Trophic transfer of differently coated zinc oxide nanoparticles using crustaceans (Daphnia magna) and zebrafish (Danio rerio) Lars Michael Skjolding*1, Margrethe Winther-Nielsen2 and Anders Baun1 1Department of Environmental Engineering, Technical University of Denmark, Building 113, 2800 Kgs. Lyngby, Denmark 2DHI, Agern Allé 5, 2970 Hørsholm, Denmark *Corresponding author: lams@env.dtu.dk INTRODUCTION METHOD Concern has been raised that nanoparticles (NP) due to their small size and novel properties can exhibit different environmental effects compared to bulk material. At present bioaccumation studies of NP are lacking in the ecotoxicological literature. Furthermore, evidence is appearing that the ecotoxicity as well as the bioaccmulation behaviour of NPs is expected to be highly influenced by coatings and funtionalizations. In this study ZnO nanoparticles with different functionalizations, produced in the FP7 project Nanopolytox, were used to: Design a testing protocol for bioaccumulation studies Study the importance of functionalization on the trophic transfer of ZnO NP from Daphnia magna to Danio rerio. Non-functionalized and functionalized ZnO nanoparticles was tested. All nanoparticles were produced and characterized by partners of the project (Table 2 and Figure 2). For ZnO NP stock suspensions (50 mg Zn/L) were prepared in 300 mL glass-vials containing M7 media. ZnO-C8H17 NP stock suspensions further contained 0.03 mL acetone/L M7 media to disperse the NP. The suspensions were ultra sonicated with a micro tip for 15 minutes (400 W) directly prior to use. Prior to the trophic transfer studies,5 days old D. magna were exposed to 1 mg/L ZnO NP (ZnO NP and ZnO-C8H17 NP) for 24h. In trophic transfer studies the D. rerio were fed for 14 days with the pre-exposed D. magna. After this exposure period the fish were fed for 7 days with non-exposed D.magna (depuration period). The daily feeding rate corresponded to 8% wet weight of the D. rerio. D. magna not eaten after 2 hours was removed and the resulting exposure loading was corrected for in the data treatment RESULTS AND DISCUSSION A: ZnO NP B: ZnO-C8H17 NP CONCLUSION Figure 1: Average content of Zn and standard deviation (n=3) in D.rerio fed with exposed D.magna for 14 days (closed symbols) and unexposed D.magna for 7 days (open symbols) Feasibility of conducting trophic transfer tests with live feed (D. magna) and predators (D.rerio) using ZnO NP yielding meaningful results. Differently functionalized ZnO NP exert different behaviour in regards to uptake and depuration thus also bioaccumulative behaviour. All BMF were below < 1 thus no biomagnification occurred of the NP Figure 1 shows rapid uptake within the first 5 days of exposure for both ZnO and ZnO-C8H17 NP. However, the Zn content increased linearly beyond day 5 for ZnO-C8H17 NP whereas the pristine ZnO NP reached a steady state after 5 days. The difference in uptake and depuration pattern indicates different bioaccumlation behaviour due to functionalization of the NP. Table 1: Body burden, BioMagnificationFactor (BMF), depuration rate and biological half-life for two differently functionalized ZnO NP feeding with exposed D.magna for 14 days, followed by feeding with unexposed D.magna for 7 days. Compound Body burden [mg Zn/ kg dw] BMF Depuration rate [d-1] Biological half-life [d] ZnO NP 887±184 0.15 0.13 5.3 ZnO-C8H17 NP 2169±414 0.42 0.32 2.2 Nanopolytox CONSORTIUM LEITAT Technological Center Glonatech S.A. Institut Català de Nanotecnologia Polyrise SAS L’Urederra Technological Center DHI The expert in water environments Laviosa Chimica Mineraria S.p.A. Lati Thermooplastic Industries S.p.A. EnvNano – ERC Starting grant Technical University of Denmark CHARACTERIZATION Table 2: Characterization differently functionalized ZnO NP used in the experiment by application of ICP-MS, BET, TEM, DLS and Zeta-potential (made by LEITAT and ICN). Technique ZnO NP ZnO-C8H17 NP ICP-MS 80.2 % Zn 54.0 % Zn BET 0.4841 cm3 /g, 12.9 nm 0.027 cm3 /g, 12.8 nm TEM 30 ± 17 nm DLS 132.9 nm (90 sec) 134.3 nm (24h) 523.8 nm (90 sec) 863.8 nm (24h) Zeta-potential 29.1 mV 42.5 mV Figure 2: TEM image of ZnO-NP dry powder. The bar indicates 500 nm. The sample might seem polydisperse due to the non-sperical particles being converted to sphere by the analysis used by the imaging program. Manual observations found high homogenity of NP along the grid (made by ICN).