory of competition between protons (H(+)) and metals for binding sites at the algal cell surface. Higher extracellular metal concentrations were observed at high pH, indicating reduced competition. Independent of pH, both extracellular and intracellular copper were directly related to growth inhibition in Chlorella sp., whereas zinc toxicity was related to cell-bound zinc only. These findings suggest that the algal cell surface may be considered as the biotic ligand in further development of a chronic BLM with microalgae. Conditional binding constants (log K) were determined experimentally (using measured intracellular metal concentrations) and theoretically (using concentration-response curves) for copper and zinc for Chlorella sp. at selected pH values. Excellent agreement was found indicating the possibility of using concentration-response data to estimate conditional metal-cell binding constants.

Juvenile rainbow trout (Oncorhynchus mykiss) were exposed to control, 3 microg/L waterborne Cd, or 500 mg/kg dietary Cd in combination with either a control (20 mg/g Ca2+ as CaCO3) or elevated (60 mg/g Ca2+) Ca2+ diet for 28 d. No mortality or growth effects were observed in response to either route of Cd exposure, although fish fed Ca2+-supplemented diets exhibited minor reductions in growth within the first few days of feeding. Waterborne and dietary Cd resulted in significant Cd accumulation in most tissues, with dietary uptake being far in excess of waterborne under the exposure conditions used. The order of Cd accumulation strongly reflected the exposure pathway, being gill and kidney > liver > gut > carcass (waterborne Cd); gut > kidney > liver > gill > carcass > bone (dietary Cd). On a whole-body basis, the net retention of Cd from the diet was < 1%, indicating that the gut wall forms an important protective barrier reducing Cd accumulation into internal tissues. Dietary Ca2+ supplementation reduced short-term whole-body uptake rates of waterborne Ca2+ and Cd by >50% and resulted in much lower chronic accumulation of Cd (via the water and diet) in target tissues. Results suggest that Ca2+ and Cd share common pathway(s)/transport mechanism(s) in the gill and gut and that increased gastrointestinal Ca2+ uptake likely caused downregulation of branchial and gastrointestinal Ca2+ and therefore Cd uptake pathways. Because nutrient metals other than Ca2+ may also influence Cd (and other metal) uptake, new regulatory approaches to metal toxicity (e.g., biotic ligand model) require understanding of the influence of dietary status on metal accumulation.

The freshwater green microalgae Chlorella sp. and Pseudokirchneriella subcapitata (P. subcapitata) were chronically (48 and 72 h, respectively) exposed to copper at various pH levels, i.e., pH 6-7.5 and pH 5.9-8.5, respectively. Concentrations resulting in 50% inhibition of exponential growth rate (EC50) were determined as dissolved Cu, estimated chemical activity of the free Cu2+ ion (as pCu = - log{Cu2+ activity as molarity}), and as external (surface-bound) Cu and internal Cu in the algal cells. With increasing pH, EC50dissolved decreased from 30 to 1.1 microg of Cu L(-1) for Chlorella sp. and from 46 to 18 microg of Cu L(-1) for P. subcapitata. The pH effect on copper toxicity was even more obvious when expressed as Cu2+ activity. The EC50pCu increased on average 1.4 pCu unit per pH unit for Chlorella sp. and 1.1 pCu unit per pH unit for P. subcapitata, thus indicating a marked increase of Cu2+ toxicity at higher pH (more than 1 order of magnitude per pH unit). In contrast, it was found that EC50 values expressed as surface bound or external copper (EC50external) and as internal copper (EC50internal) did not vary substantially when pH was increased. External Cu was operationally defined as the Cu fraction removable from the algal cell by short-term contact with ethylenediaminetetraacetic acid; internal copper was defined as the nonremovable fraction. For Chlorella sp. the EC50external varied between 5 and 10 fg of Cu/ cell (factor of 2 difference) and the EC50internal between 25 and 40 fg of Cu/cell (factor of 1.6 difference). For P. subcapitata the EC50external varied between 10 and 28 fg of Cu/cell (factor of 2.8 difference) and the EC50internal between 42 and 71 fg of Cu/cell (factor of 1.7 difference). Because the observed variation in EC50external and EC50internal is much less than the variation in EC50Cu2+, it is concluded that both external and internal copper are better predictors of copper toxicity than Cu2+ when pH is varied. From the perspective of toxicity modeling, this observation is the first step toward considering the use of the cell surface as the algal biotic ligand for Cu in a similar way as fish gills fulfill this role in the biotic ligand model for predicting metal toxicity to fish species.

Multispecies algal bioassays, suitable for assessing copper toxicity, were developed with three marine (Micromonas pusilla, Phaeodactylum tricornutum, and Heterocapsa niei) and three freshwater (Microcystis aeruginosa, Pseudokirchneriella subcapitata, and Trachelomonas sp.) microalgae. Flow cytometry was used to separate and count algal signals based on pigment fluorescence and cell size. Species were mixed together on the basis of equivalent surface areas to avoid the confounding effect on toxicity of increased biomass for metal binding. Under control conditions (no added copper), M. pusilla growth was inhibited in the presence of the other marine microalgae compared to single-species tests, while the opposite was true (i.e., growth stimulation) for M. aeruginosa and P. subcapitata in freshwater mixtures. Competition for nutrients, including CO2, and algal exudate production may account for these effects. Interactions between microalgal species also had a significant effect on copper toxicity to some species. In freshwater multispecies bioassays, the toxicity of copper to Trachelomonas sp. was greater in the presence of other species, with copper concentrations required to inhibit growth (cell division) rate by 50% (72-h [IC50]) decreasing from 9.8 to 2.8 microg Cu/L in single- and multispecies bioassays, respectively. In contrast, in marine multispecies bioassays, copper toxicity to the marine diatom P. tricornutum was reduced compared to single-species bioassays, with an increase in the 72-h IC50 value from 13 to 24 microg Cu/L. This reduction in copper toxicity was not explained by differences in the copper complexing capacity in solution (as a result of exudate production) because labile copper, measured by anodic stripping voltammetry, was similar for P. tricornutum alone and in the mixture. These results demonstrate that single-species bioassays may over- or underestimate metal toxicity in natural waters.

The individual and combined effects of copper, cadmium, and zinc on the cell division rate of the tropical freshwater alga Chlorella sp. were determined over 48 to 72 h. Metal mixtures were prepared based on multiples of their single-metal median effective concentration (EC50) values, i.e., toxic units (TU) using a triangular mixture design with five toxicant levels (0, 0.75, 1.0, 1.25, and 1.5 TU). Single-metal EC50 values after a 72-h exposure were 0.11, 0.85, and 1.4 microM for copper, cadmium, and zinc, respectively. Significant interactions were observed for all metal combinations after 48 and 72 h. An equitoxic mixture of Cu + Cd was more than concentration additive (synergistic) to the growth of Chlorella sp., while combinations of Cu + Zn, Cd + Zn, and Cu + Cd + Zn were all less than concentration additive or were antagonistic. To determine the effect of each metal on the uptake of the other, extracellular (membrane-bound) and intracellular metal concentrations, both alone and in mixtures, were compared. The increased growth inhibition observed for mixtures of Cu + Cd was due to higher concentrations of cell-bound and intracellular copper in the presence of cadmium compared with copper alone (i.e., cadmium-enhanced copper uptake). In contrast, both extra- and intracellular cadmium concentrations were reduced in the presence of copper. In mixtures of Cu + Zn, copper also inhibited the binding and cellular uptake of zinc, which resulted in decreased toxicity. Zinc had no appreciable effect on the uptake of copper by Chlorella sp. Our results suggest that all three metals share some common uptake and transport sites on Chlorella cells and that copper out competes both cadmium and zinc for cell binding. Determination of metal cell distribution coefficients (K(d)) confirmed that K(d) values for cadmium and zinc in single-metal exposures decreased in the presence of copper.

Flow cytometry is a rapid method for the quantitative measurement of light scattering and fluorescent properties of cells. Although this technique has been widely applied to biomedical and environmental studies, its potential as a tool in ecotoxicological studies has not yet been fully exploited. This article describes the application of flow cytometry to the development of bioassays with marine and freshwater algae for assessing the bioavailability of contaminants in waters and sediments.

Algal toxicity tests based on growth inhibition over 72 h have been extensively used to assess the toxicity of contaminants in natural waters. However, these laboratory tests use high cell densities compared to those found in aquatic systems in order to obtain a measurable algal response. The high cell densities and test duration can result in changes in chemical speciation, bioavailability, and toxicity of contaminants throughout the test. With the recent application of flow cytometry to ecotoxicology, it is now possible to use lower initial cell densities to minimize chemical speciation changes. The speciation and toxicity of copper in static bioassays with the tropical freshwater alga Chlorella sp. and the temperate species Selenastrum capricornutum (Pseudokirchneriella subcapitata) were investigated at a range of initial cell densities (10(2)-10(5) cells/ml). Copper toxicity decreased with increasing initial cell density. Copper concentrations required to inhibit growth (cell division) rate by 50% (72-h median effective concentration [EC50]) increased from 4.6 to 16 microg/L for Chlorella sp. and from 6.6 to 17 microg/L for S. capricornutum as the initial cell density increased from 10(2) to 10(5) cells/ml. Measurements of anodic stripping voltammetry-labile, extracellular, and intracellular copper confirmed that at higher initial cell densities, less copper was bound to the cells, resulting in less copper uptake and lower toxicity. Chemical measurements indicated that reduced copper toxicity was due primarily to depletion of dissolved copper in solution, with solution speciation changes due to algal exudates and pH playing a minor role. These findings suggest that standard static laboratory bioassays using 10(4) to 10(5) algal cells/ml may seriously underestimate metal toxicity in natural waters.

A rapid toxicity test based on inhibition of esterase activity in marine and freshwater microalgae (Selenastrum capricornutum, Chlorella sp., Dunaliella tertiolecta, Phaeodactylum tricornutum, Tetraselmis sp., Entomoneis cf. punctulata, Nitzschia cf. paleacea) was developed using flow cytometry. Uptake of fluorescein diacetate (FDA) was optimized for each species by varying the substrate concentration, incubation time, and media pH. Propidium iodide (PI) was utilized to assess membrane integrity. The optimized FDA/PI staining procedure was then used to assess the toxicity of copper in short-term exposures (1-24 h). Esterase activity was a sensitive indicator of copper toxicity in S. capricornutum and E. cf. punctulata. As copper concentrations increased, esterase activity decreased in a concentration-dependent manner. The 3- and 24-h EC50 values (based on mean activity states) were 112 microg Cu L(-1) (95% confidence limits 88-143) and 51 microg Cu L(-1) (95% confidence limits 38-70) for S. capricornutum and 47 microg Cu L(-1) (95% confidence limits 43-51) and 9.1 microg Cu L(-1) (95% confidence limits 7.6-11) for E. cf. punctulata, respectively. This enzyme inhibition endpoint showed similar sensitivity to chronic growth rate inhibition in E. cf. punctulata (48-h and 72-h EC50 values of 17 and 18 microg L(-1), respectively) but was less sensitive compared to growth for S. capricornutum (48-h and 72-h EC50 values of 4.9 and 7.5 microg L(-1), respectively). For the other five species tested, inhibition of FDA fluorescence was relatively insensitive to copper, even at copper concentrations that severely inhibited cell division rate. These short-term bioassays that detect sublethal endpoints may provide a more rapid and cost-effective way of monitoring contaminant impacts in natural waters.

Copper toxicity to the freshwater algae Selenastrum capricornutum and Chlorella sp. and the marine algae Phaeodactylum tricornutum and Dunaliella tertiolecta was investigated using different parameters measured by flow cytometry: cell division rate inhibition, chlorophyll a fluorescence, cell size (i.e., light-scattering), and enzyme activity. These parameters were assessed regarding their usefulness as alternative endpoints for acute (1-24 h) and chronic (48-72 h) toxicity tests. At copper concentrations of 10 micrograms/L or less, significant inhibition (50%) of the cell division rate was observed after 48- and 72-h exposures for Chlorella sp., S. capricornutum, and P. tricornutum. Bioassays based on increases in algal cell size were also sensitive for Chlorella sp. and P. tricornutum. Copper caused both chlorophyll a fluorescence stimulation (48-h EC50 of 10 +/- 1 micrograms Cu/L for P. tricornutum) and inhibition (48-h EC50 of 14 +/- 6 micrograms Cu/L for S. capricornutum). For acute toxicity over short exposure periods, esterase activity in S. capricornutum using fluorescein diacetate offered a rapid alternative (3-h EC50 of 90 +/- 40 micrograms Cu/L) to growth inhibition tests for monitoring copper toxicity in mine-impacted waters. For all the effect parameters measured, D. tertiolecta was tolerant to copper at concentrations up to its solubility limit in seawater. These results demonstrate that flow cytometry is a useful technique for toxicity testing with microalgae and provide additional information regarding the general mode of action of copper (II) to algal species.

Copper (Cu) and uranium (U) are of potential ecotoxicological concern to tropical freshwater organisms in northern Australia as a result of mining activity. No local data on the toxicity of these metals to tropical freshwater algae are currently available. The aim of this study was to investigate the effect of pH (5.7 and 6.5) on the toxicity of Cu and U to the green alga Chlorella sp. in a synthetic softwater representative of fresh surface waters in sandy-streams of tropical northern Australia. The effects of Cu and U on algal growth (cell division) rate after a 72-h exposure were determined. Intracellular and extracellular (membrane-bound) metal concentrations at the two selected pH values were also compared. Based on the 72-h minimum detectable effect concentrations (MDEC), Chlorella sp. was approximately 20-fold more sensitive to Cu (0.7 and 1.4 µg l(-1) at pH 6.5 and 5.7, respectively) than U (13 and 34 µg l(-1) at pH 6.5 and 5.7, respectively), and more sensitive than other Australian tropical freshwater organisms. The toxicity of Cu and U was highly pH-dependent. Copper concentrations required to inhibit growth (cell division) rate by 50% (72-h EC(50)) increased from 1.5 to 35 µg l(-1) as the pH decreased from 6.5 to 5.7. Similarly, the 72-h EC(50) values for U increased from 44 to 78 µg l(-1) over the same pH range. Calculation of Cu and U speciation using the geochemical model HARPHRQ, showed that differences in the concentrations of the free metal ions (Cu(2+) and UO(2)(2+)) were only minimal (<10%) between pH 5.7 and 6.5. The decreased toxicity at pH 5.7 was due to lower concentrations of cell-bound and intracellular Cu and U compared to those at pH 6.5. These results are explained in terms of the possible mechanism of competition between H(+) and the metal ion at the cell surface.

An insertional mutation was made in the nifX gene of Klebsiella pneumoniae. This mutation had little effect on the nitrogenase activity of the strain, as measured by acetylene reduction. However, on the addition of NH4+ or O2 (conditions which block nif protein synthesis by transcriptional and posttranscriptional mechanisms), the NifX- mutant synthesized nitrogenase proteins longer and had more accumulated nifHDKTY mRNA than did the wild-type K. pneumoniae at all time points tested. Conversely, overexpression of the wild-type nifX region blocked nif protein synthesis, protein accumulation, and nifHDKTY mRNA accumulation. These complementary results indicate that a product of the nifX region has a role in the negative regulation of nif regulation in response to NH4+ and O2.