In this study, the effect of ZnO nanoparticles and ZnCl2 on growth, reproduction and accumulation of zinc in Daphnia magna was determined in a 21-day chronic toxicity test. A variety of techniques were used to distinguish the free zinc ion, dissolved, nanoparticle and aggregated zinc fraction in the Daphnia test medium. The results showed similar chronic effects on growth, reproduction and accumulation for the ZnO nanoparticles (EC10, 20, 50 reproduction: 0.030, 0.049, 0.112 mg Zn/l) and the ZnCl2 (EC10, 20, 50 reproduction: 0.014, 0.027, 0.082 mg Zn/l). A large fraction of the nanoparticles rapidly dissolved after introduction in the exposure medium. Aggregation of nanoparticles was also observed but within 48 h of exposure most of these ZnO aggregates were dissolved. Based on the combined dissolution kinetics and toxicity results, it can be concluded that the toxicological effects of ZnO nanoparticles at the chronic level can be largely attributed to the dissolved fraction rather than the nanoparticles or initially formed aggregates.

In this study, the effects of CuO nanoparticles and CuCl2·2H2O were tested on Daphnia magna under chronic exposure scenarios. During a 21-day exposure to the nanoparticles and salt, the reproduction was followed by a daily count of the number of offspring. After the exposure, the adult Daphnia length and uptake of copper was measured. The dissolved, nanoparticle and aggregated fractions were distinguished in the exposure medium. The results showed that only a small fraction of the nanoparticles dissolved, while the majority of the particles formed large aggregates (>450 nm). The dissolved fraction of the nanoparticles corresponded with the dissolved fraction of the copper salt. The effects of the nanoparticles (reproduction EC10: 0.546 mg Cu/l, EC20: 0.693 mg Cu/l, EC50: 1.041 mg Cu/l) on reproduction and length were much lower than the effects of the copper salts (reproduction EC10: 0.017 mg Cu/l, EC20: 0.019 mg Cu/l, EC50: 0.022 mg Cu/l). Based upon total body analysis, the Daphnia copper concentration appeared much higher when exposed to the nanoparticles than when exposed to the salt. These combined results indicate that the toxicity of CuO nanoparticles to D. magna is caused by copper ions formed during dissolution of the nanoparticles in the exposure medium.

Metal oxide nanoparticles are increasingly being produced and will inevitably end up in the aquatic environment. Up till now, most papers have studied individual nanoparticle effects. However, the implementation of these data into a risk assessment tool, needed to characterise their risk to the aquatic environment, is still largely lacking. Therefore, aquatic species sensitivity distributions (SSDs) were constructed for ZnO and CuO nanoparticles and 5% hazard concentrations (HC5) were calculated in this study. The effect of individual nanoparticles on these SSDs was estimated by comparison with bulk SSDs. Additionally, the effect of nanoparticle dynamics (aggregation and dissolution) was considered by evaluating the effect of aggregate size on the toxicity, by estimation of the dissolved fraction and comparison with SSDs for ZnCl2 and CuCl2 inorganic salt. Bacteria, protozoa, yeast, rotifera, algae, nematoda, crustacea, hexapoda, fish and amphibia species were included in the analysis. The results show that algae (Zn) and crustacea (Zn, Cu) are the most sensitive species when exposed to the chemicals. Similar acute sensitivity distributions were obtained for ZnO nanoparticles (HC5: 0.06 with 90% confidence interval: 0.03-0.15 mg Zn/l; 43 data points), bulk ZnO (HC5: 0.06 with CI: 0.03-0.20 mg Zn/l; 23 dps) and ZnCl2 (HC5: 0.03 with CI: 0.02-0.05 mg Zn/l; 261 dps). CuO nanoparticles (HC5: 0.15 with CI: 0.05-0.47 mg Cu/l; 43 dps) are more toxic than the bulk materials (HC5: 6.19 with CI: 2.15-38.11 mg Cu/l; 12 dps) but less toxic than CuCl2 (HC5: 0.009 with CI: 0.007-0.012 mg Cu/l; 594 dps) to aquatic species. However, the combined dissolution and SSD results indicate that the toxicity of these nanoparticles is mainly caused by dissolved metal ions. Based on the available information, no current risk of these nanoparticles to the aquatic environment is expected.

In this study, the uptake and elimination of ZnO and CuO nanoparticles in Daphnia magna was tested. Daphnids were exposed during 10 days to sublethal concentrations of ZnO and CuO nanoparticles and corresponding metal salts (ZnCl₂ and CuCl₂.2H₂O), after which they were transferred to unexposed medium for another 10 days. At different times during the exposure and none-exposure, the total and internal zinc or copper concentration of the daphnids was determined and the nanoparticles were localized in the organism using electron microscopy. The exposure concentrations were characterized by measuring the dissolved, nanoparticle and aggregated fraction in the medium. The results showed that the ZnO nanoparticles quickly dissolved after addition to the medium. Contrarily, only a small fraction (corresponding to the dissolved metal salt) of the CuO nanoparticles dissolved, while most of these nanoparticles formed large aggregates. Despite an initial increase in zinc and copper concentration during the first 48 h to 5 day exposure, the body concentration reached a plateau level that was comparable for the ZnO nanoparticles and ZnCl₂, but much higher for the CuO nanoparticles (with visible aggregates accumulating in the gut) than CuCl₂.2H₂O. During the remaining exposure and subsequent none-exposure phase, the zinc and copper concentration decreased fast to concentrations comparable with the unexposed daphnids. The results indicate that D. magna can regulate its internal zinc and copper concentration after exposure to ZnO and CuO nanoparticles, similar as after exposure to metal salts. The combined dissolution, accumulation and toxicity results confirm that the toxicity of ZnO and CuO nanoparticles is caused by the dissolved fraction.

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