Evaluation of nano-specific toxicity of zinc oxide

J Nanopart Res (2016) 18:372 DOI 10.1007/s11051-016-3689-2

RESEARCH PAPER

Evaluation of nano-specific toxicity of zinc oxide, copper oxide, and silver nanoparticles through toxic ratio Weicheng Zhang & Xiawei Liu & Shaopan Bao & Bangding Xiao & Tao Fang

Received: 16 May 2016 / Accepted: 29 November 2016 # Springer Science+Business Media Dordrecht 2016

Abstract For safety and environmental risk assessments of nanomaterials (NMs) and to provide essential toxicity data, nano-specific toxicities, or excess toxicities, of ZnO, CuO, and Ag nanoparticles (NPs) (20, 20, and 30 nm, respectively) to Escherichia coli and Saccharomyces cerevisiae in short-term (6 h) and long-term (48 h) bioassays were quantified based on a toxic ratio. ZnO NPs exhibited no nano-specific toxicities, reflecting similar toxicities as ZnO bulk particles (BPs) (as well as zinc salt). However, CuO and Ag NPs yielded distinctly nano-specific toxicities when compared with their BPs. According to their nano-specific toxicities, the capability of these NPs in eliciting hazardous effects on humans and the environment was as follows: CuO > Ag > ZnO NPs. Moreover, long-term bioassays were more sensitive to nano-specific toxicity than short-term bioassays. Overall, nano-specific toxicity is a meaningful measurement to evaluate the environmental risk of NPs. The log Teparticle value is a useful parameter for quantifying NP nano-specific toxicity and

Electronic supplementary material The online version of this article (doi:10.1007/s11051-016-3689-2) contains supplementary material, which is available to authorized users. W. Zhang : X. Liu : S. Bao : B. Xiao : T. Fang (*) Institute of Hydrobiology, Chinese Academy of Sciences, No. 7 Donghu South Road, Wuchang District, Wuhan, Hubei Province 430072, China e-mail: [email protected] X. Liu : S. Bao Graduate University of Chinese Academy of Sciences, Beijing 100049, China

enabling comparisons of international toxicological data. Furthermore, this value could be used to determine the environmental risk of NPs. Keywords Nano-specific toxicity . ZnO nanoparticles . CuO nanoparticles . Ag nanoparticles . Toxic ratio . Environmental and health risk assessment

Introduction The health and safety risks of nanomaterials (NMs) have recently drawn the attention of the public due to the hazardous effects of NMs on the environment. Many international organizations, such as the Organization for Economic Co-operation and Development (OECD), Registration, Evaluation, Authorisation and Restriction of Chemicals legislation (REACH), NANO Safety Cluster, European Food Safety Authority (EFSA), and Environmental Protection Agency (EPA), are responsible for regulating legislation and proposing toxic endpoints for the risk assessment of NMs (Kahru and Dubourguier 2010). For example, the OECD Working Party on Manufactured Nanomaterials (WPMN) was established to promote international cooperation in guiding the human health and environmental safety aspects of manufactured nanomaterials. A number of OECD test guidelines traditionally used for non-nano chemicals have been used to assess the risks of NMs (Heinlaan et al. 2008; Xiao et al. 2015). These methods provide valuable NMs hazard and toxicity data. Moreover, in addition to the regulation of NMs use, in vivo

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methods, and reducing, refining, and replacing animal experiment principle (3Rs), in vitro and in silico standards have been proposed for nanotoxicity investigations (Burello 2015; Gajewicz et al. 2012; Gong et al. 2012; He et al. 2013; Ivask et al. 2010; Kaweeteerawat et al. 2015; Kleandrova et al. 2014; McCracken et al. 2016; Mortimer et al. 2010; Qin et al. 2008). For further information about the safety of manufactured nanomaterials, readers are referred to OECD regulations (http://www.oecd.org/science/nanosafety/). Due to the efforts of many research groups, extensive toxicological data are available for certain nanoparticles (NPs) (Bondarenko et al. 2013; Gajewicz et al. 2012; Garner et al. 2015; Golbamaki et al. 2015; Kaweeteerawat et al. 2015; Magdolenova et al. 2014). However, conflicting results are frequently reported, which are usually attributed to incomplete characterizations of the NMs, different testing conditions, diverse model organisms, and different endpoints. Due to these inconsistencies, it is difficult to draw general conclusions for nanoecotoxicities of NPs (Bondarenko et al. 2013). Importantly, comparative toxicity rank systems for NMs have yet to be established. Instead, traditional toxicity rank methods have been employed to evaluate the hazardous effects of NPs (Bondarenko et al. 2013). For instance, the toxicity of three NPs toward the microalgae Pseudokirchneriella subcapitata decreased in the order ZnO > CuO > TiO2 NPs (Aruoja et al. 2009). However, the toxicity of these NPs to the ciliated protozoan Paramecium multimicronucleatum decreased in the order CuO > ZnO > TiO2 NPs (Li et al. 2012). Indeed, the L(E)C50 values of certain NPs toward different organisms and cells could span several orders of magnitude. For example, these values range for 3.7, 2.2, and 4.9 log units for ZnO, CuO, and Ag NPs, respectively (see Table S1). Thus, the L(E)C50 data do not fully reflect NPs’ toxicities. Nano-specific toxicity is an essential property of NMs. For example, CuO NPs have shown approximately 50 times higher toxicity to Vibrio fischeri, Daphnia magna, and Thamnocephalus platyurus than bulk CuO. TiO2 NPs are known to be toxic to D. magna and zebrafish, but bulk TiO2 is not toxic (Heinlaan et al. 2008; Xiong et al. 2011). Nano-specific toxicity has been considered (Donaldson and Poland 2013; Xiu et al. 2012); however, it is usually ignored in environmental health and safety risk assessments. Subsequently, we do not know the extent of hazard effects induced by NMs compared with their bulk counterparts. To

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complement L(E)C50 data for assessing the hazard risks of NPs, the nano-specific toxicity of NPs should be considered in environmental risk assessments. Still, how to qualitatively and quantitatively evaluate nanospecific toxicity of NPs is an open question. Although our previous research introduced parameters toxicity enhancement and toxicity difference to evaluate the nano-specific toxicity of ZnO NPs (Zhang et al. 2016), there are still a lot of barriers existing, such as the different toxic test conditions (in culture medium vs. deionized water) and tested organisms (fish vs. bacterium). Herein, we try to verity the validity of toxicity enhancement for understanding the nano-specific toxicity of NPs under various test conditions and tested organisms, applied by extending tested NPs and combining our experimental data with literature data. In the present study, ZnO, CuO, and Ag NPs that served as typical metal (oxide) NPs were evaluated for nano-specific toxicity. Moreover, log Teparticle and log Teion were calculated using literature toxicity data (e.g., L(E)50) of ZnO, CuO, and Ag NPs and utilized to extensively examine the applicability of log Teparticle and log Teion, respectively. This study highlights the importance of nano-specific toxicity for NPs’ safety and environmental risk assessments and provides bacterial toxicity data for regulatory legislation.

Materials and methods Nano and bulk particles and test organisms ZnO (20 nm) and CuO (20 nm) NPs were obtained from Beijing Nachen S&T, Ltd., at purities of 99.9 %. Ag NPs (35 nm) were purchased from Nanostructured & Amorphous Materials, Inc., USA, at 99.5 % purity. Bulk CuO, CuSO 4 ·5H 2 O, and AgNO 3 were purchased from Sin opha rm Gro up C o., L td. B ulk Z nO a nd ZnSO4·7H2O were obtained from Tianjin Kemiou Chemical Reagent Co., Ltd., China. Bulk Ag was delivered from Aladdin. Transmission electron microscopy (TEM, JEM100CXII, JEOL, Ltd., Japan) was used to characterize the shapes and sizes of ZnO, CuO, and Ag particles (10 mg/L) in deionized water, and scanning electron microscopy (SEM) was unitized to visualize the shapes with solid powders. Moreover, the hydrodynamic diameters and zeta potentials of these particles at 10 mg/L were inspected at 6 and 48 h time points, respectively,


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using a Nano-Zetasizer (1000S, Malvern Instrument, Ltd., UK). The released metallic ions collected through ultrafiltrate centrifugal tubes (3 kDa, Millipore; 4000×g, 30 min) (Wang et al. 2012b) for each particle type with initial concentrations of 1 and 10 mg/L (at 30 °C) were detected using atomic absorption spectroscopy (AAS). Two model organisms of wild-type yeast Saccharomyces cerevisiae and Escherichia coli were evaluated for cytotoxicity and were donated by the Aquatic Animal Protein Engineering Lab, Institute of Hydrobiology, Chinese Academy of Sciences. The cultivation of S. cerevisiae was the same as our earlier report (Bao et al. 2015). E. coli was cultured in LB medium under the same condition but under a different temperature (37 °C). Toxicity assay Toxicity test procedure to E. coli was similar to S. cerevisiae, which had been reported previously (Bao et al. 2015), except cell density (optical density, OD600 nm) of 0.450 (±0.030) and temperature of 37 °C for E. coli were applied. Cell viability for E. coli and S. cerevisiae under non-stress condition at different time points was performed. The results reveal that cell viability for the bacteria is valid (see Fig. S1). Therefore, detected cytotoxicity during short-term (6 h) and longterm (48 h) bioassays is a result from applied materials. For each particle exposure, at least six gradients of concentrations were applied into short-term (6 h) and long-term (48 h) bioassays, and respective yielded toxicity values (EC50) were calculated through dose–response cytotoxicity curves, which was an explained four-parameter sigmoid function Eq. (1):(Schramm et al. 2011): y ¼ min þ

ðmax−minÞ −Hillslope 1 þ ECx50

ð1Þ

As been proven previously that calculated EC50 were valid, nominal EC50 were selected to examine component of particles and respective released metallic ions accounting for the toxic effect. The released metallic ions within particle suspensions were separated and collected through ultrafiltrate centrifugal tubes (3 kDa, Millipore) (Wang et al. 2012b). The cytotoxicity assay for the released ions and particle suspensions was the same with that mentioned above. Cytotoxicity contributed from released ions and particle parts was calculated

employing the following equation Eq. 2, as descried before (Wang et al. 2012a; Xiao et al. 2015): ð2Þ E ðtotalÞ ¼ 1− 1−E ðionÞ 1−E ðparticleÞ In which, the E(total) was from the nominal EC50 as total of detected toxicity effect, E(ion) was detected toxicity effect coming from dissolved metallic ions, and E(particle) could be the calculated nano or bulk particleinduced toxicity effect. Toxicity ratio (enhancement) To rank nano-specific toxicity of NPs, toxicity ratio, also named toxicity enhancement (Teparticle), was employed. Toxicity ratio as a dimensionless was defined over toxicity of NPs and BPs, as expression in Eq. 3. T particle ¼ e

EC50 ðBPsÞ EC50 ðNPsÞ

ð3Þ

Teion was served to evaluate whether toxic mechanism is dependent on metallic ions. Comparison of toxicity of ions (EC50 (ion)) and NPs (EC50 (particle)) yielded Teion in terms of: T ion e ¼

EC50 ðionÞ EC50 ðNPsÞ

ð4Þ

Theoretical and conceptual supports (Eco)toxicity is a science concerned with contaminants in the biosphere and their effects on constituents of the biosphere, which was defined as Bthe study of toxic effects, caused by natural or synthetic pollutants, to living organisms^ (Buzea et al. 2007; Kahru and Dubourguier 2010). Similarly, nano(eco)toxicity is a science of the toxicity of NPs to the biosphere, which could be defined as Ba new branch of toxicology to address the adverse health effects caused by nanoparticles^ (Buzea et al. 2007; Donaldson et al. 2004). Due to the nano-specific physicochemical properties, NPs normally show different fate in environment and excess toxicity to the biosphere compared to their bulk counterparts. The excess toxicity will become special and important for safe design and environmental risk assessment of NMs. Correspondingly, the excess toxicity elicited by NPs is essential from nano-specific properties that are governed by size of NPs only. Herein, we propose that nano-specific toxicity is particular for

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NP-induced excess toxicity or called toxicity enhancement to biosphere compared to their bulk materials (see Scheme 1). Accordingly, the nano-specific toxicity is relating with nanoscale size or nanoscale size-dependent properties, such as surface area, surface activity and energy, and so on. Toxicity that is affected by morphology, chemistry, or coating of NPs (Bai and Liu 2013; Yang et al. 2009) will be excluded in nano-specific toxicity, because these factors are nanoscale sizeindependent properties (see Scheme 1). Theoretically, nanoscale size-dependent properties also have nanospecific effects on the environmental fate of NPs, which is definitely important but not discussed in this study. Typically, metal and metal oxide NPs could dissolve in environment, and the released metallic ions will contribute to the nano(eco)toxicity. Compared to bulk particles (BPs), metal (oxide) NPs usually dissolve fast and the metallic ions will present higher concentrations (Scheme 1). Therefore, in comparison with BPs, excess toxicity of metal (oxide) NPs could be possible in the case of ion contribution to toxicity. The dissolution processes of metal (oxide) NPs correlate with both size-dependent (e.g., size and surface area) and sizeindependent features (e.g. shape and coating). In summary, nano-specific toxicity is particular part of nano(eco)toxicity, which is relating with nanoscale sizedependent properties only (Scheme 1). Thus, it is meaningful just under condition of comparison with toxicity of their bulk counterparts. Nano(eco)toxicity is particular part of (eco)toxicity, which is governed by both nanoscale size-dependent properties and size-independent properties (such as shape, chemistry, and coating) (Zhou et al. 2010; Zhou et al. 2011; Zhu et al. 2011). From an organic chemical toxicity viewpoint, toxicity enhancement (Te) is utilized to discriminate and evaluate excess toxicity (Schramm et al. 2011). It was identified that Te = 10 as a threshold was utilized to separate excess toxicity (log Te ≥ 1) from baseline toxicity (log Te < 1). The levels of toxicities are derived from reactive capacity of the hydrophobicity of chemical, respectively. Theoretically, nanotoxicity of NPs could be derived from nano-specific features that are based on bulk features of BPs with identical chemistries. Similar with toxicity of organic chemical, we propose a hypothesis that BP only exerts baseline toxicity, but NPs could exhibit excess toxicity. Therefore, Te is utilized to quantify the nano-specific toxicity of NPs, and Te = 10 serves as the cutoff to determine whether NPs possess nano-specific toxicity.


Literature toxicity data The nanotoxicity data of ZnO, CuO, and Ag nanoparticles for various species, e.g., crustaceans, algae, fish, nematodes, bacteria, yeasts/fungi, protozoa, and mammalian cells, were collected. The median lethal concentrations (LC50) and half maximal effect concentrations (EC50) were obtained. Publications without explicitly provided dose–response curves (LC50 or EC50) were excluded. For the purposes of comparison, publications that provided toxicity data for CuO and ZnO NPs, BPs, and metal salts were included. Because toxicity data for Ag BPs were scarce, the publications regarding toxicity data for Ag NPs and silver salts included chronic toxicity and genotoxicity and other toxicological response data were excluded. The collected toxicological data and basic exposure conditions were compiled and summarized in Tables S3, S4, and S5 for ZnO, CuO, and Ag NPs, respectively.

Result and discussion Characterization of nano and bulk particles Figure S2 summarized shape and size information of all nano and bulk particles in deionized water. Through inspection of these TEM images, particle sizes of most nanoparticles belong to nanoscale, although aggregations partly happened. On the contrary, particle sizes of most bulk particles settle down to micro size. Still, some of bulk particles present nanoscale size ( ZnO > CuO NPs. This was consistent with previous reports (Bondarenko et al. 2013). However, this type of classification did not consider the nano-

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specific effects of NPs. NPs’ nanotoxicities have excessive toxicities and significantly impact the environment. An excellent example would be to compare CuO NPs with BPs. CuO NPs show 50 times higher toxicity to V. fischeri, D. magna, and T. platyurus than bulk CuO particles (Heinlaan et al. 2008). Comparatively, ZnO NPs exert very similar toxicity effects as ZnO BPs (Heinlaan et al. 2008), and thus, there seems to be no more specific risks associated with ZnO NPs than BPs. However, ZnO NPs normally show higher toxic effect than CuO NPs (which have lower EC50 values) (Aruoja et al. 2009; Heinlaan et al. 2008; Kasemets et al. 2009; Mortimer et al. 2010). Furthermore, the nano-specific (excess) toxicities for CuO NPs are more serious, suggesting that higher environmental risks are associated with CuO NPs. Therefore, beside conventional toxicity data (such as E(L)C50), the nano-specific toxicities are also meaningful and should be considered when evaluating the potential toxicity effects and environmental risks of NPs. Due to the nano-specific toxicity of these NPs to the E. coli and yeast, a new toxicity ranking order appears to be CuO > Ag > ZnO NPs. Accordingly, CuO NPs will yield the most severe toxic outcome, followed by Ag and ZnO NPs. Correlation of log Teparticle and log Teion to literature data In principle, based on their nano-specific properties, the ZnO, CuO, and Ag NPs should elicit excess toxicities when compared with their BPs. Furthermore, a number of studies have concluded that these toxicities were derived from released metallic ions. To evaluate the validity of the log Te values, data from the literature were collected. The accuracy of the log Teparticle and log Teion values for each of the NPs was calculated using the log Teparticle = 1 and log Teion = −1 thresholds. As shown in Table 4, the log Teparticle and log Teion values for the ZnO NPs ranged from −0.20 to 0.67 and −0.27 to 2.10, respectively. The calculated accuracy was zero. The toxicological data from our toxicity assays were consistent with the literature data: no nano-specific toxicities were observed for the ZnO NPs, and the detected toxicities of ZnO NPs were not or only slightly dependent on the released zinc ions. Other studies have concluded that the toxicities of ZnO NPs were dependent on the dissolved zinc ions . N ev erth ele ss, this oversimplified comparison did not consider the dose or solubility ratios. In most cases, the solubility percentages of ZnO NPs were less than 30 %, and only an

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Table 4 Accuracy of log Teparticle and log Teion for ZnO, CuO, and Ag NPs was calculated according to the dataset from Tables S3, S4, and S5, employing log Teparticle = 1 and log Teion = −1 as cutoff Particles

Log Teparticle

Log Teion

n

Range

Accuracy ( %)

n

Range

Accuracy ( %)

ZnO NPs

24

–0.20 to 0.67

0.00

24

–0.27 to 2.10

0.00

CuO NPs

22

0.59 to 2.13

95.4

21

–2.06 to 0.31

38.10

Ag NPs

1

0.67

0.00

31

–2.20 to 0.60

61.29

applied dose of lower than 1 mg/L ZnO NPs could be completely dissolved within 8 h (Bian et al. 2011; Li et al. 2014). Considering only 10 % of NPs dissolved in most toxicity assays, when the metal salts exerted 10 times higher hazard effects than NPs, based on the log Teion = −1 threshold, we conclude that the detected nanotoxicity is dependent on the released ions. In a realistic toxicity assay, many factors also govern toxicity, such as exposure and toxicant bioavailability. In our study, the low accuracy of the log Teparticle calculation indicated that the ZnO NPs did not have nano-specific toxicity. The low accuracy of the log Teion calculation indicated that the ZnO NP toxicity was not association with released zinc ions or had only a minor association with released zinc ions. In the case of the CuO NPs, the accuracy of the log Teparticle value was pretty high at 95.4 %. The only outlier was a log Teparticle = 0.59 value for CuO NPs against A549 cells. CuO NPs elicited nearly four times higher toxicity against A549 cells when compared with CuO BPs (Wang et al. 2012b). Omitting the outlier, the log Teparticle values indicated that the CuO NPs induced 10 times greater toxicity than the CuO BPs and in some cases even over 100 times. However, the values of the log Teion calculations covered more than two log units from −2.06 to 0.31, suggesting that part of the detected CuO NPs’ toxicity could not be explained by dissolved copper ions. Indeed, the accuracy of the log Teion calculation was pretty low (38.10 %, see Table 4). When combined with the accuracies of the log Teparticle and log Teion calculations, the CuO NPs showed nanospecific toxicities, which were partially derived from released Cu2+. This low log Teion value accuracy could be explained by the log Teion data from bioluminescent assays on E. coli (see Table S4). However, these log Teion calculations revealed that the released Cu2+ were partially attributed to the detected toxicity, as seen by the log Teion values less than −0.5 (except log Teion = −0.38

for E. coli (JI130) 2 h test). Moreover, the solubility of CuO NPs was 20 ±5.7 %, indicating that the valid concentration of Cu2+ was dramatically lower than the applied nominal concentration of CuO NPs. Interestingly, other bioluminescent inhibition assays to V. fischeri (numbers 4 and 5, Table S4) showed similar results wherein the detected nano-specific toxicities for CuO NPs were due to released Cu2+ (log Teion = −1.93 and −1.69, respectively). The bioluminescent assay for recombinant E. coli showed less sensitivity than that for V. fischeri. Bioluminescent emissions from the bacteria require metabolic energy. However, the recombinant E. coli were used as biosensors for evaluating ROS generation caused by NPs (or their released ions) (Ivask et al. 2010). Omitting the data from these recombinant E. coli, the accuracies of the log Teparticle and log Teion calculations were 90.00 and 77.78 %, respectively. In most cases, this implies that CuO NPs could elicit nano-specific toxicities due to dissolved Cu2+. Regarding the Ag NPs, the accuracy of the log Teparticle calculations is unknown because of the lack of the data. The log Teion values range nearly 3 log units from −2.20 to 0.60. Correspondingly, the accuracy is 61.3 %. This suggests that the toxic source was not only dependent on Ag+. When the data from recombinant E. coli were omitted as mentioned above, the accuracy barely improved to 63.2 %. However, numerous studies have shown that released Ag+ from Ag NPs resulted in nano-toxic effects. This calculated accuracy is comparable for two reasons. On the one hand, PVP-coated Ag NPs released fewer Ag+ than bare Ag NPs, and hence, the calculated log Teion value was independent of the Ag+. Particularly, the smaller particle size PVP-coated Ag NPs had higher log Teparticle values (numbers 9 vs. 10 vs. 11 and numbers 12 vs. 13 vs. 14 in Table S5). In principle, the toxicity of smaller sized Ag NPs is mainly due to the particle effect. On the other hand, Ag NPs were unable to release as much Ag+ to exert a detectable


toxicity during the shortened exposure (especially at 5 min, e.g., compare numbers 15 and 16 with 17 and 18 in Table S5). Therefore, the calculated log Teion value indicated that the detected toxicities were not derived from Ag+. Toxicological investigations of NPs have suggested that the physicochemical properties of NPs, e.g., shape, size, surface charge, solubility, chemical composition, binding ability to biological sites (proteins and peptides), and their metabolic breakdown, influence the toxicity of NPs (Bondarenko et al. 2013; Golbamaki et al. 2015; He et al. 2015; Ivask et al. 2014b; McCracken et al. 2016; Pettibone and Louie 2015; von Moos et al. 2014). Based on these factors and available toxicological data, the following trends were observed. First, the log Teparticle and log Teion values increased or slightly increased with exposure time (see numbers 6 vs. 7 and number 9 vs. 10 in Table S4 for the CuO NPs), indicating that the nano-specific toxicity for NPs was more serious and that this kind of nano-specific toxicity was not due to the released metal ions in the long-term assay. This conclusion was consistent with our outcomes. The log Teparticle and log Teion values of the CuO and Ag NPs increased in the long-term bioassays when compared with the short-term bioassays. The increased log Teparticle values could be attributed to the more severe toxic effects yielded by NPs (but not BPs) for longer exposures. The increased log Teion values could be due to the lack of change in the toxic effect during the different exposure times. However, this trend was not observed for the ZnO NPs due to the lack of nano-specific toxicity. Surprisingly, the toxic effect induced by ZnO NPs to T. thermophila and S. cerevisiae did not change in the long-term exposure, as shown in Table S3. Second, an increasing particle size of the NPs decreased the log Teion values, implying that the toxic mechanisms of larger sized NPs were associated with the released ions. As shown in Table S5 (numbers 2 to 5, 9 to 11, and 12 to 14), the log Teion values of the Ag NPs against various organisms dramatically decreased as the NP size increased. Moreover, the changes to the log Teion values were gradual for particle sizes over 20 nm. Indeed, the log Teion values decreased to less than 0.25 log units as the Ag NP size increased from 30 or 20 to 50 nm (compare numbers 4 and 5, 10 and 11, and 13 and 14 in Table S5). This effect was dependent on the toxicity of the Ag NPs deriving from the dissolved ions and from the smaller size of Ag NPs more readily

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releasing Ag+ and thereby more toxicity during the assays (Xiu et al. 2012; Zhang et al. 2011). Hence, the toxicity and log Teion values of Ag NPs are size-dependent. Theoretically, the toxicity of the Ag NPs could also be due to the particle effect. Correspondingly, the log Teparticle values present the same pattern as that of the log Teion values: increasing the particle sizes of NPs decreased the log Teparticle value. However, we cannot verify this hypothesis; further studies are necessary.

Conclusions The nano-specific toxicities for NPs are meaningful and should be considered in safety and environmental risk assessments. The log Teparticle value is a useful parameter for quantifying NPs’ nano-specific toxicity and for comparing NPs’ toxicological data, regardless of species sensitivity, toxic endpoints, and exposure duration. The log Teion value can be used to determine the dependency of the nano-specific toxicity on released ions. In addition to the L(E)C50 value, the environmental risks and the nano-specific toxicities of NPs could also be helpful. In future studies, the environmental risk assessment of ZnO NPs should be evaluated for masked nanospecific toxicity. Acknowledgements We gratefully acknowledge funding support by the National Natural Science Foundation of China grant (No. 21477159). Thanks to the anonymous reviewers for their comments which greatly improved the manuscript. Compliance with ethical standards Conflict of interest There is no conflict of interest in this study.

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