1. Uptake and depuration of three differently functionalized zinc oxide nanoparticles to Daphnia magna
*Lars Michael Skjolding1, **Margrethe Winther-Nielsen2 and Anders Baun1
1Department of Environmental Engineering, Technical University of Denmark, Building 113, Kgs. Lyngby, Denmark
2DHI, Agern Allé 5, 2970 Hørsholm, Denmark
*Corresponding author:
**Corresponding author:
INTRODUCTION
METHOD
During the lastest decade the use of NPs has increased dramatically. Zinc oxide is used in a wide range of application e.g. personal care products, paints and semiconductors. Concern has been raised that nanoparticles due to their small size and novel properties can exhibit diffferent ecotoxicological effects compared to bulk material. Furthermore, due to the differences in synthesis, coating and funtionalization the versatility and possible differences in ecotoxicological effects compared to bulk material increases. Literature describing the bioaccumulative behaviour of nanoparticles is generally sparse and especially investigation of functionalized nanoparticles is lacking.
ZnO nanoparticles with different functionalizations provided through the FP7 project Nanopolytox were used to investigate the effect of functionalization of nanoparticles on their bioaccumulative behavior in Daphnia magna.
Non-functionalized as well as functionalized ZnO nanoparticles have been tested. All nanoparticles were produced and characterized by partners of the project (Table 2 and Figure 2). A protocol for nanoparticle dispersion was developed based on initial investigations and characterisation of the dispersions. Stock suspensions (50 mg Zn/L) were prepared in 300 mL glass-vials containing only M7 media for ZnO NP and ZnO-OH NP. For ZnO-octyl NP 0.03 mL acetone was added as dispergent along with M7 media. The suspensions were ultra sonicated with a micro tip for 15 minutes (400 W) directly prior to use.
Prior to the experiment the level of exposure was determined with ISO:6431 Daphnia immobilization test (Table 3). For bioaccumulation test 4-5 days old Daphnia magna were exposed to 1 mg Zn/L for 24 hours and then moved to clean media for 24 hours of depuration (Figure 1). Tests were performed in three replicates with 5 organisms in each beaker.
RESULTS AND DISCUSSION A B C
CONCLUSION D E
Table 1: BioConcentrationFactor (BCF), uptake rate and depuration rate for three differently functionalized ZnO-NP in a 24 hours bioaccumulation study with Daphnia magna exposed with 1 mg Zn/L during uptake. First order kinetics was assumed.
The investigation showed that differently functionalized ZnO NP exerted different behaviour in regards to uptake and depuration thus also bioaccumulative behaviour.
ZnO NP and ZnO-octyl NP were taken up rapidly while ZnO-OH NP did not show uptake significantly different from the control.
Lipophile ZnO-octyl NP were depurated slower than the non-functionalized ZnO NP.
Lipophile ZnO-octyl NP had a higher BCF compared to ZnO-NP.
None of the differently functionalized ZnO NP showed a BCF exceeding the level for considering a substance potentially bioaccumulative according to the EU chemical regulation (ECHA).
Compound
Uptake rate [h.1]
Depuration rate [h.1]
BCF
ZnO NP
0.11
-1.8
0.061
ZnO-octyl NP
0.35
-0.69
0.51
ZnO-OH NP
N/A
Figure 1: Uptake and depuration of differently functionalized ZnO NP in D. magna during 24 hours of exposure with a concentration of 1 mg Zn/L and 24 hours of depuration in clean media. The bars indicate the standard deviation of the measurements (n=3). * indicates that the measurement is significant different from the control using a student t-test (P<0.05). † indicates significant difference from measurement at <10 min using a student t-test (P<0.05). # indicates significant difference from pooled measurements at 30 min, 1 h, 2 h using a student t-test (P<0.05).
Figure 1A and 1D shows a rapid uptake reaching steady state within the first 2 hours of exposure with ZnO NP and ZnO-octyl NP. However, no uptake of ZnO-OH NP could be distinguished statistically from the control during the 24 hours of uptake (Figure 1C). This could indicate an effect of the functionalization due to uptake of ZnO-octyl NP but not ZnO-OH NP. Figure 1B and 1E shows rapid depuration reaching steady state within 1 hour and 2 hours respectively. The results indicate a slower depuration of ZnO-octyl NP and thus a higher BCF as also shown in Table 1. However, none of the estimated BCFs were found to exceed (>2000) the level stated by ECHA (European CHemical Agency).
CONSORTIUM
LEITAT Technological Center
Glonatech S.A.
Institut Català de Nanotecnologia
Polyrise SAS
L’Urederra Technological Center
DHI Water  Environment  Health
Laviosa Chimica Mineraria S.p.A.
Lati Thermooplastic Industries S.p.A.
CHARACTERIZATION
Table 2: Characterization of the differently functionalized ZnO NP used in the experiment by application of ICP-MS, BET, TEM, DLS and Zeta-potential (Made by LEITAT and ICN). N/A indicates that no meaningful average was obtainable due to polydispersivity along the grid.
Compound
ZnO NP
ZnO-OH NP
ZnO-octyl NP
ICP-MS
80.2 % Zn
56.6 % Zn
54.0 % Zn
BET
cm3 /g, nm
0.108 cm3 /g, nm
0.027 cm3 /g, nm
TEM
30 ± 17 nm
N/A
DLS
132.9 nm (90 sec) nm (24h)
1529 nm (90 sec) nm (24h)
523.8 nm (90 sec) nm (24h)
Zeta- potential
29.1 mV
44.2 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).