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A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials
STOTEN-21397; No of Pages 8 Science of the Total Environment xxx (2016) xxx–xxx
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials S.L. Azevedo a, T. Holz b, J. Rodrigues b, T. Monteiro b, F.M. Costa b, A.M.V.M. Soares a, S. Loureiro a,⁎ a b
Department of Biology, CESAM, University of Aveiro, 3810-193 Aveiro, Portugal Physics Department, I3N, University of Aveiro, 3810-193 Aveiro, Portugal
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• The toxicity of a ZnO/Ag nanostructure (NS) was assessed on Daphnia magna. • A mixture toxicity approach was carried to predict toxicity based on single components. • ZnO/Ag-NS showed higher toxicity than the predicted based on individual toxicity. • ZnO/Ag-NS did not present the same behavior of a mixture of ZnO-NM and Ag-NM.
a r t i c l e
i n f o
Article history: Received 18 March 2016 Received in revised form 28 October 2016 Accepted 15 November 2016 Available online xxxx Editor: D. Barcelo Keywords: Silver Zinc oxide Nanomaterials Nanostructures Mixture toxicity Daphnia magna
a b s t r a c t Nanotechnology is a rising field and nanomaterials can now be found in a vast variety of products with different chemical compositions, sizes and shapes. New nanostructures combining different nanomaterials are being developed due to their enhancing characteristics when compared to nanomaterials alone. In the present study, the toxicity of a nanostructure composed by a ZnO nanomaterial with Ag nanomaterials on its surface (designated as ZnO/Ag nanostructure) was assessed using the model-organism Daphnia magna and its toxicity predicted based on the toxicity of the single components (Zn and Ag). For that ZnO and Ag nanomaterials as single components, along with its mixture prepared in the laboratory, were compared in terms of toxicity to ZnO/Ag nanostructures. Toxicity was assessed by immobilization and reproduction tests. A mixture toxicity approach was carried out using as starting point the conceptual model of Concentration Addition. The laboratory mixture of both nanomaterials showed that toxicity was dependent on the doses of ZnO and Ag used (immobilization) or presented a synergistic pattern (reproduction). The ZnO/Ag nanostructure toxicity prediction, based on the percentage of individual components, showed an increase in toxicity when compared to the expected (immobilization) and dependent on the concentration used (reproduction). This study demonstrates that the toxicity of the prepared mixture of ZnO and Ag and of the ZnO/Ag nanostructure cannot be predicted based on the toxicity of their components, highlighting the importance of taking into account the interaction between nanomaterials when assessing hazard and risk. © 2016 Elsevier B.V. All rights reserved.
⁎ Corresponding author at: University of Aveiro, Department of Biology, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal. E-mail address: [email protected] (S. Loureiro).
http://dx.doi.org/10.1016/j.scitotenv.2016.11.095 0048-9697/© 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095
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S.L. Azevedo et al. / Science of the Total Environment xxx (2016) xxx–xxx
1. Introduction Nanotechnology is a fast growing industry and nanomaterials can today be found in a vast variety of products such as cosmetics, antibacterial substances, electronic and optoelectronic devices as light emitting diodes (LEDS) and solar cells (Piccinno et al., 2012). In the last decade, the production of nanomaterials had an exponential increase and now new research is focusing on increasing their performance and applicability. Some materials such as zinc oxide (ZnO) and titanium dioxide (TiO2), well-established semiconductors with intrinsic absorption in the ultraviolet (UV), are known to present high photocatalytic activity. Photocatalysis has several applications: in pigments, providing the white color to paper and paint, providing antibacterial properties and in water and soil remediation processes (Ullah and Dutta, 2008; Yunus et al., 2012). In the latter, photocatalysis is responsible for the oxidation of organic pollutants into nontoxic materials (Yunus et al., 2012). With the purpose of increasing the photocatalytic properties, zinc oxide nanomaterials (ZnO-NM) are now being combined with silver nanomaterials (Ag-NM) (Georgekutty et al., 2008; Zheng et al., 2008). This combination will allow the photocatalysis to occur not only with UV radiation but also with visible light due to the absorption of the silver surface plasmon resonance (Rekha et al., 2010). Several toxicity studies have demonstrated the negative effects of ZnO and Ag nanomaterials to aquatic organisms, as reported in the reviews of Ma et al. (2013) and of Fabrega et al. (2011), respectively. Although the toxicity of the individual nanomaterials is known, the toxicity of the combined component is not well understood. Taking this into account, it is important to understand if the behavior and toxicity of combined materials (also called heterostructures) can be predicted based on their individual behavior and toxicities, or if a different toxicity pattern will be observed due to potential different interactions with biotic compartments. Having this into consideration, the aim of this work was to understand the reasonableness of predicting the join toxicity of nanoheterostructures by knowing the toxicity of individual nanomaterial components. In addition, and based on the usual methodology employed for predicting mixture toxicities, a laboratorial-made binary mixture was tested to infer if results could help on this prediction. This is extremely important regarding regulatory issues, as NMs are included in the REACH regulation, meeting the regulations' substance definition. In addition, the recommendation allows also their inclusion in other European regulations, like the Classification, Labelling and Packaging (CLP) Regulation which deals directly with mixtures. For that, the toxicity of a nanostructure (NS) composed of ZnO with Ag-NM on its surface was assessed, along with the toxicity evaluation of ZnO-NM and Ag-NM alone and combined as a binary mixture. This NS was used as a model material to address the aims of the present study but also because of its wide expected application in environmental remediation processes, especially for organic pollutants' removal, which can lead to a potential exposure scenario in aquatic environments. This kind of NS can have several compositions and percentages of their individual components and therefore a mixture toxicity approach can provide a useful output. For the hazard assessment, immobilization and reproduction tests were performed with Daphnia magna and values of LC50 and EC50 were calculated from the single exposures, and used to set up the mixture toxicity experimental designs and predictions. 2. Material and methods 2.1. Nanomaterials Three nanomaterials were supplied as powders and used in the present study: zinc oxide tetrapods (ZnO-NM), a spherical silver (Ag-NM) and zinc oxide tetrapods decorated with 1–3% mol. of silver
nanomaterials (ZnO/Ag-NS). ZnO based materials were synthesized by Laser Assisted Flow Deposition (LAFD) starting from commercial powders of ZnO (AnalaR, 99.7%) and a ZnO mixture with AgNO3 (Merck, 99.8%) respectively, using a CO2 laser (λ = 10.6 μm), as reported elsewhere (Rodrigues et al., 2014; Rodrigues et al., 2012). Ag nanomaterials were synthesized by Pulsed Laser Ablation in Liquid (PLAL) technique (Soares et al., 2015a; Soares et al., 2015b) using an Ag target (Sigma Aldrich, 99.99%) immersed in acetone. The distance from the target to the surface was kept constant (10 mm) and the target surface was irradiated by a pulsed Nd:YAG laser (λ = 1064 nm) at the frequency of 10 Hz and energy per pulse of 650 mJ during 30 min. After this process the suspension was dried and the Ag particles were collected. All stock suspensions (5 mg·L−1) were prepared in ASTM moderated hard water (ASTM E729-96, 2014) with sonication in water bath (Ultrasonic SELECTA mod. 6.5 L) during 15 min. ASTM moderated hard water is constituted mainly by a set of salt solutions: calcium sulphate, potassium chloride, sodium hydrogen carbonate and magnesium sulphate. The stock suspensions were sampled for chemical analysis and characterization at time zero and after 48 h. The ZnO-NM and ZnO/AgNS stocks were analyzed through scanning electron microscope (SEM) and the Ag-NMs stock suspension through scanning transmission electron microscopy (STEM). Water samples for Zn and Ag total concentrations were acid digested; to discriminate Zn and Ag dissolution samples were also centrifuged at 2862g for 30 min using 3 kDa AMICON centrifugal filters (Merck, Millipore, Darmstadt, Germany) (Ribeiro et al., 2017). Analysis were carried out by ICP-MS at the Central Laboratory of Analysis, University of Aveiro, Portugal (certified laboratory). 2.2. Test organisms Experiments were carried out with Daphnia magna (clone Beak) as test organisms. Cultures were maintained in 1 L glass jars with artificial hard water ASTM, which was changed every other day. Organisms were fed with Raphidocelis subcapitata at a concentration of 3 × 105 cell·mL−1 with a supplement of seaweed extract (6 mL·L−1). Cultures were kept at 20 ± 1 °C and under a 16:8 h light: dark photoperiod. Neonates from the third and fourth brood were used to perform the toxicity tests. 2.3. Immobilization tests Immobilization tests were performed based on the OECD guideline 202 (OECD, 2004). Tests were carried out using five replicates per concentration and negative control, with five neonates with b24 h per replicate exposed in 50 ml glass beakers. Tests were maintained at room temperature of 20 ± 1 °C and a 16:8 h light:dark photoperiod and daphnids were not fed during the entire experiment. After 48 h, immobilization (inability to swim after gentle agitation of the beaker) and mortality were reported. The concentration that caused the immobilization of 50% of the neonates (LC50) was calculated (see below for details). Immobilized daphnids included non-mobile and dead animals. Nominal concentrations ranged from 0.5 to 1.3 mg·Zn·L−1 for the ZnO-NM treatments, 0.05 to 0.25 mg·Ag·L−1 for Ag-NM and 0.13 to 0.63 mg·ZnO/Ag·L−1 (1–3% mol. Ag) for ZnO/Ag-NS. This choice was based on available literature for ZnO and Ag-NMs toxicity to D. magna, and for a preliminary trial with ZnO/Ag-NS (data not shown). 2.4. Reproduction tests Reproduction tests were based on the OECD guideline 211 (OECD, 1998) and for each treatment 10 replicates with one neonate were used. Neonates with b24 h were transferred to 50 mL glass beakers and maintained during 21 days in similar temperature and photoperiod conditions as those described for cultures. The medium was renewed every two days and daphnids fed daily with the algae R. subcapitata. The number of offspring per brood were counted and removed from
Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095
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the beakers and the mortality of offspring and parental daphnids were also recorded. Daphnids were measured in the beginning and at the end of the test in order to assess differences on their size between treatments and the control. Dissolved oxygen, conductivity and pH were measured at the beginning, middle and end of the test in old and new medium. Toxicity tests were run with nominal concentrations ranging from: 0.1 to 0.4 mg·Zn·L−1 for ZnO-NM, 0.095 to 0.5 mg·Ag·L−1 for Ag-NM and 0.01 to 0.25 mg·ZnO/Ag·L−1 (1% Ag) for ZnO/Ag-NS. In addition, all tests were carried out with a negative control (ASTM media). As stated above concentrations were based on available literature for ZnO and Ag-NMs toxicity to D. magna, and on a preliminary trial with ZnO/Ag-NS (data not shown). 2.5. Combined exposures To perform exposures to binary mixtures of ZnO and Ag NMs, the methodology applied in the single exposures was used deferring only in the number of replicates. The number of replicates was reduced from five to three replicates in the immobilization tests, while in the reproduction tests replicates were reduced to one. This strategy provides an increase on the number of treatments used, allowing a wider range of exposure covering, without prejudice for the reliability of the results as the predictions used are based on regressions (for details see below) (Loureiro et al., 2010; Loureiro et al., 2009). The experimental design for the acute test was based on a full factorial design. For the reproduction test concentrations chosen followed a fixed ray design based on individual toxic units (TU). One TU was equal to the EC50 for each nanomaterial used and the TU never exceeded 2 aiming at avoiding mortality. Within each combined experiment an exposure to each chemical component alone was performed simultaneously using the concentrations applied in the single exposures. This approach has been implemented elsewhere in studies from the same research group where details are presented (Freitas et al., 2014; Loureiro et al., 2010). 2.6. Statistical analysis The concentration inducing 50% of immobilization (LC50) and 50% of effect (EC50) were calculated using the best fit, by a nonlinear regression using the SigmaPlot for windows version 11 (Systat, 2008). One-way analysis of variance (ANOVA) was used to identify significant differences between treatments and control (p b 0.05) (Systat, 2008), after normality and equal variance check through the ShapiroWilk test and Levene's mean test, respectively. A Dunnett's test was performed to compare each treatment to the control whenever significance was achieved in the ANOVA. The mixture set up was analyzed by the MixTox tool (Jonker et al., 2005). The MixTox tool allows to analyze binary mixtures and uses the conceptual models of Concentration addition (CA) and Independent action (IA) for toxicity prediction. Recently EFSA advised on the use of the CA model as the most conservative one, therefore this was followed in the present study to predict mixture toxicity. Deviations for synergism (more severe effect) and antagonism (less severe effect) were then evaluated by extending the mathematical equation of CA, by adding parameter a. With the addition of parameters a and also b (another extension to depict changings in the above mentioned patterns for synergism and antagonism) dose-ratio (DR) and dose-level (DL) deviations were assessed. The biological meaning of the positive or negative values of the parameters a and b can the found in Table 1. To allow further comparison with the binary mixture of ZnO-NM and Ag-NM, the results obtained from the ZnO/Ag-NS exposure were also analyzed with the MixTox tool (e.g. Loureiro et al., 2010), simulating a mixture toxicity approach by estimating each component concentration from the total. For that, Zn and Ag measured concentrations
3
were used to calculate the real exposure to both elements in the exposure of ZnO/Ag-NS (Table 2).
3. Results 3.1. Nanomaterials Fig. 1 presents the SEM and STEM images of the three nanomaterials used in the present study. ZnO-NM exhibit a wurtzite-structure and present a tetrapod morphology due to the faster growth along the normal directions to the zinc surfaces resulting in four legs preferentially oriented along the c axis direction (Rodrigues et al., 2012). In the case of the ZnO/Ag-NS, the tetrapods' surface was decorated with Ag metallic nanomaterials in the form of spherical droplets in a size smaller than 10 nm. Agglomerates (in different sizes) were observed as well as dispersed particles. For all nanomaterials the appearance did not change from time zero to 48 h after dispersion in culture medium. The ZnO/ Ag nanostructures presented a surface of ~40–50 nm, while the metallic Ag nanomaterials prepared by PLAL presented a size of ~5–12 nm. From the chemical analysis, it was observed that stock dispersions of ZnO-NM and ZnO/Ag-NS showed a recovery of 100% and 95% for Zn at the time of dispersion preparation (Table 2). After 48 h, Zn total concentration decreased approx. 41% and 9%, respectively. Zinc dissolution after 48 h was very low, corresponding only to 2.5% and 13% of the total measured in time zero, respectively (Table 2). For Ag total concentration, the stock dispersion of Ag-NM showed a recovery of 81%, while for the ZnO/Ag-NS dispersion the percentage of Ag in the NS was higher than 3% (approx. 3.5%). After 48 h, total Ag decreased to approx. 37% in the Ag-NM, while this decrease was higher (approx. 44%) for the Zn/AgNS. Dissolution of Ag was also very low, corresponding to 11% and 1.5% of the total measured in time zero for Ag-NM and Zn/Ag-NS, respectively (Table 2).
3.2. Single exposures 3.2.1. Immobilization tests All nanomaterials tested induced an increase in Daphnia magna mortality with the increasing concentration applied. For ZnO-NM the 48 h– LC50 was 1.29 mg·Zn·L−1 whereas Ag-NM presented a LC50 of 0.079 mg·Ag·L−1 (based on measured concentrations). Regarding ZnO/Ag-NS a 48 h-LC50 of 0.59 mg·ZnO/Ag·L−1 was obtained (Table 3). Table 1 Interpretation of additional parameters (a and b) that define the functional form of deviation patterns from concentration action. Adapted from Jonker et al. (2005). Deviation pattern
Concentration addition Parameter a
Synergism/antagonism Dose ratio dependent
Dose level dependent
a N 0: Antagonism a b 0: Synergism a N 0: Antagonism, except for those mixture ratios where significant negative b indicate synergism a b 0: Synergism, except for those mixture ratios where significant positive b indicate antagonism a N 0: Antagonism low dose level and synergism high dose level a b 0: Synergism low dose level and antagonism high dose level
Parameter b
bi N 0 Antagonism where the toxicity of the mixture is caused mainly by toxicant i bi b 0 Synergism where the toxicity of the mixture is caused mainly by toxicant i bDL N 1: Change at lower EC50 level bDL = 1: Change at EC50 level 0 b bDL b 1: Change at higher EC50 level bDL b 0: No change but the magnitude of S/A is DL dependent
Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095
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Table 2 Measured concentrations of Zn and Ag in a 5 mg·L−1 stock suspension (in ASTM moderate hard water) for the nanomaterials used (ZnO-NM, Ag-NM and Zn/Ag-NS) at time 0 h (after dispersion preparation) and after 48 h. The dissolved fraction of Zn and Ag was only measured after 48 h. The prediction for Ag in the ZnO/Ag-NS was of 1–3%, therefore nominal concentrations of Zn and Ag are presented as a range. Measured concentrations are expressed as average values ± in mg·L−1. Nanomaterials
Nominal concentration −1
Zn (mg·L
Zn-NM Ag-NP ZnO/Ag-NS
)
4 – 3.86–3.96
Total measured concentration −1
Ag (mg·L
−1
)
– 1.1 0.050–0.150
Zn (mg·L
)
Ag (mg·L
Dissolved fraction −1
)
Zn (mg·L−1)
Ag (mg·L−1)
0h
48 h
0h
48 h
48 h
48 h
4.01 ± 0.14 – 3.83 ± 0.02
2.36 ± 0.03 – 3.50 ± 0.29
– 0.901 ± 0.153 0.218 ± 0.52
– 0.655 ± 0.240 0.123 ± 0.60
0.150 ± 0.004 – 0.519 ± 0.35
– 0.097 ± 0.059 0.003 ± 0.002
3.2.2. Reproduction tests The EC50s for reproduction upon ZnO-NM, Ag-NM and ZnO/Ag-NS exposures can be found in Table 4. At the end of all reproduction tests, negative controls showed a mortality of parental animals always lower than 20% and the mean number of live offspring was always higher than 60 neonates per parental organisms. Therefore test validation was achieved (OECD, 1998). In addition, the exposure conditions measured validated the results, with dissolved oxygen ranging between 5.13 and 9.07 mg·L−1, conductivity between 500 and 630 μS/cm and pH between 7.66 and 8.36. For ZnO-NM significant differences in the number of neonates produced during the 21 days were observed for concentrations of 0.2, 0.3 and 0.4 mgZn·L− 1 (Fig. 2-A) (one way ANOVA, F4,41 = 83.86, p ≤ 0.001, Dunnett's method, p b 0.05). Significant differences on the daphnids' length were also observed for all concentrations used in the test (Fig. 2-B) (one way ANOVA, F4,41 = 38.37, p ≤ 0.001, Dunnett's method, p b 0.05). In the reproduction test with Ag-NM a significant decrease in the mean number of neonates produced per daphnia (Fig. 3-A) was observed at the three highest concentrations (0.25, 0.33 and 0.4 mg·Ag·L−1) (one way ANOVA, F5,48 = 21.04, p ≤ 0.001, Dunnett's method, p b 0.05). No significant differences were observed for the daphnids' length (Fig. 3-B), showing that Ag-NM did not affect this parameter when compare to the negative control. Alterations in the number of offspring and in the length of daphnids were observed in the exposures to ZnO/Ag-NS. The cumulative number of neonates produced per daphnia (Fig. 4-A) significantly decreased for the two highest concentrations (0.13 and 0.25 mg·Zn/Ag·L− 1) (one way ANOVA, F5,50 = 30.59, p ≤ 0.001, Dunnett's method, p b 0.05). The length of the adult daphnids was significantly decreased at concentrations of 0.06, 0.13 and 0.25 mg·Zn/Ag·L−1 (Fig. 4-B) (one way ANOVA, F5,50 = 10.95, p ≤ 0.001, Dunnett's method, p b 0.05). 3.3. Combined exposures approach The LC50 values derived from the single exposures to ZnO-NM and Ag-NM performed simultaneously in the combine exposures setup were 0.66 mgZn·L−1 (st.error = 0.07; r2 = 0.53) and 0.08 mg·Ag·L−1 (st.error = n.d.; r2 = 0.79), respectively. For the mixture of Ag-NM and ZnO-NM the acute toxicity results fitted the CA model (SS = 70.81; r2 = 0.76; p = 2.09 × 10−48), although a better fit was observed for the deviation pattern of dose-level (SS = 48.86; r2 = 0.84; p = 1.72 × 10−5; a = −1.48; b = 1.09), as showed in Fig. 5 response pattern. The negative a value and the positive b value close to 1 indicate synergism at low dose levels that change to antagonism at the LC50 level. The mixture toxicity concentrations for the reproduction test were designed as a fixed ray based on toxic units. Although the sum of toxic units never exceed 2 to avoid mortality, mortality was observed in thirteen combinations as shown in Fig. 6 by the white circles. The single exposures performed during the combined exposures derived EC50 values of 0.24 mg·Zn·L−1 (st.error = 0.10; r2 = 0.97) for ZnO-NM and 0.42 mg·Ag·L−1 (st.error = 0.06; r2 = 0.80) for Ag-NM.
The combined exposures fitted the CA model (SS = 5613.07; r2 = 0.66; p = 0.005), whose fit was afterwards improved by the equation extension to synergism (SS = 2107.43; r2 = 0.87; p = 2.7 × 10−5; a = −2.39) (Fig. 7). The results from the ZnO/Ag-NS toxicity output presented in Tables 3 and 4 were fractionated into the concentration of the individual components to enable toxicity comparison. From the chemical analysis ZnO/ Ag-NS presented approx. 3.5% of Ag in its composition, and such small exposure concentrations of Ag alone could not be simulated in the exposure trials. Therefore all comparisons were only carried out considering the used mixture toxicity approach, by modelling exposures and comparing observed vs predicted toxicities. Results from ZnO/Ag-NS exposures were modeled with CA reference model to predict patterns for combined exposures, based on the extrapolated concentrations for both ZnO and Ag. As an example, in the ZnO/Ag-NS acute toxicity test, the concentration of 0.13 mg·L− 1 of ZnO/Ag-NS presented 0.0998 mg·Zn·L−1 and 0.0057 mg·Ag·L−1, which were used as input for the mixture model. The results fitted the CA model explaining already 81% of the variation of the data set for the model (SS = 64.73; r2 = 0.81; p = 7.35 × 10−58). After adding parameter a to the CA equation the data fit was improved and a synergistic deviation obtained (SS = 7.93; r2 = 0.98; p = 4.8 × 10−14; a = −2.33). The results from the reproduction test with ZnO/Ag-NS exposures fitted the CA model (SS = 1891.56; r2 = 0.83; p = 1.4 × 10−3) and after the addition of parameters a and b the dose-level deviation was the one with the best fit (SS = 214.23; r2 = 0.98; p = 2.39 × 10−7; a = − 34.11, b = 0.68). Parameters interpretation showed an occurrence for synergism at low concentrations, changing to antagonism at dose levels higher than the EC50. 4. Discussion The aim of this work was to understand if it is possible to predict the toxicity of combined nanostructures based on the toxicity of its components. In addition, another goal was to infer if the toxicity of the composed nanomaterial would perform similarly to the toxicity of an “artificial” mixture of its components. In the present study single exposures as well as mixture exposures were performed with ZnO-NM and Ag-NM and the toxicity patterns compared to the one for ZnO/Ag-NS. As it is difficult to directly compare the toxicity of ZnO and Ag-NMs binary mixtures with similar concentrations of ZnO/Ag NS, considering the dilutions made, patterns for toxicity were then after compared. For ZnO-NM, negative effects were observed in the survival, reproduction and growth of Daphnia magna with the increasing concentration of ZnO-NM. Despite the tetrapod shape of the nanomaterials, the LC50 (1.29 mg·Zn·L−1) and EC50 (0.25 mg·Zn·L−1) values were in the range of those found in the literature for spherical nanomaterials. Some examples of toxicity values found for Daphnia magna exposed to ZnO spherical particles, from different sizes, ranged from 0.89 to 3.2 mg·ZnO·L− 1 for immobilization (Lopes et al., 2014; Ma et al., 2013) and from 0.26 to 0.36 mg·L− 1 for reproduction (Lopes et al., 2014). Looking also at Zn2+ concentrations as potentially responsible for the toxicity, the dissolved fraction measured after 48 h was very
Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095
STEM images of Ag-NM
SEM images of ZnO/Ag-NS
SEM images of ZnO-NM
S.L. Azevedo et al. / Science of the Total Environment xxx (2016) xxx–xxx
Fig. 1. Characterization of nanomaterials using SEM images for ZnO-NM (top image) and ZnO/Ag-NS (middle image) and STEM images for Ag-NM (bottom image).
low and therefore toxicity is mainly related to the nanoparticulated form. According to the SEM images, the ZnO tetrapods used in our study presented different sizes. Some studies regarding the toxicity of ZnONM state that the size play an important role in the toxicity, but this is not consensual. Lopes et al. (2014) observed that no significant differences were obtained when comparing the toxicity two ZnO-NM and a microsized form of ZnO (30 nm, 50–70 nm and N 200 nm) in Daphnia magna. A different result was obtained by Heinlaan et al. (2008)
5
Table 3 LC50 values of the nanomaterials tested presented in mg·Zn·L−1, mg·Ag·L−1 and in mg·ZnO/Ag·L−1. Results are expressed as mean ± standard error and are based on measured concentrations at time 0 h; R2 is the coefficient of determination; nd - not determined. Immobilization test 48 h-LC50 (mg·L−1)
Nanomaterial
ZnO-NM Ag-NP ZnO/Ag-NM
Zn
R2
Ag
R2
ZnO/Ag
R2
1.29 ± 2.15 – 0.453
0.90
– 0.079 ± nd 0.026
0.98
– – 0.59 ± 0.02
0.84
where the toxicity of the ZnO bulk material was 3-fold higher than for the ZnO-NM. Differences in results can be due to aggregation of the NMs in different exposure media used which can alter their dissolution rate and therefore their toxicity. For the immobilization effects, Ag-NMs showed higher toxicity when compared to ZnO-NM, presenting a LC50 (0.079 mg·Ag·L−1) more than ten times lower than the one for ZnO-NM (1.29 mg·Zn·L−1). Studies found in the literature regarding Ag-NMs toxicity show a large variety of results, due to high variability in the characteristics of the particles (e.g. coated or uncoated) and in the dispersion of the nanomaterials (e.g. colloids or suspensions). Colloids are usually prepared with coated nanomaterials and they are normally more stable than the suspensions. Ag-NMs suspensions tend to have a high tendency to aggregate or agglomerate which alter their surface area to volume ratio and also exhibit high sedimentation ratios (Asghari et al., 2012). These characteristics alter their toxicity and much higher LC50 values for the suspensions can be found in the literature when compared to those from colloids. Asghari et al. (2012) compared the toxicity of two Ag-NMs colloids and an Ag-NM suspension and found that Ag-NM suspensions had a LC50 of 0.187 mg·L−1 which was much higher than the values for the colloids (0.002 and 0.004 mg·L−1). The presence of food can also alter the toxicity of AgNMs. Gaiser et al. (2011) tested the toxicity of Ag-NM suspension to Daphnia magna during 96 h and obtained a LC50 of 0.1 mg·L−1. This value is similar to the one from the present study despite the differences in the duration of the test. This can be explained by the organisms' feeding during the entire experiment, which can lead to an increase on their fitness and to a decrease in the toxicity. The presence of food is known to decrease the toxicity of Ag-NMs to Daphnia magna. Ribeiro et al. (2014) showed that Ag-NMs do not enter the algae cells but rather attach to the surface of the algae. This may lead to an increase in the sedimentation and a decrease of Ag-NMs available for the organisms (Mackevica et al., 2015). The presence of food also seems to play an important role in the toxicity of Ag-NMs used in our tests since we obtained an EC50 (0.45 mg·Ag·L−1) higher than the LC50 (0.09 mg·Ag·L−1) without food, which is normally not achieved. No significant decrease in the size of the organisms was observed at the end of the test. The LC50 and EC50 values for the single exposures carried out simultaneously with the mixture trials, for both ZnO-NM and Ag-NM, were similar to the values obtained during the first tests (used to design the experimental set up of the mixture trials). These results validate our tests and organisms sensitivity and show that the reduction of replicates
Table 4 EC50 values of the nanomaterials tested presented in mg·Zn·L−1, mg·Ag·L−1 and in mg·ZnO/Ag·L−1. Results are expressed as mean ± standard error and are based on measured concentrations at time 0 h; R2 is the coefficient of determination. Reproduction test 21d-EC50 (mg·L−1)
Nanomaterial
ZnO-NM Ag-NM ZnO/Ag-NS a
Zn
R2
Ag
R2
ZnO/Ag
R2
0.25 ± 0.01 – 0.154
0.89
– 0.45 ± 0.04a 0.009
0.65
– – 0.20 ± 0.01
0.72
Value extrapolated as the prediction was higher than the highest concentration used.
Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095
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S.L. Azevedo et al. / Science of the Total Environment xxx (2016) xxx–xxx
B 140
5
120 100
*
80 60
*
40
* 0
0.1
0.2
0.3
*
*
*
0.3
0.4
3 2 1
20 0
*
4
Length (mm)
Total number of neonates
A
0
0.4
0
0.1
0.2
Zn (mg.L-1)
Zn (mg.L-1)
Fig. 2. Total number of neonates per daphnia (A) and daphnids' length (mm) (B) after a 21 day exposure to ZnO-NM. Data is expressed as mg·Zn·L−1 mean values ± st. error considering the measured concentrations of the stock dispersion at time 0 h. *p b 0.05, Dunnett test.
did not diminish the results' output accuracy. To predict the mixture toxicity of ZnO-NM and Ag-NM, the CA model was used as it has been advised by the EFSA report on the “Harmonisation of human and ecological risk assessment of combined exposure to multiple chemicals” (European Food Safety Authority, 2015). Both immobilization and reproduction data set presented deviations from the CA model. In the immobilization experiments, a synergistic pattern can be observed for low concentrations, which changed to antagonism at the level of the EC50. The antagonistic response may be a result of the agglomeration of the nanomaterials due to high number of particles in the mixture, leading possibly to sedimentation and therefore decreasing toxicity. The synergistic pattern for immobilization at low concentrations can also be observed in the reproduction test. Although the toxic units never exceed 2, mortality occurred in thirteen concentrations of twenty three. For the surviving organisms, a synergistic response was observed for the number of neonates produced. Despite this synergistic pattern in reproduction, the observed mortality indicates also an increase in toxicity more than expected from the single exposure trials. Since the predicted environmental concentrations in surface waters for ZnO and Ag nanomaterials are in the range of the ng·L−1 (Gottschalk et al., 2009), the most probable scenario to occur in the environment is the synergistic response, under low dose, as also for longer term exposures. Some studies related the toxicity of ZnO-NM and Ag-NM to the release of Zn and Ag ions (Adam et al., 2014; Jo et al., 2012). The presence of Zn2+ and Ag+ may have negative impacts in the uptake of ions that are essential to the good health of the organisms. Zn is an essential metal but it can have negative effects if present at high concentrations. Zn ions can compete with Ca2+ which can lead to a disturbance in the Ca content in the body (Muyssen et al., 2006). A reduction of Ca content in Daphnia magna can negatively affect the movement and filtration rate which will eventually lead to a reduction of growth and reproduction due to feeding impairment (Muyssen et al., 2006). Ag+ in the media
can lead to ion deregulation due to the competition with Na+ uptake (Bianchini and Wood, 2002). Ion deregulation will eventually lead to the dead of the organisms. In the present study, the percentage for dissolution for the three materials was very low, which would predict a low influence of ions on the overall toxicity. According to Lopes et al. (2016) this pattern of “dilution”, with high nanomaterial concentrations and low ionic concentrations, will direct toxicity to less deleterious effects, induced by nanoparticles than those induced by ions, which are considered more hazardous. In the literature, very few studies determine the toxic effects of exposures with more than one nanomaterial. In the above mentioned study of Lopes et al. (2016), the mixture of different Ag and ZnO NPs highlighted a synergism for immobilizations which was also obtained partly in the present study. Zhao et al. (2012) observed a synergistic effect to the survival and reproduction of Daphnia magna when combining CuO-NM and ZnO-NM. A different response is observed when the species Phaseolus vulgaris (common bean) is exposed to a mixture of CuO-NM and ZnO-NM, where higher toxicity was observed to CuONM exposures only (Dimkpa et al., 2015). The ZnO nanomaterials decorated with Ag-NMs, in a percentage of approx. 3.5 mol%, on its surface, presented lower LC50 and EC50 values than the ones of the ZnO-NM. When looking at the results expressed as Zn concentration the values for the LC50 and EC50 decreased in N2fold for the ZnO/Ag-NS when compared to the ZnO-NM alone. In addition, and based on the principles of the CA model and looking at Table 4, to have a 50% of effect on the reproductive output (eq. to 1 TU), and using a equitoxic mixture as example, one would need a mixture with a concentration of 0.125 mg Zn·L−1 (eq. to the EC25 or ½ TU) jointly with a concentration of 0.225 mg Ag·L− 1 (eq. to the EC25 or ½ TU). From Table 4, while relating the effective concentration of ZnO/Ag to their components, the concentration of ZnO/Ag inducing 50% of effects was composed by 0.154 mg Zn·L− 1 but with a much lower
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Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095
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concentration of Ag (0.009 mg Ag·L−1). This clearly indicates that less Ag was needed in the decorated ZnO to induce the same predicted effect. Metallic nanomaterials toxicity is usually linked to oxidative stress (Chang et al., 2012). NMs can cause oxidative stress by eliminating antioxidants or by direct production of reactive oxygen species (ROS) (Sánchez et al., 2011). Since the addition of Ag-NMs to the surface of ZnO-NM will increase the photocatalytic activity, this may lead to an increase in the ROS production when compared to the production by the nanomaterials alone. Oxidative stress can lead to membrane damage, lipid peroxidation or protein denaturation (Manke et al., 2013). This increase in concentration of Ag-NMs in the surface of the ZnO-NM and the consequent increase of the photocatalytic activity was demonstrated by Ren et al. (2010). These may lead to an increase of ROS production and therefore an increase in the toxicity. When analyzing the results with the MixTox tool, the synergistic patterns obtained may reflect this increase in cellular stress. Li et al. (Li et al., 2010) observed that the nanomaterials composed by Ag and gold (Au) in different percentages had different toxicity to Daphnia magna. The nanomaterials containing Ag in a percentage of 80% and 20% of Au were less toxic than expected, demonstrating that Au can reduce the toxicity of Ag-NM. When the ratio of Ag and Au is inverse (20% of Ag and 80% of Au) the nanomaterials were more toxic than expected (Li et al., 2010).
on the toxicity prediction regarding the nanomaterials and nanostructures used in the present study. As already shown in other case-studies on mixture toxicity evaluation and prediction, the CA conceptual model was not the best model to predict ZnO and Ag joint toxicity. The mixture of ZnO-NM and AgNM did not show an additive pattern but rather deviations such as dose-level and synergism. Since nanomaterials in the environment can be in a mixture with other nanomaterials it is important to understand how they will behave and more studies regarding the mixture toxicity of NMs need to be addressed. Also, ZnO-NM decorated with Ag-NM on its surface showed higher toxicity when compared with the predicted toxicity based on the results from the individual components. Therefore the toxicity of these new nanostructures needs to be addressed as a single material and not based on the toxicity of the single components. This output can be considered very important regarding the current regulations of REACH and CLP, where this ZnO/Ag NM would be treated as a binary mixture, and its toxicity predicted based on the CA model. This regulatory approach would for sure underestimate this NS's toxicity. In conclusion, more studies regarding mixture toxicity of NMs need to be conducted, and the hazard of new nanomaterials carefully evaluated before they are introduced in the market since their toxicity potential cannot be accurately predicted by the single components' toxicity.
5. Conclusion
Acknowledgements
The present study shows that the toxicity mixture approach was effective to achieve the goals of the present study and derive conclusions
Work reported here was partly funded through: the project RePulse - Responses of Daphnia magna exposed to chemical pulses and mixtures throughout generations (FCOMP-01-0124-FEDER-019321; Refª. FCT PTDC/AAC-AMB/117178/2010, by funding FEDER through COMPETE Programa Operacional Factores de Competitividade); CESAM (UID/ AMB/50017, FCT/MEC through national funds, and the co-funding by
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Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095
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the FEDER, POCI-01-0145-FEDER-007638, within the PT2020 Partnership Agreement and Compete 2020); Biomaterials for Regenerative Medicine, CENTRO-07-ST24 - FEDER – 002030. J. Rodrigues thank to FCT for their PhD grant SFRH/BD/76300/2011. References Adam, N., Leroux, F., Knapen, D., Bals, S., Blust, R., 2014. The uptake of ZnO and CuO nanoparticles in the water-flea Daphnia magna under acute exposure scenarios. Environ. Pollut. 194:130–137. http://dx.doi.org/10.1016/j.envpol.2014.06.037. Asghari, S., Johari, S.A., Lee, J.H., Kim, Y.S., Jeon, Y.B., Choi, H.J., Moon, M.C., Yu, I.J., 2012. Toxicity of various silver nanoparticles compared to silver ions in Daphnia magna. J. Nanobiotechnol. http://dx.doi.org/10.1186/1477-3155-10-14. ASTM E729-96, 2014. Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes. Macroinvertebrates, and Amphibians. ASTM International, West Conshohocken, PA. www.astm.org. Bianchini, A., Wood, C.M., 2002. Physiological effects of chronic silver exposure in Daphnia magna. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 133:137–145. http://dx. doi.org/10.1016/S1532-0456(02)00088-1. Chang, Y.-N., Zhang, M., Xia, L., Zhang, J., Xing, G., 2012. The toxic effects and mechanisms of CuO and ZnO nanoparticles. Materials (Basel) 5:2850–2871. http://dx.doi.org/10. 3390/ma5122850. Dimkpa, C.O., McLean, J.E., Britt, D.W., Anderson, A.J., 2015. Nano-CuO and interaction with nano-ZnO or soil bacterium provide evidence for the interference of nanoparticles in metal nutrition of plants. Ecotoxicology 24:119–129. http://dx.doi.org/10. 1007/s10646-014-1364-x. European Food Safety Authority, 2015. Harmonisation of human and ecological risk assessment of combined exposure to multiple chemicals. EFSA Support. Publ., EN-784 39. Fabrega, J., Luoma, S.N., Tyler, C.R., Galloway, T.S., Lead, J.R., 2011. Silver nanoparticles: behaviour and effects in the aquatic environment. Environ. Int. http://dx.doi.org/10. 1016/j.envint.2010.10.012. Freitas, E.C., Pinheiro, C., Rocha, O., Loureiro, S., 2014. Can mixtures of cyanotoxins represent a risk to the zooplankton? The case study of Daphnia magna Straus exposed to hepatotoxic and neurotoxic cyanobacterial extracts. Harmful Algae 31:143–152. http://dx.doi.org/10.1016/j.hal.2013.11.004. Gaiser, B.K., Biswas, A., Rosenkranz, P., Jepson, M.A., Lead, J.R., Stone, V., Tyler, C.R., Fernandes, T.F., 2011. Effects of silver and cerium dioxide micro- and nano-sized particles on Daphnia magna. J. Environ. Monit. 13:1227–1235. http://dx.doi.org/10.1039/ c1em10060b. Georgekutty, R., Seery, M.K., Pillai, S.C., 2008. A highly efficient Ag-ZnO photocatalyst: synthesis, properties, and mechanism. J. Phys. Chem. C 112:13563–13570. http:// dx.doi.org/10.1021/jp802729a. Gottschalk, F., Sonderer, T., Scholz, R.W., Nowack, B., 2009. Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, fullerenes) for different regions. Environ. Sci. Technol. 43:9216–9222. http://dx.doi.org/10.1021/es9015553. Heinlaan, M., Ivask, A., Blinova, I., Dubourguier, H.C., Kahru, A., 2008. Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus. Chemosphere 71:1308–1316. http://dx.doi. org/10.1016/j.chemosphere.2007.11.047. Jo, H.J., Choi, J.W., Lee, S.H., Hong, S.W., 2012. Acute toxicity of Ag and CuO nanoparticle suspensions against Daphnia magna: the importance of their dissolved fraction varying with preparation methods. J. Hazard. Mater. 227-228:301–308. http://dx.doi.org/ 10.1016/j.jhazmat.2012.05.066.
Jonker, M.J., Svendsen, C., Bedaux, J.J.M., Bongers, M., Kammenga, J.E., 2005. Significance testing of synergistic/antagonistic, dose level-dependent, or dose-ratio-dependent effects in mixture dose-response analysis. Environ. Toxicol. Chem. 24, 2701–2713. Li, F., Yuan, Y., Luo, J., Qin, Q., Wu, J., Li, Z., Huang, X., 2010. Synthesis and characterization of ZnO-Ag core-shell nanocomposites with uniform thin silver layers. Appl. Surf. Sci. 256:6076–6082. http://dx.doi.org/10.1016/j.apsusc.2010.03.123. Lopes, S., Ribeiro, F., Wojnarowicz, J., Lojkowski, W., Jurkschat, K., Crossley, A., Soares, A.M.V.M., Loureiro, S., 2014. Zinc oxide nanoparticles toxicity to Daphnia magna: size-dependent effects and dissolution. Environ. Toxicol. Chem. 33:190–198. http:// dx.doi.org/10.1002/etc.2413. Lopes, S., Pinheiro, C., Soares, A.M.V.M., Loureiro, S., 2016. Joint toxicity prediction of nanoparticles and ionic counterparts: simulating toxicity under a fate scenario. J. Hazard. Mater. 320:1–9. http://dx.doi.org/10.1016/j.jhazmat.2016.07.068. Loureiro, S., Amorim, M.J.B., Campos, B., Rodrigues, S.M.G., Soares, A.M.V.M., 2009. Assessing joint toxicity of chemicals in Enchytraeus albidus (Enchytraeidae) and Porcellionides pruinosus (Isopoda) using avoidance behaviour as an endpoint. Environ. Pollut. 157:625–636. http://dx.doi.org/10.1016/j.envpol.2008.08.010. Loureiro, S., Svendsen, C., Ferreira, A.L., Pinheiro, C., Ribeiro, F., Soares, A.M., 2010. Toxicity of three binary mixtures to Daphnia magna: comparing chemical modes of action and deviations from conceptual models. Environ. Toxicol. Chem. 29, 1716–1726. Ma, H., Williams, P.L., Diamond, S.A., 2013. Ecotoxicity of manufactured ZnO nanoparticles - a review. Environ. Pollut. 172:76–85. http://dx.doi.org/10.1016/j.envpol.2012.08. 011. Mackevica, A., Skjolding, L.M., Gergs, A., Palmqvist, A., Baun, A., 2015. Chronic toxicity of silver nanoparticles to Daphnia magna under different feeding conditions. Aquat. Toxicol. 161C:10–16. http://dx.doi.org/10.1016/j.aquatox.2015.01.023. Manke, a., Wang, L.Y., Rojanasakul, Y., 2013. Mechanisms of nanoparticle-induced oxidative stress and toxicity. Biomed. Res. Int. 2013:15. http://dx.doi.org/10.1155/2013/ 942916. Muyssen, B.T., De Schamphelaere, K.A., Janssen, C.R., 2006. Mechanisms of chronic waterborne Zn toxicity in Daphnia magna. Aquat. Toxicol. 77:393–401. http://dx.doi.org/10. 1016/j.aquatox.2006.01.006. OECD, 1998. Test No. 211: Daphnia magna Reproduction Test. OECD Guidel. Test. Chem. OECD, 2004. Test No. 202: Daphnia sp., acute immobilization test. OECD Guidel. Test. Chem. Piccinno, F., Gottschalk, F., Seeger, S., Nowack, B., 2012. Industrial production quantities and uses of ten engineered nanomaterials in Europe and the world. J. Nanopart. Res. 14:1–11. http://dx.doi.org/10.1007/s11051-012-1109-9. Rekha, K., Nirmala, M., Nair, M.G., Anukaliani, A., 2010. Structural, optical, photocatalytic and antibacterial activity of zinc oxide and manganese doped zinc oxide nanoparticles. Phys. B Condens. Matter 405:3180–3185. http://dx.doi.org/10.1016/j.physb. 2010.04.042. Ren, C., Yang, B., Wu, M., Xu, J., Fu, Z., Lv, Y., Guo, T., Zhao, Y., Zhu, C., 2010. Synthesis of Ag/ ZnO nanorods array with enhanced photocatalytic performance. J. Hazard. Mater. 182:123–129. http://dx.doi.org/10.1016/j.jhazmat.2010.05.141. Ribeiro, F., Gallego-Urrea, J.A., Goodhead, R.M., Van Gestel, C.A.M., Moger, J., Soares, A.M.V.M., Loureiro, S., 2014. Uptake and elimination kinetics of silver nanoparticles and silver nitrate by Raphidocelis subcapitata: the influence of silver behaviour in solution. Nanotoxicology 5390:1–10. http://dx.doi.org/10.3109/17435390.2014. 963724. Ribeiro, F., Van Gestel, C.A.M., Pavlaki, M.D., Azevedo, S., Soares, A.M.V.M., Loureiro, S., 2017. Bioaccumulation of silver in Daphnia magna: waterborne and dietary exposure to nanoparticles and dissolved silver. Sci. Total Environ. 574:1633–1639. http://dx. doi.org/10.1016/j.scitotenv.2016.08.204. Rodrigues, J., Soares, M.R.N., Carvalho, R.G., Fernandes, A.J.S., Correia, M.R., Monteiro, T., Costa, F.M., 2012. Synthesis, structural and optical characterization of ZnO crystals grown in the presence of silver. Thin Solid Films. Elsevier B.V.:pp. 4717–4721 http://dx.doi.org/10.1016/j.tsf.2011.10.208. Rodrigues, J., Fernandes, A.J.S., Mata, D., Holz, T., Carvalho, R.G., Fath Allah, R., Ben, T., Gonzalez, D., Silva, R.F., da Cunha, A.F., Correia, M.R., Alves, L.C., Lorenz, K., Neves, A.J., Costa, F.M., Monteiro, T., 2014. ZnO micro/nanocrystals grown by laser assisted flow deposition. SPIE Photonics West 2014-OPTO: Optoelectronic Devices and Materials:p. 89871F http://dx.doi.org/10.1117/12.2039907. Sánchez, A., Recillas, S., Font, X., Casals, E., González, E., Puntes, V., 2011. Ecotoxicity of, and remediation with, engineered inorganic nanoparticles in the environment. TrAC Trends Anal. Chem. 30:507–516. http://dx.doi.org/10.1016/j.trac.2010.11.011. Soares, M.R.N., Ferro, M., Costa, F.M., Monteiro, T., 2015a. Upconversion luminescence and blackbody radiation in tetragonal YSZ co-doped with Tm(3+) and Yb(3+). Nanoscale 7:19958–19969. http://dx.doi.org/10.1039/c5nr04052c. Soares, M.R.N., Holz, T., Oliveira, F., Costa, F.M., Monteiro, T., 2015b. Tunable green to red ZrO 2 :Er nanophosphors. RSC Adv. 5:20138–20147. http://dx.doi.org/10.1039/ C5RA00189G. Systat, 2008. Systat Software, Incorporation. SigmaPlot for Windows version 11.0. Ullah, R., Dutta, J., 2008. Photocatalytic degradation of organic dyes with manganesedoped ZnO nanoparticles. J. Hazard. Mater. 156:194–200. http://dx.doi.org/10.1016/ j.jhazmat.2007.12.033. Yunus, I.S., Harwin, A.K., Adityawarman, D., Indarto, A., 2012. Nanotechnologies in water and air pollution treatment. Environ. Technol. Rev. 2515:1–13. http://dx.doi.org/10. 1080/21622515.2012.733966. Zhao, H., Lu, G., Xia, J., Jin, S., 2012. Toxicity of Nanoscale CuO and ZnO to Daphnia magna. Chem. Res. Chin. Univ. 28, 209–213. Zheng, Y., Chen, C., Zhan, Y., Lin, X., Zheng, Q., Wei, K., Zhu, J., 2008. Photocatalytic activity of Ag/ZnO heterostructure nanocatalyst: correlation between structure and property. J. Phys. Chem. C 112:10773–10777. http://dx.doi.org/10.1021/jp8027275.
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Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials S.L. Azevedo a, T. Holz b, J. Rodrigues b, T. Monteiro b, F.M. Costa b, A.M.V.M. Soares a, S. Loureiro a,⁎ a b
Department of Biology, CESAM, University of Aveiro, 3810-193 Aveiro, Portugal Physics Department, I3N, University of Aveiro, 3810-193 Aveiro, Portugal
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• The toxicity of a ZnO/Ag nanostructure (NS) was assessed on Daphnia magna. • A mixture toxicity approach was carried to predict toxicity based on single components. • ZnO/Ag-NS showed higher toxicity than the predicted based on individual toxicity. • ZnO/Ag-NS did not present the same behavior of a mixture of ZnO-NM and Ag-NM.
a r t i c l e
i n f o
Article history: Received 18 March 2016 Received in revised form 28 October 2016 Accepted 15 November 2016 Available online xxxx Editor: D. Barcelo Keywords: Silver Zinc oxide Nanomaterials Nanostructures Mixture toxicity Daphnia magna
a b s t r a c t Nanotechnology is a rising field and nanomaterials can now be found in a vast variety of products with different chemical compositions, sizes and shapes. New nanostructures combining different nanomaterials are being developed due to their enhancing characteristics when compared to nanomaterials alone. In the present study, the toxicity of a nanostructure composed by a ZnO nanomaterial with Ag nanomaterials on its surface (designated as ZnO/Ag nanostructure) was assessed using the model-organism Daphnia magna and its toxicity predicted based on the toxicity of the single components (Zn and Ag). For that ZnO and Ag nanomaterials as single components, along with its mixture prepared in the laboratory, were compared in terms of toxicity to ZnO/Ag nanostructures. Toxicity was assessed by immobilization and reproduction tests. A mixture toxicity approach was carried out using as starting point the conceptual model of Concentration Addition. The laboratory mixture of both nanomaterials showed that toxicity was dependent on the doses of ZnO and Ag used (immobilization) or presented a synergistic pattern (reproduction). The ZnO/Ag nanostructure toxicity prediction, based on the percentage of individual components, showed an increase in toxicity when compared to the expected (immobilization) and dependent on the concentration used (reproduction). This study demonstrates that the toxicity of the prepared mixture of ZnO and Ag and of the ZnO/Ag nanostructure cannot be predicted based on the toxicity of their components, highlighting the importance of taking into account the interaction between nanomaterials when assessing hazard and risk. © 2016 Elsevier B.V. All rights reserved.
⁎ Corresponding author at: University of Aveiro, Department of Biology, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal. E-mail address: [email protected] (S. Loureiro).
http://dx.doi.org/10.1016/j.scitotenv.2016.11.095 0048-9697/© 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095
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1. Introduction Nanotechnology is a fast growing industry and nanomaterials can today be found in a vast variety of products such as cosmetics, antibacterial substances, electronic and optoelectronic devices as light emitting diodes (LEDS) and solar cells (Piccinno et al., 2012). In the last decade, the production of nanomaterials had an exponential increase and now new research is focusing on increasing their performance and applicability. Some materials such as zinc oxide (ZnO) and titanium dioxide (TiO2), well-established semiconductors with intrinsic absorption in the ultraviolet (UV), are known to present high photocatalytic activity. Photocatalysis has several applications: in pigments, providing the white color to paper and paint, providing antibacterial properties and in water and soil remediation processes (Ullah and Dutta, 2008; Yunus et al., 2012). In the latter, photocatalysis is responsible for the oxidation of organic pollutants into nontoxic materials (Yunus et al., 2012). With the purpose of increasing the photocatalytic properties, zinc oxide nanomaterials (ZnO-NM) are now being combined with silver nanomaterials (Ag-NM) (Georgekutty et al., 2008; Zheng et al., 2008). This combination will allow the photocatalysis to occur not only with UV radiation but also with visible light due to the absorption of the silver surface plasmon resonance (Rekha et al., 2010). Several toxicity studies have demonstrated the negative effects of ZnO and Ag nanomaterials to aquatic organisms, as reported in the reviews of Ma et al. (2013) and of Fabrega et al. (2011), respectively. Although the toxicity of the individual nanomaterials is known, the toxicity of the combined component is not well understood. Taking this into account, it is important to understand if the behavior and toxicity of combined materials (also called heterostructures) can be predicted based on their individual behavior and toxicities, or if a different toxicity pattern will be observed due to potential different interactions with biotic compartments. Having this into consideration, the aim of this work was to understand the reasonableness of predicting the join toxicity of nanoheterostructures by knowing the toxicity of individual nanomaterial components. In addition, and based on the usual methodology employed for predicting mixture toxicities, a laboratorial-made binary mixture was tested to infer if results could help on this prediction. This is extremely important regarding regulatory issues, as NMs are included in the REACH regulation, meeting the regulations' substance definition. In addition, the recommendation allows also their inclusion in other European regulations, like the Classification, Labelling and Packaging (CLP) Regulation which deals directly with mixtures. For that, the toxicity of a nanostructure (NS) composed of ZnO with Ag-NM on its surface was assessed, along with the toxicity evaluation of ZnO-NM and Ag-NM alone and combined as a binary mixture. This NS was used as a model material to address the aims of the present study but also because of its wide expected application in environmental remediation processes, especially for organic pollutants' removal, which can lead to a potential exposure scenario in aquatic environments. This kind of NS can have several compositions and percentages of their individual components and therefore a mixture toxicity approach can provide a useful output. For the hazard assessment, immobilization and reproduction tests were performed with Daphnia magna and values of LC50 and EC50 were calculated from the single exposures, and used to set up the mixture toxicity experimental designs and predictions. 2. Material and methods 2.1. Nanomaterials Three nanomaterials were supplied as powders and used in the present study: zinc oxide tetrapods (ZnO-NM), a spherical silver (Ag-NM) and zinc oxide tetrapods decorated with 1–3% mol. of silver
nanomaterials (ZnO/Ag-NS). ZnO based materials were synthesized by Laser Assisted Flow Deposition (LAFD) starting from commercial powders of ZnO (AnalaR, 99.7%) and a ZnO mixture with AgNO3 (Merck, 99.8%) respectively, using a CO2 laser (λ = 10.6 μm), as reported elsewhere (Rodrigues et al., 2014; Rodrigues et al., 2012). Ag nanomaterials were synthesized by Pulsed Laser Ablation in Liquid (PLAL) technique (Soares et al., 2015a; Soares et al., 2015b) using an Ag target (Sigma Aldrich, 99.99%) immersed in acetone. The distance from the target to the surface was kept constant (10 mm) and the target surface was irradiated by a pulsed Nd:YAG laser (λ = 1064 nm) at the frequency of 10 Hz and energy per pulse of 650 mJ during 30 min. After this process the suspension was dried and the Ag particles were collected. All stock suspensions (5 mg·L−1) were prepared in ASTM moderated hard water (ASTM E729-96, 2014) with sonication in water bath (Ultrasonic SELECTA mod. 6.5 L) during 15 min. ASTM moderated hard water is constituted mainly by a set of salt solutions: calcium sulphate, potassium chloride, sodium hydrogen carbonate and magnesium sulphate. The stock suspensions were sampled for chemical analysis and characterization at time zero and after 48 h. The ZnO-NM and ZnO/AgNS stocks were analyzed through scanning electron microscope (SEM) and the Ag-NMs stock suspension through scanning transmission electron microscopy (STEM). Water samples for Zn and Ag total concentrations were acid digested; to discriminate Zn and Ag dissolution samples were also centrifuged at 2862g for 30 min using 3 kDa AMICON centrifugal filters (Merck, Millipore, Darmstadt, Germany) (Ribeiro et al., 2017). Analysis were carried out by ICP-MS at the Central Laboratory of Analysis, University of Aveiro, Portugal (certified laboratory). 2.2. Test organisms Experiments were carried out with Daphnia magna (clone Beak) as test organisms. Cultures were maintained in 1 L glass jars with artificial hard water ASTM, which was changed every other day. Organisms were fed with Raphidocelis subcapitata at a concentration of 3 × 105 cell·mL−1 with a supplement of seaweed extract (6 mL·L−1). Cultures were kept at 20 ± 1 °C and under a 16:8 h light: dark photoperiod. Neonates from the third and fourth brood were used to perform the toxicity tests. 2.3. Immobilization tests Immobilization tests were performed based on the OECD guideline 202 (OECD, 2004). Tests were carried out using five replicates per concentration and negative control, with five neonates with b24 h per replicate exposed in 50 ml glass beakers. Tests were maintained at room temperature of 20 ± 1 °C and a 16:8 h light:dark photoperiod and daphnids were not fed during the entire experiment. After 48 h, immobilization (inability to swim after gentle agitation of the beaker) and mortality were reported. The concentration that caused the immobilization of 50% of the neonates (LC50) was calculated (see below for details). Immobilized daphnids included non-mobile and dead animals. Nominal concentrations ranged from 0.5 to 1.3 mg·Zn·L−1 for the ZnO-NM treatments, 0.05 to 0.25 mg·Ag·L−1 for Ag-NM and 0.13 to 0.63 mg·ZnO/Ag·L−1 (1–3% mol. Ag) for ZnO/Ag-NS. This choice was based on available literature for ZnO and Ag-NMs toxicity to D. magna, and for a preliminary trial with ZnO/Ag-NS (data not shown). 2.4. Reproduction tests Reproduction tests were based on the OECD guideline 211 (OECD, 1998) and for each treatment 10 replicates with one neonate were used. Neonates with b24 h were transferred to 50 mL glass beakers and maintained during 21 days in similar temperature and photoperiod conditions as those described for cultures. The medium was renewed every two days and daphnids fed daily with the algae R. subcapitata. The number of offspring per brood were counted and removed from
Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095
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the beakers and the mortality of offspring and parental daphnids were also recorded. Daphnids were measured in the beginning and at the end of the test in order to assess differences on their size between treatments and the control. Dissolved oxygen, conductivity and pH were measured at the beginning, middle and end of the test in old and new medium. Toxicity tests were run with nominal concentrations ranging from: 0.1 to 0.4 mg·Zn·L−1 for ZnO-NM, 0.095 to 0.5 mg·Ag·L−1 for Ag-NM and 0.01 to 0.25 mg·ZnO/Ag·L−1 (1% Ag) for ZnO/Ag-NS. In addition, all tests were carried out with a negative control (ASTM media). As stated above concentrations were based on available literature for ZnO and Ag-NMs toxicity to D. magna, and on a preliminary trial with ZnO/Ag-NS (data not shown). 2.5. Combined exposures To perform exposures to binary mixtures of ZnO and Ag NMs, the methodology applied in the single exposures was used deferring only in the number of replicates. The number of replicates was reduced from five to three replicates in the immobilization tests, while in the reproduction tests replicates were reduced to one. This strategy provides an increase on the number of treatments used, allowing a wider range of exposure covering, without prejudice for the reliability of the results as the predictions used are based on regressions (for details see below) (Loureiro et al., 2010; Loureiro et al., 2009). The experimental design for the acute test was based on a full factorial design. For the reproduction test concentrations chosen followed a fixed ray design based on individual toxic units (TU). One TU was equal to the EC50 for each nanomaterial used and the TU never exceeded 2 aiming at avoiding mortality. Within each combined experiment an exposure to each chemical component alone was performed simultaneously using the concentrations applied in the single exposures. This approach has been implemented elsewhere in studies from the same research group where details are presented (Freitas et al., 2014; Loureiro et al., 2010). 2.6. Statistical analysis The concentration inducing 50% of immobilization (LC50) and 50% of effect (EC50) were calculated using the best fit, by a nonlinear regression using the SigmaPlot for windows version 11 (Systat, 2008). One-way analysis of variance (ANOVA) was used to identify significant differences between treatments and control (p b 0.05) (Systat, 2008), after normality and equal variance check through the ShapiroWilk test and Levene's mean test, respectively. A Dunnett's test was performed to compare each treatment to the control whenever significance was achieved in the ANOVA. The mixture set up was analyzed by the MixTox tool (Jonker et al., 2005). The MixTox tool allows to analyze binary mixtures and uses the conceptual models of Concentration addition (CA) and Independent action (IA) for toxicity prediction. Recently EFSA advised on the use of the CA model as the most conservative one, therefore this was followed in the present study to predict mixture toxicity. Deviations for synergism (more severe effect) and antagonism (less severe effect) were then evaluated by extending the mathematical equation of CA, by adding parameter a. With the addition of parameters a and also b (another extension to depict changings in the above mentioned patterns for synergism and antagonism) dose-ratio (DR) and dose-level (DL) deviations were assessed. The biological meaning of the positive or negative values of the parameters a and b can the found in Table 1. To allow further comparison with the binary mixture of ZnO-NM and Ag-NM, the results obtained from the ZnO/Ag-NS exposure were also analyzed with the MixTox tool (e.g. Loureiro et al., 2010), simulating a mixture toxicity approach by estimating each component concentration from the total. For that, Zn and Ag measured concentrations
3
were used to calculate the real exposure to both elements in the exposure of ZnO/Ag-NS (Table 2).
3. Results 3.1. Nanomaterials Fig. 1 presents the SEM and STEM images of the three nanomaterials used in the present study. ZnO-NM exhibit a wurtzite-structure and present a tetrapod morphology due to the faster growth along the normal directions to the zinc surfaces resulting in four legs preferentially oriented along the c axis direction (Rodrigues et al., 2012). In the case of the ZnO/Ag-NS, the tetrapods' surface was decorated with Ag metallic nanomaterials in the form of spherical droplets in a size smaller than 10 nm. Agglomerates (in different sizes) were observed as well as dispersed particles. For all nanomaterials the appearance did not change from time zero to 48 h after dispersion in culture medium. The ZnO/ Ag nanostructures presented a surface of ~40–50 nm, while the metallic Ag nanomaterials prepared by PLAL presented a size of ~5–12 nm. From the chemical analysis, it was observed that stock dispersions of ZnO-NM and ZnO/Ag-NS showed a recovery of 100% and 95% for Zn at the time of dispersion preparation (Table 2). After 48 h, Zn total concentration decreased approx. 41% and 9%, respectively. Zinc dissolution after 48 h was very low, corresponding only to 2.5% and 13% of the total measured in time zero, respectively (Table 2). For Ag total concentration, the stock dispersion of Ag-NM showed a recovery of 81%, while for the ZnO/Ag-NS dispersion the percentage of Ag in the NS was higher than 3% (approx. 3.5%). After 48 h, total Ag decreased to approx. 37% in the Ag-NM, while this decrease was higher (approx. 44%) for the Zn/AgNS. Dissolution of Ag was also very low, corresponding to 11% and 1.5% of the total measured in time zero for Ag-NM and Zn/Ag-NS, respectively (Table 2).
3.2. Single exposures 3.2.1. Immobilization tests All nanomaterials tested induced an increase in Daphnia magna mortality with the increasing concentration applied. For ZnO-NM the 48 h– LC50 was 1.29 mg·Zn·L−1 whereas Ag-NM presented a LC50 of 0.079 mg·Ag·L−1 (based on measured concentrations). Regarding ZnO/Ag-NS a 48 h-LC50 of 0.59 mg·ZnO/Ag·L−1 was obtained (Table 3). Table 1 Interpretation of additional parameters (a and b) that define the functional form of deviation patterns from concentration action. Adapted from Jonker et al. (2005). Deviation pattern
Concentration addition Parameter a
Synergism/antagonism Dose ratio dependent
Dose level dependent
a N 0: Antagonism a b 0: Synergism a N 0: Antagonism, except for those mixture ratios where significant negative b indicate synergism a b 0: Synergism, except for those mixture ratios where significant positive b indicate antagonism a N 0: Antagonism low dose level and synergism high dose level a b 0: Synergism low dose level and antagonism high dose level
Parameter b
bi N 0 Antagonism where the toxicity of the mixture is caused mainly by toxicant i bi b 0 Synergism where the toxicity of the mixture is caused mainly by toxicant i bDL N 1: Change at lower EC50 level bDL = 1: Change at EC50 level 0 b bDL b 1: Change at higher EC50 level bDL b 0: No change but the magnitude of S/A is DL dependent
Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095
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Table 2 Measured concentrations of Zn and Ag in a 5 mg·L−1 stock suspension (in ASTM moderate hard water) for the nanomaterials used (ZnO-NM, Ag-NM and Zn/Ag-NS) at time 0 h (after dispersion preparation) and after 48 h. The dissolved fraction of Zn and Ag was only measured after 48 h. The prediction for Ag in the ZnO/Ag-NS was of 1–3%, therefore nominal concentrations of Zn and Ag are presented as a range. Measured concentrations are expressed as average values ± in mg·L−1. Nanomaterials
Nominal concentration −1
Zn (mg·L
Zn-NM Ag-NP ZnO/Ag-NS
)
4 – 3.86–3.96
Total measured concentration −1
Ag (mg·L
−1
)
– 1.1 0.050–0.150
Zn (mg·L
)
Ag (mg·L
Dissolved fraction −1
)
Zn (mg·L−1)
Ag (mg·L−1)
0h
48 h
0h
48 h
48 h
48 h
4.01 ± 0.14 – 3.83 ± 0.02
2.36 ± 0.03 – 3.50 ± 0.29
– 0.901 ± 0.153 0.218 ± 0.52
– 0.655 ± 0.240 0.123 ± 0.60
0.150 ± 0.004 – 0.519 ± 0.35
– 0.097 ± 0.059 0.003 ± 0.002
3.2.2. Reproduction tests The EC50s for reproduction upon ZnO-NM, Ag-NM and ZnO/Ag-NS exposures can be found in Table 4. At the end of all reproduction tests, negative controls showed a mortality of parental animals always lower than 20% and the mean number of live offspring was always higher than 60 neonates per parental organisms. Therefore test validation was achieved (OECD, 1998). In addition, the exposure conditions measured validated the results, with dissolved oxygen ranging between 5.13 and 9.07 mg·L−1, conductivity between 500 and 630 μS/cm and pH between 7.66 and 8.36. For ZnO-NM significant differences in the number of neonates produced during the 21 days were observed for concentrations of 0.2, 0.3 and 0.4 mgZn·L− 1 (Fig. 2-A) (one way ANOVA, F4,41 = 83.86, p ≤ 0.001, Dunnett's method, p b 0.05). Significant differences on the daphnids' length were also observed for all concentrations used in the test (Fig. 2-B) (one way ANOVA, F4,41 = 38.37, p ≤ 0.001, Dunnett's method, p b 0.05). In the reproduction test with Ag-NM a significant decrease in the mean number of neonates produced per daphnia (Fig. 3-A) was observed at the three highest concentrations (0.25, 0.33 and 0.4 mg·Ag·L−1) (one way ANOVA, F5,48 = 21.04, p ≤ 0.001, Dunnett's method, p b 0.05). No significant differences were observed for the daphnids' length (Fig. 3-B), showing that Ag-NM did not affect this parameter when compare to the negative control. Alterations in the number of offspring and in the length of daphnids were observed in the exposures to ZnO/Ag-NS. The cumulative number of neonates produced per daphnia (Fig. 4-A) significantly decreased for the two highest concentrations (0.13 and 0.25 mg·Zn/Ag·L− 1) (one way ANOVA, F5,50 = 30.59, p ≤ 0.001, Dunnett's method, p b 0.05). The length of the adult daphnids was significantly decreased at concentrations of 0.06, 0.13 and 0.25 mg·Zn/Ag·L−1 (Fig. 4-B) (one way ANOVA, F5,50 = 10.95, p ≤ 0.001, Dunnett's method, p b 0.05). 3.3. Combined exposures approach The LC50 values derived from the single exposures to ZnO-NM and Ag-NM performed simultaneously in the combine exposures setup were 0.66 mgZn·L−1 (st.error = 0.07; r2 = 0.53) and 0.08 mg·Ag·L−1 (st.error = n.d.; r2 = 0.79), respectively. For the mixture of Ag-NM and ZnO-NM the acute toxicity results fitted the CA model (SS = 70.81; r2 = 0.76; p = 2.09 × 10−48), although a better fit was observed for the deviation pattern of dose-level (SS = 48.86; r2 = 0.84; p = 1.72 × 10−5; a = −1.48; b = 1.09), as showed in Fig. 5 response pattern. The negative a value and the positive b value close to 1 indicate synergism at low dose levels that change to antagonism at the LC50 level. The mixture toxicity concentrations for the reproduction test were designed as a fixed ray based on toxic units. Although the sum of toxic units never exceed 2 to avoid mortality, mortality was observed in thirteen combinations as shown in Fig. 6 by the white circles. The single exposures performed during the combined exposures derived EC50 values of 0.24 mg·Zn·L−1 (st.error = 0.10; r2 = 0.97) for ZnO-NM and 0.42 mg·Ag·L−1 (st.error = 0.06; r2 = 0.80) for Ag-NM.
The combined exposures fitted the CA model (SS = 5613.07; r2 = 0.66; p = 0.005), whose fit was afterwards improved by the equation extension to synergism (SS = 2107.43; r2 = 0.87; p = 2.7 × 10−5; a = −2.39) (Fig. 7). The results from the ZnO/Ag-NS toxicity output presented in Tables 3 and 4 were fractionated into the concentration of the individual components to enable toxicity comparison. From the chemical analysis ZnO/ Ag-NS presented approx. 3.5% of Ag in its composition, and such small exposure concentrations of Ag alone could not be simulated in the exposure trials. Therefore all comparisons were only carried out considering the used mixture toxicity approach, by modelling exposures and comparing observed vs predicted toxicities. Results from ZnO/Ag-NS exposures were modeled with CA reference model to predict patterns for combined exposures, based on the extrapolated concentrations for both ZnO and Ag. As an example, in the ZnO/Ag-NS acute toxicity test, the concentration of 0.13 mg·L− 1 of ZnO/Ag-NS presented 0.0998 mg·Zn·L−1 and 0.0057 mg·Ag·L−1, which were used as input for the mixture model. The results fitted the CA model explaining already 81% of the variation of the data set for the model (SS = 64.73; r2 = 0.81; p = 7.35 × 10−58). After adding parameter a to the CA equation the data fit was improved and a synergistic deviation obtained (SS = 7.93; r2 = 0.98; p = 4.8 × 10−14; a = −2.33). The results from the reproduction test with ZnO/Ag-NS exposures fitted the CA model (SS = 1891.56; r2 = 0.83; p = 1.4 × 10−3) and after the addition of parameters a and b the dose-level deviation was the one with the best fit (SS = 214.23; r2 = 0.98; p = 2.39 × 10−7; a = − 34.11, b = 0.68). Parameters interpretation showed an occurrence for synergism at low concentrations, changing to antagonism at dose levels higher than the EC50. 4. Discussion The aim of this work was to understand if it is possible to predict the toxicity of combined nanostructures based on the toxicity of its components. In addition, another goal was to infer if the toxicity of the composed nanomaterial would perform similarly to the toxicity of an “artificial” mixture of its components. In the present study single exposures as well as mixture exposures were performed with ZnO-NM and Ag-NM and the toxicity patterns compared to the one for ZnO/Ag-NS. As it is difficult to directly compare the toxicity of ZnO and Ag-NMs binary mixtures with similar concentrations of ZnO/Ag NS, considering the dilutions made, patterns for toxicity were then after compared. For ZnO-NM, negative effects were observed in the survival, reproduction and growth of Daphnia magna with the increasing concentration of ZnO-NM. Despite the tetrapod shape of the nanomaterials, the LC50 (1.29 mg·Zn·L−1) and EC50 (0.25 mg·Zn·L−1) values were in the range of those found in the literature for spherical nanomaterials. Some examples of toxicity values found for Daphnia magna exposed to ZnO spherical particles, from different sizes, ranged from 0.89 to 3.2 mg·ZnO·L− 1 for immobilization (Lopes et al., 2014; Ma et al., 2013) and from 0.26 to 0.36 mg·L− 1 for reproduction (Lopes et al., 2014). Looking also at Zn2+ concentrations as potentially responsible for the toxicity, the dissolved fraction measured after 48 h was very
Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095
STEM images of Ag-NM
SEM images of ZnO/Ag-NS
SEM images of ZnO-NM
S.L. Azevedo et al. / Science of the Total Environment xxx (2016) xxx–xxx
Fig. 1. Characterization of nanomaterials using SEM images for ZnO-NM (top image) and ZnO/Ag-NS (middle image) and STEM images for Ag-NM (bottom image).
low and therefore toxicity is mainly related to the nanoparticulated form. According to the SEM images, the ZnO tetrapods used in our study presented different sizes. Some studies regarding the toxicity of ZnONM state that the size play an important role in the toxicity, but this is not consensual. Lopes et al. (2014) observed that no significant differences were obtained when comparing the toxicity two ZnO-NM and a microsized form of ZnO (30 nm, 50–70 nm and N 200 nm) in Daphnia magna. A different result was obtained by Heinlaan et al. (2008)
5
Table 3 LC50 values of the nanomaterials tested presented in mg·Zn·L−1, mg·Ag·L−1 and in mg·ZnO/Ag·L−1. Results are expressed as mean ± standard error and are based on measured concentrations at time 0 h; R2 is the coefficient of determination; nd - not determined. Immobilization test 48 h-LC50 (mg·L−1)
Nanomaterial
ZnO-NM Ag-NP ZnO/Ag-NM
Zn
R2
Ag
R2
ZnO/Ag
R2
1.29 ± 2.15 – 0.453
0.90
– 0.079 ± nd 0.026
0.98
– – 0.59 ± 0.02
0.84
where the toxicity of the ZnO bulk material was 3-fold higher than for the ZnO-NM. Differences in results can be due to aggregation of the NMs in different exposure media used which can alter their dissolution rate and therefore their toxicity. For the immobilization effects, Ag-NMs showed higher toxicity when compared to ZnO-NM, presenting a LC50 (0.079 mg·Ag·L−1) more than ten times lower than the one for ZnO-NM (1.29 mg·Zn·L−1). Studies found in the literature regarding Ag-NMs toxicity show a large variety of results, due to high variability in the characteristics of the particles (e.g. coated or uncoated) and in the dispersion of the nanomaterials (e.g. colloids or suspensions). Colloids are usually prepared with coated nanomaterials and they are normally more stable than the suspensions. Ag-NMs suspensions tend to have a high tendency to aggregate or agglomerate which alter their surface area to volume ratio and also exhibit high sedimentation ratios (Asghari et al., 2012). These characteristics alter their toxicity and much higher LC50 values for the suspensions can be found in the literature when compared to those from colloids. Asghari et al. (2012) compared the toxicity of two Ag-NMs colloids and an Ag-NM suspension and found that Ag-NM suspensions had a LC50 of 0.187 mg·L−1 which was much higher than the values for the colloids (0.002 and 0.004 mg·L−1). The presence of food can also alter the toxicity of AgNMs. Gaiser et al. (2011) tested the toxicity of Ag-NM suspension to Daphnia magna during 96 h and obtained a LC50 of 0.1 mg·L−1. This value is similar to the one from the present study despite the differences in the duration of the test. This can be explained by the organisms' feeding during the entire experiment, which can lead to an increase on their fitness and to a decrease in the toxicity. The presence of food is known to decrease the toxicity of Ag-NMs to Daphnia magna. Ribeiro et al. (2014) showed that Ag-NMs do not enter the algae cells but rather attach to the surface of the algae. This may lead to an increase in the sedimentation and a decrease of Ag-NMs available for the organisms (Mackevica et al., 2015). The presence of food also seems to play an important role in the toxicity of Ag-NMs used in our tests since we obtained an EC50 (0.45 mg·Ag·L−1) higher than the LC50 (0.09 mg·Ag·L−1) without food, which is normally not achieved. No significant decrease in the size of the organisms was observed at the end of the test. The LC50 and EC50 values for the single exposures carried out simultaneously with the mixture trials, for both ZnO-NM and Ag-NM, were similar to the values obtained during the first tests (used to design the experimental set up of the mixture trials). These results validate our tests and organisms sensitivity and show that the reduction of replicates
Table 4 EC50 values of the nanomaterials tested presented in mg·Zn·L−1, mg·Ag·L−1 and in mg·ZnO/Ag·L−1. Results are expressed as mean ± standard error and are based on measured concentrations at time 0 h; R2 is the coefficient of determination. Reproduction test 21d-EC50 (mg·L−1)
Nanomaterial
ZnO-NM Ag-NM ZnO/Ag-NS a
Zn
R2
Ag
R2
ZnO/Ag
R2
0.25 ± 0.01 – 0.154
0.89
– 0.45 ± 0.04a 0.009
0.65
– – 0.20 ± 0.01
0.72
Value extrapolated as the prediction was higher than the highest concentration used.
Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095
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B 140
5
120 100
*
80 60
*
40
* 0
0.1
0.2
0.3
*
*
*
0.3
0.4
3 2 1
20 0
*
4
Length (mm)
Total number of neonates
A
0
0.4
0
0.1
0.2
Zn (mg.L-1)
Zn (mg.L-1)
Fig. 2. Total number of neonates per daphnia (A) and daphnids' length (mm) (B) after a 21 day exposure to ZnO-NM. Data is expressed as mg·Zn·L−1 mean values ± st. error considering the measured concentrations of the stock dispersion at time 0 h. *p b 0.05, Dunnett test.
did not diminish the results' output accuracy. To predict the mixture toxicity of ZnO-NM and Ag-NM, the CA model was used as it has been advised by the EFSA report on the “Harmonisation of human and ecological risk assessment of combined exposure to multiple chemicals” (European Food Safety Authority, 2015). Both immobilization and reproduction data set presented deviations from the CA model. In the immobilization experiments, a synergistic pattern can be observed for low concentrations, which changed to antagonism at the level of the EC50. The antagonistic response may be a result of the agglomeration of the nanomaterials due to high number of particles in the mixture, leading possibly to sedimentation and therefore decreasing toxicity. The synergistic pattern for immobilization at low concentrations can also be observed in the reproduction test. Although the toxic units never exceed 2, mortality occurred in thirteen concentrations of twenty three. For the surviving organisms, a synergistic response was observed for the number of neonates produced. Despite this synergistic pattern in reproduction, the observed mortality indicates also an increase in toxicity more than expected from the single exposure trials. Since the predicted environmental concentrations in surface waters for ZnO and Ag nanomaterials are in the range of the ng·L−1 (Gottschalk et al., 2009), the most probable scenario to occur in the environment is the synergistic response, under low dose, as also for longer term exposures. Some studies related the toxicity of ZnO-NM and Ag-NM to the release of Zn and Ag ions (Adam et al., 2014; Jo et al., 2012). The presence of Zn2+ and Ag+ may have negative impacts in the uptake of ions that are essential to the good health of the organisms. Zn is an essential metal but it can have negative effects if present at high concentrations. Zn ions can compete with Ca2+ which can lead to a disturbance in the Ca content in the body (Muyssen et al., 2006). A reduction of Ca content in Daphnia magna can negatively affect the movement and filtration rate which will eventually lead to a reduction of growth and reproduction due to feeding impairment (Muyssen et al., 2006). Ag+ in the media
can lead to ion deregulation due to the competition with Na+ uptake (Bianchini and Wood, 2002). Ion deregulation will eventually lead to the dead of the organisms. In the present study, the percentage for dissolution for the three materials was very low, which would predict a low influence of ions on the overall toxicity. According to Lopes et al. (2016) this pattern of “dilution”, with high nanomaterial concentrations and low ionic concentrations, will direct toxicity to less deleterious effects, induced by nanoparticles than those induced by ions, which are considered more hazardous. In the literature, very few studies determine the toxic effects of exposures with more than one nanomaterial. In the above mentioned study of Lopes et al. (2016), the mixture of different Ag and ZnO NPs highlighted a synergism for immobilizations which was also obtained partly in the present study. Zhao et al. (2012) observed a synergistic effect to the survival and reproduction of Daphnia magna when combining CuO-NM and ZnO-NM. A different response is observed when the species Phaseolus vulgaris (common bean) is exposed to a mixture of CuO-NM and ZnO-NM, where higher toxicity was observed to CuONM exposures only (Dimkpa et al., 2015). The ZnO nanomaterials decorated with Ag-NMs, in a percentage of approx. 3.5 mol%, on its surface, presented lower LC50 and EC50 values than the ones of the ZnO-NM. When looking at the results expressed as Zn concentration the values for the LC50 and EC50 decreased in N2fold for the ZnO/Ag-NS when compared to the ZnO-NM alone. In addition, and based on the principles of the CA model and looking at Table 4, to have a 50% of effect on the reproductive output (eq. to 1 TU), and using a equitoxic mixture as example, one would need a mixture with a concentration of 0.125 mg Zn·L−1 (eq. to the EC25 or ½ TU) jointly with a concentration of 0.225 mg Ag·L− 1 (eq. to the EC25 or ½ TU). From Table 4, while relating the effective concentration of ZnO/Ag to their components, the concentration of ZnO/Ag inducing 50% of effects was composed by 0.154 mg Zn·L− 1 but with a much lower
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Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095
S.L. Azevedo et al. / Science of the Total Environment xxx (2016) xxx–xxx
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Fig. 4. Total number of neonates per daphnia (A) and daphnids' length (mm) (B) after a 21 day exposure to ZnO/Ag-NS. Data is expressed as mg·ZnO/Ag·L−1 mean values ± st. error of nominal concentrations. *p b 0.05, Dunnett test.
concentration of Ag (0.009 mg Ag·L−1). This clearly indicates that less Ag was needed in the decorated ZnO to induce the same predicted effect. Metallic nanomaterials toxicity is usually linked to oxidative stress (Chang et al., 2012). NMs can cause oxidative stress by eliminating antioxidants or by direct production of reactive oxygen species (ROS) (Sánchez et al., 2011). Since the addition of Ag-NMs to the surface of ZnO-NM will increase the photocatalytic activity, this may lead to an increase in the ROS production when compared to the production by the nanomaterials alone. Oxidative stress can lead to membrane damage, lipid peroxidation or protein denaturation (Manke et al., 2013). This increase in concentration of Ag-NMs in the surface of the ZnO-NM and the consequent increase of the photocatalytic activity was demonstrated by Ren et al. (2010). These may lead to an increase of ROS production and therefore an increase in the toxicity. When analyzing the results with the MixTox tool, the synergistic patterns obtained may reflect this increase in cellular stress. Li et al. (Li et al., 2010) observed that the nanomaterials composed by Ag and gold (Au) in different percentages had different toxicity to Daphnia magna. The nanomaterials containing Ag in a percentage of 80% and 20% of Au were less toxic than expected, demonstrating that Au can reduce the toxicity of Ag-NM. When the ratio of Ag and Au is inverse (20% of Ag and 80% of Au) the nanomaterials were more toxic than expected (Li et al., 2010).
on the toxicity prediction regarding the nanomaterials and nanostructures used in the present study. As already shown in other case-studies on mixture toxicity evaluation and prediction, the CA conceptual model was not the best model to predict ZnO and Ag joint toxicity. The mixture of ZnO-NM and AgNM did not show an additive pattern but rather deviations such as dose-level and synergism. Since nanomaterials in the environment can be in a mixture with other nanomaterials it is important to understand how they will behave and more studies regarding the mixture toxicity of NMs need to be addressed. Also, ZnO-NM decorated with Ag-NM on its surface showed higher toxicity when compared with the predicted toxicity based on the results from the individual components. Therefore the toxicity of these new nanostructures needs to be addressed as a single material and not based on the toxicity of the single components. This output can be considered very important regarding the current regulations of REACH and CLP, where this ZnO/Ag NM would be treated as a binary mixture, and its toxicity predicted based on the CA model. This regulatory approach would for sure underestimate this NS's toxicity. In conclusion, more studies regarding mixture toxicity of NMs need to be conducted, and the hazard of new nanomaterials carefully evaluated before they are introduced in the market since their toxicity potential cannot be accurately predicted by the single components' toxicity.
5. Conclusion
Acknowledgements
The present study shows that the toxicity mixture approach was effective to achieve the goals of the present study and derive conclusions
Work reported here was partly funded through: the project RePulse - Responses of Daphnia magna exposed to chemical pulses and mixtures throughout generations (FCOMP-01-0124-FEDER-019321; Refª. FCT PTDC/AAC-AMB/117178/2010, by funding FEDER through COMPETE Programa Operacional Factores de Competitividade); CESAM (UID/ AMB/50017, FCT/MEC through national funds, and the co-funding by
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Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095
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the FEDER, POCI-01-0145-FEDER-007638, within the PT2020 Partnership Agreement and Compete 2020); Biomaterials for Regenerative Medicine, CENTRO-07-ST24 - FEDER – 002030. J. Rodrigues thank to FCT for their PhD grant SFRH/BD/76300/2011. References Adam, N., Leroux, F., Knapen, D., Bals, S., Blust, R., 2014. The uptake of ZnO and CuO nanoparticles in the water-flea Daphnia magna under acute exposure scenarios. Environ. Pollut. 194:130–137. http://dx.doi.org/10.1016/j.envpol.2014.06.037. Asghari, S., Johari, S.A., Lee, J.H., Kim, Y.S., Jeon, Y.B., Choi, H.J., Moon, M.C., Yu, I.J., 2012. Toxicity of various silver nanoparticles compared to silver ions in Daphnia magna. J. Nanobiotechnol. http://dx.doi.org/10.1186/1477-3155-10-14. ASTM E729-96, 2014. Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes. Macroinvertebrates, and Amphibians. ASTM International, West Conshohocken, PA. www.astm.org. Bianchini, A., Wood, C.M., 2002. Physiological effects of chronic silver exposure in Daphnia magna. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 133:137–145. http://dx. doi.org/10.1016/S1532-0456(02)00088-1. Chang, Y.-N., Zhang, M., Xia, L., Zhang, J., Xing, G., 2012. The toxic effects and mechanisms of CuO and ZnO nanoparticles. Materials (Basel) 5:2850–2871. http://dx.doi.org/10. 3390/ma5122850. Dimkpa, C.O., McLean, J.E., Britt, D.W., Anderson, A.J., 2015. Nano-CuO and interaction with nano-ZnO or soil bacterium provide evidence for the interference of nanoparticles in metal nutrition of plants. Ecotoxicology 24:119–129. http://dx.doi.org/10. 1007/s10646-014-1364-x. European Food Safety Authority, 2015. Harmonisation of human and ecological risk assessment of combined exposure to multiple chemicals. EFSA Support. Publ., EN-784 39. Fabrega, J., Luoma, S.N., Tyler, C.R., Galloway, T.S., Lead, J.R., 2011. Silver nanoparticles: behaviour and effects in the aquatic environment. Environ. Int. http://dx.doi.org/10. 1016/j.envint.2010.10.012. Freitas, E.C., Pinheiro, C., Rocha, O., Loureiro, S., 2014. Can mixtures of cyanotoxins represent a risk to the zooplankton? The case study of Daphnia magna Straus exposed to hepatotoxic and neurotoxic cyanobacterial extracts. Harmful Algae 31:143–152. http://dx.doi.org/10.1016/j.hal.2013.11.004. Gaiser, B.K., Biswas, A., Rosenkranz, P., Jepson, M.A., Lead, J.R., Stone, V., Tyler, C.R., Fernandes, T.F., 2011. Effects of silver and cerium dioxide micro- and nano-sized particles on Daphnia magna. J. Environ. Monit. 13:1227–1235. http://dx.doi.org/10.1039/ c1em10060b. Georgekutty, R., Seery, M.K., Pillai, S.C., 2008. A highly efficient Ag-ZnO photocatalyst: synthesis, properties, and mechanism. J. Phys. Chem. C 112:13563–13570. http:// dx.doi.org/10.1021/jp802729a. Gottschalk, F., Sonderer, T., Scholz, R.W., Nowack, B., 2009. Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, fullerenes) for different regions. Environ. Sci. Technol. 43:9216–9222. http://dx.doi.org/10.1021/es9015553. Heinlaan, M., Ivask, A., Blinova, I., Dubourguier, H.C., Kahru, A., 2008. Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus. Chemosphere 71:1308–1316. http://dx.doi. org/10.1016/j.chemosphere.2007.11.047. Jo, H.J., Choi, J.W., Lee, S.H., Hong, S.W., 2012. Acute toxicity of Ag and CuO nanoparticle suspensions against Daphnia magna: the importance of their dissolved fraction varying with preparation methods. J. Hazard. Mater. 227-228:301–308. http://dx.doi.org/ 10.1016/j.jhazmat.2012.05.066.
Jonker, M.J., Svendsen, C., Bedaux, J.J.M., Bongers, M., Kammenga, J.E., 2005. Significance testing of synergistic/antagonistic, dose level-dependent, or dose-ratio-dependent effects in mixture dose-response analysis. Environ. Toxicol. Chem. 24, 2701–2713. Li, F., Yuan, Y., Luo, J., Qin, Q., Wu, J., Li, Z., Huang, X., 2010. Synthesis and characterization of ZnO-Ag core-shell nanocomposites with uniform thin silver layers. Appl. Surf. Sci. 256:6076–6082. http://dx.doi.org/10.1016/j.apsusc.2010.03.123. Lopes, S., Ribeiro, F., Wojnarowicz, J., Lojkowski, W., Jurkschat, K., Crossley, A., Soares, A.M.V.M., Loureiro, S., 2014. Zinc oxide nanoparticles toxicity to Daphnia magna: size-dependent effects and dissolution. Environ. Toxicol. Chem. 33:190–198. http:// dx.doi.org/10.1002/etc.2413. Lopes, S., Pinheiro, C., Soares, A.M.V.M., Loureiro, S., 2016. Joint toxicity prediction of nanoparticles and ionic counterparts: simulating toxicity under a fate scenario. J. Hazard. Mater. 320:1–9. http://dx.doi.org/10.1016/j.jhazmat.2016.07.068. Loureiro, S., Amorim, M.J.B., Campos, B., Rodrigues, S.M.G., Soares, A.M.V.M., 2009. Assessing joint toxicity of chemicals in Enchytraeus albidus (Enchytraeidae) and Porcellionides pruinosus (Isopoda) using avoidance behaviour as an endpoint. Environ. Pollut. 157:625–636. http://dx.doi.org/10.1016/j.envpol.2008.08.010. Loureiro, S., Svendsen, C., Ferreira, A.L., Pinheiro, C., Ribeiro, F., Soares, A.M., 2010. Toxicity of three binary mixtures to Daphnia magna: comparing chemical modes of action and deviations from conceptual models. Environ. Toxicol. Chem. 29, 1716–1726. Ma, H., Williams, P.L., Diamond, S.A., 2013. Ecotoxicity of manufactured ZnO nanoparticles - a review. Environ. Pollut. 172:76–85. http://dx.doi.org/10.1016/j.envpol.2012.08. 011. Mackevica, A., Skjolding, L.M., Gergs, A., Palmqvist, A., Baun, A., 2015. Chronic toxicity of silver nanoparticles to Daphnia magna under different feeding conditions. Aquat. Toxicol. 161C:10–16. http://dx.doi.org/10.1016/j.aquatox.2015.01.023. Manke, a., Wang, L.Y., Rojanasakul, Y., 2013. Mechanisms of nanoparticle-induced oxidative stress and toxicity. Biomed. Res. Int. 2013:15. http://dx.doi.org/10.1155/2013/ 942916. Muyssen, B.T., De Schamphelaere, K.A., Janssen, C.R., 2006. Mechanisms of chronic waterborne Zn toxicity in Daphnia magna. Aquat. Toxicol. 77:393–401. http://dx.doi.org/10. 1016/j.aquatox.2006.01.006. OECD, 1998. Test No. 211: Daphnia magna Reproduction Test. OECD Guidel. Test. Chem. OECD, 2004. Test No. 202: Daphnia sp., acute immobilization test. OECD Guidel. Test. Chem. Piccinno, F., Gottschalk, F., Seeger, S., Nowack, B., 2012. Industrial production quantities and uses of ten engineered nanomaterials in Europe and the world. J. Nanopart. Res. 14:1–11. http://dx.doi.org/10.1007/s11051-012-1109-9. Rekha, K., Nirmala, M., Nair, M.G., Anukaliani, A., 2010. Structural, optical, photocatalytic and antibacterial activity of zinc oxide and manganese doped zinc oxide nanoparticles. Phys. B Condens. Matter 405:3180–3185. http://dx.doi.org/10.1016/j.physb. 2010.04.042. Ren, C., Yang, B., Wu, M., Xu, J., Fu, Z., Lv, Y., Guo, T., Zhao, Y., Zhu, C., 2010. Synthesis of Ag/ ZnO nanorods array with enhanced photocatalytic performance. J. Hazard. Mater. 182:123–129. http://dx.doi.org/10.1016/j.jhazmat.2010.05.141. Ribeiro, F., Gallego-Urrea, J.A., Goodhead, R.M., Van Gestel, C.A.M., Moger, J., Soares, A.M.V.M., Loureiro, S., 2014. Uptake and elimination kinetics of silver nanoparticles and silver nitrate by Raphidocelis subcapitata: the influence of silver behaviour in solution. Nanotoxicology 5390:1–10. http://dx.doi.org/10.3109/17435390.2014. 963724. Ribeiro, F., Van Gestel, C.A.M., Pavlaki, M.D., Azevedo, S., Soares, A.M.V.M., Loureiro, S., 2017. Bioaccumulation of silver in Daphnia magna: waterborne and dietary exposure to nanoparticles and dissolved silver. Sci. Total Environ. 574:1633–1639. http://dx. doi.org/10.1016/j.scitotenv.2016.08.204. Rodrigues, J., Soares, M.R.N., Carvalho, R.G., Fernandes, A.J.S., Correia, M.R., Monteiro, T., Costa, F.M., 2012. Synthesis, structural and optical characterization of ZnO crystals grown in the presence of silver. Thin Solid Films. Elsevier B.V.:pp. 4717–4721 http://dx.doi.org/10.1016/j.tsf.2011.10.208. Rodrigues, J., Fernandes, A.J.S., Mata, D., Holz, T., Carvalho, R.G., Fath Allah, R., Ben, T., Gonzalez, D., Silva, R.F., da Cunha, A.F., Correia, M.R., Alves, L.C., Lorenz, K., Neves, A.J., Costa, F.M., Monteiro, T., 2014. ZnO micro/nanocrystals grown by laser assisted flow deposition. SPIE Photonics West 2014-OPTO: Optoelectronic Devices and Materials:p. 89871F http://dx.doi.org/10.1117/12.2039907. Sánchez, A., Recillas, S., Font, X., Casals, E., González, E., Puntes, V., 2011. Ecotoxicity of, and remediation with, engineered inorganic nanoparticles in the environment. TrAC Trends Anal. Chem. 30:507–516. http://dx.doi.org/10.1016/j.trac.2010.11.011. Soares, M.R.N., Ferro, M., Costa, F.M., Monteiro, T., 2015a. Upconversion luminescence and blackbody radiation in tetragonal YSZ co-doped with Tm(3+) and Yb(3+). Nanoscale 7:19958–19969. http://dx.doi.org/10.1039/c5nr04052c. Soares, M.R.N., Holz, T., Oliveira, F., Costa, F.M., Monteiro, T., 2015b. Tunable green to red ZrO 2 :Er nanophosphors. RSC Adv. 5:20138–20147. http://dx.doi.org/10.1039/ C5RA00189G. Systat, 2008. Systat Software, Incorporation. SigmaPlot for Windows version 11.0. Ullah, R., Dutta, J., 2008. Photocatalytic degradation of organic dyes with manganesedoped ZnO nanoparticles. J. Hazard. Mater. 156:194–200. http://dx.doi.org/10.1016/ j.jhazmat.2007.12.033. Yunus, I.S., Harwin, A.K., Adityawarman, D., Indarto, A., 2012. Nanotechnologies in water and air pollution treatment. Environ. Technol. Rev. 2515:1–13. http://dx.doi.org/10. 1080/21622515.2012.733966. Zhao, H., Lu, G., Xia, J., Jin, S., 2012. Toxicity of Nanoscale CuO and ZnO to Daphnia magna. Chem. Res. Chin. Univ. 28, 209–213. Zheng, Y., Chen, C., Zhan, Y., Lin, X., Zheng, Q., Wei, K., Zhu, J., 2008. Photocatalytic activity of Ag/ZnO heterostructure nanocatalyst: correlation between structure and property. J. Phys. Chem. C 112:10773–10777. http://dx.doi.org/10.1021/jp8027275.
Please cite this article as: Azevedo, S.L., et al., A mixture toxicity approach to predict the toxicity of Ag decorated ZnO nanomaterials, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.11.095