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Abstract: natural organic matter (NOM) has a profound effect on the colloidal stability of discharged C₆₀ nanoparticles in the water environment, which influences the environmental behaviors and risks of C₆₀ and therefore merits more specific studies. This study investigates the effects of humic acid (HA), as a model NOM, on the aqueous stabilization of C₆₀ powder and the colloidal stability of a previously suspended C₆₀ suspension (aqu/nC₆₀) with variations of pH values and ionic strengths. Our results reveal that HA could disperse C₆₀ powder in water to some degree, but was unable to stably suspend them. The aqu/nC₆₀ could remain stable at pH>4 but was destabilized at lower pH values. However, the colloidal stability of aqu/nC₆₀ in the presence of HA was insensitive to pH 3–11, owing to the adsorption of HA onto nC₆₀ and the increased electrosteric repulsions among nC₆₀ aggregates. The colloidal stability of aqu/nC₆₀, with and without HA, decreased as we increased the valence and concentration of the added cations. HA was found to mitigate the destabilization effect of Na⁺ on the colloidal stability of aqu/nC₆₀ by increasing the critical coagulation concentration (CCC) of Na⁺, while HA lowered the CCCs of Ca²⁺ and La³⁺ probably by the bridging effect of nC₆₀ with HA aggregates formed through the intermolecular bridging of the HA macromolecules via cation complexation at high concentrations of cations with high valences.

腐殖酸对富勒烯C60的悬浮作用

研究目的：腐殖酸（HA）对富勒烯（C60）粉末的悬浮作用以及pH、离子强度对HA-C60悬浮性能的影响。
创新要点：研究水质条件对C60悬浮性能的影响。
研究方法：测定C60粉末在HA溶液中的zeta电位，水力学粒径和悬浮浓度；HA存在下，C60悬浮体系的zeta电位与水力学粒径随pH的变化及C60悬浮体系团聚动力学随离子强度的变化。
重要结论：HA对C60粉末起到一定的分散作用，但不能使其长时间稳定悬浮于水中。当pH〈4时，C60水悬液开始沉淀；而当HA存在时，C60水悬液在pH3-11范围内都保持稳定，这是由于HA吸附于C60表面，通过静电排斥和空间位阻作用，促进C60分散悬浮。C60水悬液的稳定性随盐离子价位和浓度升高而降低。HA会抑制Na⁺对C60水悬液的脱稳作用；但高价离子Ca²⁺和La³⁺存在时，HA与C60之间会发生桥联从而促进C60水悬液脱稳沉淀。
富勒烯；腐殖酸；胶体稳定性；天然有机质；纳米材料

1.  Introduction

 Fullerene C₆₀ nanoparticles have triggered much attention by their unique physicochemical, electrical, and mechanical properties, which enable their numerous potential applications in electronics (Zhang et al., 2013), biomedicine (Colvin, 2003; Nakamura and Isobe, 2003), cosmetics (Takada et al., 2006), etc. C₆₀ is inevitably released into the aquatic environment with its widespread production and usage (van Wezel et al., 2011), and may thus threaten aquatic organisms and human health as well through food chains (Nel et al., 2006; Oberdörster et al., 2006; Britto et al., 2012).

 Once released into the water environment, the hydrophobic C₆₀ will be presented as aggregates (nC₆₀). Ubiquitous natural organic matter (NOM), such as humic acid (HA), is likely to interact with the discharged nC₆₀ and influence the aggregation/dispersion behavior and ecotoxicity of nC₆₀ (Bhatt and Tripathi, 2011; Kim et al., 2012). Therefore, it is essential to take into account the interactions between NOM and nC₆₀ when elucidating the behavior of nC₆₀ in natural water bodies.

 Several studies have investigated the interaction between NOM and nC₆₀ and the effect of NOM on the aggregation/dispersion behavior of nC₆₀. Xie et al. (2008) observed significant changes in the aggregate size and morphology of nC₆₀ with the addition of NOM. Li et al. (2009) revealed that NOM could effectively stabilize nC₆₀ in the aqueous phase and nC₆₀ nanoparticles may have been chemically modified under illumination. Mashayekhi et al. (2012) attributed the increased stability of nC₆₀ to the transformation of surface properties through the HA adsorption. The adsorption of HA can increase electrostatic and steric repulsions among nC₆₀ aggregates, and thus facilitate dispersion and stabilization of nC₆₀ in water (Chen and Elimelech, 2007; Mashayekhi et al., 2012; Zhang et al., 2013). Moreover, environmental conditions (e.g., water quality parameters) may have profound effects on the interaction between HA and nC₆₀ (Isaacson and Bouchard, 2010; Qu et al., 2010; Yang et al., 2013), which has not been well examined. Chen and Elimelech (2007) found that HA could increase the C₆₀ nanoparticle stability in NaCl or MgCl₂ electrolytes, but HA enhanced the aggregation at high concentrations of CaCl₂. Therefore, water quality parameters, such as ionic strength and pH, are likely to have significant effects on the colloidal stability of C₆₀ in the presence of HA, which merit further investigations.

 This work is aimed to study the capabilities of HA in dispersing and stabilizing C₆₀ powder in water and in maintaining the colloidal stability of nC₆₀ suspension (aqu/nC₆₀) as affected by variations of pH and ionic strength. The changes of zeta potentials and hydrodynamic sizes of the C₆₀ powder and aqu/nC₆₀ under different pH values and the impact of ionic strength on the aggregation kinetics of the aqu/nC₆₀ in the absence and presence of HA were specifically examined.

2.  Materials and methods

2.1.  Preparation and characterization of HA

 HA was purchased from Sigma-Aldrich. HA was purified before use and was characterized following the procedures detailed in Lin et al. (2012a; 2012b). HA of 500 mg was dissolved in 10 ml 1 mol/L NaOH solution, and adjusted to pH 7.0 using 1 mol/L HCl. The supernatant was obtained by centrifugation at 3000 r/min for 15 min, and then was diluted to 1 L using ultra-pure water and used as the stock HA solution (500 mg/L). NaN₃ (200 mg/L) was added to prevent HA from microbial interference. The HA solution was scanned with an ultraviolet (UV) spectrophotometer (SHIMADZU, UV-2450, Japan).

2.2.  Preparation and characterization of aqu/nC₆₀

 C₆₀ powder was purchased from Nanjing XFNANO Materials Tech Co., Ltd., China. The elemental contents were measured using an elemental analyzer (Vario ELIII, Germany). Aqueous suspension of C₆₀ was prepared following a solvent exchange procedure modified from previous works (Deguchi et al., 2001; Chen and Elimelech, 2006; Kim et al., 2010). C₆₀ powder (120 mg) was dispersed in 60 ml of high-performance liquid chromatography (HPLC)-grade toluene by stirring for several hours, forming a clear dark purple mixture. The mixture was then added into ultra-pure water at a volume ratio of 1:15, resulting in two distinct phases. The resultant mixture was sonicated with a sonifier cell disrupter (KBS-150, Kunshan, China) for more than 4 h, allowing for the evaporation of toluene. The stabilized C₆₀ suspension (aqu/nC₆₀) was obtained by centrifugation at 2000 r/min for 15 min, and then stored at room temperature in the dark. The morphology and aggregate size of the aqu/nC₆₀ were examined with a transmission electron microscope (TEM; JEM-1230, JEOL, Japan). The hydrodynamic size and zeta potential were determined using a Zetasizer (Nano ZS90, Malvern Instruments, UK) at 25 °C (Tian et al., 2010). The aqu/C₆₀ suspension was also scanned using the UV spectrophotometer.

2.3.  Suspension experiment

 A suspension experiment was performed to study the effect of HA on the aqueous stabilization of C₆₀ powder. C₆₀ powder (10 mg) was added into 20 ml of HA solutions (pH 7.0, 10 mg/L of NaCl) with initial HA concentrations of 0, 5, 10, 20, and 40 mg/L. The mixtures were sonicated (240 W, 40 KHz) for 1 h and were subsequently kept in a thermostat shaker (150 r/min, 25 °C) for 1 d. The zeta potential and hydrodynamic size of the samples were measured right after the shaking process with the Zetasizer at 25 °C. Each concentration was run in triplicate. The absorbance at 800 nm (UV800) was considered to be capable of quantifying the stabilized multi-walled carbon nanotubes (MWCNTs) (Lin et al., 2010; 2012a). Similarly, UV800 of the C₆₀ suspension was in a positive correlation with the concentration of suspended C₆₀ determined by a total organic carbon (TOC) analyzer (SHIMADZU, TOC-VCPH, Japan), and the HA solution had no absorbance at 800 nm (Fig. 1). Therefore, the UV800 of the supernatant of the C₆₀-HA suspension after settling for 0–2 d was used to measure the stability of the nC₆₀ suspensions in the presence of different concentrations of HA.

2.4.  Effect of pH on stability of aqu/nC₆₀ with and without HA

 The stock suspension of aqu/nC₆₀ (10 ml) was added into 22-ml glass vials containing 10 ml of ultra-pure water or 20 mg/L HA with 20 mg/L NaCl. The pH values of the suspensions were then adjusted to 3.0–11.0 with several drops of 1 mol/L NaOH or HCl solutions. The mixtures were equilibrated (150 r/min) for 1 d, and then their zeta potentials, hydrodynamic sizes, and final pH values were measured. Each pH value was run in triplicate.

2.5.  Aggregation kinetics of aqu/nC₆₀ with and without HA

 Aggregation kinetics of the aqu/nC₆₀ in the presence and absence of HA were investigated under various ionic strengths using a time-resolved dynamic light scattering (DLS) technique. A portion of the stock aqu/nC₆₀ of pH 7.0 with or without 10 mg/L HA was added into a polystyrene measurement cell and the hydrodynamic sizes were recorded by the Zetasizer at an interval of 1 min for 30 min at 25 °C. Various ionic strengths were obtained by mixing 0.5 ml of NaCl, CaCl₂, or LaCl₃ solutions of different concentrations into the cells containing aqu/nC₆₀ prior to the hydrodynamic size measurements. At the early stage of aggregation, the initial aggregation rate of the aqu/nC₆₀ can be expressed as (1) , where D _h(t) is the hydrodynamic size (nm) of nC₆₀ at time t, N ₀ is the initial particle concentration (mg/L), and k ₁₁ is the initial aggregation rate constant (Chen and Elimelech, 2006; Chen et al., 2006). The initial rate of increase in D _h(t) is obtained by determining the initial slope up to the point where the hydrodynamic size reaches 1.25D _h(0).

 The attachment efficiency (α) under different ionic strengths was calculated to quantify the aggregation kinetics of the aqu/nC₆₀: (2) , where (k ₁₁)_fast refers to k ₁₁ in the diffusion controlled regime (Mashayekhi et al., 2012).

3.  Results and discussion

3.1.  Characteristics of aqu/nC₆₀ and HA

 Selected properties of the C₆₀ powder and HA are listed in Table 1. The aggregate size of aqu/nC₆₀ as determined using TEM was (34.8±5.9) nm (Fig. 1). The transparent yellow aqu/nC₆₀ remained stable for several months during and after the experiments. The concentration of the stock aqu/nC₆₀ was 8.0 mg C/L with the zeta potential of −40.8 mV, hydrodynamic size of 172 nm, and pH value of 7.0. Fig. 1 shows the UV-vis absorbance spectrum of the aqu/nC₆₀. The aqu/nC₆₀ had absorption peaks at 269 and 350 nm as well as a broad band between 410 and 550 nm, which were identified to be characteristics of nC₆₀ suspension as previously reported (Hyung and Kim, 2009; Hwang and Li, 2010; Navarro et al., 2013).

3.2.  Effect of HA on aqueous stabilization of C₆₀

 Fig. 2 shows the changes of zeta potential, hydro-dynamic size, and UV800 of the C₆₀ powder sonicated in water against a concentration of HA. The zeta potentials of the C₆₀ powder in the presence of 0–40 mg/L HA were all lower than −50 mV, and the electronegativity initially increased with increasing the HA concentration and then leveled off at about −65 mV (Fig. 2a). The greater electronegativity of the C₆₀ powder in the presence of HA indicated a greater electrostatic repulsion among the C₆₀ particles, which was evidenced by the smaller hydrodynamic sizes in the HA solutions (Fig. 2a). The hydrodynamic size of the C₆₀ powder was (953±145) nm in the absence of HA, which however decreased to (516±31) nm in the presence of 40 mg/L HA, suggesting that HA could disperse the C₆₀ powder to some degree in water.




 The increased electrostatic repulsion between C₆₀ aggregates accompanied by decreased hydrodynamic size with increasing HA concentrations led to the enhanced aqueous suspension of C₆₀ in the HA solutions, which was confirmed by the increased UV800 of the C₆₀ powder in the HA solutions. UV800 of the C₆₀-HA suspension was positively correlated with the HA concentration after settling for 0−2 d, which was in accordance with the changes of zeta potential and hydrodynamic size against the HA concentration (Fig. 2b). However, the UV800 of the C₆₀-HA suspensions decreased with increasing settling time. After settling for 2 d, all of the C₆₀-HA suspensions became transparent, suggesting the severe agglomeration and precipitation of the C₆₀ aggregates. The above result implies that after being discharged, the hydrophobic C₆₀ powder could hardly be dispersed and stabilized in the water environment even in the presence of HA.

3.3.  Effect of pH on aqueous stability of aqu/nC₆₀ with and without HA

 The stability of aqu/nC₆₀ in the absence and presence of HA was investigated under various pH values. Dramatic increases in electronegativity of the aqu/nC₆₀ with and without HA were observed as pH increased from 3 to 6, while the increases leveled off over pH 6 (Fig. 3). This suggests that the neutralization reaction between H⁺ and the acidic surfaces of aqu/nC₆₀ occurred at low pH (Yang et al., 2013), thereby reducing the electronegativity and electrostatic repulsion among the nC₆₀ aggregates and consequently causing the aggregation and destabilization of the aqu/nC₆₀ in the absence of HA. The aqu/nC₆₀ had a lower zeta potential in the presence of 10 mg/L HA, and the differences between the zeta potentials with and without HA became more significant under acidic conditions (pH<6). HA could be adsorbed by the nC₆₀ aggregates (Mashayekhi et al., 2012), and the dissociation of –COOH and phenolic –OH groups of the surface-bound HA (Table 1) could impart negative charges on the nC₆₀ aggregates, causing the difference in the zeta potential of the aqu/nC₆₀ with and without HA.




 The aqu/nC₆₀ retained its hydrodynamic size of (186±10) nm at pH>4 with and without HA (Fig. 3). The hydrodynamic size of the aqu/nC₆₀ in the absence of HA sharply increased to (1028±100) nm at pH=3, whereas it remained largely unchanged in the presence of HA (Fig. 3). The differences in the hydrodynamic size of the aqu/nC₆₀ with and without HA at pH=3 could be explained by the differences in the zeta potential. At pH=3, the zeta potential of the aqu/nC₆₀ increased to (−18±1) mV in the absence of HA while it remained lower than −30 mV in the presence of HA (Fig. 3). Colloids with absolute zeta potential >30 mV are considered to have strong electrostatic repulsions and tend to remain dispersed, while with the absolute zeta potential lower than 30 mV, they are prone to aggregation and destabilization due to the weak electrostatic repulsions among them (Lin et al., 2009; 2012a). The adsorbed HA could mitigate the neutralization reaction between H⁺ and the acidic surfaces of the aqu/nC₆₀, preventing the nC₆₀ from aggregation, owing to the decreased electrostatic repulsion at low pH values. Moreover, the adsorbed HA could also enhance the aqueous stability of aqu/nC₆₀ via the increased steric hindrance among the nC₆₀ aggregates. From the above results, we can see that the aqu/nC₆₀ would remain stabilized in the water environment with broad pH values, but would be subject to aggregation and destabilization at extremely low pH values; the coexisting HA is likely to help to stabilize the aqu/nC₆₀, especially at low pH values.

3.4.  Effect of ionic strength on aqueous stability of aqu/nC₆₀ with and without HA

3.4.1.  Aggregation kinetics of the aqu/nC₆₀ in the absence of HA

 Aggregation of the aqu/nC₆₀ occurred after the addition of various concentrations of the electrolytes as evidenced by the increase in its hydrodynamic size (Fig. 4). The cations at high concentrations immediately increased the aggregate size after their addition to the aqu/nC₆₀. Fig. 4a shows the increasing of the aggregation rate of the aqu/nC₆₀ by increasing Na⁺ concentration from 50 to 400 mmol/L during 30 min. The hydrodynamic size remained constant when 50 mmol/L Na⁺ was added. But the aggregation rate markedly increased with the concentration of Na⁺ increasing from 50 to 150 mmol/L, and then remained steady with further addition of Na⁺. The attachment efficiencies (α) of the aqu/nC₆₀ at each Na⁺ concentration were calculated according to Eq. (2) (Fig. 5a). The increase in Na⁺ concentration could screen the surface charge of nC₆₀, reduce the electrostatic energy barrier, and subsequently lead to the fast aggregation in the reaction controlled regime where α linearly increased with the increasing Na⁺ concentration. However, once the electrolyte concentration was beyond the critical coagulation concentration (CCC), the energy barrier was completely eliminated and the diffusion controlled aggregation began. Under the diffusion controlled regime, α no longer responded to the increasing Na⁺ concentration. Based on the data in Fig. 5a, the CCC, the intersection of the reaction and diffusion controlled regimes, was determined to be 142 mmol/L for Na⁺.







 The DLS measurement was also applied to study the aggregation kinetics of aqu/nC₆₀ in the presence of Ca²⁺ and La³⁺. The increases in the hydrodynamic size with time were observed starting at 3 and 0.07 mmol/L of Ca²⁺ (Fig. 4c) and La²⁺ (Fig. 4e), respectively, revealing that much lower concentrations of the cations with higher valence were needed to destabilize the aqu/nC₆₀. This is because cations with higher valence have much higher capability of eliminating the energy barrier between the dispersed nanoparticles and thereby promoting the aggregation (Chen et al., 2006). The increase in aggregation rate of the aqu/nC₆₀ leveled off (i.e., α=1) with the concentrations of Ca²⁺ and La³⁺ over 5 and 0.1 mmol/L, respectively. Figs. 5b and 5c show variations of α of the aqu/nC₆₀ with the concentrations of Ca²⁺ and La³⁺, where their CCCs were calculated to be 5.30 and 0.110 mmol/L, respectively. The ratio of CCCs of the three electrolytes NaCl:CaCl₂:AlCl₃ was 140:5.30: 0.110 (i.e., 1^−4.78:2^−4.78:3^−4.78) (Fig. 5d), which was close to the ratio indicated by the Schulze-Hardy rule (i.e., 1⁻⁶:2⁻⁶:3⁻⁶) (Li and Huang, 2010).

 Our CCC of NaCl is comparably higher than that determined by Chen and Elimelech (2006) for fullerene nanoparticles synthesized using the same technique (120 mmol/L). Their reported CCC of CaCl₂ was 4.8 mmol/L, which was close to our obtained result. It was noteworthy that the CCC values were quite similar, regardless of the 10 times higher suspension concentration which we employed, implying that the initial concentration of aqu/nC₆₀ may play an insignificant role in its aggregation.

3.4.2.  Aggregation of aqu/nC₆₀ in the presence of HA

 The effects of HA on the aggregation rate of aqu/nC₆₀ at different concentrations of NaCl, CaCl₂, and LaCl₃ are shown in Figs. 4b, 4d, and 4f, respectively. aqu/nC₆₀ started to aggregate with the hydrodynamic size increasing with time at Na⁺ concentrations higher than 600 mmol/L and did not reach its maximum aggregation rate until 1670 mmol/L (CCC) of NaCl (Fig. 5a). Clearly, the presence of HA effectively prevented the aqu/nC₆₀ from homoaggregation and forming bigger aggregates as affected by NaCl. This was in accordance with the result of a previous study that NOM increased the stability of nC₆₀ suspension (Duncan et al., 2008). It was supposed that the adsorption of NOM including HA on the nC₆₀ aggregates could increase electrosteric repulsions and thereby enhance stabilization of the nC₆₀ suspension (Chen and Elimelech, 2007). Environmental concentrations of Na⁺ are generally much lower than 1670 mmol/L, therefore the Na⁺ effect on the stability of aqu/nC₆₀ could be ignored in the presence of 10 mg/L HA.

 The aggregation rate of aqu/nC₆₀ started to increase at 3.2 and 0.02 mmol/L of Ca²⁺ (Fig. 4d) and La³⁺ (Fig. 4f) and the increase leveled off at 4.80 and 0.032 mmol/L of Ca²⁺ (Fig. 5b) and La³⁺ (Fig. 5c), respectively. According to Figs. 5b and 5c, the calculated CCCs were 4.90 and 0.035 mmol/L of Ca²⁺ and La³⁺, respectively, which were surprisingly lower than their respective CCCs in the absence of HA. The negative effect of HA on the stability of aqu/nC₆₀ as affected by Ca²⁺ was also observed by Chen and Elimelech (2007). The accelerated aggregation in the presence HA was attributed to the bridging of nC₆₀ by the HA aggregates formed through the intermolecular bridging of HA macromolecules via cation complexation at high concentrations of cations with high valences (Chen and Elimelech, 2007). The ratio of CCCs of the three electrolytes NaCl:CaCl₂:AlCl₃ in the presence of HA was 1670:4.90:0.035 (i.e., 1^−8.42: 2^−8.42:3^−8.42) (Fig. 5d), which was also largely comparable to the precipitation by the Schulze-Hardy rule (i.e., 1⁻⁶:2⁻⁶:3⁻⁶) (Li and Huang, 2010) but was different from the ratio in the absence of HA. This differences were owing to the different effects of the HA on the colloidal stability of the aqu/nC₆₀ as affected by the three electrolytes as addressed above.

4.  Conclusions

 The effects of HA on the aqueous stabilization of C₆₀ powder and on the colloidal stability of aqu/nC₆₀ as affected by variations of pH and ionic strength were studied. C₆₀ powder could be dispersed to some degree but hardly be stably suspended in the presence of HA. Low pH and high ionic strength would affect the colloidal stability of aqu/nC₆₀. Big aggregates formed at pH<4, owing to the screening of surface charges of the aqu/nC₆₀ at such low pH. However, the colloidal stability of aqu/nC₆₀ in the presence of HA was insensitive to pH 3–11. It was supposed that HA could be adsorbed onto the surfaces of nC₆₀ and the surface-bound HA increased the surface electronegativity of the nC₆₀, and thereby mitigated the aggregation through electrosteric repulsion at low pH values. Cation valence and concentration had a profound effect on the destabilization of the aqu/nC₆₀. The CCCs of Na⁺, Ca²⁺, and La³⁺ in the presence and absence of HA were exponentially decreased with the increase in cationic valence. The presence of HA could mitigate the impact of Na⁺ on the colloidal stability of aqu/nC₆₀, but enhanced the destabilization effect of Ca²⁺ and La³⁺ on the aqu/C₆₀.

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* Project supported by the National Natural Science Foundation of China (No. 21337004), the National Basic Research Program (973) of China (No. 2014CB441104), the Zhejiang Provincial Natural Science Foundation of China (No. LR12B07001), the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP) (No. 20130101110132), the Fujian Provincial Natural Science Foundation of China (No. 2014J01053), and the Fundamental Research Funds for the Central Universities (No. 2014FZA6009), China