Interaction of silver nanoparticles with algae and fish cells: a side by side comparison.

BACKGROUND: Silver nanoparticles (AgNP) are widely applied and can, upon use, be released into the aquatic environment. This raises concerns about potential impacts of AgNP on aquatic organisms. We here present a side by side comparison of the interaction of AgNP with two contrasting cell types: algal cells, using the algae Euglena gracilis as model, and fish cells, a cell line originating from rainbow trout (Oncorhynchus mykiss) gill (RTgill-W1). The comparison is based on the AgNP behavior in exposure media, toxicity, uptake and interaction with proteins. RESULTS: (1) The composition of exposure media affected AgNP behavior and toxicity to algae and fish cells. (2) The toxicity of AgNP to algae was mediated by dissolved silver while nanoparticle specific effects in addition to dissolved silver contributed to the toxicity of AgNP to fish cells. (3) AgNP did not enter into algal cells; they only adsorbed onto the cell surface. In contrast, AgNP were taken up by fish cells via endocytic pathways. (4) AgNP can bind to both extracellular and intracellular proteins and inhibit enzyme activity. CONCLUSION: Our results showed that fish cells take up AgNP in contrast to algal cells, where AgNP sorbed onto the cell surface, which indicates that the cell wall of algae is a barrier to particle uptake. This particle behaviour results in different responses to AgNP exposure in algae and fish cells. Yet, proteins from both cell types can be affected by AgNP exposure: for algae, extracellular proteins secreted from cells for, e.g., nutrient acquisition. For fish cells, intracellular and/or membrane-bound proteins, such as the Na+/K+-ATPase, are susceptible to AgNP binding and functional impairment.

Linking toxicity and adaptive responses across the transcriptome, proteome, and phenotype of Chlamydomonas reinhardtii exposed to silver.

Understanding mechanistic and cellular events underlying a toxicological outcome allows the prediction of impact of environmental stressors to organisms living in different habitats. A systems-based approach aids in characterizing molecular events, and thereby the cellular pathways that have been perturbed. However, mapping only adverse outcomes of a toxicant falls short of describing the stress or adaptive response that is mounted to maintain homeostasis on perturbations and may confer resistance to the toxic insult. Silver is a potential threat to aquatic organisms because of the increasing use of silver-based nanomaterials, which release free silver ions. The effects of silver were investigated at the transcriptome, proteome, and cellular levels of Chlamydomonas reinhardtii. The cells instigate a fast transcriptome and proteome response, including perturbations in copper transport system and detoxification mechanisms. Silver causes an initial toxic insult, which leads to a plummeting of ATP and photosynthesis and damage because of oxidative stress. In response, the cells mount a defense response to combat oxidative stress and to eliminate silver via efflux transporters. From the analysis of the perturbations of the cell's functions, we derived a detailed mechanistic understanding of temporal dynamics of toxicity and adaptive response pathways for C. reinhardtii exposed to silver.

Effects of Differently Coated Silver Nanoparticles on the Photosynthesis of Chlamydomonas reinhardtii.

Various factors have been invoked to explain the toxicity of silver nanoparticles (AgNP) to microorganisms including particle size and the nature of stabilizing coatings as well as the amount of dissolved silver occurring in AgNP suspensions. In this study we have assessed the effects of nine differently coated AgNP (chitosan, lactate, polyvinylpyrrolidone, polyethelene glycol, gelatin, sodium dodecylbenzenesulfonate, citrate, dexpanthenol, and carbonate) and AgNO3 on the photosynthesis of the freshwater algae Chlamydomonas reinhardtii. We have thus examined how AgNP effects on algae relate to particle size, measured dissolved silver (Agd), and bioavailable silver (Agbioav). Agbioav was indirectly estimated in toxicity experiments by cysteine-silver complexation at the EC50. The EC50 calculated as a function of measured Agd concentrations showed for some coatings values similar to that of dissolved Ag, whereas other coated AgNP displayed lower EC50 values. In all cases, excess cysteine completely prevented effects on photosynthetic yield, confirming the role of Agd as a cause of the observed effect on the photosynthesis. Toxicity was related neither to particle size nor to the coatings. For all differently coated AgNP suspensions, the EC50 values calculated as a function of Agbioav were comparable to the value of AgNO3. Depending on the coatings Agbioav was comparable to or higher than measured Agd.

Impact of chronic lead exposure on metal distribution and biological effects to periphyton.

Chronic Pb exposure microcosm studies were carried out on two different periphyton communities over the course of 3 weeks to link Pb distribution to biological effects in periphyton. We show that three-week exposures of periphyton to 20.6 ± 0.4 µM PbT (330 nM Pb(2+)) did not have observable biological effects on photosynthesis, respiration, extracellular enzymatic activities, or biomass accrual. Metal distribution studies showed that the majority of Pb was associated with the operationally defined sorbed and non-EDTA-exchangeable fractions, and relatively little with extracellular polymeric substances (EPS). No significant effects of Pb on Fe and Mn distributions were observed, whereas higher Cu accumulation occurred from increased free Cu(2+) in the exposure medium. High Fe:C and Mn:C ratios indicated the presence of inorganic Fe and Mn material associated with the non-EDTA-exchangeable fraction, which likely sequesters Pb and explains the absence of measurable biological effects. Although no toxic effects of Pb were observed on the periphytic organisms themselves, periphyton can be a significant source of Pb to grazing organisms in aquatic ecosystems.

Effect of TiO2 nanoparticles and UV radiation on extracellular enzyme activity of intact heterotrophic biofilms.

When introduced into the aquatic environment, TiO2 NP are likely to settle from the water column, which results in increased exposure of benthic communities. Here, we show that the activity of two extracellular enzymes of intact heterotrophic biofilms, ß-glucosidase (carbon-cycling) and l-leucin aminopeptidase (nitrogen-cycling), was reduced following exposure to surface functionalized TiO2 NP and UV radiation, depending on the particles' coating. This reduction was partially linked to ROS production. Alkaline phosphatase (phosphorus-cycling) activity was not affected, however in contrast, an alkaline phosphatase isolated from E. coli was strongly inhibited at lower concentrations of TiO2 NP than the intact biofilms. These results indicate that enzymes present in the biofilm matrix are partly protected against exposure to TiO2 NP and UV radiation. Impairment of extracellular enzymes which mediate the uptake of nutrients from water may affect ecosystem function.

Chemical Aspects of Nanoparticle Ecotoxicology.

Nanoecotoxicology strives to understand the processes and mechanisms by which engineered nanoparticles (ENP) may exert toxic effects on aquatic organisms. Detailed knowledge of the chemical reactions of nanoparticles in the media and of their interactions with organisms is required to understand these effects. The processes of agglomeration of nanoparticles, of dissolution and release of toxic metal ions, and of production of reactive oxygen species (ROS) are considered in this article. Important questions concern the role of uptake of nanoparticles in various organisms, in contrast to uptake of ions released from nanoparticles and to nanoparticle attachment to organism surfaces. These interactions are illustrated for effects of silver nanoparticles (AgNP), cerium oxide (CeO2 NP) and titanium dioxide (TiO2 NP), on aquatic organisms, including algae, biofilms, fish cells and fish embryos.

Diuron sorbed to carbon nanotubes exhibits enhanced toxicity to Chlorella vulgaris.

Carbon nanotubes (CNT) are more and more likely to be present in the environment, where they will associate with organic micropollutants due to strong sorption. The toxic effects of these CNT-micropollutant mixtures on aquatic organisms are poorly characterized. Here, we systematically quantified the effects of the herbicide diuron on the photosynthetic activity of the green alga Chlorella vulgaris in presence of different multiwalled CNT (industrial, purified, pristine, and oxidized) or soot. The presence of carbonaceous nanoparticles reduced the adverse effect of diuron maximally by <78% (industrial CNT) and <34% (soot) at 10.0 mg CNT/L, 5.0 mg soot/L, and diuron concentrations in the range 0.73-2990 µg/L. However, taking into account the measured dissolved instead of the nominal diuron concentration, the toxic effect of diuron was equal to or stronger in the presence of CNT by a factor of up to 5. Sorbed diuron consequently remained partially bioavailable. The most pronounced increase in toxicity occurred after a 24 h exposure of algae and CNT. All results point to locally elevated exposure concentration (LEEC) in the proximity of algal cells associated with CNT as the cause for the increase in diuron toxicity.

Colloidal stability of carbonate-coated silver nanoparticles in synthetic and natural freshwater.

To gain important information on fate, mobility, and bioavailability of silver nanoparticles (AgNP) in aquatic systems, the influence of pH, ionic strength, and humic substances on the stability of carbonate-coated AgNP (average diameter 29 nm) was systematically investigated in 10 mM carbonate and 10 mM MOPS buffer, and in filtered natural freshwater. Changes in the physicochemical properties of AgNP were measured using nanoparticle tracking analysis, dynamic light scattering, and ultraviolet-visible spectroscopy. According to the pH-dependent carbonate speciation, below pH 4 the negatively charged surface of AgNP became positive and increased agglomeration was observed. Electrolyte concentrations above 2 mM Ca(2+) and 100 mM Na(+) enhanced AgNP agglomeration in the synthetic media. In the considered concentration range of humic substances, no relevant changes in the AgNP agglomeration state were measured. Agglomeration of AgNP exposed in filtered natural freshwater was observed to be primarily controlled by the electrolyte type and concentration. Moreover, agglomerated AgNP were still detected after 7 days of exposure. Consequently, slow sedimentation and high mobility of agglomerated AgNP could be expected under the considered natural conditions. A critical evaluation of the different methods used is presented as well.

Intracellular silver accumulation in Chlamydomonas reinhardtii upon exposure to carbonate coated silver nanoparticles and silver nitrate.

The intracellular silver accumulation ({Ag}(in)) upon exposure to carbonate coated silver nanoparticles (AgNP, 0.5-10 µM, average diameter 29 nm) and silver nitrate (20-500 nM) was examined in the wild type and in the cell wall free mutant of the green alga Chlamydomonas reinhardtii at pH 7.5. The {Ag}(in) was measured over time up to 1 h after a wash procedure to remove silver ions (Ag(+)) and AgNP from the algal cell surface. The {Ag}(in) increased with increasing exposure time and with increasing AgNP and AgNO(3) concentrations in the exposure media, reaching steady-state concentrations between 10(-5) and 10(-3) mol L(cell)(-1). According to estimated kinetic parameters, high Ag(+) bioconcentration factors were calculated (>10(3) L L(cell)(-1)). Higher accumulation rate constants were assessed in the cell wall free mutant, indicating a protective role of the cell wall in limiting Ag(+) uptake. The bioavailability of AgNP was calculated to be low in both strains relative to Ag(+), suggesting that AgNP internalization across the cell membrane was limited.

Are carbon nanotube effects on green algae caused by shading and agglomeration?

Due to growing production, carbon nanotubes (CNT) may soon be found in a broad range of products and thus in the environment. In this work, an algal growth test was developed to determine effects of pristine and oxidized CNT on the green algae Chlorella vulgaris and Pseudokirchneriella subcapitata. CNT suspensions were prepared in algal test medium and characterized taking into account the suspension age, the reduced light transmittance of nanoparticle suspensions defined as shading of CNT and quantified by UV/vis spectroscopy, and the agglomeration of the CNT and of the algal cells. Growth inhibition and photosynthetic activity were investigated as end points. Growth of C. vulgaris was inhibited with effect concentrations of 50% (EC(50)) values of 1.8 mg CNT/L and of 24 mg CNT/L in well dispersed and in agglomerated suspensions, respectively, and 20 mg CNT/L and 36 mg CNT/L for P. subcapitata, respectively. However, the photosynthetic activity was not affected. Growth inhibition was highly correlated with the shading of CNT and the agglomeration of algal cells. This suggests that the reduced algal growth might be caused mainly by indirect effects, i.e. by reduced availability of light and different growth conditions caused by the locally elevated algal concentration inside of CNT agglomerates.

Cytotoxicity, Accumulation and Translocation of Silver and Silver Sulfide Nanoparticles in contact with Rainbow Trout Intestinal Cells.

Silver nanoparticles (Ag NPs) are widely used in consumer products especially because of their antimicrobial properties. However, this wide usage of Ag NPs is accompanied by their release into the environment where they will be rapidly transformed to other silver species - especially silver sulfide (Ag2S). In the present study, we synthesized Ag NPs and sulfidized them to obtain a core-shell system Ag@Ag2S NPs. Both types of particles form stable dispersions with hydrodynamic diameters of less than 100 nm when diluted in water, but tend to form micrometer-sized agglomerates in biological exposure media. Application of Ag and Ag@Ag2S NPs to rainbow trout intestinal cells (RTgutGC) resulted in a concentration-dependent cytotoxicity for both types of particles, as assessed by a three-endpoint assay for metabolic activity, membrane integrity and lysosomal integrity. The Ag NPs were shown to be slightly more toxic than the Ag@Ag2S NPs. Adding Ag or Ag@Ag2S NPs to RTgutGC cells, grown on a permeable membrane to mimic the intestinal barrier, revealed considerable accumulation of silver for both types of particles. Indeed, the cells significantly attenuated the NP translocation, allowing only a fraction of the metal to translocate across the intestinal epithelium. These findings support the notion that the intestine constitutes an important sink for Ag NPs and that, despite the reduced cytotoxicity of a sulfidized NP form, the particles can enter fish where they may constitute a long-term source for silver ion release and cytotoxicity.

Kinetics of cadmium accumulation in periphyton under freshwater conditions.

The aim of the present study was to investigate the kinetics of cadmium (Cd) accumulation (total and intracellular) in periphyton under freshwater conditions in a short-term microcosm experiment. Periphyton was precolonized in artificial flow-through channels supplied with natural freshwater and then exposed for 26.4 h to nominal Cd concentrations of 5 and 20 nM added to natural freshwater. Labile Cd in water determined with diffusion gradient in thin films was 60 to 69% of total dissolved Cd in the exposure channels and 11% in the control channel. Intracellular Cd concentrations in periphyton increased rapidly and linearly during the first 71 min. Initial intracellular uptake rates were 0.05 and 0.18 nmol of Cd/g of dry weight x min in the 5 nM and 20 nM exposures, respectively. The subsequent intracellular uptake was slower, approaching steady state at the end of Cd exposure. Uptake kinetics of Cd was slower when compared to experiments with planktonic algal cultures, probably due to diffusion limitations. Intracellular Cd uptake during the entire exposure was modeled with a nonlinear, one-compartment model from which uptake and clearance rate constants, as well as bioconcentration factors, were obtained. The release of Cd from periphyton after the end of Cd exposure was slow when compared to the initial uptake rates.

Aerobic methane oxidation under copper scarcity in a stratified lake.

Aerobic methane-oxidizing bacteria (MOB) substantially reduce methane fluxes from freshwater sediments to the atmosphere. Their metalloenzyme methane monooxygenase (MMO) catalyses the first oxidation step converting methane to methanol. Its most prevalent form is the copper-dependent particulate pMMO, however, some MOB are also able to express the iron-containing, soluble sMMO under conditions of copper scarcity. So far, the link between copper availability in different forms and biological methane consumption in freshwater systems is poorly understood. Here, we present high-resolution profiles of MOB abundance and pMMO and sMMO functional genes in relation to copper, methane and oxygen profiles across the oxic-anoxic boundary of a stratified lake. We show that even at low nanomolar copper concentrations, MOB species containing the gene for pMMO expression are present at high abundance. The findings highlight the importance of copper as a micronutrient for MOB species and the potential usage of copper acquisition strategies, even under conditions of abundant iron, and shed light on the spatial distribution of these microorganisms.

Uptake and effects of cerium(III) and cerium oxide nanoparticles to Chlamydomonas reinhardtii.

Cerium (Ce) and cerium oxide nanoparticles (CeO2 NP) are increasingly used in different applications. Upon their release into the aquatic environment, the exposure of aquatic organisms becomes likely. In this study, the uptake of CeO2 NP and Ce3+ into the wild type and cell wall free mutant of Chlamydomonas reinhardtii was examined upon short term exposure. Separation of CeO2 NP and Ce3+ not taken up or loosely bound to the cells was performed by washing algae with EDTA. Despite a concentration and time dependent increase of cellular Ce upon exposure to CeO2 NP with the maximal calculated Ce concentration corresponding to 1.1 CeO2 NP per cell, an internalization of CeO2 NP with a mean size of 140 nm in C. reinhardtii was excluded. In contrast, dissolved Ce3+ (1 and 10 µM) was taken up both in the wild type and cell wall free mutant of C. reinhardtii, with a linear increase of cellular Ce within 1-2 h and maximal cellular Ce of 6.04 × 10-4 mol Lcell-1 (wild type) and 9.0 × 10-5 mol Lcell-1 (cell wall free mutant). Based on competition with Ca2+ for Ce3+ uptake, on the comparison of the wild type and the cell wall free mutant and on inhibition of photosynthetic yield, we suggest that no efficient uptake routes for Ce3+ are available in C. reinhardtii and that a fraction of the cellular Ce in the wild type strongly sorbs to the algal cell wall.

Competition among zinc, manganese, and cadmium uptake in the freshwater alga Scenedesmus vacuolatus.

In the present study, Zn and Mn competition with Cd uptake was investigated in the freshwater alga Scenedesmus vacuolatus. Scenedesmus vacuolatus was exposed to experimental media with Cd and either Zn or Mn in short-term experiments; long-term experiments were undertaken to investigate the effect of growth on Cd accumulation. Cadmium accumulation in S. vacuolatus could be detected at very low exposure concentrations (free Cd2+, 2 x 10(-14) to 1 x 10(-11) M), and uptake was proportional to the free-Cd2+ concentration. Zinc was an effective competitive inhibitor of Cd uptake when the Zn2+ to Cd2+ ratio was greater than 14 in the exposure medium, whereas Mn competed with Cd for uptake above a Mn2+ to Cd2+ ratio of greater than 10,000. Binding constants for Cd and Zn affinity to the transport sites were determined (KZn and KCd). Values for KZn were slightly higher (log K = 9.4-9.8) than values for KCd (log K = 8.9-9.8). In contrast, Cd seemed not to compete with the Mn-binding sites for uptake over the Mn concentration range from 1 x 10(-10) to 1 x 10(-8) M. Determined values for the binding constants of Zn and Cd show that a simple model can be applied to predict Cd uptake at known Zn and Cd concentrations. The environmental implications of these results are discussed with respect to potential Cd toxicity for aquatic organisms.

Influence of daylight on the fate of silver and zinc oxide nanoparticles in natural aquatic environments.

Nanoparticles, such as silver (Ag-NP) and zinc oxide (ZnO-NP), are increasingly used in many consumer products. These nanoparticles (NPs) will likely be exposed to the aquatic environment (rain, river, lake water) and to light (visible and UV) in the products where they are applied, or after those products are discharged. Dissolution of Ag-NP and ZnO-NP is an important process because the dissolved Ag+ and Zn2+ are readily available and toxic for aquatic organisms. The objective of this study was to investigate the role of daylight (UV and visible) for the fate of engineered Ag-NP and ZnO-NPs in different types of natural waters. Ag-NP and ZnO-NP were exposed to rainwater, river Rhine, and lake waters (Greifen, Lucerne, Cristallina, Gruère) under different light conditions (no light, UV 300-400 nm and visible light 400-700 nm) for up to 8 days. Stronger agglomeration of Ag-NP was observed in the waters with higher ionic strength in comparison to those with lower ionic strength. Visible light tended to increase the dissolution of Ag-NP under most natural water conditions in comparison to dark conditions, whereas UV-light led to decreased dissolved Ag+ after longer exposure time. These effects illustrate the dynamic interactions of Ag-NP with light, which may lead both to increased oxidation and to increased reduction of Ag+ by organic compounds under UV-light. In the case of ZnO-NP, agglomeration occurred at higher ionic strength, but the effects of pH were predominant for dissolution, which occurred up to concentrations close to the solubility limit of ZnO(s) at pH around 8.2 and to nearly complete dissolution of ZnO-NP at lower pH (pH 4.8-6.5), with both visible and UV-light facilitating dissolution. This study thus shows that light conditions play an important role in the dissolution processes of nanoparticles.

Thiols in Scenedesmus vacuolatus upon exposure to metals and metalloids.

Phytochelatins are intracellular metal ligands produced by algae when exposed to elevated metal concentrations. In freshwater ecosystems, algae are exposed to a wide range of metals and metalloids. The aim of this study was thus to investigate phytochelatin induction in freshwater algae upon metal and metalloid exposure. To that purpose, the unicellular green alga Scenedesmus vacuolatus, was exposed to Cu, Zn, Ni, Pb and Ag, as well as to As(III), As(V), Sb(III) and Sb(V), and examined for its thiol content (gamma-glutamylcysteine, glutathione and phytochelatins). Glutathione content was found to decrease upon the exposure to Zn and to increase upon the exposure to Pb and Ag. Phytochelatins were only induced by Cu (at [Cu2+] = 8x10(-11) M) and Pb (at [Pb2+] = 8x10(-11) to 8x10(-10) M), where [Cu2+] and [Pb2+] are computed free metal ion concentrations. Glutathione content also decreased upon the exposure to Sb(V) whereas an increase was observed as a result as the exposure to As(III) and As(V). The metalloids As(III), As(V) and Sb(III) in the concentration range from 8x10(-6) to 2x10(-4) M (total concentrations of oxyanions) were inducing phytochelatins. Glutathione and phytochelatin content in S. vacuolatus do thus sensitively respond to exposure to a number of metals and metalloids.

Silver nanoparticles inhibit fish gill cell proliferation in protein-free culture medium.

While short-term exposures of vertebrate cells, such as from fish, can be performed in defined, serum-free media, long-term cultures generally require addition of growth factors and proteins, normally supplied with a serum supplement. However, proteins are known to alter nanoparticle properties by binding to nanoparticles. Therefore, in order to be able to study nanoparticle-cell interactions for extended periods, the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, was adapted to proliferate in a commercial, serum-free medium, InVitrus VP-6. The newly adapted cell strain was named RTgill-W1-pf (protein free). These cells proliferate at a speed similar to the RTgill-W1 cells cultured in a fully supplemented medium containing 5% fetal bovine serum. As well, they were successfully cryopreserved in liquid nitrogen and fully recovered after thawing. Yet, senescence set in after about 10 passages in InVitrus VP-6 medium, revealing that this medium cannot fully support long-term culture of the RTgill-W1 strain. The RTgill-W1-pf cell line was subsequently applied to investigate the effect of silver nanoparticles (AgNP) on cell proliferation over a period of 12 days. Indeed, cell proliferation was inhibited by 10 µM AgNP. This effect correlated with high levels of silver being associated with the cells. The new cell line, RTgill-W1-pf, can serve as a unique representation of the gill cell-environment interface, offering novel opportunities to study nanoparticle-cell interactions without serum protein interference.

Rapid evolutionary loss of metal resistance revealed by hatching decades-old eggs.

We investigated the evolutionary response of an ecologically important freshwater crustacean, Daphnia, to a rapidly changing toxin environment. From the 1920s until the 1960s, the use of leaded gasoline caused the aquatic concentration of Pb to increase at least fivefold, presumably exerting rapid selective pressure on organisms for resistance. We predicted that Daphnia from this time of intense pollution would display greater resistance than those hatched from times of lower pollution. This question was addressed directly using the resurrection ecology approach, whereby dormant propagules from focal time periods were hatched and compared. We hatched several Daphnia genotypes from each of two Swiss lakes, during times of higher (1960s /1980s) and lower (2000s) lead stress, and compared their life histories under different laboratory levels of this stressor. Modern Daphnia had significantly reduced fitness, measured as the population growth rate (λ), when exposed to lead, whereas those genotypes hatched from times of high lead pollution did not display this reduction. These phenotypic differences contrast with only slight differences measured at neutral loci. We infer that Daphnia in these lakes were able to rapidly adapt to increasing lead concentrations, and just as rapidly lost this adaptation when the stressor was removed.

Natural water as the test medium for Ag and CuO nanoparticle hazard evaluation: An interlaboratory case study.

Engineered nanoparticles (NPs) have realistic potential of reaching natural waterbodies and of exerting toxicity to freshwater organisms. The toxicity may be influenced by the composition of natural waters as crucial NP properties are influenced by water constituents. To tackle this issue, a case study was set up in the framework of EU FP7 NanoValid project, performing an interlaboratory hazard evaluation of NPs in natural freshwater. Ag and CuO NPs were selected as model NPs because of their potentially high toxicity in the freshwater. Daphnia magna (OECD202) and Danio rerio embryo (OECD236) assays were used to evaluate NP toxicity in natural water, sampled from Lake Greifen and Lake Lucerne (Switzerland). Dissolution of the NPs was evaluated by ultrafiltration, ultracentrifugation and metal specific sensor bacteria. Ag NP size was stable in natural water while CuO NPs agglomerated and settled rapidly. Ag NP suspensions contained a large fraction of Ag(+) ions and CuO NP suspensions had low concentration of Cu(2+) ions. Ag NPs were very toxic (48 h EC50 1-5.5 µg Ag/L) to D. magna as well as to D. rerio embryos (96 h EC50 8.8-61 µg Ag/L) in both standard media and natural waters with results in good agreement between laboratories. CuO NP toxicity to D. magna differed significantly between the laboratories with 48 h EC50 0.9-11 mg Cu/L in standard media, 5.7-75 mg Cu/L in Lake Greifen and 5.5-26 mg Cu/L in Lake Lucerne. No toxicity of CuO NP to zebrafish embryos was detected up to 100 mg/L independent of the medium used. The results show that Ag and CuO NP toxicity may be higher in natural water than in the standard media due to differences in composition. NP environmental hazard evaluation can and should be carried out in natural water to obtain more realistic estimates on the toxicity.