Accepted Manuscript Title: Toxicity Assessment Of Tio2 Nanoparticles In Zebrafish Embryos Under Different Exposure Conditions Author: Z. Clemente V.L.S.S. Castro M.A.M. Moura C.M. Jonsson L.F. Fraceto PII: DOI: Reference:
S0166-445X(13)00374-3 http://dx.doi.org/doi:10.1016/j.aquatox.2013.12.024 AQTOX 3719
To appear in:
Aquatic Toxicology
Received date: Revised date: Accepted date:
31-10-2013 13-12-2013 18-12-2013
Please cite this article as: Clemente, Z., Castro, V.L.S.S., Moura, M.A.M., Jonsson, C.M., Fraceto, L.F.,Toxicity Assessment Of Tio2 Nanoparticles In Zebrafish Embryos Under Different Exposure Conditions, Aquatic Toxicology (2014), http://dx.doi.org/10.1016/j.aquatox.2013.12.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
TOXICITY ASSESSMENT OF TiO2 NANOPARTICLES IN ZEBRAFISH
2
EMBRYOS UNDER DIFFERENT EXPOSURE CONDITIONS
ip t
3 Clemente, Z.a,b; Castro, V.L.S.S.a; Moura, M.A.M.c; Jonsson, C.M.a; Fraceto, L.F.b,d
4
cr
5 6 a
Laboratório de Ecotoxicologia e Biossegurança, Embrapa CNPMA, Jaguariúna, SP, Brazil.
8
b
Programa de Pós-graduação em Biologia Funcional e Molecular, UNICAMP, Campinas, SP,
9
Brazil.
10
c
11
Brazil.
12
d
15 16 17 18
M
d
Departamento de Engenharia Ambiental, UNESP, Sorocaba, SP, Brazil.
te
14
Laboratório da Ciência das Plantas Daninhas, Instituto Biológico, APTA/SAA, Campinas, SP,
Ac ce p
13
an
us
7
*Corresponding author. E-mail: [email protected], [email protected]
Page 1 of 40
18 ABSTRACT
20
The popularity of TiO2 nanoparticles (nano-TiO2) lies in their wide range of
21
nanotechnological applications, together with low toxicity. Meanwhile, recent studies have
22
shown that the photocatalytic properties of this material can result in alterations in their behavior
23
in the environment, causing effects that have not yet been fully elucidated. The objective of this
24
study was to evaluate the toxicity of two formulations of nano-TiO2 under different illumination
25
conditions, using an experimental model coherent with the principle of the three Rs of alternative
26
animal experimentation (reduction, refinement, and replacement). Embryos of the fish Danio
27
rerio were exposed for 96 h to different concentrations of nano-TiO2 in the form of anatase (TA)
28
or an anatase/rutile mixture (TM), under either visible light or a combination of visible and
29
ultraviolet light (UV). The acute toxicity and sublethal parameters evaluated included survival
30
rates, malformation, hatching, equilibrium, and overall length of the larvae, together with
31
biochemical biomarkers (specific activities of catalase (CAT), glutathione S-transferase (GST),
32
and acid phosphatase (AP)). Both TA and TM caused accelerated hatching of the larvae. Under
33
UV irradiation, there was greater mortality of the larvae of the groups exposed to TM, compared
34
to those exposed to TA. Exposure to TM under UV irradiation altered the equilibrium of the
35
larvae. Alterations in the activities of CAT and GST were indicative of oxidative stress, although
36
no clear dose-response relationship was observed. The effects of nano-TiO2 appeared to depend
37
on both the type of formulation and the illumination condition. The findings contribute to
38
elucidation of the factors involved in the toxicity of these nanoparticles, as well as to the
39
establishment of protocols for risk assessments of nanotechnology.
Ac ce p
te
d
M
an
us
cr
ip t
19
40 41
Key words: FET, ultraviolet, nanotoxicology, biomarker, oxidative stress. Page 2 of 40
42
1. INTRODUCTION
43 Titanium dioxide nanoparticles (nano-TiO2) are used industrially in the areas of
45
cosmetics and pharmaceuticals, amongst others, as well as in environmental applications, and are
46
increasingly encountered in daily life. The use of these materials can often be beneficial, while at
47
the same time questions remain concerning the environmental risks of nanotechnology. The fast
48
development of nanotechnology requires practical ecotoxicological tools to correctly evaluate its
49
safety. On the other hand, scientific research using animals has been much discussed in terms of
50
bioethics, and there have been renewed efforts to identify alternative techniques that do not
51
require the use of animals.
M
an
us
cr
ip t
44
Tests employing fish embryos (the Fish Embryo Test, FET) fits between traditional
53
studies employing cell cultures and those that use mammalian models (Lin et al., 2013). The
54
results obtained with FET have a strong correlation with the outcomes of acute toxicity tests
55
using adult fish (Knobel et al., 2012), and it is likely that the embryos do not have the same
56
perception of pain as the adults, due to the immaturity of the nervous system (Lammer, 2009).
57
The zebrafish (Danio rerio) is widely used in FET because of its large numbers of eggs laid,
58
rapid development, the transparency of the eggs, and its similarity with human genome
59
(Braunbeck and Lammer, 2006; Howe et al., 2013). To date, there have been few studies that
60
have applied the FET method in nanoecotoxicological assessments. However, its use is
61
promising, especially considering the wide variety of nanomaterials that have emerged over a
62
very short space of time, the small volumes involved in the tests, and the low levels of waste
63
generation.
Ac ce p
te
d
52
64
There is evidence that nano-TiO2 can adhere to the chorion of the embryo, where it can
65
be absorbed and then distributed uniformly throughout the tissues of the fish, without any tissuePage 3 of 40
specificity (Bar-Ilan et al., 2012). The accumulation of Ti has also been observed in tissues of the
67
larvae of D. rerio, especially in the intestine (associated with the microvilli), the gill lamellae, the
68
liver, and the skeletal muscle (Bar-Ilan et al., 2013). Studies with aquatic organisms such as
69
hydra, microcrustaceans and fish have indicated that the toxicity of nano-TiO2 is negligible
70
(Blaise et al., 2008; Griffith et al., 2008; Yeo and Kang, 2010; Xiong et al., 2011; Clemente et al.,
71
2013). Similar findings have been reported for fish embryos (Zhu et al., 2008; Paterson et al.,
72
2011; Ma et al., 2012b). Nevertheless, in these bioassays employing aquatic organisms, the
73
circadian cycle is usually established using fluorescent lamps, which mainly emit visible light.
74
TiO2 is a semiconductor with the important property of being able to be photoactivated, which
75
makes it especially attractive for use in processes based on heterogeneous photocatalysis to
76
degrade a variety of organic and inorganic compounds (Nogueira and Jardim, 1998; Gaya and
77
Abdullah, 2008). Exposure of TiO2 to ultraviolet radiation (UV) in the wavelength range 300-388
78
nm results in the production of reactive oxygen species (ROS) that can cause damage to
79
biomolecules. The photocatalytic properties of TiO2 are enhanced when the compound is present
80
in the form of nanoparticles, which can increase its toxicity to aquatic organisms under
81
environmental conditions with exposure to solar UV radiation (Ma et al., 2012a; Marcone et al.,
82
2012; Clemente et al., 2013; Xiong et al., 2013).
cr
us
an
M
d
te
Ac ce p
83
ip t
66
TiO2 occurs in different crystal phases, being the photocatalytic properties and toxicity
84
of anatase higher than rutile (Malato et al., 2009; Allouni et al., 2012). Evidence has been found
85
for synergism between the phases, with anatase/rutile mixtures being more photoactive than the
86
pure phases. The nano-TiO2 known as P25®, manufactured by Evonik Degussa, is the mixture
87
that is most commonly used in photocatalytic processes (Nogueira and Jardim, 1998; Malato et
88
al., 2009). Earlier work in our research group indicated that exposure to nano-TiO2 can cause
89
sublethal effects in fish and microcrustaceans (Clemente et al., 2013), depending on the crystal Page 4 of 40
phase, concentration, and illumination conditions. Nonetheless, there remain a number of
91
uncertainties, as a result of which further work is needed to fully evaluate the toxicity (especially
92
sublethal effects) of nano-TiO2 in the fish embryos.
ip t
90
Effects on reproduction, as well as on the development and survival of new generations,
94
could lead to serious impacts on ecosystems. There is therefore a need for careful scrutiny of any
95
effects of nano-TiO2 on embryos and larvae exposed to the substance. To this end, biomarkers
96
can be used as effective early warning systems, and are often more useful than direct
97
measurements of a chemical agent in the organism. Since oxidative stress is probably the main
98
cause of the toxicity of nano-TiO2, the investigation of biomarkers associated with this effect, as
99
antioxidant enzymes, should be used in the case of organisms exposed to the material. While
100
some studies have reported no adverse effects, others have described changes in the activities of
101
the antioxidant enzymes catalase, superoxide dismutase, glutathione S-transferase, and
102
peroxidase in aquatic organisms exposed to nano-TiO2 (Federici et al., 2007; Hao et al., 2009;
103
Scown et al., 2009; Kim et al., 2010). On the other hand, equally important enzymes such as
104
phosphatases, which are involved in a variety of transphosphorylation reactions, and can be
105
affected by metals and ROS (Aoyama et al., 2003), have received little attention in terms of the
106
effects of nano-TiO2. It has been reported that exposure to nano-TiO2 can affect the growth and
107
size of microcrustaceans and fish (Zhu et al., 2010; Chen et al., 2011a; Fouqueray et al., 2012;
108
Campos et al., 2013). However, the published data are often contradictory, and comparison of
109
results is hindered by insufficient information as well as the absence of standardized
110
nanoecotoxicological protocols.
Ac ce p
te
d
M
an
us
cr
93
111
Although there have been many studies concerning the effects of UV radiation on
112
aquatic organisms, no standardized ecotoxicological protocols exist for the evaluation of
113
photosensitive compounds. Charron et al. (2000) reported a 75% survival rate of D. rerio Page 5 of 40
embryos exposed to 0.15 W/m2 of UVB for up 30 h. Dong et al. (2007) obtained LD50 values of
115
2.32 and 855.3 J/cm2 of UVB and UVA, respectively, for embryos exposed during the mid-
116
gastrula period. The exposure of tadpoles to 4 mW/cm2 of UVA for 14 days increased the toxicity
117
of nano-TiO2 (Zhang et al., 2012). Increased toxicity of nano-TiO2 was also observed in the
118
larvae of medaka (Oryzias latipes) exposed daily for 4 h to a UV dose of 6.12 W/cm2/h
119
(equivalent to a total of 97 W/cm2 over 4 days) (Ma et al., 2012b). The lamps and UV irradiation
120
intensities employed in ecotoxicological experiment have varied widely, so that it is difficult to
121
compare results.
an
us
cr
ip t
114
In summary, there is a need to evaluate in detail the safety of nano-TiO2, as well as to
123
standardize nanoecotoxicological protocols, including tests designed to assess the effects of UV
124
irradiation. The objective of the present work was to investigate the toxicity of different nano-
125
TiO2 formulations to D. rerio embryos exposed under different illumination conditions, and to
126
establish suitable experimental protocols. The parameters evaluated reflected acute toxicity and
127
sublethal effects, and included survival rates, malformation, hatching, overall length of the larvae,
128
and biochemical biomarkers.
130 131 132
d
te
Ac ce p
129
M
122
2. MATERIALS AND METHODS
2.1 Characterization of the NPs and their stability in suspension
133 134
Toxicity evaluation of the nano-TiO2 to D. rerio embryos employed titanium (IV) oxide
135
nanopowders, either 100% anatase, with primary particle size
S0166-445X(13)00374-3 http://dx.doi.org/doi:10.1016/j.aquatox.2013.12.024 AQTOX 3719
To appear in:
Aquatic Toxicology
Received date: Revised date: Accepted date:
31-10-2013 13-12-2013 18-12-2013
Please cite this article as: Clemente, Z., Castro, V.L.S.S., Moura, M.A.M., Jonsson, C.M., Fraceto, L.F.,Toxicity Assessment Of Tio2 Nanoparticles In Zebrafish Embryos Under Different Exposure Conditions, Aquatic Toxicology (2014), http://dx.doi.org/10.1016/j.aquatox.2013.12.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
TOXICITY ASSESSMENT OF TiO2 NANOPARTICLES IN ZEBRAFISH
2
EMBRYOS UNDER DIFFERENT EXPOSURE CONDITIONS
ip t
3 Clemente, Z.a,b; Castro, V.L.S.S.a; Moura, M.A.M.c; Jonsson, C.M.a; Fraceto, L.F.b,d
4
cr
5 6 a
Laboratório de Ecotoxicologia e Biossegurança, Embrapa CNPMA, Jaguariúna, SP, Brazil.
8
b
Programa de Pós-graduação em Biologia Funcional e Molecular, UNICAMP, Campinas, SP,
9
Brazil.
10
c
11
Brazil.
12
d
15 16 17 18
M
d
Departamento de Engenharia Ambiental, UNESP, Sorocaba, SP, Brazil.
te
14
Laboratório da Ciência das Plantas Daninhas, Instituto Biológico, APTA/SAA, Campinas, SP,
Ac ce p
13
an
us
7
*Corresponding author. E-mail: [email protected], [email protected]
Page 1 of 40
18 ABSTRACT
20
The popularity of TiO2 nanoparticles (nano-TiO2) lies in their wide range of
21
nanotechnological applications, together with low toxicity. Meanwhile, recent studies have
22
shown that the photocatalytic properties of this material can result in alterations in their behavior
23
in the environment, causing effects that have not yet been fully elucidated. The objective of this
24
study was to evaluate the toxicity of two formulations of nano-TiO2 under different illumination
25
conditions, using an experimental model coherent with the principle of the three Rs of alternative
26
animal experimentation (reduction, refinement, and replacement). Embryos of the fish Danio
27
rerio were exposed for 96 h to different concentrations of nano-TiO2 in the form of anatase (TA)
28
or an anatase/rutile mixture (TM), under either visible light or a combination of visible and
29
ultraviolet light (UV). The acute toxicity and sublethal parameters evaluated included survival
30
rates, malformation, hatching, equilibrium, and overall length of the larvae, together with
31
biochemical biomarkers (specific activities of catalase (CAT), glutathione S-transferase (GST),
32
and acid phosphatase (AP)). Both TA and TM caused accelerated hatching of the larvae. Under
33
UV irradiation, there was greater mortality of the larvae of the groups exposed to TM, compared
34
to those exposed to TA. Exposure to TM under UV irradiation altered the equilibrium of the
35
larvae. Alterations in the activities of CAT and GST were indicative of oxidative stress, although
36
no clear dose-response relationship was observed. The effects of nano-TiO2 appeared to depend
37
on both the type of formulation and the illumination condition. The findings contribute to
38
elucidation of the factors involved in the toxicity of these nanoparticles, as well as to the
39
establishment of protocols for risk assessments of nanotechnology.
Ac ce p
te
d
M
an
us
cr
ip t
19
40 41
Key words: FET, ultraviolet, nanotoxicology, biomarker, oxidative stress. Page 2 of 40
42
1. INTRODUCTION
43 Titanium dioxide nanoparticles (nano-TiO2) are used industrially in the areas of
45
cosmetics and pharmaceuticals, amongst others, as well as in environmental applications, and are
46
increasingly encountered in daily life. The use of these materials can often be beneficial, while at
47
the same time questions remain concerning the environmental risks of nanotechnology. The fast
48
development of nanotechnology requires practical ecotoxicological tools to correctly evaluate its
49
safety. On the other hand, scientific research using animals has been much discussed in terms of
50
bioethics, and there have been renewed efforts to identify alternative techniques that do not
51
require the use of animals.
M
an
us
cr
ip t
44
Tests employing fish embryos (the Fish Embryo Test, FET) fits between traditional
53
studies employing cell cultures and those that use mammalian models (Lin et al., 2013). The
54
results obtained with FET have a strong correlation with the outcomes of acute toxicity tests
55
using adult fish (Knobel et al., 2012), and it is likely that the embryos do not have the same
56
perception of pain as the adults, due to the immaturity of the nervous system (Lammer, 2009).
57
The zebrafish (Danio rerio) is widely used in FET because of its large numbers of eggs laid,
58
rapid development, the transparency of the eggs, and its similarity with human genome
59
(Braunbeck and Lammer, 2006; Howe et al., 2013). To date, there have been few studies that
60
have applied the FET method in nanoecotoxicological assessments. However, its use is
61
promising, especially considering the wide variety of nanomaterials that have emerged over a
62
very short space of time, the small volumes involved in the tests, and the low levels of waste
63
generation.
Ac ce p
te
d
52
64
There is evidence that nano-TiO2 can adhere to the chorion of the embryo, where it can
65
be absorbed and then distributed uniformly throughout the tissues of the fish, without any tissuePage 3 of 40
specificity (Bar-Ilan et al., 2012). The accumulation of Ti has also been observed in tissues of the
67
larvae of D. rerio, especially in the intestine (associated with the microvilli), the gill lamellae, the
68
liver, and the skeletal muscle (Bar-Ilan et al., 2013). Studies with aquatic organisms such as
69
hydra, microcrustaceans and fish have indicated that the toxicity of nano-TiO2 is negligible
70
(Blaise et al., 2008; Griffith et al., 2008; Yeo and Kang, 2010; Xiong et al., 2011; Clemente et al.,
71
2013). Similar findings have been reported for fish embryos (Zhu et al., 2008; Paterson et al.,
72
2011; Ma et al., 2012b). Nevertheless, in these bioassays employing aquatic organisms, the
73
circadian cycle is usually established using fluorescent lamps, which mainly emit visible light.
74
TiO2 is a semiconductor with the important property of being able to be photoactivated, which
75
makes it especially attractive for use in processes based on heterogeneous photocatalysis to
76
degrade a variety of organic and inorganic compounds (Nogueira and Jardim, 1998; Gaya and
77
Abdullah, 2008). Exposure of TiO2 to ultraviolet radiation (UV) in the wavelength range 300-388
78
nm results in the production of reactive oxygen species (ROS) that can cause damage to
79
biomolecules. The photocatalytic properties of TiO2 are enhanced when the compound is present
80
in the form of nanoparticles, which can increase its toxicity to aquatic organisms under
81
environmental conditions with exposure to solar UV radiation (Ma et al., 2012a; Marcone et al.,
82
2012; Clemente et al., 2013; Xiong et al., 2013).
cr
us
an
M
d
te
Ac ce p
83
ip t
66
TiO2 occurs in different crystal phases, being the photocatalytic properties and toxicity
84
of anatase higher than rutile (Malato et al., 2009; Allouni et al., 2012). Evidence has been found
85
for synergism between the phases, with anatase/rutile mixtures being more photoactive than the
86
pure phases. The nano-TiO2 known as P25®, manufactured by Evonik Degussa, is the mixture
87
that is most commonly used in photocatalytic processes (Nogueira and Jardim, 1998; Malato et
88
al., 2009). Earlier work in our research group indicated that exposure to nano-TiO2 can cause
89
sublethal effects in fish and microcrustaceans (Clemente et al., 2013), depending on the crystal Page 4 of 40
phase, concentration, and illumination conditions. Nonetheless, there remain a number of
91
uncertainties, as a result of which further work is needed to fully evaluate the toxicity (especially
92
sublethal effects) of nano-TiO2 in the fish embryos.
ip t
90
Effects on reproduction, as well as on the development and survival of new generations,
94
could lead to serious impacts on ecosystems. There is therefore a need for careful scrutiny of any
95
effects of nano-TiO2 on embryos and larvae exposed to the substance. To this end, biomarkers
96
can be used as effective early warning systems, and are often more useful than direct
97
measurements of a chemical agent in the organism. Since oxidative stress is probably the main
98
cause of the toxicity of nano-TiO2, the investigation of biomarkers associated with this effect, as
99
antioxidant enzymes, should be used in the case of organisms exposed to the material. While
100
some studies have reported no adverse effects, others have described changes in the activities of
101
the antioxidant enzymes catalase, superoxide dismutase, glutathione S-transferase, and
102
peroxidase in aquatic organisms exposed to nano-TiO2 (Federici et al., 2007; Hao et al., 2009;
103
Scown et al., 2009; Kim et al., 2010). On the other hand, equally important enzymes such as
104
phosphatases, which are involved in a variety of transphosphorylation reactions, and can be
105
affected by metals and ROS (Aoyama et al., 2003), have received little attention in terms of the
106
effects of nano-TiO2. It has been reported that exposure to nano-TiO2 can affect the growth and
107
size of microcrustaceans and fish (Zhu et al., 2010; Chen et al., 2011a; Fouqueray et al., 2012;
108
Campos et al., 2013). However, the published data are often contradictory, and comparison of
109
results is hindered by insufficient information as well as the absence of standardized
110
nanoecotoxicological protocols.
Ac ce p
te
d
M
an
us
cr
93
111
Although there have been many studies concerning the effects of UV radiation on
112
aquatic organisms, no standardized ecotoxicological protocols exist for the evaluation of
113
photosensitive compounds. Charron et al. (2000) reported a 75% survival rate of D. rerio Page 5 of 40
embryos exposed to 0.15 W/m2 of UVB for up 30 h. Dong et al. (2007) obtained LD50 values of
115
2.32 and 855.3 J/cm2 of UVB and UVA, respectively, for embryos exposed during the mid-
116
gastrula period. The exposure of tadpoles to 4 mW/cm2 of UVA for 14 days increased the toxicity
117
of nano-TiO2 (Zhang et al., 2012). Increased toxicity of nano-TiO2 was also observed in the
118
larvae of medaka (Oryzias latipes) exposed daily for 4 h to a UV dose of 6.12 W/cm2/h
119
(equivalent to a total of 97 W/cm2 over 4 days) (Ma et al., 2012b). The lamps and UV irradiation
120
intensities employed in ecotoxicological experiment have varied widely, so that it is difficult to
121
compare results.
an
us
cr
ip t
114
In summary, there is a need to evaluate in detail the safety of nano-TiO2, as well as to
123
standardize nanoecotoxicological protocols, including tests designed to assess the effects of UV
124
irradiation. The objective of the present work was to investigate the toxicity of different nano-
125
TiO2 formulations to D. rerio embryos exposed under different illumination conditions, and to
126
establish suitable experimental protocols. The parameters evaluated reflected acute toxicity and
127
sublethal effects, and included survival rates, malformation, hatching, overall length of the larvae,
128
and biochemical biomarkers.
130 131 132
d
te
Ac ce p
129
M
122
2. MATERIALS AND METHODS
2.1 Characterization of the NPs and their stability in suspension
133 134
Toxicity evaluation of the nano-TiO2 to D. rerio embryos employed titanium (IV) oxide
135
nanopowders, either 100% anatase, with primary particle size
