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Keywords:

  • Nanoparticles;
  • Fullerene;
  • Colloids;
  • Toxicity;
  • Daphnia magna

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

The present study assessed the toxic effects of stable aqueous colloidal suspensions of gallic-acid-stabilized C70 fullerene on Daphnia magna. The suspensions were stabilized through noncovalent surface modification with gallic acid. In addition to whole-organism responses, changes in antioxidative processes in D. magna were quantified. Acute toxicity was observed with 96LC50 for C70-gallic acid of 0.4 ± 0.1 mg/L C70. Daphnia magna fecundity was significantly reduced in 21-d bioassays at C70-gallic aqcid concentrations below quantifiable limits. Antioxidant enzyme activities of glutathione peroxidase and superoxide dismutase as well as lipid peroxidation suggested that exposed organisms experienced oxidative stress. Microscopic techniques used to determine cellular toxicity via apoptosis proved unsuccessful. Environ. Toxicol. Chem. 2012;31:215–220. © 2011 SETAC


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

The burgeoning development of nanotechnology allows for a wide range of applications, with potentially exponential growth in production and use, leading to considerable discharge of nanomaterials into the environment. Understanding how these materials interact in the environment is of particular importance in determining bioavailability to organisms. Carbon nanomaterials discharged into aquatic systems can become functionalized or derivatized by biomolecules and natural organic matter (NOM) 1. Salonen et al. 2 showed that gallic acid (GA), a ubiquitously occurring component of NOM, self-assembled with the fullerene C70 to form stable aqueous suspensions. Furthermore, these authors demonstrated translocation of these surface-modified fullerenes into mammalian cells and observed consequential contraction of the cell membranes. These observations laid the foundation for the present research.

Much of the existing research on the ecotoxicology of fullerenes has focused on the effects of C60 and derivatives of C603. The effects of the fullerene C70 are not as well characterized. Fullerenes, such as C70, may have toxicological effects similar to those of C60 because they share similar closely related physical and chemical properties.

Conflicting opinions exist about the toxicity of C60 in aquatic organisms. Suspended C60 has been observed to induce oxidative damage in human cells and in aquatic organisms 4, 5. Other research has indicated, however, that, depending on surface modification and method of preparation, these C60 can actually act as highly effective antioxidants by radical scavenging 6–8. Foley et al. 9 reported that although fullerenes can quench reactive oxygen species (ROS), they will produce singlet oxygen when illuminated with ultraviolet (UV) radiation. Therefore, oxidative stress might be a significant contributing factor to C70 toxicity, but might not be the exclusive cause.

The ability of different colloidal C60 suspensions to produce ROS depends on the solvent used for their preparation and environmental conditions (that is, UV radiation) 5, 10. However, evidence also shows that C60, depending on derivatization and method of suspension, can act as an antioxidant 11–13. In cells exposed to C60, cell death occurs because of lipid oxidation caused by generating oxygen radicals; highly derivatized C60 systems do not generate these species as readily and thus have lower cellular toxicity 5. Both lipid-soluble and water-soluble C60 derivatives effectively prevent lipids from radical-initiated peroxidation and the breakdown of membrane integrity 13.

To minimize oxidative damage to cellular components resulting from ROS production, organisms have developed antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GPX) 14, 15. Upregulation of these antioxidant enzymes within organisms can occur in response to elevated ROS production: differences in how these enzymes are regulated can be observed as biomarkers of ROS levels 16. Failure to moderate excessive ROS can lead to deleterious effects such as enzyme inactivation, protein degradation, DNA damage, and lipid peroxidation 15. Lipid peroxidation is of particular concern as a cause of tissue damage, which can lead to disrupting essential cellular functions 17. Malondialdehyde, a byproduct of lipid peroxidation, can be measured as an oxidative stress biomarker 18. Daphnia magna, a widely used test organism for aquatic risk assessment, has been shown to express such biomarkers as changes in enzyme regulation and lipid peroxidation resulting from exposure to UV light, redox cycling compounds, and transition metals 19, 20.

These antioxidative measures require energy, which would otherwise be used for physiological functions such as reproduction. These functions may also be impaired by carbon nanomaterials via physical stress. For instance, carbon nanomaterials such as carbon nanotubes can accumulate in the gut lumen, possibly reducing efficient absorption of food 21, 22. Therefore, in addition to oxidative damage, C70 potentially causes other sublethal effects on organism fitness.

The goal of the present study was to assess the response of D. magna to exposures of GA-modified C70 fullerene. To accomplish this, we established and met the following objectives: characterize the response of D. magna to acute and chronic exposures of C70-gallic acid (C70-GA) and quantify the oxidative damage following sublethal C70-GA exposure.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

Suspension of C70-gallic acid

Fullerene C70 was purchased from SES Research, and its purity was reported by the distributor to be 95%. To prepare suspensions of C70-GA, 5 mg C70 and 25 mg GA were combined in a 100-ml conical glass tube. Increments of synthetic laboratory moderately hard freshwater (MHW) were added to obtain a final volume of 100 ml 23. The contents of the tube were probe sonicated for 1 h. Suspensions were allowed to settle for 1 h, and the supernatant was transferred to a clean glass tube using a glass pipet. Care was taken not to transfer settled particles. All exposure suspensions were prepared assuming a nominal stock concentration of C70-GA (50 mg/L C70). Because the suspension procedure was not 100% effective, exposure concentrations were quantified as described below.

Characterization of particle size

Test suspensions of C70-GA complex (nominal concentration 10 mg/L C70) were analyzed for particle size using dynamic light scattering. A Beckman Coulter Photon Correlation Spectroscopy submicrometer particle size analyzer was used for dynamic light scattering analysis. A Zeta Sizer Z (Malvern) was used to measure zeta potential of C70 and C70-GA suspensions. For each characterization, care was taken to avoid samples containing unsuspended materials.

Quantification of C70-GA test suspensions

Samples of test suspensions were obtained prior to each daily medium renewal to determine the C70 concentration. Twenty-five-milliliter samples were shaken vigorously with 5 ml hexane to extract the C70. A single aliquot was obtained from each daily-prepared treatment suspension concentration. Extracts were stored in 7-ml glass vials. Concentrations of C70 in hexane extracts were quantified spectrophotometrically (λ = 550 nm) using a Molecular Devices SpectraMAX Gemini UV spectrophotometer.

A standard curve was prepared (Supplemental Data) using stock C70-GA dissolved in hexane. C70-GA concentrations were calculated as milligrams C70 detected per liter aqueous test suspension. The lowest concentration used for the standard curve was 0.015 mg/L C70 in hexane, so calculated suspension concentrations less than 0.03 mg/L C70 were considered beneath the detectable limit of quantification. Concentrations were averaged over the duration of each experiment.

Daphnid acute bioassays

Daphnia magna neonates were obtained from an in-house laboratory stock maintained at the Institute of Environmental Toxicology, Clemson University (CU-ENTOX). Routine reference acute toxicity tests have been performed previously with this culture to ensure consistent culture sensitivity to sodium chloride. Results of these reference toxicity tests are available through CU-ENTOX by contacting the corresponding author. Tests were performed based on U.S. Environmental Protection Agency standard methods using synthetic freshwater 23. Test volumes and the number of organisms per replicate were altered to compensate for the limited supply of C70. This synthetic freshwater was used to prepare C70-GA suspensions of the following nominal test concentrations: 10, 8, 4, 2, and 1 mg/L C70 by serial dilution for acute toxicity bioassays. Test suspensions were prepared daily.

Acute bioassay methods followed standard procedures 23. Daphnia magna neonates aged less than 24 h were exposed in static renewal acute toxicity tests in 30-ml glass beakers containing 25-ml test solutions at 25 ± 1°C. Three replicates per treatment were tested, and each treatment included five neonates. Mortality was observed at 24-h intervals with organisms fed a diet of algae (Selenastrum capricornutum) and yeast-cereal-trout chow. After allowing organisms to feed for 1 h, all living organisms were transferred to test chambers containing fresh test solutions.

Daphnid chronic bioassays

Chronic bioassay methods followed standard procedures 23, 24, with modifications. Test suspensions of C70-GA of nominal test concentrations 2, 1, 0.5, 0.25, and 0.125 mg/L C70 were prepared by serial dilution of stock suspension with synthetic freshwater for the 21-d bioassays. Neonates aged less than 24 h were exposed in static renewal tests in 500-ml polyethylene beakers containing 400-ml test solutions at 25 ± 1°C. Three replicates of five individuals were tested per treatment. Mortality and reproduction were observed at 24-h intervals. At this time, all offspring were counted and discarded, and remaining living organisms were transferred to test chambers containing fresh test solutions. After daily transfer, organisms were fed a diet of algae (S. capricornutum) and yeast-cereal-trout chow.

Lipid peroxidation

For each treatment, 20 D. magna neonates (24 h old) were placed in each of three 500-ml polyethylene beakers containing 400 ml of appropriate test solutions: C70-GA (0.5 and 2.5 mg C70), 10 mg/L GA, and control. Synthetic MHW and a solution of 50 mg/L GA were each used as controls. Each treatment was carried out in three replicates. After static exposure for 24 h, organisms from each experiment were subsampled for analysis of lipid peroxidation. Lipids were extracted from whole-body daphnids (pooled wet mass 100–200 mg, n = 3), with the extent of lipid peroxidation determined by the thiobarbituric acid-reactive species (TBARS) assay according to the procedure of Barata et al. 20. Measurements were carried out with a fluorescence spectrometer.

Enzyme activity and protein determination

Juvenile D. magna (4–5 d old) were exposed for 24 h as in the test described in the Lipid peroxidation section above. Treatments included 10 mg/L GA solution, a C70-GA suspension (nominal concentration of 2 mg/L C70), and 20 µg/L Cu solution. Copper was used as a positive control as a known oxidative stressor that results in changes in TBARS and antioxidant enzyme expressions 20. Three replicate tests were performed for each treatment. Gallic acid and copper solutions were prepared using MHW. Juveniles (pooled wet mass 50–100 mg) from each sample were homogenized in 1:4 wet weight:buffer volume ratio in 4°C 100 mM KCl, pH 7.4, and 1 mM ethylenediaminetetraacetic acid.

Homogenates were centrifuged at 10,000 g for 10 min and the supernatants removed for immediate enzyme activity analysis. Measurements were carried out on a plate reader spectrophotometer (Molecular Devices SpectraMax 190) at 25°C. Superoxide dismutase activity was measured by SOD assay kit (Cayman Chemical) according to kit instructions. Glutathione peroxidase activity was determined using a GPX assay kit (Cayman Chemical) according to kit instructions. Protein concentrations in the supernatants were measured using a modified Lowry Protein Assay kit (Pierce).

Apoptosis

Juvenile D. magna were exposed to C70-GA (nominal concentration of 2 mg/L C70) for 24 h. For each treatment and control, 10 individuals were fixed in 10% buffered paraformaldehyde. Samples were embedded in paraffin, and then cross-sections were prepared by ultramicrotome. Samples were prepared and analyzed according to the manufacturer's instructions for the ApopTag ISOL Dual Fluorescence Apoptosis Detection kit (Chemicon). Analysis was performed on a Zeiss 510 laser scanning confocal fluorescent microscope.

Statistical analysis

Survival data from the 96-h test were analyzed using the trimmed Spearman–Karber method to derive 96-h median lethal concentration (LC50). Data sets from the 21-d toxicity bioassays, lipid peroxidation, and enzyme activity levels were each analyzed for significance by one-way ANOVA with Tukey's post hoc test in SAS Software (SAS Institute). Apoptosis data were analyzed for significance using Student's t test. Significant differences were established at p < 0.05.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

C70-GA suspension characterization

We produced colloidal suspensions of C70-GA in MHW to use in the D. magna toxicity bioassays by modifying the method of Salonen et al. 2 for coating C70 with GA. While quantification methods did not account for 100% of the C70 placed in suspension, we were able to quantify the average C70 concentrations in treatments by hexane extraction. Measured concentrations were approximately 10% of nominal concentrations, indicating that a considerable amount of C70 did not remain suspended after sonication of the stock suspensions. The mean particle size of a C70-GA test solution 2 h after preparation was calculated as 1,432 ± 690 nm (Fig. 1). The size distribution corresponds to the tens of nanometers up to micrometers range of particle sizes reported by Salonen et al. 2 for unfiltered C70-GA in deionized water.

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Figure 1. Particle size distribution of C70-gallic acid determined by dynamic light scattering within 2 h of preparation. Mean particle size of 1,432 ± 690 nm.

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Zeta potentials for C70 and C70-GA suspended in MHW were −29 ± 7.8 mV and −32 ± 7.2 mV, respectively. These values are not significantly different and indicate that both methods produced suspensions considered incipiently to moderately stable. However, a visible portion of C70 had precipitated from the suspension without GA prior to zeta potential analysis.

On visual inspection of suspensions prepared in MHW compared with those prepared as described by Salonen et al. 2 with deionized water, larger aggregates were observed in suspensions prepared in MHW. A possible explanation for the increase in the suspensions in the present study is the greater ionic strength of MHW compared with distilled water. Ionic strength has been shown to increase aggregation of fullerene suspensions in water 25.

Acute toxicity of C70-GA

A dose-dependent decrease in survival was observed for D. magna neonates with a 96-h LC50 value of 0.4 ± 0.1 mg/L (Fig. 2). Microscopic examination of the gut tract of exposed organisms indicated substantial amounts of nanoparticles, which coincides with previous data showing that D. magna collected nanomaterial aggregates within their gut tract 21, 22. We could not confirm, however, that C70-GA migrated beyond the epithelia of the gut tract.

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Figure 2. Mean percentage survival ( ± SD) of Daphnia magna exposed to C70-gallic acid complex (C70-GA) in a 96-h static renewal test. The x axis concentrations represent average [C70] of suspensions calculated from milligrams C70 extracted in hexane per liter of aqueous suspension.

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Time-course micrographs (Supplemental Data) indicated rapid accumulation of fullerenes within the gut tract, followed by clearance after the individual was placed in clean MHW. Depuration times of greater than 12 h were required for the material to clear completely from the gut tract. This time is greater than that reported for clearance of suspended clay by D. magna26, but shorter than the 28 h reported for multiwalled carbon nanotubes suspended in NOM 22. However, given that C70-GA in the present study was more toxic than the multiwalled carbon nanotubes reported by Edgington et al. 22, it is likely that something beyond gut tract clogging and interference with food processing is causing the toxicity.

Chronic toxicity of C70-GA

Daphnia magna exposed to C70-GA for 21 d exhibited significant mortality at fullerene concentrations ≥1 mg/L (Fig. 3). Sublethal effects of a 21-d C70-GA exposure on D. magna indicate a significant decrease in fecundity at concentrations <0.1 mg/L (Fig. 4). The use of organic solvents such as tetrahydrofuran in preparing aqueous fullerene suspensions has been controversial, because this solvent has been shown to exhibit neurotoxicity 27. However, GA is a widely used antioxidant and did not exhibit significant toxicity when tested alone in this study. The surface-modified fullerenes tested in the present study exhibited toxicity to D. magna similar to that of the C60 tested by Oberdörster et al. 4 prepared with tetrahydrofuran (5 ppm C60 clusters).

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Figure 3. Mean percentage survival ( ± SD) of Daphnia magna exposed to C70-gallic acid complex (C70-GA) in a 21-d static renewal bioassay. The x axis concentrations represent average [C70] of suspensions calculated from milligrams C70 extracted in hexane per liter of aqueous suspension. Concentrations below the limits of the standard curve (0.03 mg/L) are labeled as ND. *Significantly greater than control (p < 0.05).

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Figure 4. Mean number of neonates per adult ( ± SD) of Daphnia magna exposed to C70-gallic acid complex (C70-GA) in a 21-d static renewal test. The x axis concentrations represent average [C70] of suspensions calculated from milligrams C70 extracted in hexane per liter of aqueous suspension. Concentrations below the limits of the standard curve (0.03 mg/L) are labeled as ND. *Significantly greater than control (p < 0.05).

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Few other studies have reported the effects of chronic exposure of fullerenes to aquatic organisms. For example, juvenile carp exposed for 32 d to 0.2 mg/L C60 exhibited reduced body length and weight 28. To the best of our knowledge, this is the first study to report a significant reproductive effect on a eukaryotic organism resulting from fullerene exposure. Indeed, Ringwood et al. 29 postulated that fullerene exposures greater than 10 µg/L could reduce the reproductive success of adult oysters.

The observed decrease in fecundity of D. magna exposed to C70-GA could be a result of energetic effects caused by interference with food processing as previously proposed for single-walled nanotube aggregates 21. In addition, should the observed elevated rates of enzyme activity continue over the course of the Daphnia life cycle, the energy required for reproduction could be reallocated to maintain these enzymatic pathways.

Lipid peroxidation

The presence of malondialdehyde, a product of lipid peroxidation that reacts with thiobarbituric acid, was determined for each treatment by the TBARS assay. No significant difference in lipid peroxidation was observed with the GA treatment. C70-GA significantly increased lipid peroxidation, but the effect was not dose dependent (Fig. 5).

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Figure 5. Mean thiobarbituric acid-reactive species detected as relative to mass of lipid extracts ( ± SD) of Daphnia magna exposed to C70-gallic acid (C70-GA) in a 24-h static test (n = 3). Treatments include moderately hard water control, GA, and nominal concentrations of C70-GA: 0.5 and 2 mg/L C70. *Significantly greater than control (p < 0.05).

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From the data, no dependence on concentration could be determined, suggesting that at least one limiting factor exists for the induction of lipid peroxidation in D. magna by C70-GA. A contributing factor that might constrain lipid peroxidation could be that much of the C70-GA is retained unabsorbed within the gut tract. If C70-GA was absorbed by D. magna, the amount could be limited by the gut epithelium.

Antioxidant enzyme activity

Daphnia magna exposed to C70-GA exhibited increased SOD and GPX activity (Figs. 6 and 7, respectively). Juveniles exposed to GA alone were observed to have a decreased SOD activity response. This decreased activity could be due to the fact that GA can act as an antioxidant. However, no significant change in GPX activity was observed in D. magna juveniles exposed to GA alone. Because GA was observed to have an opposite action on GPX activity, the GA may have an ameliorative effect counter to the apparent oxidative stress caused by the C70-GA.

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Figure 6. Activity of superoxide dismutase (SOD) in D. magna juveniles exposed to gallic acid, C70-gallic acid (C70-GA), and Cu (n = 3). Values are expressed as mean ± SD normalized to protein concentration. ‡Significantly less than control (p < 0.05). *Significantly greater than control (p < 0.05).

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Figure 7. Activity of glutathione peroxidase (GPX) in D. magna juveniles exposed to gallic acid, C70-gallic acid, and Cu (n = 3). Values are expressed as mean ± SD normalized to protein concentration. *Significantly greater than control (p < 0.05).

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Apoptosis

The ApopTag ISOL dual fluorescence apoptosis detection kit (Chemicon) assay is designed to detect DNA fragmentation resulting from two DNase classes. However, autofluorescence of D. magna tissue samples was detected in the same fluorescence wavelength range as the DNase type II label probe carboxyfluoroscein. Therefore, only Cal Fluor Red 590-labeled probes bound to fragmented DNA resulting from type I nucleases were detected.

Fluorescence micrographs of cross-sections of both control and C70-GA-exposed D. magna juveniles were observed for fluorescence associated with label probes. Only cells of the gut tract were considered for analysis for ease of tissue identification and proximity to C70-GA aggregates. There was no significant difference between mean apoptotic cell counts between control and C70-GA-exposed D. magna juveniles (Supplemental Data). From the data, no evidence emerged of a significant induction of apoptosis in D. magna gut tissue by the C70-GA complex.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

Results of this work demonstrate that chronic exposure to C70-GA can cause deleterious effects on fecundity in D. magna at concentrations below 0.03 mg/L C70, which was the detection limit of our quantification assay. These chronic effects may result from physical stress of fullerene aggregates in the gut, impeding the ability of individuals to feed efficiently. It is likely, however, that this physical effect is not the only adverse outcome, because C70-GA exhibited toxicity at lower concentrations despite having a shorter gut tract clearance time 22. This mechanism could be oxidative stress, as suggested by the results of the biochemical assays in the present study. Previous studies examining fullerene-induced oxidative stress might have been confounded by the use of solvents such as tetrahydrofuran and dimethylsulfoxide to produce the aqueous suspensions 30. Whereas these solvents were inherently toxic, the GA used in the present study to stabilize C70 was not toxic. Results of the present study underscore the need for additional chronic studies with nanomaterials on aquatic organisms.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

The authors acknowledge the generous support of the Clemson University Public Service Activities, technical contribution 5801 of the Clemson University Experiment Station, and T. Bruce of the Clemson University Jordan Imaging facility. This research received financial support from grants R833886 and R834092 from the U.S. Environmental Protection Agency's Science to Achieve Results program. This is Technical Contribution No. 5992 of the Clemson University Experiment Station.

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  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSIONS
  7. SUPPLEMENTAL DATA
  8. Acknowledgements
  9. REFERENCES
  10. Supporting Information

Additional Supporting information may be found in the online version of this article.

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etc_727_sm_suppdata.pdf1127KSupplementary Data

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