Nanomatériaux et nanoparticules manufacturées : Risques environnementaux /
Jean‐Yves Bottero, and
Mélanie Auffan, Jérôme Rose, Céline Botta, Jérôme Labille, Armand Masion, N Solovitch‐Vella, E.M Hotze
And the US and French members of the GDR‐I Consortium for the Environmental Implications of Nano Technology
IRSN
21 Septembre 2010
Quelques chiffres: Nombre de produits répertoriés en 2009 Source: Program Emerging Nanotechnology
En 2010: plus de 2000 Source Nanowerk
Catégories
Dans le domaine dit « du bien être »
Les éléments les plus présents
From Ch Robichaud PhD DUKE Univ Decembre 2010
Understanding AgNPs formation/ transformation
in wastewater treatment
Targeted National Sewage sludge Survey
Statistical Analysis Report
(Released in Jan 2009)
ƒ 74 plants across the States
ƒ Total metal contents
ƒ Pharmaceuticals, steroids, and hormones
Sludge ID 68349 (from Midwest region)
Elemental Analysis
Blaser, S. A. et al., Science of the Total Environment
(2008).
Element
(mg kg-1)
Mg
13500
Ag
856
Mn
1070
Al
57300
Na
6080
Ca
98900
P
57200
Cu
1720
Ti
4510
Fe
51000
Zn
1530
Spécificités extrinsèques des nanos: Relations taille, nombre, surface
Specifities « intrinèsques » des Nanos
Tout n’est pas NANO
!
1
NP < 30 nm méritent une considération spéciale en écotoxicité…
30 nm
?
Strong increase of the surface atom number
Strong surface reactivity
< 30 nm
M.Auffan et al., NATURE nano, 09- 2009
Environmental behavior and (eco)toxicity
• Trophic transfer via
food webs
A terrestrial
food chain
A marine
food chain
Quaternary consumers
Hawk
Killer whale
Snake
Tertiary consumers
Cod
Secondary consumers
Mouse Herring
Transformation?
Primary consumers
Grasshoper Zooplancton
Producers
Flowers Phytoplancton bacterias..
http://www.pearsonsuccessnet.com/iText/products/0‐13‐115075‐8/text/chapter36/concept36.1.html
Before bioavailability:
A-Transformation from
products: speciation,
surface properties and
stability
B-transfer
diffusion
Ionic stren
gth
coagulation
pH
Inorganic colloïds
III. D-Biodegradation
Toxicity
bioavailability
Organic molecules
pollutants
diffusion
Biofilms
C-Interaction with
matter
floculation
A- Transformation
Ex 1:Fate of C60 fullerenes in water
from hydrophobic to hydrophilic material
stirring
ultra
ce
io
ugat
f
i
r
t
n
n
stable dispersion of nanometric
crystallites (nC60) Evidence of C60 hydrophilic character
gravimetric measurement of H2O vapour adsorption
at 30°C, and ambient pressure H2O
C60 fullerite
stirring
number of H 2O monolayers adsorbed
10
9
8
7
6
5
4
3
2
1
0
0
Previous outgassing (110°C, 18h)
0.2
0.4
0.6
0.8
1
P (H2O) / P 0
multi‐layer adsorption of H2O
irreversible modification of the surface
Brant et al., 2007 JCIS
Labille et al., 2006 Fullerenes Nanotubes and Carbon Nanostructures
Labile et al., 2009, Langmuir, in press
Chemical characterisation of AQU/nC60s
FTIR
0.4
CO‐H
H2O
Absorbance
nC60
pure C60
H2O
C‐OH
0.6
0.2
1H SS NMR
0.0
fullerol
4000
3000
2000
1000
-1
Wave number (cm )
hydroxylation of the nC60 surface gives
hydrophilic surface
AQU/nC60
C60
14
Labille et al., langmuir, in press 2009
12
10
8
6
ppm
4
2
0
-2
-4
Ex 2: Case of Titanium dioxide-based nanocomposite
• Nano‐TiO2 in sunscreen
TiO2
CH3
Si
CH3
O
n
PDMS
AlOOH
AlOOH
14-16 nm
50 nm
PDMS
Leaching of the surface layers may
lead to a direct contact between
water and TiO2. TiO2 can then
generate ROS (O2• O2- OH •)
TiO2
UV
TiO2
O2• O2- OH •
J Labille et al (Env Pollution) 2010 and M Auffan et al, ES and T et al, ES and T 2010
UV alteration of PDMS coating ?
B‐Transfert and diffusion in porous media Case of TiO2 in sand
Case of Nano Ag°
0.6
NaCl
attachment coefficient
0.5
CaCl2
NaCl + gellan
0.4
NaCl + A. tannic
0.3
0.2
0.1
0
1.E-04
1.E-03
1.E-02
1.E-01
1.E+00
salt concentration (mol/L)
N Solovitch et al, Environmental Science
And Technology 2010
Sand Sand and FeOOH
Shihong Lin et al , Duke
C‐Effect of the surface functionalisation on the NP dispersion
Coating is often used by suppliers to improve NP dispersion, but is rarely specified.
electrostatic or sterric repulsions
favour dispersion
bio‐
available
stabilisation
ionic strength
pH
no surface charge
favours aggregation
not
bio‐
available
aggregation
C‐Aggregation kinetic vs adsorption onto biological surface: CeO2 + salt + cells
In water : stable NPs
Initial conditions:
3 mg/l NPs 108 cells/ml
only adsorption
In medium :
unstable NPs =
adsorption + aggregation
t ~ 1s
Zeyons et al, ES and T 2009
Adsorption of CeO2 onto biological membrane and floculation
Synechocystis PCC 6803
E. coli
Zeyon, Thill et al
Forte affinité vis à vis du vivant Ex E Coli or Synechocystis cells mais pas de toxicité si
absence de contact direct
Zeyon, Thill et a ES and T 2008 et Langmuir 2009
D‐ Biodisponibility:
Strong affinity for Eucaryotes cells and endocytosis: ex γFe2O3 or CeO2
M.Auffan et al, ES and T 2006; M Auffan et al, Nanotoxicology 2009
Toxicité: mécanismes
Adapted from A Nel UCLA 2009
Environmental behavior and (eco)toxicity
• Trophic transfer via
food webs
A terrestrial
food chain
A marine
food chain
Quaternary consumers
Hawk
Killer whale
Snake
Tertiary consumers
Cod
Secondary consumers
Mouse Herring
Transformation?
Primary consumers
Grasshoper Zooplancton
Majorité des travaux
Producers
Flowers Phytoplancton bacterias..
http://www.pearsonsuccessnet.com/iText/products/0‐13‐115075‐8/text/chapter36/concept36.1.html
Toxicité vis à vis des micro‐organismes bactériens
Dissolution/aggregation
NP Reactivity
ROS production
Membrane Integrity
test Rouge neutre
O2‐, O2•‐, OH•
Cristalline Defects
TEM, Diffraction
mitochondrial
activity
ROS detection
WST1 test
Surface
Spéciation
and reactivity
EXAFS, XANES, RMN, FTIR
Internalisation
TEM; Micro‐fluorescence..
oxydant
stress
Anti‐oxydants, mutant strains
ADN
synchrotron
ROS
Cytotoxicity
comets tests
Chromosomes
Micronuclei tests
Génotoxicity
Réglons la question: Est ce que l'agrégation contrôle la toxicité ? Nano‐γFe2O3
enrobées
Nano‐CeO2
Toxique
Non toxique
50‐100 nm
Cellules
Eucaryotes
2.5‐3 μm
Two examples
CeO2 = Slow Chemical solubility Fe° = Very weak stability
Interactions NP / procaryote cells on mutant and wild strains
Fe0
CeO2
E.coli
7nm
γFe2O3
E.coli
< 20nm
E.coli
6nm
No cytotoxicity
Cytotoxicity ≤ 10 mg/L
30% of Ce(IV) ‐> Ce(III) on
the surface of Cerine Ce4+
e‐,ROS production
Cytotoxic > 70 mg/L
No structiural modification
Oxydation of Fe°
Dissolution / precipitation
Fe0
No cytotoxic
γFeOOH
γFe23+O3
Ce3+
Fe3O4
Fe2+, ROS
Damage membrane
Toxicity is associated to the
chemical instability of NP
Redox cycle (Ce3+/Ce4+) Fenton Reaction (Fe2+)
Stress oxydatif through ROS production
stable
Zeyons et al, ES and T 2008; M Auffan et al, Nanotoxicology 2009
Interactions NP with eucaryote cells
CeO2
7nm
Ce4+
Reduction of Ce(IV) in
Ce(III) e‐,ROS
Ce3+
Stress oxydatif
ADN and chromosomic
Damages
60 mg/L
0,06 mg/L
control
micronoyaux
No cytotoxic effects but …
… génotoxicity at very low
concentrations > 0,06 mg/L
Xanes at L3 edge of Ce = 30% of reduced surface atoms M.Auffan et al, ES and T 2006;
M Auffan et al, Nanotoxicology 2009
Environmental behavior and (eco)toxicity
• Trophic transfer via
food webs
A terrestrial
food chain
A marine
food chain
Quaternary consumers
Hawk
Killer whale
Snake
Tertiary consumers
Cod
Secondary consumers
Mouse Herring
Travaux qui se développent:
cf I‐CEINT = mésocosmes
Primary consumers
Grasshoper Zooplancton
Producers
Flowers Phytoplancton bacterias..
http://www.pearsonsuccessnet.com/iText/products/0‐13‐115075‐8/text/chapter36/concept36.1.html
Physical-chemical characterization and
ecotoxicity of residues from alteration of
engineered nanomaterials
Ex: Ag°, Solar creams with TiO2
Toxicity of coated‐silver nanoparticles : C.elegans
PVP coated Nano‐Ag0
21 ± 17 nm
Ag0
Caenorhabditis
elegans
Wild and transgenic strains
Mtl2: deficient in protein involved in metal regulation and detoxification
M Auffan In collaboration with J. Meyer (DUKE university, USA)
Toxicity of coated‐silver nanoparticles : C.elegans
Dissolved Ag effect
24h
Mtl2 strain more sensitive than the wild
24h
48h
48h
72h
72h
Wild
Mtl2
PVP‐nanoAg
In collaboration with J. Meyer (DUKE university, USA)
Toxicity across a salinity gradient : Fundulus heteroclitus
Simulated silver speciation
Fundulus heteroclitus
Assessing toxicity across a salinity gradient
Sea Water
Tolerate
Low
Salt
PVP coated nanoAg0
Gum Arabic coated nanoAg0
21 ± 17 nm
7 ± 3 nm
In collaboration with C. Matson, R. Digiulio (DUKE university, USA)
Toxicity across a salinity gradient
Embryotoxicity of PVP‐nanoAg0: ‘V‐curve’ shape
Match
Match
Amount of dissolved Ag after interaction with the embryo
Embryotoxicity: GumArabic‐nanoAg0 > PVP‐nanoAg0
In collaboration with C. Matson, R. Digiulio (DUKE university, USA)
Example: Nanocomposite in sunscreen
PDMS = polydimethylsiloxane
Al(OH)3
TiO2
Al(OH)3
9 thin amorphous layer
9 improve PDMS coating
9 protection against TiO2
photocatalytic effects
PDMS = polydimethylsiloxane
9 hydrophobic properties
9enhance dispersion of the nanomaterial CH3
Si
14‐20nm
1O
2, O2
y‐, OHy
CH3
O
n
Alteration of nanocomposites: conclusion
UV/Vis
UV, stirring, water
X
Hydrophobic
surface
Hydrophilic
surface
No photocatalytic properties
Localization and toxicity on biological
organisms ?
O2
-
Introduction into chain food
CCD Camera
¾Adsorption of Ti on algae
Titanium map
X-Ray beam size: 10 mm
X-Ray source: Rh
X-Ray tube voltage: 30 kV
Counting time: 9x360
Localization and ecotoxic studies
CCD image
Adults Daphnia
9d exposure
Concentration: 10 mg/l
Calcium map
Titanium map
Combined map
XGT5000 Horiba Jobin Yvon
X-Ray beam size: 10 mm
X-Ray source: Rh
X-Ray tube voltage: 30 kV
Counting time: 20x360
Ecotoxicity results on Danio rerio
% hatching
Danio rerio embryos hatching time
Days of exposure (post fertilization)
¾ Premature hatching of embryos for all tested concentrations
¾ 100% hatching at 3 days post fertilization in contaminated media
¾ Living larvae
Ecotoxicity results on Danio rerio
% of alive larvae
Danio rerio larvae survival time
Days of exposure
¾For all tested concentrations: significant effect on survival time/control
¾First mortalities observed in contaminated solutions from 8d contact
Conclusion and future
• The complexity of the studies come from the complexity of the NM or NP in term of the chemistry, stability and reactivity.
M Auffan et al, Nature Nano 09‐2009
If the mechanisms of the toxicity must be studied on « laboratory » NP, the researches must focused also on engineered products containing NP knowing that the formulations are complexe. As the studies on the ecosystems are at the beginning we need to
coordinate the researches at the national level to have a systemic
approach allowing to assess the toxicity in food webs and Prioritize in vivo testing at increasing trophic levels
Try to be predicitive from the:
‐ knowledge and modelling of the interaction mechanisms of « nano »
with water, components, biota
‐Modelling of the kinetic nteractions of NP in the aqueous media (water molecules, solutes, aggregation ……
Study the interactions with biota within mesocosms managed by competent people and gathering the best labs around them.
La toxicité des résidus des nanomatériaux s’étudiera via des chaines strophiques
A terrestrial
food chain
* GDR‐I I‐CEINT = CEA‐CNRS‐
5 Univ US * Projet MESONNET ANR P2N
(2011‐2014)
* FR ECCOREV
A marine
food chain
Quaternary consumers
Hawk
Killer whale
Snake
Tertiary consumers
Cod
Secondary consumers
Mouse Herring
Primary consumers
Grasshoper Zooplancton
Producers
Flowers Phytoplancton bacterias..
http://www.pearsonsuccessnet.com/iText/products/0‐13‐115075‐8/text/chapter36/concept36.1.html
Thanks to the french partners in I‐CEINT and particularly to:
C Chanéac; J.P Jolivet; A Thill; A Botta, J.L Hazeman, O Proux….
And all the partners from CIRIMAT (Toulouse), ECOLAB (Toulouse) , HYGHES 5starsbourg), LCBM (Grenoble), , LBME (Marseille); LIEBE (Metz); IMEP (Marseille); IBEB (Cadarache)
Inst Neel (Grenoble); ESRF (Fame beam line)
Thanks to CNRS and CEA
And also thanks to the american partners in I‐CEINT:
M.R Wiesner (Duke); G Lowry (Carnegie‐Mellon); P Bertsch (Kentuky University); G Brown (Stanford); P Vikesland ……and many others