Toxicity of high-purity silver and gold nanoparticles to the aquatic plant Lemna minor Panagiotis Minogiannis1, Marco Velenti2, Vaya Kati3, Olga I. Kalantzi1* and George Biskos1,2 Department of Environment, University of the Aegean, Mytilene 81100, Greece 2 Faculty of Applied Sciences, Delft University of Technology, Delft 2628 BL, The Netherlands 3 Benaki Phytopathological Institute, Athens 14561, Greece 1
INTRODUCTION AgNPs
50
AuNPs
AgNPs
40
Frond Number
AgNP1 AgNP2
30
Lemna minor was purchased from the Federal Environmental Agency (UBA, Germany). The nutrient solution used for Lemna minor growth was prepared according to the ISO 2079 guidelines. The ENPs used in the tests were synthesized using an innovative aerosol-based method (namely the spark discharge generator), which produces nanoparticles of very high purity and provides good control over their size, morphology and composition (cf. Figure 1).
10 0
Control
5 10 20 Concentration (μg/L)
20 0
40
Inhibition of Growth (%)
Inhibition of Growth (%)
AgNPs
100
CPC
0
5 10 20 Concentration (μg/L)
40
Spherical nanoparticles with diameters in the range of 20 to 80 nm were produced in high purity Ar gas and inserted in deionised water. Tested concentrations of AgNPs and AuNPs were 0, 5, 10, 20 and 40 μg L-1. The culture medium used in the tests was sterile modified APHA (Moody and Miller 2005). All tested NP concentrations had 5 replicates.
RESULTS After 7 days of exposure to AgNPs, Lemna cultures showed a statistically significant (p < 0,05) inhibition of frond numbers for all tested concentrations (cf. Figure 2). AuNPs on the other hand, had a positive effect on frond number at 5 and 10 μg L-1, but at 20 and 40 μg L-1 the measurements were similar to the control (Figure 2). The AgNPs affected the inhibition of growth rate (IGR) in Lemna for both frond number and dry weight, for all concentrations (cf. Figure 3). In general IGR based on dry weight was greater than the equivalent based on frond numbers. AuNPs exhibited higher IGRs as particle mass concentration increased. However, for the lower concentrations (5 and 10 μg L-1) AuNPs showed an unexpected enhancement of frond numbers and dry weight. As shown in Figure 4, relative growth rate (RGR) of frond area was inhibited for all concentrations of AgNPs after 4 days of the 7-day treatment. On the contrary, AuNPs reduced RGR at higher concentrations (20 and 40 μg L-1), but enhanced frond area at lower concentrations (5 and 10 μg L-1).
60 40
Dry Weight
20 0
Frond Number
0
5
10 15 20 25 30 35 40 45 Concentration (μg/L)
AuNPs 5
10 15 20 25 30 35 40 45 Dry Weight Frond Number
Concentration (μg/L)
Relative Growth Rate AgNPs 4,8
3
6,3
20 μg/L 10 μg/L 5 μg/L
2
Control
1
0
2
4
AuNPs
40 μg/L
6
Days of Treatment
7
7
Relative Growth Rate
Figure 1: The Spark Discharge is a generator in which repeated spark discharges are formed between to conductive electrodes (Au or Ag in our case). Vapors produced from the electrodes due to the high temperature of the spark are cooled down to form ENPs which are carried out of the reactor by the N2 quenching flow. The airborne nanoparticles are captured in the liquid samples by passing them through a bubbler. The size distributions of the resulting particles monitored by an electrical mobility spectrometer consisting of an electrostatic neutralizer, a differential mobility analyzer (DMA), and a condensation particle
80
40 20 0 0 -20 -40 -60 -80
Figure 3: AgNPs Inhibition of Growth rate (%) for frond number (green dots) and dry weight (red dots) with logistic trendlines (left) and (right) AuNPs Inhibition of Growth Rate (%) for frond number (green dots) and dry weight (red dots) with logistic trendlines
Relative Growth Rate
Neutralizer
40
Inhibition of Growth Rate DMA
3-way valve
Ar flow
60
Figure 2: Frond number of Lemna minor when exposed to (a) AgNPs with diameter from 20 to 80 nm (this study) and (b) ionic silver, AgNP1 with diameter at 30 nm, AgNP2 with diameter at 100 nm (Gubbins et al., 2011) (left), and (right) AuNPs with diameter from 20 to 80 nm (this study)
Bubbler
Spark Discharge
Control
80
Ionic Ag
20
MATERIALS AND METHODS
AuNPs
100
Frond Number
The increasing use and applications of engineered nanoparticles (ENPs) in consumer products has raised many questions with regard to their potential toxic behavior. Although an increasing number of studies is focusing on the toxicity of ENPs, little information is available on their toxic effects on aquatic plants. This study assesses the effects of silver and gold nanoparticles (AgNPs and AuNPs, respectively) on Lemna minor under a modified version of the ISO 2079 protocol.
Frond Number
6 5 4 3 2 1 0
2
4 6 Days of Treatment
7
Figure 4: AgNPs (left) and AuNPs (right). Relative Growth Rate for frond area (color bars) for 2nd, 4th, 6th, and 7th days and control (green bars)
CONCLUSIONS • AgNPs cause toxicity to Lemna minor at exposures as low as 5 μg L-1. • This is in agreement with previous studies and can be attributed to the toxicity of ionic silver (Gubbins et al., 2011). • AuNPs exhibited an unexpected positive effect on Lemna minor growth at lower concentrations, which may be an indication of hormesis and requires further investigation.
REFERENCES Table 1: EC50 values for AgNPs (this study), AgNP1 & AgNP2 (Gubbins et al., 2011), based on dry weight and frond numbers (Figure 2) EC50 AgNPs AgNP1 AgNP2 Dry Weight
2,38
63,7
61,6
Frond Number
8,89
140
125
SETAC Basel May 2014
Gubbins, E.J., Batty, L.C. and Lead, J.R. (2011), "Phytotoxicity of silver nanoparticles to Lemna minor L", Environmental Pollution, 159, 1551–1559. Moody, M. and Miller, J. (2005), "Lemna Minor Growth Inhibition Test", Book Chapter, Small-scale Freshwater Toxicity Investigations, Toxicity Test Methods, Springer, The Netherlands,1, 271–298. ISO 20079 (2005), "Water quality-Determination of the toxic effect of water constituents and waste water to Duckweed (Lemna minor)-Duckweed growth inhibition", Geneva.
*Email contact:
[email protected]
