Giftighet av nanosølv for fisk
I norske vann og vassdrag
Eivind Farmen
18.03.2013
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Silver - the most common material
used in commercial nano-products
Eivind Farmen
18.03.2013
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Nanosilver from a washing machine
Concentration in effluent:
2,7 µg/L to 25 µg/L
Average: 10,522 µg/L
Large variability!
Particles found with TEM:
Size range: 6 – 11 nm
Farkas (2011) Environment international 37 (6), 1057-1062
Objectives
• Determine acute and sublethal toxicity of AgNP to fish & fish cells
• Determine AgNP characteristics in various matrixes
• Compare toxic potential of AgNP to ionic silver and AuNP
• Assess risk to fish under ecologicaly relevant exposure scenarios
Eivind Farmen
18.03.2013
In vitro experimental model
What are the possible effects of
nanoparticles on fish cells used as an in
vitro test system?
Exposure
(48h)
Primary hepatocytes
Liver
Gill
Cytotoxicity
Reactive oxygen species (ROS) production
Epithelial integrity
Primary gill epithelium cells
Eivind Farmen
18.03.2013
Particle characteristics by TEM
AgNPs
Roundish to oval
particle shape
Water
cell media
In–house synthesized
citrate-coated NPs
cell media
+DOC
AuNPs
ROS production & Cytotoxicity
(hepatocytes)
Gold NPs
Silver NPs
ROS
Cytotoxicity
Farkas et al. 2009. Effects of silver and gold nanoparticles on rainbow trout (Oncorhynchus mykiss)
hepatocytes. Aquatic toxicol.
Eivind Farmen
18.03.2013
Effects on primary gill cells
Ag is less cytotoxic to gill cells than to hepatocytes
AuNPs were not cytotoxic
Eivind Farmen
18.03.2013
What happens in natural water?
Commercial AgNP
In-house
4 nmAgNP
1:10 in natural lake
water
1:10 in natural lake
water
Eivind Farmen
18.10.2012
220 nm
9
Effects Ag nanoparticles
Ag in gills
(n=15)
*
30
(n=13)
20
*
(n=15)
*
Control
(n=14)
1 g/L
AgNP
20 g/L 100 g/L 20 g/L
AgNP AgNP
Ag+
100 g/L
AgNP2
*
10
(n=15) (n=15) (n=15)
µg
/L
µg
/L
10
A
g+
0
µg
/L
N
P2
20
10
0
µg
/L
L
20
µg
/
1
ro
l
T0
0
C
on
t
Ag (mg/kg gill tissue)
40
?
?
Eivind Farmen
18.03.2013
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GO analysis AgNP 20 µg/L
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18.03.2013
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GO analysis
Eivind Farmen
18.10.2012
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Transcriptional UP-regulation (Ag)
100 ug/L AgNP
20 ug/L Ag+
GTPase IMAP
family member 4
Na/H exchanger 9
20 ug/L AgNP
Protein-Glutamine
-glutamyltransferase
Heme-binding protein 2
Eivind Farmen
18.03.2013
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Transcriptional Down-regulation (Ag)
MHC Class II
Interferon gamma
100 ug/L AgNP
20 ug/L Ag+
Carbonic anhydrase 7
Na/K-ATPase
20 ug/L AgNP
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18.03.2013
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Mode of action Ag
Blood
Gill
Water
Na+
Cl
K
Na
Na+
K+
NaKATPase
H+ (NH4+)
*
K+
Cl-
(n=14)
6
120
125
HCO3Cl-
150
155
(n=14)
(n=1)
(n=9)
(n=9)
10
(n=9)
(n=14)
(n=14) (n=14)
(n=13)
140
145
(n=13)
(n=9)
8 (n=9)
(n=10)
(n=10)
(n=14)
(n=10) (n=13)
130
135
(n=9)
m
mol/L
m mol/L
mol/L
Na+
Cl-
4
110
115
90
0
95
C
on
nttr
roo
ll
11 µ
gg//L
L
20
0µ
µgg
/
10 /LL
000
µµgg
220
/
µµgg /LL
//LL
110
AAg
g++
00 µ
µgg
//LL
NNP
P22
Carbonic
anhydrase
CO2 + H2O =>
H+ + HCO3-
*
T0
0
CO2
(n=1)
**
(n=4)
2
100
105
CO2
*
*
Eivind Farmen
18.03.2013
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Hazard/risk assessment
100 ug/L
AgNP
20 ug/L
Ag+
20 ug/L
AgNP
Control
Toxicity of AgNP due to acting as reservoir for Ag+?
Toxicity of AgNP higher in soft waters !
Eivind Farmen
18.03.2013
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Effects Ag nanoparticles
Ag in gills
Ag (mg/kg gill tissue)
40
(n=15)
*
30
(n=13)
20
(n=15)
*
*
Control
(n=14)
1 g/L
AgNP
20 g/L 100 g/L 20 g/L
AgNP AgNP AgNP
100 g/L
AgNP2
*
10
(n=15) (n=15) (n=15)
on
tr
ol
1
µg
/L
20
µg
/L
10
0
µg
20
/L
µg
/L
10
A
g+
0
µg
/L
N
P2
C
T0
0
Osmoregulation
Necrosis/
apoptosis
Eivind Farmen
18.03.2013
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Eivind Farmen
18.03.2013
10
0
/L
g
g
/L
/L
N
P2
g+
A
N
P1
N
P1
N
P1
5
g
/L
g
/L
6
20
10
0
20
ro
l
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
g
0.0
on
t
1.0
1
*
T0
*
Mean norm expr
3.0
C
1.5
on
tr
1
ol
g
/L
N
20
 g P1
/L
10
N
0
P1
g
/L
N
20
P1
g
/L
10
A
0
g+
g
/L
N
P2
2.5
T0
3.5
C
0.5
µg
2Mean
norm
expr
0
/L
µg
/L
10
A
g+
0
µg
/L
N
P2
µg
/L
µg
/L
2.0
10
0
20
1
ro
l
on
t
C
T0
Ag (mg/kg gill tissue)
Work in progress
Ag in liver
*
*
HSP70
MT-A
*
4
3
2
1
0
18
Differences Ag-NPs
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1
C
on
tr
ol
g
/L
N
20
 g P1
/L
10
N
P1
0
g
/L
N
20
P1
g
/L
10
A
0
g+
g
/L
N
P2
*
T0
µM GSSG/mg liver (ww)
GSSG (µM/mg)
Eivind Farmen
18.03.2013
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Conclusion
• High toxicity of Ag-NPs in natural
Norwegian waters
• Ag-NP effect mediated by Ag+ ions?
• Effects in gills/osmoregulation
• Modulation of immune system
• Effect in gills > liver
Eivind Farmen
18.03.2013
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Acknowledgements
• Farkas J, Mikkelsen HN, Evensen Ø, Heier LS,
Einset J, Salbu B, Rosseland BO, Oughton DH,
Tollefsen KE, Paul Christian, Julián Alberto
Gallego-Urrea, Norbert Roos, Martin Hassellöv,
Kevin V Thomas, Mie Jareid, Hanne Fossnes,
Hans-Christian Teien
• Funding:
– Norwegian Research Council
» Nanotrace Project
» NIVA, SIP project 24024 NEWPOLL
Thank you for listening!
Eivind Farmen
18.03.2013
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