Aquatic nanotoxicolgy

Environnement Environment
Canada
Canada
Aquatic nanotoxicolgy
Research perspectives
Gagné , F., Gagnon, C., Blaise, C., Eullafroy, P.
Fluvial Ecosystem Research, AEPRD,
Water Science & Technology
Environment Canada, Montréal, Qc.
Presentation outline
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Preamble – nanotechnology
Program
g
description,
p
,p
partners and funding…
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Highlights on exposure characterization
Aquatic
q
toxicity
y tests
Different aspects of nanoparticle toxicity
Biomarkers research
Perspectives
Nanotechnology
Preamble
1) Nanomaterials (NM) are materials associated to the colloidal phase i.e., have properties
of fine particles and dissolved matter with high surface area;
2) NM refers to macromolecules where at least one dimension is in the 1
1-100
100 nm range;
3) It is estimated that investments in nanotechnology will reach 1 trillion $ by 2012;
4) The pollution picture will likely change and will require to revise,
revise or re
re-consider
consider some aspects
at least of, our current risk assessment paradigm.
« These NM will likely benefit our quality of life in many aspects such as
medical and industrial applications, biotechnology and even remediation
processes, they are not exempt of potential toxicity to humans and wildlife
(Moore 2006)
(Moore,
2006).»
»
Problems for the environment ?
(Believed it or not; here it comes
comes…))
A written survey of 40 companies working with nanomaterials
in Germanyy and Switzerland revealed that :
1) The nanomaterials examined exhibited such a diversity of properties that a
categorization according to risk and material issues could not be made;
2) Twenty-six companies (65%) indicated that they did not perform any risk
assessment of their nanomaterials and 13 companies (32.5%) performed
risk assessments sometimes or always;
y
3) Fate of nanomaterials in the use and disposal stage received little attention by
industry and the majority of companies did not foresee unintentional release of
nanomaterials throughout the life cycle
cycle.
Helland et al., Environ. Sci. Technol. 2008, 42, 640–646
Nanotechnology
Preamble
« The space domain of these NM is congruent to the space where
biological macromolecules interact to assist life-related processes »
Nanotechnology will likely increase pollution at this scale with perhaps
unprecedented toxic effects.
The nanotoxicology research program at Montréal
Ecotoxicology
of nanomatérials
(NM)
Occurrence and persistence
in aquatic ecoystems
(C. Gagnon, P. Turcotte)
Size fractionation of NM
Occurrence and fate
in effluents and
surface waters
Bioavailability in fish
and mussels
Microbiotests
(Blaise, C., M.Douville,
S. Trépanier)
Biomarkers research
(J. Auclair, F. Gagné)
Toxicity of NMs
(microbioassays)
Solid and liquid phases
Allosteric toxicity
Oxidative stress
Genotoxicity
Effects of particle size and
surface properties
Effects studies in mussels
exposed to ME
Toxicogenomics
(G. vanAggelen,
K. Bull, F. Gagné)
Trout genomics
Bivalve genomics
Partners….
1) St-Lawrence River toxicology research network (CIRE)
- A network of university and governmental researchers dedicated to
ecotoxicology
gy of the SLR;
- Aquatic nanotoxicology node (FG) was created in 2007
http://www.ecotox.uquebec.ca/programmes.htm;
2) Prof. Michel Fournier, Immunotoxicology of nanotechnology (IAF-INRS)
3) Dr Tibor Kovacs and Pierre Martel. Pulp and paper research institute of Canada
(FPinnovations-PAPRICAN) on nanocrystalline cellulose (tensil strenght > steel);
4)) Dusica Maysinger,
y g Pharmacology
gy Department,
p
Mc Gill University
y
- Ageing effects on the cytotoxicity of coated CdTe quantum dots to fish liver cells;
5) Geoffrey Sunahara, Bernard Lachance, Pierres-Yves Robidoux, Biotechnology Research
Institute, Montréal
- Evaluation of CdTe quantum dots to human cell lines; effects of size of the
nanoparticle;
6) Sébastien Sauvé, Chemistry dept, Montréal University
- Bioavailability and toxicity of CdTe quantum dots in freshwater mussels;
7) Graham vanAggelen at EC in vancouver (PESC)
- Toxicogenomics of CdTe quantum dots in rainbow trout (DNA microarrays).
OECD list: a proposed working plan
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Fullerenes (C60)
(
)
SWCNTs
MWCNTs
Silver nanoparticles
I
Iron
nanoparticles
ti l
Carbon black
Titanium dioxide
Aluminum dioxide
Cerium oxide
Zinc oxide
Silicon dioxide
Polystyrene
Dendrimers
Nanoclays
We should try to focus on the NMs that are:
1) likely to be found in water column/suspended
matter;
2) produced at sufficient quantity ;
3) analytical feasibility;
4) th
those acting
ti as ““endocrine
d i di
disrupters”
t ” ii.e.
producing effects at very low concentrations.
Exposure characterization
in aquatic ecosystems
Issues
1) Where will NM partition in the aquatic environment?
Solubility of nanomaterials in distilled water1.
Nanomaterials
Chemical
compositon
Solubility2
(%)
Solubility
rank
Copper zinc iron oxide
CuZnFe2O4
2
4
Nickel zinc iron oxide
NiZnFe2O4
2
4
Yttrium iron oxide
Y3Fe5O12
4
3
Titanium oxide
TiO2
2
4
Fulleren-C60
C60
9
2
Strontium ferrite
SrFe12O19
4
3
Indium-tin oxide
In2O3 • (SnO2)x9
0.8
5
Samarium oxide
Sm2O3
3
3
Erbium oxide
Er2O3
0.9
5
Holmium oxide
Ho2O3
2
4
Single-walled carbon nanotube
C
2
4
Quantum dots
CdTe
20
1
1. A suspension
p
of the nanopowders
p
were resuspended
p
in distilled water and allowed to mix for 24 h
at room temperature in the dark. After this mixing period, the solution was filtered through a 0.22 µm
pore membrane and filtrate materials were analyzed by gravimetry.
2. Colloidal partition coefficient was determined by the dry weight of the eluate/dry weight of the
suspension expressed in percentage.
Issues
1) Where will NM partition in the aquatic environment?....continued.
NOM caused disaggregation of nC60 crystals and aggregates under typical solution conditions of
natural water, leading to significant changes in particle size and morphology;
these effects increased with increasing NOM concentration (Xie et al., 2008. Environ. Sci.
Technol. 42, 2853–2859).
Environmental fate of nanomaterials (C. Gagnon)
Exposure characterization
in aquatic ecosystems
Nanoparticles
- Processes
P
in
i A
Aquatic
ti E
Environment:
i
t
Ex.: Quantum dots
Aggregation
CdTe
Dissociation
~ 5 nm
Cd
Te
Cd2+, Cd-L
Cd
Membrane
permeable
Metal associations
Bioaccumulation,
Toxicity
Particle size fractionation by
ultrafiltration CdTe
100
%C
Cd per fractio
on
Milli-Q water
Surface water
10
1
Total
< 0,45
0 45 µm < 0,1
0 1 µm
< 30 KDa
Size distribution
< 10 KDa
< 1 KDa
Aquatic toxicity tests
1) Toxicity of 11 NMs using a test battery (bacteria, algae, microcrustacean,
Hydra and trout hepatocytes)
Toxicity classification of nanopowders based on European Union Commission Guideline 93/67/EEC
and the most sensitive bioassay measurement endpoint values (i.e., LCx/ECx/ICx, etc).
Extremely
Toxic
(< 0.1 mg/L)
Very Toxic
(0.1 – 1 mg/L)
Toxic
(1-10 mg/L)
Not Toxic
Harmful
(10-100 mg/L) (> 100 mg/L)
NiZnFeO
TiO
YFeO
SmO
SrFeO
ErO
Ful-C60
CuZnFeO
InSnO
CNSW
HoO
Toxic spread index
Median
Min-Max
(155)
CNTSW
(19)
Ful-C60
(25)
Nanopro
oducts
ErO
SmO
(108)
(100)
InSnO
(6.5)
SrFeO
(229)
HoO
(48)
TiO
YtFeO
(16)
NiZnFeO
(134)
(71)
CuZnFeO
1
5
50
Concentration (mg/L)
500
Biomarker research activities
Fundamental aspects of nanomaterials.
The introduction of nanoparticles in aquatic ecosystems will bring about and new potentially toxic interactions
in exposed organisms.
Primary interaction
from the leaching of
constitutive molecules
Size effects
(aggregation)
Surface properties
local electric/ magnetic fields,
energy transfers leading to
reactive oxygen species
Adducts
Add
t
Detoxication mechanism
Elimination
Change in protein folding,
Loss of structure-function equilibrium,
DNA destabilization,
Protein turnover (ubiquitinylation)
Modulation of electric field gradients
in membranes for energy production,
electrolyte balance and electric nerve
current and muscle tonic contraction
x
x
x
Vector function
Carrier of chemical
pollutants
Gagné, F., Gagnon, C., Blaise, C. (2008) Aquatic Nanotoxicology: a review. Research Trends:
current topics in toxicology, In press.
1) Bioreactivity of CdTe quantum dots in rainbow trout liver extracts
P
Parameter
t
R t off change/hour
Rate
h
/h
S12
S12,
NADPH
S12
CdTe
S12,
CdTe,
Ob
Observations
ti
NADPH
S12,
CdTe,
MT
S12,
CdTe,
H2O2/
Per
NAD(P)H
- 0.002
-5.7
0.10
5.1
-0.07
0.091
CdTe blocks the oxidation of
NAD(P)H and this is reversed by
the addition of MT and addition of
H2O2
Qdot
fluorescence
0
0
-1.6
-1.9
-2.12
-1.53
The addition of S12 decreases
Qdot fluorescence where the drop
is enhanced by MT and lowered
by H2O2
Labile zinc
0.0002
0.046
- 0.18
- 0.15
-0.091
-0.032
The addition of CdTe decreases
labile zinc and this is hindered by
MT > H2O2 > NADPH
Experiment conditions: S12 proteins: 250 ug/mL; NADPH: 100 uM; MT: 10 ug/mL; H2O2: 100 uM; peroxidase (100 ug/mL)
Incubation temperature: 15oC
Change in surface charge density
CdTe are semisemi-conductors
M+
Electron dense
NADP+
« Filled » holes
NADPH
Electronic holes
M+
Metal
mobilization
The presence of electronic holes on the surface enables to trap
external charges and reduce radiative emission intensity (indeed,
a drop of green fluorescence was observed)
2) Change in heavy metals metabolism, oxidative stress, DNA damage and
heat shock proteins by QD exposed to rainbow trout hepatocytes
Discriminant analysis of QD cytotoxicity effects in rainbow trout hepatocyte
hepatocyte.
5
4
Root 2
(DNA>Hsp7
72>MT)
3
2
*
1
*
0
*
-1
*
-2
0.4
2
10
50
250
Control
Cd2+
*
*
-3
-4
4
-8
*
-6
-4
-2
0
2
4
6
8
Root 1
(Via>MT>Zn/Cd)
Discriminant analyses were performed on CdTe QD. The effect endpoints in parentheses are the
principal components of the axis. The cumulative variance explained 95% respectively. Asterisks (*)
indicate the centre of gravity of each treatment group. Evidence of toxic steric interactions (size
related effects).
3) Toxicogenomics of CdTe in rainbow trout (96h exposure):
analysis in the liver.
CdTe
(colloidal)
50 genes
-Vtg (!)
- Vn envelop
p
- Dop D2 receptor
- CYP1A2
- C4 gene
- chemokine receptor
- Glu DHase
- Gln
Gl synthase
h
- cystatin C
- cathepsin L
- ubiquitin
…
CdSO4
(dissolved)
8
g
genes
-MT 1A
- actin
- Apolipoprot
- Cyt. c oxidase
- CYP2K1, 3A27
….
17 genes
-COX (prostaglandin)
- cathepsin
p
D
- DNA exotransferase
- cystatin C
….
1) Over the 207 genes analyzed, 21 % of them were affected by CdTe while 6 % were influenced
by dissolved Cd;
2) CdTe produced a larger spectrum of effects than dissolved Cd
Cd, 12 % of the expressed genes
were expressed by both colloidal and molecular Cd;
3) The QD was able to induced Vtg and vitellin coat suggested an endocrine disrupting activity;
4) The inflammatory properties was evident for dissolved Cd in the liver.
F t
Future
perspectives
ti
1) Preliminary data on NM solubility suggest that they tend to aggregate and should
favor the sediment compartment over the water compartments
- organic matter or other matrix effects could change the equilibrium…
- ingestion through feeding should also be considered;
2) The NM are expected to degrade in the environment and are biologically reactive;
3) The toxic effects of NM are not only related to its chemical constituents but to
the size, shape and surface properties as well;
4) For CdTe QDs, they were cytotoxic and induced protein chaperones
(first biomarkers for field monitoring studies ?); Moreover, the responses pattern were
not always related to free cadmium ions;
5) Toxicogenomic analysis revealed that colloidal CdTe revealed a different pattern of
gene expression than dissolved Cd. Moreover, CdTe was able to induce the expression
vitellogenin (a biomarker for estrogenic effects), however the mechanism remains to be
elucidated.
elucidated
Are NMs potential endocrine disrupters ?
L’équipe EC du programme
Impacts environnementaux en nanotechnologie
1) Chimie colloïdale environnementale (nanochimie aquatique)
Christian Gagnon
(Spéciation de la nanotechnologie)
Patrice Turcotte:
chimie des colloïdes
2) Nanotoxicologie Aquatique (microbioessais et biomarqueurs)
Christian Blaise
(microbioessais)
Philippe Eullafroy
(Phyto-toxicologie)
Joelle Auclair
(Biomarqueurs
physiologiques)
Kimberly Bull
(Toxicogénomique)
Thank you !
Publications in nanotoxicology:
1) Gagné, F., Auclair, J., Turcotte, P., Fournier, M., Gagnon, C., Sauvé, S., Blaise, C. (2008) Ecotoxicity of
CdTe quantum dots to freshwater mussels: Impacts on immune system, oxidative stress and
genotoxicity.Aquatic Toxicol. 86, 333-340.
2) Blaise, C., Gagné, F., Férard, J.F., Eullafroy, P. (2008) Ecotoxicity of selected nano
nano-materials
materials to aquatic
organisms. Environ Toxicol., In press.
3) Gagné, F., Maysinger, D., André, C., Blaise, C. (2008) Cytotoxicity of aged cadmium-telluride
quantum dots to rainbow trout hepatocytes. Nanotoxicology, In press.
4) Gagné, F., Gagnon, C., Blaise, C. (2008) Aquatic Nanotoxicology: a review. Research Trends: current
topics in toxicology, In press.
5)) Santos, M.A., Monteiro, R. T. R., C. Blaise, F. Gagné,
g Bull, K.2008. Influence of sediment g
grain size on
elutriate toxicity of inorganic nano-materials. Water Res. J. Canada, Submitted.
6) Gagné, F., Auclair, J., Turcotte, P., Gagnon, C. (2008). Bioreactivity and sublethal effects of CdTe quantum
dots to rainbow trout hepatocytes.Comp. Biochem Physiol, In preparation.
7) Peyrot, C., Gagnon, C., Gagné, F., Willkinson, K.J., Turcotte, P., Sauvé, S. 2008 Effects of cadmium telluride quantum
dots upon the bioaccumulation of cadmium and the methallothionein production of the freshwater mussels
Elliptio complanata. Environ Toxicol. Chem., In preparation.