Ecotoxicity of silica
nanoparticles to the green alga
Pseudokirchneriella
subcapitata: importance of
surface area
Karen Van Hoecke
Karel A.C. De Schamphelaere
Colin Janssen
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Introduction
Search for research papers in Web of Science*:
Results:
95
90
85
91
Total
“Nanoparticles AND tox*” in “article title”
1
“surface” +
ecotoxicology
5
Eco
25
“surface”
80
75
86
Human
70
65
60
55
50
NP ecotoxicology is a small research field of nanotoxicology in general
“Surface” is an important issue in nanotoxicology
* The search was conducted on 14th February
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Surface area in nanotoxicology
L. Yang and D. J. Watts. (2005). Toxicology letters, 158, p. 122-132.
D. B. Warheit et al. (2006). Toxicological sciences, 91 (1), p. 227-236
T. Stoeger et al. (2007).
Environmental health
perspectives, 115 (6),
p. A291-A292.
D. Fubini (2007). Platform presentation. Beltox Annual
Meeting, 28th November 2007, Namur, Belgium.
R. Duffin et al. (2007). Inhalation toxicology, 19, p. 849-856.
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Importance of particle diameter
Surface = 4 π R2
Volume = 4/3 π R3
Surface to volume ratio ~ 1/R
Oberdörster et al. (2005). Environmental Health Perspectives
113: 823.
For diameters < 100 nm, a huge amount of surface area is available for
potential toxicological interaction
Are smaller nanoparticles more toxic than larger nanoparticles?
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Research hypothesis
Based on inhalation toxicology studies:
Rat
Öberdorster et al. (2005). Environmental Health Perspectives 113: 823.
When concentration is expressed as mass, smaller
nanoparticles are more toxic than larger nanoparticles.
However, when concentration is expressed as surface
area, the difference in toxicity disappears.
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Testorganism and test design
Testorganism
Unicellular freshwater green alga
Pseudokirchneriella subcapitata
Test design
Chronic 72 h growth inhibition test (OECD TGD No. 201)
Test medium: described in OECD guideline + 750 mg/l MOPS* buffer
Toxicity endpoint: % decrease in average specific growth rate, relative to
control
*3-(N-Morpholino)propanesulfonic acid
OECD test guidelines at:
http://www.oecd.org/document/40/0,3343,en_2649_34377_37051368_1_1_1_1,00.html
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Materials: LUDOX® silica particles
Commercial LUDOX® silica (SiO2) nanoparticles of 2 different sizes:
LUDOX® LS
LUDOX® TM40
Average diameter: 12 nm
Surface area: 236 m2/g
Average diameter: 22 nm
Surface area: 135 m2/g
800 nm
500 nm
http://www.gracedavison.com/products/ludox/techinfo.htm.
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Particle characterization
1. Stability of the particles in test medium
Particle size distribution, using dynamic light scattering
Are the nominal diameter and surface area of the particles
respresentative for their behaviour in test medium?
2. Dissolution of the particles in test medium
Colorimetric analysis of reactive silica (SiO32-) in particle
suspensions
Is toxicity due to (partial) dissolution of the particles
or to the particles themselves?
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1. Particle size distribution
12
®
LUDOX LS
®
LUDOX TM40
% Intensity
10
8
6
4
2
0
1
10
Diameter (nm)
100
LUDOX® LS
LUDOX® TM40
Mean diameter (std. dev.)
12.5 (0.2)
27.0 (0.5)
Polydispersity index (std.dev.)
0.26 (0.02)
0.17 (0.02)
SiO2 particles are monodisperse
Nominal diameter and surface area are representative
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2. Particle dissolution
Concentration of reactive silica
Reactive (=dissolved) SiO2 (mg/L)
Total SiO2 (mg/L)
LUDOX LS (12.5 nm)
LUDOX TM40 (27 nm)
4.6
< MDL
< MDL
10
0.21
< MDL
22
0.22
< MDL
46
0.56
0.35
100
1.01
0.47
220
2.04
1.09
460
4.14
1.58
EC10 of reactive SiO2 to P. subcapitata = 55 mg SiO2/L
If toxicity is observed, it is not due to (partial) dissolution of the
silica particles
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Concentration-response curves (1)
-1
Average specific growth rate µ (d )
Concentration expressed as mass
1.8
LS (12.5 nm)
TM40 (27.0 nm)
control response
1.6
1.4
1.2
1.0
0.8
0.6
1
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100
-1
Mass concentration (mg.l SiO2)
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Concentration-response curves (2)
-1
Average specific growth rate µ (d )
Concentration expressed as surface area
1.8
LS (12.5 nm)
1.6
TM40 (27.0 nm)
1.4
control response
1.2
1.0
0.8
0.6
1
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100
Surface concentration (m².l-1)
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Statistical analysis
Experiment was repeated four times ⇒ 4 EC20 values for both particles
30
EC20 (mg.l-1)
25
<
6
27.9
4.30
5
18.2
EC20 (m².l-1)
35
20
15
4
=
3.77
3
2
10
LS (12.5 nm)
5
TM40 (27.0 nm)
0
1
0
LS (12.5 nm)
TM40 (27.0 nm)
T-test for dependent samples (α ≤ 0.05)
Research hypothesis is valid
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TEM research of algal cells
70 nm slices of algal cells were placed on a copper grid and examined
with a Jeol JSM 7500 F
Control
400 nm
600 nm
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SiO2 nanoparticle-cell interaction (1)
Algal cells exposed to LUDOX® LS (12.5) SiO2 nanoparticles
100 nm
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SiO2 nanoparticle-cell interaction (2)
Algal cells exposed to LUDOX® TM40 (27.0) SiO2 nanoparticles
100 nm
400 nm
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Conclusion
• LUDOX® silica particles are stable in aqueous suspension and do not
dissolve readily
• Smaller nanoparticles are more toxic than larger ones when
concentration is expressed as mass
• Difference in toxicity disappears when concentration is expressed as
surface area
• Silica nanoparticles are adsorbed to the cell wall of algal cells
=
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Acknowledgement
Institute for the Promotion of Innovation through Science and
Technology (IWT-Vlaanderen)
Flanders Science Foundation (FWO-Vlaanderen)
Research group Particle & Interface Chemistry (UGent)
Laboratoire d’Analyse par Réactions Nucléaires (FUNDP)
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