Colloids and Surfaces B: Biointerfaces 116 (2014) 277–283
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Impact of agglomeration on the bioaccumulation of sub-100 nm sized TiO2 Dongwook Kwon, Soo Kyung Jeon, Tae Hyun Yoon ∗ Laboratory of Nanoscale Characterization & Environmental Chemistry, Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea
a r t i c l e
i n f o
Article history: Received 20 August 2013 Received in revised form 1 November 2013 Accepted 18 December 2013 Available online 18 January 2014 Keywords: Nanoparticle toxicity Dose–response Agglomeration Effective dose Bioaccumulation
a b s t r a c t To improve our understanding on the impact of extrinsic properties of NPs on their bioaccumulation and toxicity, we have investigated the bioaccumulation of sub 100 nm sized P25 TiO2 nanoparticles (NPs) by Daphnia magna (D. magna) in toxicity testing media. Based on our quantitative ICP-MS measurements as well as spectromicroscopic observations, we found that the bioaccumulation by D. magna were strongly influenced by the extrinsic properties of NPs as well as the biological uptake characteristics of D. magna. New sets of effective dosimetry parameters well correlated with the amount of NPs bioaccumulated within D. magna were also proposed. Based on these findings, we suggested that the extrinsic physicochemical properties of NPs (e.g., interfacial and colloidal properties of NPs) and biological characteristics for NPs (e.g., uptake cutoff sizes of testing organisms) should be considered and included when developing alternative dosimetry of NPs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Recent advances in nanoscience and nanotechnology have resulted in the widespread application of nanoparticles (NPs), including drug delivery, medical diagnostics, solar cells, and environmental catalysts [1–4]. However, there are also increasing concerns for their potential hazards on human health and ecosystems [5–7]. As a response to these concerns, the number of publications on the toxicity of NPs has recently increased dramatically. Nonetheless, many challenges and limitations still exist in the current methods of NP toxicity test and the development of unbiased testing strategies for impartial assessment of NPs toxicity is urgently needed [8–15]. According to recent reports, current challenges and limitations in NP toxicity assessments can be largely ascribed to (1) incomplete information about the widely varying physicochemical properties of NPs and (2) the lack of well-defined dosing metrics for the establishment of appropriate dose–response relationships [7–9,14–16]. To overcome these limitations, a comprehensive characterization of their physicochemical properties (e.g., chemical composition, crystallite phase, surface area, hydrodynamic size, etc.) is now considered as a prerequisite for toxicity testing of manufactured nanomaterials (MNs). Additionally, international collaborative activities for reference MNs and characterization
∗ Corresponding author. Tel.: +82 2 2220 4593; fax: +82 2 2299 0762. E-mail addresses: [email protected], [email protected] (T.H. Yoon). 0927-7765/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.12.047
protocols are actively in progress (e.g., OECD WPMN and ISO) [17,18]. On the other hand, while intrinsic (or static) physicochemical properties (e.g., total mass, number, and surface area) of NP are frequently used as alternative dosing metrics of NP [7,15], many other extrinsic (or dynamic) physicochemical properties, such as agglomeration and gravitational settling rate were also reported to influence the toxic responses of NPs and should be considered as key factors for alternative dosing metrics of NPs [19–23]. For instance, even for the reference NPs with clear identification and quantification of their intrinsic physicochemical properties, they may strongly interact with the components of toxicity testing media, and agglomerate and sediment differently according to their interfacial and colloidal properties as well as the characteristics of surrounding media [24–27]. These extrinsic properties are thought to strongly influence the delivered dose of NPs and may result in significant changes in bioaccumulation and acute/chronic toxicity of NPs. However, lack of appropriate control over these extrinsic properties often cause serious difficulties in performing the reliable and reproducible toxicity assessment of NPs. The purpose of this study was to improve our knowledge on the impact of extrinsic properties of NPs on the effective dose and bioaccumulation of NPs for Daphnia magna (D. magna) in toxicity testing media. To achieve this goal, extrinsic physicochemical properties of sub-100 nm sized TiO2 (TiO2 P25–70 ) were carefully monitored in reconstituted freshwater as a function of exposure time and surface properties ([Cit]/[TiO2 ]). Then, based on observations of a well-known planktonic invertebrate species, D. magna, bioaccumulation of TiO2 NPs were correlated with their
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physicochemical and biological properties to find out the most relevant dosing metrics of NPs in toxicity testing media.
3. Results and discussion
levels of citrate ions ([Cit]/[TiO2 ] = 0–14) were exposed to MHW media and their agglomeration and sedimentation behavior were monitored by measuring hydrodynamic size distributions and nort0 malized optical density at 320 nm (i.e., nOD320 = ODt1 320 /OD320 ). Agglomeration and sedimentation behavior of TiO2 dispersion in MHW media is presented in Fig. 1. The contour plot shown in Fig. 1a display the temporal variations in normalized optical density (nOD320 ) as a function of [Cit]/[TiO2 ], which clearly demonstrated the effect of surface charge on the sedimentation of NPs in the presence of this ecotoxicity test medium. Blue to purple colors (left side of the contour plot) indicate strong sedimentation of NPs under this condition (nOD320 < 0.9), while the green (right side of the contour plot) color implies no significant sedimentation or agglomeration under these conditions (nOD320 = 1.0 ± 0.1). The yellow to orange colors found in the central region indicate enhancement in the nOD320 > 1.1 range due to enhanced light scattering by slightly agglomerated but still well-suspended NPs under these conditions. Based on this color-coded contour plot of nOD320 , the colloidal stability (i.e., progress of sedimentation) of NP dispersion can be visualized and categorized into three subgroups: “unstable”, “partially stable”, and “stable” dispersions. Temporal variations of hydrodynamic size distribution were also monitored (Fig. 1b–d), which confirmed aforementioned variations in NP agglomeration. Under the stable dispersion condition, the hydrodynamic size distribution was maintained under 100 nm during the entire observation period (Fig. 1d), which is mostly due to the enhanced surface charge of the TiO2 NPs via adsorption of multiply-charged citrate ions [29,30]. As a consequence, TiO2 doses, at least in terms of their intrinsic properties (e.g., total mass, total number, and total surface area of NPs), were maintained at constant levels throughout the whole observation period under this “stable” dispersion condition. Under “partially stable” dispersion conditions, no significant sedimentations were observed, however, slight but significant enhancements in nOD320 and hydrodynamic size were observed, indicating significant progress in TiO2 NPs agglomeration process under this condition. Although this condition may allow a constant level of NP dose in terms of total mass of NPs, the progress in agglomeration will cause significant decreases in total number of NPs. In contrast to the above two conditions, TiO2 dispersions showed rapid agglomeration followed by heavy sedimentation within a few hours of exposure under “unstable” dispersion conditions. These agglomeration and sedimentation processes, which were frequently encountered during sample preparation for NP toxicity testing, have been considered the most serious obstacles in obtaining reliable and reproducible toxicity test results. Agglomeration and sedimentation observed in this unstable dispersion condition have been reported to cause completely different effective doses and biological uptakes for various biological species [19–23,31].
3.1. Preparation of TiO2 NPs with various dispersion conditions in test media
3.2. Microscopic and spectroscopic observation of TiO2 NP uptake by D. magna
The TiO2 used in this study may represent a well-suspended, nanosized fraction of bulk P25 TiO2 in environmental media. As we previously reported, it is comprised of aggregates or agglomerates of primary particles with diameters of about 21 nm and hydrodynamic size distributions centered around 70 nm [26]. It is stable in DIW for over a week with minimal size variation, although agglomeration followed by sedimentation were observed in typical in vivo ecotoxicity test media (MHW) and in vitro cytotoxic test media (Dulbecco’s modified Eagle’s medium, DMEM) [26,27]. In this study, to test the impact of extrinsic properties (surface charge, hydrodynamic size and agglomeration rate), we used citrate ions as surface modifying ligands of TiO2 in reconstituted fresh water media (MHW). TiO2 dispersions containing different
Both qualitative and quantitative studies on the bioaccumulation of TiO2 NPs were performed with D. magna, a well-known freshwater invertebrate species commonly used as a standard model organism of in vivo ecotoxicity assessments [28]. The absorption (or uptake), distribution, and excretion of TiO2 NPs by D. magna were monitored using bright field optical microscopy, Raman microspectrometry and ICP-MS measurements. In bright field optical images of D. magna (Fig. 2a–f), considerable bioaccumulations of TiO2 NPs were observed for unstable (Fig. 2b) and partially stable (Fig. 2c and d) dispersions, while no significant accumulations of NPs were visually observed for those exposed to stable TiO2 NP dispersions (Fig. 2e and f). Raman spectra shown in Fig. 2g confirmed that the local accumulation of TiO2 in the gut
2. Materials and methods 2.1. Nanoparticles preparation The aeroxide P25 TiO2 used in this study was provided by the manufacturer (lot no. 4168112198, Evonik GmbH, Germany). Stock solutions of 10 g/L P25 TiO2 powder were prepared by dispersing bulk powder in deionized water (DIW) and sonicated for 10 min using a probe sonicator (420 W, 20 kHz, Sonosmasher, Ulsso Hitech, Korea). Sodium citrate (99.0%, Cat. No. S4641, Sigma, MO, USA) was used as an electrostatic stabilizer of NPs. The sub-100 nm fractions of the TiO2 suspension were prepared by centrifugation at 6800 g for 20 min (Mega 17R, Hanil Science Industrial, Korea), from which the supernatant was carefully removed [26]. 2.2. Physicochemical characterization of TiO2 nanoparticles To monitor colloidal stability of TiO2 suspension in moderately hard synthetic freshwater (MHW), the optical density (OD) at 320 nm was monitored using an UV/vis spectrometer (Optizen2120UV, Mecasys, Korea). The hydrodynamic sizes of NPs in MHW were measured by using dynamic light scattering (DLS) instrument (Scatteroscope I, Qudix Inc, Korea). To measure the Ti concentration, samples were digested with mixed acid containing HNO3 , HCl, and HF and then analyzed using ICP-MS (Elan 6100, Perkin Elmer, CT, USA). 2.3. Characterization of TiO2 accumulation by D. magna Daphnia magna were cultured according to the U.S. EPA standard operating procedure [28]. Non-effect concentration value of 45 mg/L TiO2 was determined from preliminary acute toxicity tests and it was chosen for the exposure condition used in this accumulation study. Ten D. magna were used per group and experiments were conducted in 100 mL beakers. After 24 h exposure to 45 mg/L TiO2 , D. magna were removed and placed in fresh MHW. For the observation of TiO2 localization within D. magna, optical microscopy (IX51, Olympus, Japan) and Raman spectroscopy (LabRam HR, Jobin-Yvon, France) were used. To determine Ti bioaccumulation, group of D. magna were digested (0.5 mL 69% HNO3 for 30 min at 60 ◦ C and overnight at room temperature), and then Ti contents were measured using ICP-MS with mixed acid solution (HNO3 , HCl, and HF)
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Fig. 1. (a) Agglomeration and sedimentation of TiO2 at different citrate concentrations. Hydrodynamic size distributions at (b) unstable, (c) partially stable and (d) stable conditions.
and/or gill regions of D. magna are strongly affected by the dispersion stability of NPs. For instance, under the conditions of unstable NP dispersion, Raman peaks of TiO2 were observed for both gut and gill regions of D. magna, while similar peaks were observed only for the gut region of D. magna exposed to partially stable NP dispersions. In contrast, no Raman peaks of TiO2 NPs were observed for specimens exposed to stable dispersions of TiO2 NPs and control medium. By using ICP-MS analysis followed by acid digestion of carefully sampled D. magna, the total amounts of TiO2 accumulated within D. magna were also measured and presented in Table S1. For the TiO2 of unstable condition, the concentration of TiO2 was increased to 42.42 ug/ea after 24 h, which is about 40 times higher than those of “partially stable” dispersion condition (1.03 ug/ea) and “stable” dispersion condition (1.45 ug/ea) after 24 h. These quantitative measurements using ICP-MS technique also agree
well with the results from qualitative imaging techniques, such as optical microscopic and Raman spectroscopic observations. These observations regarding the impact of agglomeration on the bioaccumulation of NPs can be partially attributable to the unique uptake characteristics of D. magna. For this planktonic invertebrate species, the most likely pathway of NP uptake is through their active filter feeding. Previously, it was reported that D. magna actively filters and feeds on particles larger than their mean filter mesh sizes of 200 [28,32] and 280 nm [33]. Since the hydrodynamic size of TiO2 under the “stable” dispersion conditions is quite below this uptake cutoff size, it is less bioavailable for D. magna. However, under unstable dispersion conditions, TiO2 quickly agglomerates to form particles over about 200 nm and is available for uptake by D. magna. Thus, the agglomeration of sub-100 nm sized TiO2 particles may have caused a dramatic increase in the total amount of TiO2
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Fig. 2. Light microscope images of TiO2 exposure at different citrate concentrations. (a) Without TiO2 , (b) 0, (c) 4.88, (d) 6.03, (e) 12.07, (f) 14.30 (citrate mM/TiO2 mM). (g) Raman spectra of D. magna. (1, gut tract of (b) D. magna; 2, gill tract of (b) D. magna; 3, gut tract of (c) D. magna; 4, gill tract of (c) D. magna,; 5, gut tract of (f) D. magna; 6, gill tract of (f) D. magna).
accumulation in D. magna. Moreover, the kinetics of agglomeration also appeared to affect the biological uptake of NPs. Compared to the slow agglomeration kinetics under partially stable dispersion conditions, heavier accumulation of TiO2 were observed for D. magna under unstable dispersion conditions. 3.3. Impact of agglomeration on the uptake of TiO2 NPs by D. magna So far, we have shown that both extrinsic physicochemical properties (i.e., surface charge, hydrodynamic size distribution and etc.) and biological characteristics (i.e., uptake cutoff size) have strong influence on the bioaccumulation of TiO2 NPs by D. magna. By considering these physicochemical and biological characteristics, we have calculated the effective TiO2 NP doses and exposures bioavailable for D. magna under given dispersion conditions and their corresponding temporal variations are presented in Fig. 3. Even under the same nominal dose of TiO2 ([TiO2 ] = 45 mg/L), the variation in their physicochemical properties (e.g., surface charge) resulted in completely different hydrodynamic size distributions as well as the total amount of suspended TiO2 NPs (Fig. 3a). Furthermore, as shown in Fig. 3b and c, effective doses over cutoff sizes of 200 and 280 nm TiO2 (i.e., [TiO2 eff ], total amount of TiO2 NPs with hydrodynamic sizes over these uptake cutoff sizes of 200 and 280 nm) were calculated. Although it was previously reported that D. magna usually consume particles up to about 50 m [34], we did not consider particles larger than 10 m in these calculations, because that the DLS measurement is not reliable for this large size range. The effective doses were continuously varied under the “unstable” and “partially stable” conditions, while it was kept nearly constant under the “stable” dispersion condition during the whole exposure period. On the other hand, the effective exposures
of TiO2 (i.e., [TiO2 eff ], integrated sum of effective dose over exposure time) showed continuously increasing curves with different slopes depending on the dispersion stabilities of NPs. Under the “unstable” dispersion condition, the effective dose and effective exposure of TiO2 have similar values for both cutoff sizes (i.e., 200 and 280 nm). However, in “partially stable” and “stable” dispersion conditions, [TiO2 eff ] and [TiO2 eff ] change, these values are reduced for cutoff size of 280 nm [33]. In Fig. 4, relationships between the TiO2 NP exposures and their bioaccumulation were plotted. As previously noted, the nominal dose of TiO2 is the same for all experimental conditions ([TiO2 ] = 45 mg/L) and the corresponding TiO2 exposure was increased in proportion with the exposure time, but the measured bioaccumulation of TiO2 was completely independent of the nominal doses and exposures of NPs (Fig. 4a). In contrast, the [TiO2 eff ], in which hydrodynamic size distribution as well as biological uptake cutoff size were taken into account, had a positive relationship with the bioaccumulation of TiO2 by D. magna. For the cutoff size from literature (i.e., 200 nm, see Fig. 4b) [28,32], the correlation curve resulted in R2 value of 0.900. Moreover, much better correlation (R2 = 0.980) was observed for the cutoff size of 280 nm (see Fig. 4c), which was found as optimum cutoff size in a parallel modeling study [33]. These results confirmed us that new sets of dosing metrics (effective dose and exposures) involving extrinsic physicochemical properties of NPs (i.e., hydrodynamic size distribution) and biological characteristics of test species (i.e., uptake cutoff size), as well as intrinsic physicochemical properties (i.e., total mass concentration) provided a better understanding of the observed bioaccumulation of NPs and might show better predictability on the ecotoxicity of NPs. In addition to the effective exposure of TiO2 in mass basis, similar sets of parameters based on the number or surface area of NPs can be estimated and
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Fig. 3. (a) Mass (%) of TiO2 by hydrodynamic size at conditions. Effective dose for D. magna with cutoff size of (b) 200 nm and (c) 280 nm (left; unstable, middle; partially stable and right; stable condition). [TiO2 eff ], the amount of suspended TiO2 with hydrodynamic size above the uptake cutoff size of D. magna, in mg/L; [TiO2 eff ], integrated sum of effective dose over the exposure period, in hours mg/L; particle size of larger than cutoff size can be absorbed by D. magna.
compared as potential candidates for dosing metrics to find the most appropriate dose–response relationships with the observed NP accumulations or resultant toxicities of D. magna. 3.4. General discussion As far as we know, this is the first study that quantitatively correlates extrinsic physicochemical properties of NPs with their
bioaccumulation. However, there are several limitations in the current approach that need further improvements in future studies. One of the drawbacks in this approach is the limited capability of DLS technique in measuring hydrodynamic size distributions. Agglomeration process occurring in the “partially stable” state dispersions are typically very fast and involve multimodal size distribution of NP agglomerates. Although DLS is one of the most commonly used techniques for the measurement of hydrodynamic
Fig. 4. Relationships between NP doses and bioaccumulation were plotted. (b) Normal exposure, effective exposure with cutoff size of (b) 200 nm and (c) 280 nm TiO2 . The effective exposure (i.e., [TiO2 eff ]) was integrated sum of effective dose over the exposure period, and bioaccumulation was 45 mg/L TiO2 exposed concentration of D. magna.
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diameter, but it has limited capability to characterize dynamically changing colloids with multi-modal size distributions [35]. Additionally, due to the high scattering efficiency of micron-sized particles, presence of a small number of large aggregates may skew the measured hydrodynamic sizes towards a larger particle size distribution. Therefore, further studies using improved analytical techniques (e.g., nanoparticle tracking analysis) are necessary. Additionally, in the current study, we take the reductionist approach and focused only on the relationship between the physicochemical properties and the bioaccumulation of NPs rather than their toxicity. Although we have carefully performed current study under the non-toxic exposure condition based on the results of previous studies [36–38], it is also necessary to further expand current findings to better understand quantitative relationship between the physicochemical properties and the biological toxicity of NPs. Although our study was conducted for a single model organism, recently published studies also suggested that the bioaccumulation and toxicity depends on agglomeration and sedimentation properties of NPs in other biological species. With the increasing concentrations of sedimented NPs, enhancement in the bioaccumulation of Al2 O3 NPs was observed for sediment-dwelling organism (e.g., Hyalella azteca, Lumbriculus variegates and Corbicula fluminea) [39]. For the adherently grown mammalian cells, which typically used for in vitro toxicity test, sedimentation of NPs causes accumulation of NPs on top of the cell surface with much higher delivered (or effective) doses than nominal dose, which may lead to enhancement in cellular NP uptake and resultant cytotoxicity [23]. Thus, cell viability seems to depends on the type of mammalian cells, the cell viabilities of adherent cells (e.g., macrophages and kidney cell) were significantly decreased compared to those of floating cells (e.g., monocyte) [40]. In the case of the intravenous injection of agglomerated and well-dispersed multiwalled carbon nanotubes (MWCNTs) to mice, the agglomerated MWCNTs were accumulated in the lungs with inflammatory responses, while the well-suspended ones did not [41]. These results suggested that the extrinsic physicochemical properties (i.e., hydrodynamic size, agglomeration and sedimentation) of NP play important role in their bioaccumulation and toxicity. Further studies on the effects of these extrinsic parameters are urgently needed. Particularly, the concepts of effective dose and exposure involving extrinsic properties as well as biological characteristics should be further applied to various biological systems to improve our understanding on nanotoxicity mechanisms and included as parts of alternative dosing metrics of NPs for the unbiased assessment of nanotoxicity.
Acknowledgments This work was supported by the Korea Ministry of Environment and The Eco-Technopia 21 Project (091-091-081). We also thank Prof. Kyungho Choi’s Environmental Toxicology Laboratory of Seoul National University (Seoul, Korea) for providing D. magna for this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.12.047. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
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4. Conclusions In this study, we have investigated the impact of extrinsic properties of NPs on the bioaccumulation of sub-100 nm sized TiO2 dispersions with different surface charges, hydrodynamic size distributions and agglomeration kinetics. The effective doses of NPs and their bioaccumulation by D. magna were strongly influenced by the extrinsic properties of NPs as well as biological characteristics of D. magna. Based on our findings, we proposed that the physicochemical properties of NPs, including intrinsic (e.g., mass, number, and surface area of NPs) and extrinsic (e.g., interfacial and colloidal properties of NPs) properties as well as biological characteristics for the uptake of NPs (e.g., uptake cutoff sizes of testing organisms) should be considered and included when developing alternative dosimetry of NPs.
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Impact of agglomeration on the bioaccumulation of sub-100 nm sized TiO2 Dongwook Kwon, Soo Kyung Jeon, Tae Hyun Yoon ∗ Laboratory of Nanoscale Characterization & Environmental Chemistry, Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791, Republic of Korea
a r t i c l e
i n f o
Article history: Received 20 August 2013 Received in revised form 1 November 2013 Accepted 18 December 2013 Available online 18 January 2014 Keywords: Nanoparticle toxicity Dose–response Agglomeration Effective dose Bioaccumulation
a b s t r a c t To improve our understanding on the impact of extrinsic properties of NPs on their bioaccumulation and toxicity, we have investigated the bioaccumulation of sub 100 nm sized P25 TiO2 nanoparticles (NPs) by Daphnia magna (D. magna) in toxicity testing media. Based on our quantitative ICP-MS measurements as well as spectromicroscopic observations, we found that the bioaccumulation by D. magna were strongly influenced by the extrinsic properties of NPs as well as the biological uptake characteristics of D. magna. New sets of effective dosimetry parameters well correlated with the amount of NPs bioaccumulated within D. magna were also proposed. Based on these findings, we suggested that the extrinsic physicochemical properties of NPs (e.g., interfacial and colloidal properties of NPs) and biological characteristics for NPs (e.g., uptake cutoff sizes of testing organisms) should be considered and included when developing alternative dosimetry of NPs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Recent advances in nanoscience and nanotechnology have resulted in the widespread application of nanoparticles (NPs), including drug delivery, medical diagnostics, solar cells, and environmental catalysts [1–4]. However, there are also increasing concerns for their potential hazards on human health and ecosystems [5–7]. As a response to these concerns, the number of publications on the toxicity of NPs has recently increased dramatically. Nonetheless, many challenges and limitations still exist in the current methods of NP toxicity test and the development of unbiased testing strategies for impartial assessment of NPs toxicity is urgently needed [8–15]. According to recent reports, current challenges and limitations in NP toxicity assessments can be largely ascribed to (1) incomplete information about the widely varying physicochemical properties of NPs and (2) the lack of well-defined dosing metrics for the establishment of appropriate dose–response relationships [7–9,14–16]. To overcome these limitations, a comprehensive characterization of their physicochemical properties (e.g., chemical composition, crystallite phase, surface area, hydrodynamic size, etc.) is now considered as a prerequisite for toxicity testing of manufactured nanomaterials (MNs). Additionally, international collaborative activities for reference MNs and characterization
∗ Corresponding author. Tel.: +82 2 2220 4593; fax: +82 2 2299 0762. E-mail addresses: [email protected], [email protected] (T.H. Yoon). 0927-7765/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.12.047
protocols are actively in progress (e.g., OECD WPMN and ISO) [17,18]. On the other hand, while intrinsic (or static) physicochemical properties (e.g., total mass, number, and surface area) of NP are frequently used as alternative dosing metrics of NP [7,15], many other extrinsic (or dynamic) physicochemical properties, such as agglomeration and gravitational settling rate were also reported to influence the toxic responses of NPs and should be considered as key factors for alternative dosing metrics of NPs [19–23]. For instance, even for the reference NPs with clear identification and quantification of their intrinsic physicochemical properties, they may strongly interact with the components of toxicity testing media, and agglomerate and sediment differently according to their interfacial and colloidal properties as well as the characteristics of surrounding media [24–27]. These extrinsic properties are thought to strongly influence the delivered dose of NPs and may result in significant changes in bioaccumulation and acute/chronic toxicity of NPs. However, lack of appropriate control over these extrinsic properties often cause serious difficulties in performing the reliable and reproducible toxicity assessment of NPs. The purpose of this study was to improve our knowledge on the impact of extrinsic properties of NPs on the effective dose and bioaccumulation of NPs for Daphnia magna (D. magna) in toxicity testing media. To achieve this goal, extrinsic physicochemical properties of sub-100 nm sized TiO2 (TiO2 P25–70 ) were carefully monitored in reconstituted freshwater as a function of exposure time and surface properties ([Cit]/[TiO2 ]). Then, based on observations of a well-known planktonic invertebrate species, D. magna, bioaccumulation of TiO2 NPs were correlated with their
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physicochemical and biological properties to find out the most relevant dosing metrics of NPs in toxicity testing media.
3. Results and discussion
levels of citrate ions ([Cit]/[TiO2 ] = 0–14) were exposed to MHW media and their agglomeration and sedimentation behavior were monitored by measuring hydrodynamic size distributions and nort0 malized optical density at 320 nm (i.e., nOD320 = ODt1 320 /OD320 ). Agglomeration and sedimentation behavior of TiO2 dispersion in MHW media is presented in Fig. 1. The contour plot shown in Fig. 1a display the temporal variations in normalized optical density (nOD320 ) as a function of [Cit]/[TiO2 ], which clearly demonstrated the effect of surface charge on the sedimentation of NPs in the presence of this ecotoxicity test medium. Blue to purple colors (left side of the contour plot) indicate strong sedimentation of NPs under this condition (nOD320 < 0.9), while the green (right side of the contour plot) color implies no significant sedimentation or agglomeration under these conditions (nOD320 = 1.0 ± 0.1). The yellow to orange colors found in the central region indicate enhancement in the nOD320 > 1.1 range due to enhanced light scattering by slightly agglomerated but still well-suspended NPs under these conditions. Based on this color-coded contour plot of nOD320 , the colloidal stability (i.e., progress of sedimentation) of NP dispersion can be visualized and categorized into three subgroups: “unstable”, “partially stable”, and “stable” dispersions. Temporal variations of hydrodynamic size distribution were also monitored (Fig. 1b–d), which confirmed aforementioned variations in NP agglomeration. Under the stable dispersion condition, the hydrodynamic size distribution was maintained under 100 nm during the entire observation period (Fig. 1d), which is mostly due to the enhanced surface charge of the TiO2 NPs via adsorption of multiply-charged citrate ions [29,30]. As a consequence, TiO2 doses, at least in terms of their intrinsic properties (e.g., total mass, total number, and total surface area of NPs), were maintained at constant levels throughout the whole observation period under this “stable” dispersion condition. Under “partially stable” dispersion conditions, no significant sedimentations were observed, however, slight but significant enhancements in nOD320 and hydrodynamic size were observed, indicating significant progress in TiO2 NPs agglomeration process under this condition. Although this condition may allow a constant level of NP dose in terms of total mass of NPs, the progress in agglomeration will cause significant decreases in total number of NPs. In contrast to the above two conditions, TiO2 dispersions showed rapid agglomeration followed by heavy sedimentation within a few hours of exposure under “unstable” dispersion conditions. These agglomeration and sedimentation processes, which were frequently encountered during sample preparation for NP toxicity testing, have been considered the most serious obstacles in obtaining reliable and reproducible toxicity test results. Agglomeration and sedimentation observed in this unstable dispersion condition have been reported to cause completely different effective doses and biological uptakes for various biological species [19–23,31].
3.1. Preparation of TiO2 NPs with various dispersion conditions in test media
3.2. Microscopic and spectroscopic observation of TiO2 NP uptake by D. magna
The TiO2 used in this study may represent a well-suspended, nanosized fraction of bulk P25 TiO2 in environmental media. As we previously reported, it is comprised of aggregates or agglomerates of primary particles with diameters of about 21 nm and hydrodynamic size distributions centered around 70 nm [26]. It is stable in DIW for over a week with minimal size variation, although agglomeration followed by sedimentation were observed in typical in vivo ecotoxicity test media (MHW) and in vitro cytotoxic test media (Dulbecco’s modified Eagle’s medium, DMEM) [26,27]. In this study, to test the impact of extrinsic properties (surface charge, hydrodynamic size and agglomeration rate), we used citrate ions as surface modifying ligands of TiO2 in reconstituted fresh water media (MHW). TiO2 dispersions containing different
Both qualitative and quantitative studies on the bioaccumulation of TiO2 NPs were performed with D. magna, a well-known freshwater invertebrate species commonly used as a standard model organism of in vivo ecotoxicity assessments [28]. The absorption (or uptake), distribution, and excretion of TiO2 NPs by D. magna were monitored using bright field optical microscopy, Raman microspectrometry and ICP-MS measurements. In bright field optical images of D. magna (Fig. 2a–f), considerable bioaccumulations of TiO2 NPs were observed for unstable (Fig. 2b) and partially stable (Fig. 2c and d) dispersions, while no significant accumulations of NPs were visually observed for those exposed to stable TiO2 NP dispersions (Fig. 2e and f). Raman spectra shown in Fig. 2g confirmed that the local accumulation of TiO2 in the gut
2. Materials and methods 2.1. Nanoparticles preparation The aeroxide P25 TiO2 used in this study was provided by the manufacturer (lot no. 4168112198, Evonik GmbH, Germany). Stock solutions of 10 g/L P25 TiO2 powder were prepared by dispersing bulk powder in deionized water (DIW) and sonicated for 10 min using a probe sonicator (420 W, 20 kHz, Sonosmasher, Ulsso Hitech, Korea). Sodium citrate (99.0%, Cat. No. S4641, Sigma, MO, USA) was used as an electrostatic stabilizer of NPs. The sub-100 nm fractions of the TiO2 suspension were prepared by centrifugation at 6800 g for 20 min (Mega 17R, Hanil Science Industrial, Korea), from which the supernatant was carefully removed [26]. 2.2. Physicochemical characterization of TiO2 nanoparticles To monitor colloidal stability of TiO2 suspension in moderately hard synthetic freshwater (MHW), the optical density (OD) at 320 nm was monitored using an UV/vis spectrometer (Optizen2120UV, Mecasys, Korea). The hydrodynamic sizes of NPs in MHW were measured by using dynamic light scattering (DLS) instrument (Scatteroscope I, Qudix Inc, Korea). To measure the Ti concentration, samples were digested with mixed acid containing HNO3 , HCl, and HF and then analyzed using ICP-MS (Elan 6100, Perkin Elmer, CT, USA). 2.3. Characterization of TiO2 accumulation by D. magna Daphnia magna were cultured according to the U.S. EPA standard operating procedure [28]. Non-effect concentration value of 45 mg/L TiO2 was determined from preliminary acute toxicity tests and it was chosen for the exposure condition used in this accumulation study. Ten D. magna were used per group and experiments were conducted in 100 mL beakers. After 24 h exposure to 45 mg/L TiO2 , D. magna were removed and placed in fresh MHW. For the observation of TiO2 localization within D. magna, optical microscopy (IX51, Olympus, Japan) and Raman spectroscopy (LabRam HR, Jobin-Yvon, France) were used. To determine Ti bioaccumulation, group of D. magna were digested (0.5 mL 69% HNO3 for 30 min at 60 ◦ C and overnight at room temperature), and then Ti contents were measured using ICP-MS with mixed acid solution (HNO3 , HCl, and HF)
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Fig. 1. (a) Agglomeration and sedimentation of TiO2 at different citrate concentrations. Hydrodynamic size distributions at (b) unstable, (c) partially stable and (d) stable conditions.
and/or gill regions of D. magna are strongly affected by the dispersion stability of NPs. For instance, under the conditions of unstable NP dispersion, Raman peaks of TiO2 were observed for both gut and gill regions of D. magna, while similar peaks were observed only for the gut region of D. magna exposed to partially stable NP dispersions. In contrast, no Raman peaks of TiO2 NPs were observed for specimens exposed to stable dispersions of TiO2 NPs and control medium. By using ICP-MS analysis followed by acid digestion of carefully sampled D. magna, the total amounts of TiO2 accumulated within D. magna were also measured and presented in Table S1. For the TiO2 of unstable condition, the concentration of TiO2 was increased to 42.42 ug/ea after 24 h, which is about 40 times higher than those of “partially stable” dispersion condition (1.03 ug/ea) and “stable” dispersion condition (1.45 ug/ea) after 24 h. These quantitative measurements using ICP-MS technique also agree
well with the results from qualitative imaging techniques, such as optical microscopic and Raman spectroscopic observations. These observations regarding the impact of agglomeration on the bioaccumulation of NPs can be partially attributable to the unique uptake characteristics of D. magna. For this planktonic invertebrate species, the most likely pathway of NP uptake is through their active filter feeding. Previously, it was reported that D. magna actively filters and feeds on particles larger than their mean filter mesh sizes of 200 [28,32] and 280 nm [33]. Since the hydrodynamic size of TiO2 under the “stable” dispersion conditions is quite below this uptake cutoff size, it is less bioavailable for D. magna. However, under unstable dispersion conditions, TiO2 quickly agglomerates to form particles over about 200 nm and is available for uptake by D. magna. Thus, the agglomeration of sub-100 nm sized TiO2 particles may have caused a dramatic increase in the total amount of TiO2
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Fig. 2. Light microscope images of TiO2 exposure at different citrate concentrations. (a) Without TiO2 , (b) 0, (c) 4.88, (d) 6.03, (e) 12.07, (f) 14.30 (citrate mM/TiO2 mM). (g) Raman spectra of D. magna. (1, gut tract of (b) D. magna; 2, gill tract of (b) D. magna; 3, gut tract of (c) D. magna; 4, gill tract of (c) D. magna,; 5, gut tract of (f) D. magna; 6, gill tract of (f) D. magna).
accumulation in D. magna. Moreover, the kinetics of agglomeration also appeared to affect the biological uptake of NPs. Compared to the slow agglomeration kinetics under partially stable dispersion conditions, heavier accumulation of TiO2 were observed for D. magna under unstable dispersion conditions. 3.3. Impact of agglomeration on the uptake of TiO2 NPs by D. magna So far, we have shown that both extrinsic physicochemical properties (i.e., surface charge, hydrodynamic size distribution and etc.) and biological characteristics (i.e., uptake cutoff size) have strong influence on the bioaccumulation of TiO2 NPs by D. magna. By considering these physicochemical and biological characteristics, we have calculated the effective TiO2 NP doses and exposures bioavailable for D. magna under given dispersion conditions and their corresponding temporal variations are presented in Fig. 3. Even under the same nominal dose of TiO2 ([TiO2 ] = 45 mg/L), the variation in their physicochemical properties (e.g., surface charge) resulted in completely different hydrodynamic size distributions as well as the total amount of suspended TiO2 NPs (Fig. 3a). Furthermore, as shown in Fig. 3b and c, effective doses over cutoff sizes of 200 and 280 nm TiO2 (i.e., [TiO2 eff ], total amount of TiO2 NPs with hydrodynamic sizes over these uptake cutoff sizes of 200 and 280 nm) were calculated. Although it was previously reported that D. magna usually consume particles up to about 50 m [34], we did not consider particles larger than 10 m in these calculations, because that the DLS measurement is not reliable for this large size range. The effective doses were continuously varied under the “unstable” and “partially stable” conditions, while it was kept nearly constant under the “stable” dispersion condition during the whole exposure period. On the other hand, the effective exposures
of TiO2 (i.e., [TiO2 eff ], integrated sum of effective dose over exposure time) showed continuously increasing curves with different slopes depending on the dispersion stabilities of NPs. Under the “unstable” dispersion condition, the effective dose and effective exposure of TiO2 have similar values for both cutoff sizes (i.e., 200 and 280 nm). However, in “partially stable” and “stable” dispersion conditions, [TiO2 eff ] and [TiO2 eff ] change, these values are reduced for cutoff size of 280 nm [33]. In Fig. 4, relationships between the TiO2 NP exposures and their bioaccumulation were plotted. As previously noted, the nominal dose of TiO2 is the same for all experimental conditions ([TiO2 ] = 45 mg/L) and the corresponding TiO2 exposure was increased in proportion with the exposure time, but the measured bioaccumulation of TiO2 was completely independent of the nominal doses and exposures of NPs (Fig. 4a). In contrast, the [TiO2 eff ], in which hydrodynamic size distribution as well as biological uptake cutoff size were taken into account, had a positive relationship with the bioaccumulation of TiO2 by D. magna. For the cutoff size from literature (i.e., 200 nm, see Fig. 4b) [28,32], the correlation curve resulted in R2 value of 0.900. Moreover, much better correlation (R2 = 0.980) was observed for the cutoff size of 280 nm (see Fig. 4c), which was found as optimum cutoff size in a parallel modeling study [33]. These results confirmed us that new sets of dosing metrics (effective dose and exposures) involving extrinsic physicochemical properties of NPs (i.e., hydrodynamic size distribution) and biological characteristics of test species (i.e., uptake cutoff size), as well as intrinsic physicochemical properties (i.e., total mass concentration) provided a better understanding of the observed bioaccumulation of NPs and might show better predictability on the ecotoxicity of NPs. In addition to the effective exposure of TiO2 in mass basis, similar sets of parameters based on the number or surface area of NPs can be estimated and
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Fig. 3. (a) Mass (%) of TiO2 by hydrodynamic size at conditions. Effective dose for D. magna with cutoff size of (b) 200 nm and (c) 280 nm (left; unstable, middle; partially stable and right; stable condition). [TiO2 eff ], the amount of suspended TiO2 with hydrodynamic size above the uptake cutoff size of D. magna, in mg/L; [TiO2 eff ], integrated sum of effective dose over the exposure period, in hours mg/L; particle size of larger than cutoff size can be absorbed by D. magna.
compared as potential candidates for dosing metrics to find the most appropriate dose–response relationships with the observed NP accumulations or resultant toxicities of D. magna. 3.4. General discussion As far as we know, this is the first study that quantitatively correlates extrinsic physicochemical properties of NPs with their
bioaccumulation. However, there are several limitations in the current approach that need further improvements in future studies. One of the drawbacks in this approach is the limited capability of DLS technique in measuring hydrodynamic size distributions. Agglomeration process occurring in the “partially stable” state dispersions are typically very fast and involve multimodal size distribution of NP agglomerates. Although DLS is one of the most commonly used techniques for the measurement of hydrodynamic
Fig. 4. Relationships between NP doses and bioaccumulation were plotted. (b) Normal exposure, effective exposure with cutoff size of (b) 200 nm and (c) 280 nm TiO2 . The effective exposure (i.e., [TiO2 eff ]) was integrated sum of effective dose over the exposure period, and bioaccumulation was 45 mg/L TiO2 exposed concentration of D. magna.
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diameter, but it has limited capability to characterize dynamically changing colloids with multi-modal size distributions [35]. Additionally, due to the high scattering efficiency of micron-sized particles, presence of a small number of large aggregates may skew the measured hydrodynamic sizes towards a larger particle size distribution. Therefore, further studies using improved analytical techniques (e.g., nanoparticle tracking analysis) are necessary. Additionally, in the current study, we take the reductionist approach and focused only on the relationship between the physicochemical properties and the bioaccumulation of NPs rather than their toxicity. Although we have carefully performed current study under the non-toxic exposure condition based on the results of previous studies [36–38], it is also necessary to further expand current findings to better understand quantitative relationship between the physicochemical properties and the biological toxicity of NPs. Although our study was conducted for a single model organism, recently published studies also suggested that the bioaccumulation and toxicity depends on agglomeration and sedimentation properties of NPs in other biological species. With the increasing concentrations of sedimented NPs, enhancement in the bioaccumulation of Al2 O3 NPs was observed for sediment-dwelling organism (e.g., Hyalella azteca, Lumbriculus variegates and Corbicula fluminea) [39]. For the adherently grown mammalian cells, which typically used for in vitro toxicity test, sedimentation of NPs causes accumulation of NPs on top of the cell surface with much higher delivered (or effective) doses than nominal dose, which may lead to enhancement in cellular NP uptake and resultant cytotoxicity [23]. Thus, cell viability seems to depends on the type of mammalian cells, the cell viabilities of adherent cells (e.g., macrophages and kidney cell) were significantly decreased compared to those of floating cells (e.g., monocyte) [40]. In the case of the intravenous injection of agglomerated and well-dispersed multiwalled carbon nanotubes (MWCNTs) to mice, the agglomerated MWCNTs were accumulated in the lungs with inflammatory responses, while the well-suspended ones did not [41]. These results suggested that the extrinsic physicochemical properties (i.e., hydrodynamic size, agglomeration and sedimentation) of NP play important role in their bioaccumulation and toxicity. Further studies on the effects of these extrinsic parameters are urgently needed. Particularly, the concepts of effective dose and exposure involving extrinsic properties as well as biological characteristics should be further applied to various biological systems to improve our understanding on nanotoxicity mechanisms and included as parts of alternative dosing metrics of NPs for the unbiased assessment of nanotoxicity.
Acknowledgments This work was supported by the Korea Ministry of Environment and The Eco-Technopia 21 Project (091-091-081). We also thank Prof. Kyungho Choi’s Environmental Toxicology Laboratory of Seoul National University (Seoul, Korea) for providing D. magna for this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.12.047. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
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4. Conclusions In this study, we have investigated the impact of extrinsic properties of NPs on the bioaccumulation of sub-100 nm sized TiO2 dispersions with different surface charges, hydrodynamic size distributions and agglomeration kinetics. The effective doses of NPs and their bioaccumulation by D. magna were strongly influenced by the extrinsic properties of NPs as well as biological characteristics of D. magna. Based on our findings, we proposed that the physicochemical properties of NPs, including intrinsic (e.g., mass, number, and surface area of NPs) and extrinsic (e.g., interfacial and colloidal properties of NPs) properties as well as biological characteristics for the uptake of NPs (e.g., uptake cutoff sizes of testing organisms) should be considered and included when developing alternative dosimetry of NPs.
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