Universidade Federal de Santa Catarina
COMPARATIVE EFFECTS OF ZINC OXIDE TOXICITY IN BULK,
NANOPARTICLE AND NANORODS FORMS TO
Daphnia magna and Aliivibrio fischeri
Cristiane Funghetto Fuzinatto, Dra.
Bianca Vicente Oscar, Eng.
Renata Amanda Gonçalves, Msc.
Sílvia Pedroso Melegari, Dra.
Denice Schulz Vicentini, Dra.
William Gerson Matias, Dr.
Introduction
1
Introduction
o
Nanotechnology
Engineered nanoparticles (ENs), defined as particles smaller than 100nm
in at least one dimensions with properties different from their bulk
materials.
They are classified into five groups based on their chemical composition:
Hair
•
•
•
•
•
carbon related materials;
metal containing products (including metal oxides);
Virus
semiconductor nanocrystals;
Carbon
nanotubes
zero-valent metals;
Atoms and
dendrimers.
molecules
nanometer
(nm)
Erythorcytes
micron
(µm)
milimeter
(mm)
Source: Toma, 2004. Modified by Cristiane Fuzinatto.
2
Introduction
o
Nanotechnology
Worldwide consumption of Zinc Oxide (ZnO)
Because of its diverse properties, both chemical and physical, ZnO is
widely used in many areas.
It plays an important role in a very wide range of applications, ranging from
tyres to ceramics, from pharmaceuticals to agriculture, and from paints to
chemicals.
40
38%
35
31%
30
25
18%
20
15
10
7%
5
2%
4%
0
Far East
Africa
Near East
South
America
Europe
North
America
Adapted from Kołodziejczak-Radzimska and Jesionowski (2014).
3
Introduction
Nanotechnology
Application of ZnO NMs
TEXTILE INDUSTRY
Absorver of radiation
RUBBER INDUSTRY
Filters, activator of rubber
compounds
PHARMACEUTICAL AND COSMETIC
INDUSTRY: Component of creams,
powders, dental pastes, etc., absorver of
UV radiation
PHOTOCATALYSIS
used as
Photocatalyst
ELECTRONICS AND
ELETROTECHNOLOGY INDUSTRIES:
photoelectronics,
field
emitters,
sensors, UV lasers, solar cells etc.
MISCELLANEOUS APLICATIONS
Used in: production of zinc silicates,
biosensor, process of producing and
packing meat and vegetables products etc.
4
Introduction
o Increased production of ZnO NMs
x Biological impacts of ZnO NMs
Potential for release and exposure to nanoscale substances
Nanomaterial Synthesis Feedstock
Human Exposure
(Occupational)
Product Manufactures
Products
Products
Consumers
Attrition
Products
Human Exposure
(Public)
Disposal
Environment
Ecological
Exposure
Adapted from: Tsuji et al., 2006. Research Strategies for Safety Evaluation of Nanomaterials, Part IV: Risk Assessment of
Nanoparticles. TOXICOLOGICAL SCIENCES 89(1), 42–50.
5
Introduction
o
ZnO NMs toxicity
There is an urgent need to understand their toxicity to organisms and the
environment.
Adapted from Bondarenko et al., 2013.
6
Introduction
o
ZnO NMs toxicity
Modification on particle properties: NPs’ characteristics that
can be controlled could influence toxicity
TOXIC
Adapted from Chang et al., 2012.
7
Introduction
Goals
To elucidate the environmental fate and potential toxicity of ZnO NMs the
present research has two aims:
• Synthesize and characterize the fundamental properties two different
shapes of ZnO NM (ZnO as nanoparticles - ZnO NP - and ZnO as
nanoroads - ZnO NR).
• Evaluate the potential acute toxicity of different morphologies of ZnO
to the microcrustacean Daphnia magna and the marine bacteria
Aliivibrio fischeri, in comparison to ZnO bulk form.
8
Materials and Methods
9
Materials and Methods
o
Synthesis of
ZnO NP and ZnO NR
ZnO NP
ZnO NR
7 g of Citric Acid
50 mL of H2O
13,20 g of Zinc
Acetate
60 mL of MeOH
Heated at 60°C
Acid solution
1,5g of ZnO
Heated at 70°C
Metalic solution
1 g of
Ethyleneglycol
7,4 g of KOH
40 mL of MeOH
Shake
Mix of solubilized
products
Shake and Reflux, 60°C
Heat until concentrate sample
Polymeric resine
Calcinate 350°C for 1h
Expanded resine
1 - Mashed
2 - Calcinate 500°C for 1h
ZnO NR
1 – Wash with H2O (3x);
2 – Wash with EtOH (3x);
3 – Dry, 50°C, 24h.
ZnO NR
ZnO NP
(Yang & Liu, 2011)
Polymeric Precursors Method (Costa et al., 2007)
10
Materials and Methods
o
Characterization of
ZnO-NP and ZnO-NR
Characterization
Method
Specifications
Location of the
laboratory
Size and morfology
TEM
TEM JEM 1011
LCME, UFSC (Brazil)
Chemical purity
XRD
X’Pert, Philips
LCME, UFSC (Brazil)
Zeta Potential
Electrophoretic
mobility
ZetaSizer, Malvern
GEIMM, UFSC (Brazil)
Hydrodynamic size
DLS
ZetaSizer, Malvern
GEIMM, UFSC (Brazil)
11
Materials and Methods
o
Toxicity Tests
D. magna
Test Organism: D. magna Straus, 1823.
Principle of the test: Inhibition of swimming ability
according to ISO 6341 (ISO, 1996) and OECD 202
(OECD, 2004).
NMs ZnO tested:
- Bulk;
- NP;
- NR.
Acute toxicity test with D. magna
Negative controls
Dilutions
Becker
50 mL
Medium test: ISO
Results: EC50,48h
10 D. magna neonates per becker
12
Materials and Methods
o
Toxicity Tests
A. fischeri
Test Organism: Bacteria suspension of A. fischeri
Principle of the test: Luminescence inhibition test performed
according to ISO 11348-3 (ISO, 1998).
Equipment: Microtox 500©
NMs ZnO tested:
- Bulk;
- NP;
- NR.
Medium test: NaCl 2%
Results: EC50,15min
13
Results and Discussion
14
Results and discussion
Characterization of Nanoparticles
ZnO NR
ZnO NP
TRANSMISSION ELECTRON MICROSCOPY (TEM)
TEM images confirmed the elongated
morphology of 2D-type ZnO NR with
diameters between 30 and 40 nm and lengths
between.100 – 200 nm.
X-RAY DIFRACTION (XRD)
ZnO NP with diameters between 20
and 50 nm.
Intensidade u.a
NP ZnO
10
ZnO NR
20
30
40
50
60
70
80
2
The diffractogram obtained showed the purity of the crystalline phase of hexagonal ZnO NP
synthesized. The structure of NP was identified as international database card (JCPDS No. 361451 CARD).
15
Results and discussion
Acute toxicity to D. magna
NM
Suspension
pH
CE50,48h
(mg/L)
Zn+2
(mg/L)
DLS
(nm)
Zeta
(mv)
Bulk ZnO
7,90
NT
-
-
-
ZnO NP
7,88
2,7±0,22
0,38±0,04
235,72±58,34
12,61
ZnO NR
7,73
2,9±0,18
0,39±0,01
392,78 ±148,26
11,35





Similar toxicity to different morphologies
Zeta potential indicate instability of both suspensions
DLS size much higher for NP and NR powder
Medium agglomerations of NM on media
Toxicity can be attributed to Zn+2 dissolved in medium test
16
Results and discussion
Inhibition luminescent test to A. fischeri
NM
Suspension
pH
CE50,15min
(mg.L-1)
Zn+2
(mg.L-1)
DLS
(nm)
Zeta
(mv)
Bulk ZnO
7,32
19,21±2,39
6,70±0,55
796,59±205,28
-14,25±0,91
ZnO NP
7,50
12,3±1,43
1,03±0,13
538,16±133,50
-8,74±1,10
ZnO NR
7,33
28,8±2,52
0,45±0,04
1248,14 ±161,01
-9,35±1,34




ZnO NR more toxic than Bulk and NP
Zeta potential indicate instability of NMs suspension
DLS size of NR much higher than NP
Large agglomerations of NM on media
17
Conclusions
18
Conclusions
 For D. magna both ENMs showed higher toxicity when compared to
Bulk form.
 Different morphologies of NM showed similar toxicity.
 The toxicity were attributed to instability of NM solution and the Zn+2
dissolved in the medium test.
ZnO + H2O → Zn+2 + H2O2
 For A. fischeri, the morphology of ZnO affected in the toxicity:
NR were more toxic than NP and Bulk forms
 Further researches are being done to understand the behavior of ZnO in
different medium tests.
 Studies related to the chronic effect and penetration of ZnO in the
organism D. magna are being conducted.
19
Acknowledgements
Rede cooperativa de pesquisas em nanotoxicologia aplicada a
nanopartículas de interesse da indústria petrolífera e de tintas.
Projeto CAPES/PNPD Institucional 2590/2011 Nanotoxicologia: métodos
toxicológicos, genéticos e epigenéticos como uma estratégia para avaliar
o risco de nanopartículas ao ambiente.
Equipe:
William Gerson Matias, Dr –
Coordenador do LABTOX
Ana Letícia O. F. Rossetto, MSc –
Doutoranda
Bianca V.Oscar, IC
Cristiane Funghetto Fuzinatto, Dra
Denice S. Vicentini, Dra.
Karine G.de Oliveira, Mestranda
Martina Garcia de Cezar, IC
Miriam Arl, bolsista AT
Paula Magro, IC
Renata A.Gonçalves, MScDoutoranda
Rodrigo C. Puerari, Mestrando
Silvia P.Melegari, Dra
Apresentação disponível em nosso website: www.labtox.ufsc.br
20
Acknowledgements
21
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