Eivind Farmen 118.03.2013

Giftighet av nanosølv for fisk I norske vann og vassdrag

Eivind Farmen 2

Silver - the most common material used in commercial nano-products

18.03.2013

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

18.03.2013Eivind Farmen

In vitro experimental modelWhat are the possible effects of nanoparticles on fish cells used as an in vitro test system?

Liver

Gill

Exposure

(48h)

Cytotoxicity

Reactive oxygen species (ROS) production

Epithelial integrity

Primary hepatocytes

Primary gill epithelium cells

18.03.2013Eivind Farmen

Particle characteristics by TEM

AgNPs

Water

cell media

cell media +DOC

Roundish to oval particle

shape

AuNPs

In–house synthesized citrate-coated NPs

ROS production & Cytotoxicity(hepatocytes)Silver NPs Gold NPs

ROS

Cytotoxicity

Farkas et al. 2009. Effects of silver and gold nanoparticles on rainbow trout (Oncorhynchus mykiss) hepatocytes. Aquatic toxicol.

18.03.2013Eivind Farmen

Effects on primary gill cells

Ag is less cytotoxic to gill cells than to hepatocytesAuNPs were not cytotoxic

18.03.2013Eivind Farmen

Eivind Farmen 918.10.2012

What happens in natural water?

Commercial AgNP1:10 in natural lake water

In-house AgNP1:10 in natural lake water

4 nm

220 nm

Eivind Farmen 10

Effects Ag nanoparticles

? ?

Ag in gills

0

10

20

30

40

* **

*

(n=15) (n=15) (n=15)

(n=15)

(n=15)

(n=13)

(n=14)

Ag

(m

g/k

g g

ill

tiss

ue)

Control 1 g/LAgNP

20 g/LAgNP

100 g/LAgNP

20 g/LAg+

100 g/LAgNP2

18.03.2013

Eivind Farmen 1118.03.2013

GO analysis AgNP 20 µg/L

Eivind Farmen 1218.10.2012

GO analysis

Eivind Farmen 13

Transcriptional UP-regulation (Ag)

100 ug/L AgNP 20 ug/L Ag+

20 ug/L AgNP

Heme-binding protein 2Protein-Glutamine -glutamyltransferase

GTPase IMAP family member 4Na/H exchanger 9

18.03.2013

Eivind Farmen 14

Transcriptional Down-regulation (Ag)100 ug/L AgNP 20 ug/L Ag+

20 ug/L AgNP

Carbonic anhydrase 7Na/K-ATPase

Interferon gammaMHC Class II

18.03.2013

Eivind Farmen 15

Mode of action AgBlood Gill Water

HCO3-

Cl-Cl-

NaK-ATPase

H+ (NH4+)

Na+

K+

Na+

Carbonic anhydrase

CO2 + H2O =>H+ + HCO3

-

CO2

Na+

Na

95

105

115

125

135

145

155

*

*

(n=9)

(n=14)

(n=14)

(n=9)

(n=4)

(n=10)

(n=13)

m m

ol/

L

K+

K

T0

Control

1 µg

/L

20 µ

g/L

100

µg/L

20 µ

g/L A

g+

100

µg/L

NP2

0

2

4

6

8

10

*

*(n=9)

(n=14)

(n=14)

(n=9)

(n=1)

(n=10) (n=13)

m m

ol/

L

Cl-

CO2

Cl

T0

Control

1 µg

/L

20 µ

g/L

100

µg/L

20 µ

g/L A

g+

100

µg/L

NP2

90

100

110

120

130

140

150

**

(n=9) (n=14) (n=14)(n=9)

(n=1)

(n=10)

(n=13)

m m

ol/

L

18.03.2013

Eivind Farmen 16

Hazard/risk assessment

100 ug/LAgNP

20 ug/LAg+

20 ug/LAgNP

Control

Toxicity of AgNP due to acting as reservoir for Ag+?

Toxicity of AgNP higher in soft waters !

18.03.2013

Eivind Farmen 17

Effects Ag nanoparticlesAg in gills

0

10

20

30

40

* **

*

(n=15) (n=15) (n=15)

(n=15)

(n=15)

(n=13)

(n=14)

Ag

(m

g/k

g g

ill

tiss

ue)

Control 1 g/LAgNP

20 g/LAgNP

100 g/LAgNP

20 g/LAgNP

100 g/LAgNP2

18.03.2013

Osmo-regulation

Necrosis/apoptosis

Eivind Farmen 18

Work in progress

18.03.2013

T0

Control

1 µg

/L

20 µ

g/L

100

µg/L

20 µ

g/L A

g+

100

µg/L

NP2

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

*

**

*Ag in liver

Ag

(m

g/k

g g

ill

tiss

ue)

HSP70

T0

Control

g/L N

P1

1

g/L N

P1

20

g/L

NP1

100

g/L A

g+

20

g/L

NP2

100

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5M

ean

no

rm e

xpr

T0

Control

g/L N

P1

1

g/L N

P1

20

g/L

NP1

100

g/L A

g+

20

g/L

NP2

100

0

1

2

3

4

5

6 *

MT-A

Mea

n n

orm

exp

r

Eivind Farmen 19

Differences Ag-NPs

18.03.2013

GSSG (µM/mg)

T0

Control

g/L N

P1

1

g/L N

P1

20

g/L

NP1

100

g/L A

g+

20

g/L

NP2

100

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9*

µM

GS

SG

/mg

liv

er (

ww

)

Eivind Farmen 2018.03.2013

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 2118.03.2013

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

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