under carbon nanotube exposure - Wiley Online Library

Environmental Toxicology and Chemistry, Vol. 34, No. 12, pp. 2824–2832, 2015 # 2015 SETAC Printed in the USA

REDUCED CADMIUM ACCUMULATION AND TOXICITY IN DAPHNIA MAGNA UNDER CARBON NANOTUBE EXPOSURE JIE LIU and WEN-XIONG WANG* Environmental Science Program, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (Submitted 1 May 2015; Returned for Revision 28 May 2015; Accepted 15 June 2015) Abstract: With increasing application and commercial production, carbon nanotubes (CNTs) will inevitably be released into aquatic environments and affect the transport and toxicity of toxic metals in ecosystems. The present study examined how CNTs affected the biokinetics and toxicity of a toxic metal, cadmium (Cd), in the freshwater zooplankton Daphnia magna. The authors quantified the dissolved uptake and the 50% lethal concentration (LC50, 48 h and 72 h) of Cd in daphnids in the presence of functionalized multiwalled nanotubes (F-CNTs) with different lengths (10–30 mm vs 0.5–2 mm) and concentrations (4 mg/L and 8 mg/L). Compared with the control treatment without CNTs, both CNTs slowed down the accumulation rate of Cd in D. magna over 8 h of exposure and further reduced the accumulation thereafter. Mechanisms for the reduced Cd uptake were mainly related to the influences of CNTs on the physiological activity of daphnids. The LC50 of D. magna in the presence of Cd and shorter CNTs was almost the same as that of the control group without CNTs. However, the LC50 of the groups with normal CNTs was significantly higher than that of the control group (i.e., F-CNTs decreased Cd toxicity significantly). Meanwhile, CNTs also decreased the tolerance of D. magna to Cd. The present study suggests that different physical properties of CNTs, such as length, need to be considered in the environmental risk assessment of CNTs. Environ Toxicol Chem 2015;34:2824–2832. # 2015 SETAC Keywords: Carbon nanotube

Daphnia magna

Cadmium

Uptake

Toxicity

magna, thus making the suspensions less stable [20]. Later, Alpatova et al. [22] and Yu and Wang [23] achieved stable aqueous suspensions of multiwalled CNTs by surface coating them with polyvinylpyrrolidone (PVP). Yu and Wang [23] showed that the PVP-CNT suspension could maintain a stable state even with tested organisms. They then examined the biokinetics (uptake from the dissolved phase and assimilation efficiency from the dietary phase) of Cd and zinc (Zn) in D. magna, with the presence of CNTs. However, whether the CNT uptake would affect the toxicity of Cd is unknown. Carbon nanotubes may behave similarly to natural organic matter (NOM) and possibly decrease the dissolved uptake of metals by binding with free metal ions. It is also possible that CNTs may act at the sorption sites for metals and become carriers for metal uptake in animals. Earlier studies investigated the combined effects of copper (Cu) or lead (Pb) with CNTs in D. magna [24,25] and found that CNTs enhanced metal concentration in the body when the metal concentrations were low. In the present study, we specifically examined whether there were any relationships between metal accumulation and toxicity in the presence of CNTs. Also, we examined how physical factors, such as length of CNTs, would affect the metal accumulation and toxicity in daphnids. The present study can therefore provide important information on the accumulation and toxicity of Cd in the presence of CNTs of different lengths.

INTRODUCTION

Multiwalled carbon nanotubes (CNTs) as 1 category of CNTs are macromolecules consisting of many layers of rolled graphene sheets [1]. In general, multiwalled CNTs may have 2 to 30 layers and their outer diameters may vary from 20 nm to 30 nm [1–3]. Since the invention of multiwalled CNTs, their unique characteristics have led to a wide range of applications in daily life [4–6]. Given these widespread applications, a considerable amount of CNTs will eventually be released into the environment. A computational estimation showed that CNT concentrations in soil treated with sewage sludge may increase by 74 ng/kg/yr [6–8]. Because of the mobility of CNTs among different compartments, they will probably enter the food chain [9]; and the bioaccumulation of CNTs as a result of their hydrophobicity has raised considerable interest. Once CNTs enter aquatic environments they can interact with some inorganic or organic substances, such as cadmium (Cd), a toxic metal that can cause osteoporosis, anemia, and renal damage [10,11]. Because of the large specific surface area of CNTs, they are also applied as adsorbents for trace metals. Earlier works suggested that CNTs performed better if they were functionalized beforehand (i.e., adding some carboxyl groups onto the surface) [12–15]. This may further increase the risk of toxicity in case of accidental exposures of these materials to organisms. To gain a better understanding of the biological effects of CNTs on organisms, diverse surfactants have been provided to achieve a relatively stable suspension [16–21]. For example, some studies used lysophosphatidylcholine as a surfactant to stabilize CNTs in solutions. However, lysophosphatidylcholine may be potentially utilized by the freshwater flea Daphnia

MATERIALS AND METHODS

CNTs

Functionalized-multiwalled CNTs (F-CNTs) and short functionalized-multiwalled CNTs (SF-CNTs), containing carboxyl groups, were obtained from Timesnano (Chengdu Organic Chemicals, Chinese Academy of Sciences), which specializes in producing CNTs. All CNTs were stored in the dryer and used as soon as received. These 2 types of CNTs (F-CNTs and SF-CNTs)

* Address correspondence to [email protected] Published online 19 June 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.3122 2824

Cd uptake and toxicity under carbon nanotube exposure

were produced by catalytic chemical vapor deposition, with the following technical data: –COOH content of 2.0%, purity of >95%, inner diameter of 5 nm to 10 nm, outer diameter of 10 nm to 20 nm, specific surface area of >200 m2/g, and electric conductivity of >100 s/cm. The lengths of F-CNTs and SF-CNTs were 10 mm to 30 mm and 0.5 mm to 2.0 mm, respectively. Transmission electronic microscopy (TEM; JEOL 10CXII) was used for CNT characterization. For the TEM sample preparation, CNTs were ultrasonicated for 15 min to avoid aggregation. Then, the dilutions were dripped onto a carbon film–covered copper grid with an absorbent paper underneath. After being dried in the dryer, the samples were placed under the TEM in a vacuum environment. Energy dispersive X-ray spectroscopy (EDX) was used to analyze all the elements contained in the CNTs, especially their catalyst species. Organisms, medium, and radioisotopes

The tested organisms used in the present study were clones of D. magna cultured in the laboratory for more than 14 yr. These D. magna were reared in filtered (GF/C Whatman) water collected from a pond in the Hong Kong University of Science of Technology at a density of 1 individual/10 mL before the experiment and fed green alga (Chlamydomonas reinhardtii) at 5 104 cells/mL (neonates 3 d old) or 105 cells/mL (adults >3 d old), which were cultured in WC medium beforehand [26]. Both green algae and cladocerans were maintained at 23.5 8C with a 14:10-h light:dark cycle in a thermostatic chamber. For all experiments 7-d-old D. magna were chosen because of their relatively large body sizes and before reaching the reproduction stage. To avoid the potential influence of high Ca concentration on the stability of CNT solutions, simplified Elendt M7 medium (SM7, containing CaCl2 2H2O, 20 mg/L; MgSO4 7H2O, 123.3 mg/L; K2HPO4, 0.184 mg/L; KH2PO4, 0.143 mg/L; NaNO3, 0.274 mg/L; NaHCO3, 64.8 mg/L; Na2SiO3 9H2O, 10 mg/L; H3BO3, 0.715 mg/L; and KCl, 5.8 mg/L, without disodium ethylenediaminetetraacetic acid [EDTA], trace metals, and vitamins) was used in all experiments [26]. Daphnids were not acclimated to SM7 medium before experiments. The medium pH was maintained at 8.0 0.2; 109CdCl2 was used as a radiotracer for its accumulation in daphnids, and its effect on daphnids was considered negligible during the short-term uptake period. A Wallac 1480 NaI (T1) gamma detector was used to determine the radioactivity of 109Cd at 88 keV. Counting time was from 1 min to 3 min to yield a propagated counting error of less than 5%. Dispersion of CNTs and stability in suspension

One surfactant, PVP (Sigma-Aldrich), was used to disperse CNTs and dissolved in 18 MΩ cm–1 MilliQ water. An earlier study demonstrated that PVP did not have any toxic effects on 7-d-old D. magna even when the concentration reached as high as 5 g/L [23]. A suspension of CNTs with PVP was prepared according to the method described by Alpatova et al. [22]. Briefly, 4.0 mg CNTs were added into 40 mL of 1-g/L PVP solution in a 50-mL tube. The mixture was ultrasonicated for 15 min using a probe sonicator at an intensity of 60 W with 5-s intervals and then settled for 12 h to obtain the surfactant-coated CNTs. A zeta-potential analyzer (Brookhaven Instruments; ZetaPlus) was used to measure the effective diameter, polydispersity, zeta potential, and mobility of suspensions. All measurements were made at room temperature. To ensure that the concentrations of CNTs in each batch of experiments were maintained the same, ultraviolet (UV)-visible

Environ Toxicol Chem 34, 2015

2825

spectrophotometry (8500 II) was applied to determine the concentrations of CNTs in the solutions at 500 nm [27–30]. A standard curve was established by diluting 20 mg/L CNT-PVP suspension (prepared as the procedure mentioned above) to 1 mg/L, 2.5 mg/L, 5 mg/L, and 10 mg/L; and their absorption at 500 nm was then recorded. The standard curve was used for quality control of all experiments. Meanwhile, the absorption at 500 nm was used to determine whether D. magna affected the stability of solutions. Explicitly, 0 mg/L, 4 mg/L, and 8 mg/L CNT-PVP suspensions (100 mL) were prepared with 3 replicates, respectively; and 10 D. magna (7 d old) were added into each replicated beaker under experimental conditions (23.5 8C, with a 14:10-h light:dark cycle). All tested solutions were not renewed, and D. magna were purged of their gut contents for 4 h prior to the experiments and not fed during the period. Absorptions at 500 nm were recorded at 0 h, 6 h, 12 h, 24 h, 48 h, and 72 h. The percentage of CNTs remaining in suspension was calculated by the absorption at different time points divided by the adsorption at 0 h. Cd uptake by D. magna

This experiment quantified the uptake of Cd by daphnids in the presence of F-CNTs and SF-CNTs. There were a total of 5 groups: Cd only (control group), Cd with 4 mg/L F-CNTs, Cd with 8 mg/L F-CNTs, Cd with 4 mg/L SF-CNTs, and Cd with 8 mg/L SF-CNTs. The 2 concentrations of CNTs were chosen to produce observable effects on Cd uptake and toxicity. These concentrations were below lethal levels, although they were not environmentally relevant. In each group, the Cd concentration treatments included 2 mg/L, 5 mg/L, 10 mg/L, 15 mg/L, and 20 mg/L. Ultrasonication of CNTs was conducted 1 d before the experiment, and Cd (with radioactive 109Cd) was then spiked, allowing equilibrium between Cd and the CNT mixture. Prior to the exposure, D. magna of similar sizes were removed from the primary culture containers and transferred to clean SM7 for depuration for 4 h. Afterward, 30 individual daphnids were equally divided into triplicated beakers of each treatment containing 100 mL of suspension (e.g., n ¼ 3, with 10 individuals in each replicate). No food was added, to avoid other pathways of Cd uptake. In the first experiment, daphnids were exposed to Cd for a total of 8 h. At 2 h, 4 h, 6 h, and 8 h, they were pipetted onto a mesh and rinsed with SM7 to remove the loosely adsorbed Cd and CNTs from their carapaces [23]. After their radioactivity was measured by the gamma counter, the animals were returned to their original beakers immediately. By the end of exposure, all animals from each treatment were separately collected and dried at 80 8C overnight to measure their dry weights. Based on the radioactivity measurements, the influx rate (I, micrograms per gram per hour) of Cd was calculated as the slope of linear regression between the accumulated Cd and the exposure time [26]. It was assumed that the efflux was negligible over the short-term uptake period. In another experiment, daphnids were exposed to Cd for a longer period (72 h), using the same experimental procedures. The water was renewed every 24 h to ensure a relatively constant exposure of daphnids to Cd and CNTs. During the uptake experiment, metal adsorption onto CNTs was also measured simultaneously. At 0 h, 24 h, 48 h, and 72 h, duplicated 0.5-mL solutions were collected from each beaker. One sample was used to measure the radioactivity directly, another was filtered through a 0.22-mm polycarbonate membrane; and the radioactivity of the filter was measured. The percentage of Cd adsorbed onto CNTs was then calculated.

2826


Furthermore, because Co and Ni were used as the catalysts for CNTs, we also quantified the dissolved cobalt (Co) and nickel (Ni) concentrations in the uptake medium. Specifically, CNTs were prepared as described in the section Dispersion of CNTs and stability in suspension and suspended in the uptake medium at normal pH (8.0) or acidic pH (5.5). After 24 h of suspension, the water was filtered through a 0.22-mm polycarbonate membrane and the dissolved concentrations of Co and Ni were quantified using inductively coupled plasma-mass spectroscopy (NexION 300X). Cd toxicity to D. magna

Seven-day-old D. magna were used for the toxicity testing at different Cd concentrations (0 mg/L, 2 mg/L, 5 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, 40 mg/L) with 2 types of CNTs (F-CNTs and SF-CNTs). Except for the control group without CNTs, 2 concentrations of CNTs (4 mg/L and 8 mg/L) were used; thus, there was a total of 5 groups. In the preliminary experiments, both CNTs themselves showed no toxic effect on 7-d-old D. magna even when the concentration reached as high as 10 mg/L. Death rate was used as an endpoint for toxicity testing, with 3 replicates for each treatment. Ten individual daphnids were added to 100 mL SM7 solutions containing PVPcoated CNTs and Cd in a 200-mL high-density polyethylene beaker. The mortality of D. magna after 12 h, 24 h, 48 h, and 72 h was counted and plotted against the tested concentrations; and the data were analyzed by the statistical probit method (SPSS) to calculate the 50% lethal concentrations (LC50) and their 95% confidence intervals. During the 72-h period, no food was provided and water was renewed every 24 h to ensure a relatively constant exposure of daphnids to Cd and CNTs. RESULTS AND DISCUSSION

Characterization of CNTs

Transmission electron microscopic graphs confirmed that F-CNTs were longer than SF-CNTs (Figure 1). The UV-visible absorption spectrum of F-CNTs and SF-CNTs in the PVP-dispersed SM7 solutions at 500 nm was quantified. According to Beer’s law, A ¼ eLc where A is absorbance, e is the apparent absorption coefficient (milliliters per milligram per centimeter), L is the distance that the light traveled through the suspension (1 cm in this experiment), and c is the concentration of CNTs (milligrams per liter). Absorbances for the control group (no CNTs) and the

J. Liu and W.X. Wang

4 mg/L SF-CNT, 8 mg/L SF-CNT, 4 mg/L F-CNT, and 8 mg/L F-CNT groups were 0, 0.0516, 0.1032, 0.1516, and 0.3032, respectively. Thus, e can be calculated from the linear regression of the above equation [30]. For F-CNTs and SF-CNTs, the apparent absorption coefficients were 37.9 mL/mg/cm and 12.9 mL/mg/cm, respectively, indicating that F-CNTs were much darker than SF-CNTs at the same concentration. The solutions of each type of CNT became darker with increasing concentrations. The e value of F-CNTs (37.9 mL/mg/cm) was very close to the results for monodispersed multiwalled CNTs (39.9 mL/mg/cm) obtained by Li et al. [30] and those for polybutadiene–dispersed multiwalled CNTs in chloroform (42.2 mL/mg/cm) obtained by Baskaran et al. [31]. However, the e value of another type of CNT used in the present study (SF-CNTs) was much smaller than those of the multiwalled CNTs mentioned above, although all 4 types of CNTs were obtained from the same chemical vapor deposition method. From EDX images (not shown), different catalysts were found for different types of CNTs. For F-CNTs, Co was used as catalyst, whereas for SF-CNTs, the catalyst was Ni. Several studies also have demonstrated that the metals as catalyst could not be avoided even after washing with nitric acid [32,33]. It was thus important to measure the dissolved Ni (for SF-CNTs) and Co (for F-CNTs) concentrations in the medium. The measured initial dissolved Co concentrations in the medium after the suspension of CNTs were 13.7 0.87 mg/L and 25.9 2.91 mg/L for the 4 mg/L and 8 mg/L F-CNT treatments, respectively. After 24-h suspension, the dissolved Co concentrations were 88% to 102% of the initial values at normal pH or pH 5.5, suggesting that there was no further desorption of Co from F-CNTs. No measurable Co was detected for the SF-CNT treatments. The initial dissolved Ni concentrations in the medium were 3.83 0.08 mg/L and 8.38 0.28 mg/L for the 4 mg/L and 8 mg/L SF-CNT treatments, respectively. After 24-h suspension, the dissolved Ni concentrations were 90% to 108% of the initial values at normal pH or pH 5.5, again suggesting that there was no further desorption of Ni from SF-CNTs. Very low dissolved Ni concentrations were measured for the F-CNT treatments (0.34 mg/L and 0.75 mg/L for the 4 mg/L and 8 mg/L F-CNTs treatments, respectively). In the present study, no mortality of daphnids was observed at the added CNT concentrations; therefore, the potential influences of catalysts on Cd uptake and toxicity were ignored. An earlier study also suggested that the 48-h 50% effective concentration (EC50) of Ni in the immobilization test with D. magna was in the range of 1.8 mg/L to 5.5 mg/L under

Figure 1. Typical transmission electron microscopic images of multiwalled nanotubes dispersed in SM7-polyvinylpyrrolidone medium. (A) Functionalized carbon nanotubes; (B) short functionalized carbon nanotubes.



2827

Figure 2. Effective diameter, zeta potential, polydispersity, and mobility of carbon nanotubes over 72 h of suspension. SF-CNT ¼ short functionalized carbon nanotube; F-CNT ¼ functionalized carbon nanotube.

different magnesium (Mg), Ca, Na, and pH conditions [34]. The 48-h LC50 of Co in D. magna ranged from 1.1 mg/L to 5.2 mg/L, depending on hardness [35]. Stability of CNTs in suspension

Previous studies suggested that the dispersibility, dispersion stability, and biocompatibility of surfactants played an important role in the toxicity evaluation of CNTs [19]. Nanomaterials had a strong tendency of aggregation, and nanoscaled materials differed from bulk materials because of the change of their physicochemical properties. Diverse surfactants were therefore used to form a stable and compatible test system for CNTs, such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, bovine serum albumin, Tween 20, Tween 80, lysophosphatidylcholine, polyethyleneimine, and pyrrolidone [16–23]. Alpatova et al. [22] and Yu and Wang [23] proved that among these surfactants PVP performed the best in the CNT toxicity testing with D. magna. The physical characteristics of the 2 types of CNTs are shown in Figure 2. Particle sizes were measured 3 times under irradiation with laser. These sizes were considered as the effective diameters instead of actual diameters because the method provided more precise results when the objectives were circle-shaped rather than of long tube–shaped. The SF-CNTs showed a minor trend of aggregation, whereas the F-CNTs stayed stable in the PVP-facilitated solution for as long as 72 h. The zeta potential of the CNTs was measured twice using a palladium electrode, with 40 circles each; and data were analyzed by Smoluchowski-type modeling. The stability of PVP-coated CNT suspensions is also shown in Figure 2. The PVP coating did not result in significant differences in the surface properties of the 2 CNT types. We then further tested the stability of CNTs in the presence of daphnids (Figure 3). After 72-h exposure, the percentages of CNTs remaining in the suspension were approximately 95% to 97% and 93% to 95% for SF-CNTs and F-CNTs, respectively, indicating that D. magna had little effect on the PVP-coated solutions, consistent with the results of Yu and Wang [23].

Adsorption of Cd onto CNTs

To better understand the interaction between Cd and CNTs, the adsorption of Cd onto CNTs was considered. In preliminary adsorption and desorption experiments, radioactive 109Cd was used to quantify the Cd adsorbed onto CNTs; and the results showed little adsorption during the 72-h period. During the uptake experiment, Cd adsorption onto CNTs was measured under different concentrations of CNTs and Cd. Figure 4 shows the percentage and amount of Cd adsorbed onto CNTs. In general, a very small percentage of Cd was adsorbed on the

Figure 3. Percentage of carbon nanotubes remaining in suspension with addition of Daphnia magna. SF-CNT ¼ short functionalized carbon nanotube; F-CNT ¼ functionalized carbon nanotube.

2828


Figure 4. Adsorption of Cd onto carbon nanotubes at different Cd dissolved concentrations. Data are mean standard deviation (n ¼ 3). SF-CNT ¼ short functionalized carbon nanotube; F-CNT ¼ functionalized carbon nanotube.

CNTs, especially at the higher concentration (4 mg/L, 2 times higher than that in the control group, suggesting that F-CNTs decreased the toxicity of Cd by approximately 2 times compared with multiwalled SF-CNTs. The difference between the F-CNTs and the control or SF-CNTs was consistent with the difference of the Cd influx, as well as the UV-visible absorption. Therefore, F-CNTs decreased the dissolved Cd uptake rate to a greater degree than SF-CNTs did, and the lower bioaccumulation of Cd resulted in less toxicity to the animals.

Figure 9. Mortality of Daphnia magna after 72-h exposure to different concentrations of Cd with and without CNTs. SF-CNT ¼ short functionalized carbon nanotube; F-CNT ¼ functionalized carbon nanotube; CNT ¼ carbon nanotube.

In an earlier study, Kim et al. [39] investigated the bioavailability and acute toxicity of Cu to D. magna and found that Cu toxicity increased in the NOM dispersed multiwalled CNT solutions. In a subsequent study [24], Cu toxicity to D. magna also increased in the presence of lysophosphatidylcholine-coated single-walled CNTs, as a result of increasing Cu accumulation with increasing concentration of lysophosphatidylcholine-CNTs. The surfactant lysophosphatidylcholine could be directly ingested by daphnids and increased the release of Cu ions in the gut, therefore further accelerating the mortality of D. magna. Roberts et al. [20] also provided evidence that D. magna utilized lysophosphatidylcholine-CNTs as a food source. In the present study, PVP was not digested as lysophosphatidylcholine in the gut of D. magna and the CNTs were blocked in the guts, which resulted in less Cd release and reduced toxicity. For other metals, Qin et al. [25] found that in the presence of OH-multiwalled CNTs, the toxicity of Pb(II) to D. magna increased with increasing CNT concentrations from 0 mg/L to 1.0 mg/L. In addition, Cu adsorbed onto nano-TiO2 was subsequently ingested and accumulated in D. magna, causing toxic effects as a result of the enhanced oxidative stress and physiological damage induced by nano-TiO2 [43]. A similar phenomenon was observed on the combined toxicity of TiO2 nanoparticles and Cd and Zn [44]. The main difference between nano-TiO2 and CNTs was that nano-TiO2 could be purged [44] by D. magna, whereas CNTs tended to remain in the guts of daphnids. Based on the Cd accumulation in daphnids at different Cd concentrations in water, it was possible to calculate the 72-h



Table 1. Calculated 48-h and 72-h 50% lethal concentrations of Cd in Daphnia magna exposed to different carbon nanotubesa Treatments Cd only 4 mg/L SFCNTs 8 mg/L SFCNTs 4 mg/L FCNTs 8 mg/L FCNTs

48-h LC50 (mg/L)

72-h LC50 (mg/L)

72-h LT30 (mg/g)

72-h LT50 (mg/g)

36.8 (27.5–63.5) 33.6 (26.4–49.3) 28.6 (22.3–40.4) 77.4 (58.1–130) 84.9 (62.0–164)

24.7 (19.4–33.6) 25.2 (20.1–33.8) 24.6 (19.1–34.4) 56.7 (44.7–77.5) 51.6 (39.6–73.4)

408

574

333

461

213

300

511

ND

342

ND

a

Values are given with their 95% confidence intervals. Calculated 72-h 30% and 50% lethal tissue concentrations are also shown. The median lethal tissue concentration values for 4 mg/L and 8 mg/L functionalized-multiwalled carbon nanotubes are not given because their median lethal concentration value was beyond the maximum tested concentrations. LC50 ¼ 50% lethal concentration; LT30/50 ¼ 30% and 50% lethal tissue concentrations; SF-CNT ¼ short functionalized-multiwalled carbon nanotube; F-CNT ¼ functionalized-multiwalled carbon nanotube; ND ¼ not determined.

lethal Cd accumulated tissue concentrations (30% [LT30] and 50% [LT50]) in the animals at LC30 and LC50 (Table 1). For the 4 mg/L and 8 mg/L SF-CNT groups, the calculated Cd LT50 values were 461 mg/g and 300 mg/g, respectively, which were lower than that of the control group (574 mg/g), in contrast to the comparable LC50 values for these treatments (24.6–25.2 mg/L). For F-CNTs, the difference in LT30 with the control treatment (342–511 mg/g vs 408 mg/g) was also much smaller than the difference in LC50 (>2 times). These data suggested that although SF-CNTs slowed down the Cd influx and bioaccumulation into the body of D. magna, the tolerance of the animals was actually reduced. Such a decrease in Cd tolerance may have been the result of a combined effect of CNTs on the physiological activity of the animals. For example, the CNTs may have attached onto the external carapace and thoracopods and subsequently reduced the filtering and swimming activity of D. magna. In conclusion, the present study demonstrated that different lengths of F-CNTs performed differently in affecting the toxicity of Cd to D. magna. The longer CNTs significantly decreased Cd toxicity, whereas the shorter CNTs did not obviously affect Cd toxicity. The decrease of Cd toxicity was mainly related to the reduced Cd bioaccumulation by the animals. Ingestion of CNTs may have greatly affected the physiological activity of the animals and subsequently inhibited Cd uptake. Nevertheless, the tolerance of daphnids to Cd was also reduced under CNT exposure. The present study suggests that the complex interaction of CNTs and metals needs to be carefully considered in future ecotoxicological studies. Acknowledgment—We thank the anonymous reviewers for their comments on the present study. The present study was supported by the General Research Fund from the Hong Kong Research Grants Council (663011; to W.-X. Wang). Data availability—Data, associated metadata, and calculation tools are available on request from the authors ([email protected]). REFERENCES 1. Ong YT, Ahmad AL, Hussein S, Zein S, Tan SH. 2010. A review on carbon nanotubes in an environmental protection and green engineering perspective. Carbon Nanotubes 27:227–242.

2831

2. Li Z, Hulderman T, Salmen R, Chapman R, Leonard SS, Young SH, Shvedova A, Luster MI, Simeonova PP. 2007. Cardiovascular effects of pulmonary exposure to single-wall carbon nanotubes. Environ Health Perspect 115:377–382. 3. Walther JH, Jaffe RL, Kotsalis EM, Werder T, Halicioglu T, Koumoutsakos P. 2004. Hydrophobic hydration of C60 and carbon nanotubes in water. Carbon 42:1185–1194. 4. Chung H, Son Y, Yoon TK, Kim S, Kim W. 2011. The effect of multiwalled carbon nanotubes on soil microbial activity. Ecotoxicol Environ Saf 74:569–575. 5. Sarma SJ, Bhattacharya I, Brar SK, Tyagi RD, Surampalli RY. 2014. Carbon nanotube-bioaccumulation and recent advances in environmental monitoring. Crit Rev Environ Sci Technol 45:905–938. 6. Sayes CM, Liang F, Hudson JL, Mendez J, Guo W, Beach JM, Moore VC, Doyle CD, West JL, Billups WE, Ausman KD, Colvin VL. 2006. Functionalization density dependence of single-walled carbon nanotubes cytotoxicity in vitro. Toxicol Lett 161:135–142. 7. Gottschalk F, Sondere T, Schols R, Nowack B. 2009. Modeled environmental concentrations of engineered nanomaterials for different regions. Environ Sci Technol 43:9216–9222. 8. Larue C, Pinault M, Czarny B, Georgin D, Jaillard D, Bendiab N, Mayne-L’Hermite M, Taran F, Dive V, Carriere M. 2012. Quantitative evaluation of multi-walled carbon nanotube uptake in wheat and rapeseed. J Hazard Mater 227–228:155–163. 9. Petersen EJ, Akkanen J, Kukkonen JVK, Weber WJ. 2009. Biological uptake and depuration of carbon nanotubes by Daphnia magna. Environ Sci Technol 43:2969–2975. 10. Hamilton MA, Rode PW, Merchant ME, Sneddon J. 2008. Determination and comparison of heavy metals in selected seafood, water, vegetation and sediments by inductively coupled plasma-optical emission spectrometry from an industrialized and pristine waterway in southwest Louisiana. Microchem J 88:52–55. 11. Hawari AH, Mulligan CN. 2006. Biosorption of lead(II), cadmium(II), copper(II) and nickel(II) by anaerobic granular biomass. Bioresour Technol 97:692–700. 12. Kuo CY. 2009. Prevenient dye-degradation mechanisms using UV/ TiO2/carbon nanotubes process. J Hazard Mater 163:239–244. 13. Kuo CY, Lin HY. 2009. Adsorption of aqueous cadmium(II) onto modified multi-walled carbon nanotubes following microwave/chemical treatment. Desalination 249:792–796. 14. Liu J, Zubiri MRI, Vigolo B, Dossot M, Fort Y, Ehrhardt JJ, McRae E. 2007. Efficient microwave-assisted radical functionalization of single-wall carbon nanotubes. Carbon 45:885–891. 15. Wu CH. 2007. Studies of the equilibrium and thermodynamics of the adsorption of Cu2þ onto as-produced and modified carbon nanotubes. J Colloid Interface Sci 311:338–346. 16. Bihari P, Vippola M, Schultes S, Praetner M, Khandoga AG, Reichel CA, Coester C, Tuomi T, Rehberg M, Krombach F. 2008. Optimized dispersion of nanoparticles for biological in vitro and in vivo studies. Part Fibre Toxicol 5:14. 17. Buford MC, Hamilton RF, Holian A. 2007. A comparison of dispersing media for various engineered carbon nanoparticles. Part Fibre Toxicol 4:6. 18. Foucaud L, Wilson MR, Brown DM, Stone V. 2007. Measurement of reactive species production by nanoparticles prepared in biologically relevant media. Toxicol Lett 174:1–9. 19. Kim JS, Song KS, Lee JH, Yu IJ. 2011. Evaluation of biocompatible dispersants for carbon nanotube toxicity tests. Arch Toxicol 85:1499–1508. 20. Roberts AP, Mount AS, Seda B, Souther J, Qiao R, Lin S, Lin SJ, Ke PC, Rao AM, Klaine SJ. 2007. In vivo biomodification of lipid-coated carbon nanotubes by Daphnia magna. Environ Sci Technol 41:3028–3029. 21. Wick P, Manser P, Limbach LK, Dettlaff-Weglikowska U, Krumeich F, Roth S, Stark WJ, Bruinink A. 2007. The degree and kind of agglomeration affect carbon nanotube cytotoxicity. Toxicol Lett 168:121–131. 22. Alpatova AL, Shan W, Babica P, Upham BL, Rogensues AR, Masten SJ, Drown D, Mohanty AK, Alocilja EC, Tarabara VV. 2010. Single-walled carbon nanotubes dispersed in aqueous media via non-covalent functionalization: Effect of dispersant on the stability, cytotoxicity, and epigenetic toxicity of nanotube suspensions. Water Res 44:505–520. 23. Yu ZG, Wang W-X. 2013. Influences of ambient carbon nanotubes on toxic metals accumulation in Daphnia magna. Water Res 47:4179–4187. 24. Kim KT, Klaine SJ, Lin S, Ke PC, Kim SD. 2010. Acute toxicity of a mixture of copper and single-walled carbon nanotubes to Daphnia magna. Environ Toxicol Chem 29:122–126. 25. Qin L, Huang Q, Wei Z, Wang L, Wang Z. 2014. The influence of hydroxyl-functionalized multi-walled carbon nanotubes and pH levels

2832

26. 27. 28. 29. 30.

31.

32.

33. 34.


on the toxicity of lead to Daphnia magna. Environ Toxicol Pharmacol 38:199–204. Tan Q-G, Wang W-X. 2008. The influences of ambient and body calcium on cadmium and zinc accumulation in Daphnia magna. Environl Toxicol Chem 27:1605–1613. Lin D, Xing B. 2008. Adsorption of phenolic compounds by carbon nanotubes: Role of aromaticity and substitution of hydroxyl groups. Environ Sci Technol 42:7254–7259. Bahr JL, Mickelson ET, Bronikowski MJ, Smalley RE, Tour JM. 2001. Dissolution of small diameter single-wall carbon nanotubes in organic solvents? Chem Commun (Camb) 2:193–194. Lee JU, Huh J, Kim KH, Park C, Jo WH. 2007. Aqueous suspension of carbon nanotubes via non-covalent functionalization with oligothiophene-terminated poly(ethylene glycol). Carbon 45:1051–1057. Li ZF, Luo GH, Zhou WP, Wei F, Xiang R, Liu YP. 2006. The quantitative characterization of the concentration and dispersion of multi-walled carbon nanotubes in suspension by spectrophotometry. Nanotechnology 17:3692–3698. Baskaran D, Mays JW, Bratcher MS, Uni V, Hall B, Knox V. 2005. Noncovalent and nonspecific molecular interactions of polymers with multiwalled carbon nanotubes with multiwalled carbon nanotubes. Chem Mater 17:3389–3397. Pumera M. 2007. Carbon nanotubes contain residual metal catalyst nanoparticles even after washing with nitric acid at elevated temperature because these metal nanoparticles are sheathed by several graphene sheets. Langmuir 23:6453–6458. Liu Y, Zhao Y, Sun B, Chen C. 2013. Understanding the toxicity of carbon nanotubes. Acc Chem Res 46:702–713. Deleebeeck NME, De Schamphelaere KAC, Heijerick DG, Bossuyt BTA, Janssen CR. 2009. The acute toxicity of nickel to Daphnia magna: Predictive capacity of bioavailability models in artificial and natural waters. Ecotoxicol Environ Saf 70:67–78.

J. Liu and W.X. Wang 35. Diamond JM, Winchester EL, Mackler DG, Rasnake WJ, Fanelli JK, Gruber D. 1992. Toxicity of cobalt to freshwater indicator species as a function of water hardness. Aquat Toxicol 22:163–180. 36. Li YH, Wang S, Luan Z, Ding J, Xu C, Wu D. 2003. Adsorption of cadmium(II) from aqueous solution by surface oxidized carbon nanotubes. Carbon 41:1057–1062. 37. Guo L, Hunt BJ, Santschi PH, Ray SM. 2001. Effect of dissolved organic matter on the uptake of trace metals by American oysters. Environ Sci Technol 35:885–893. 38. Tian X, Zhou S, Zhang Z, He X, Yu M, Lin D. 2010. Metal impurities dominate the sorption of a commercially available carbon nanotube for Pb(II) from water. Environ Sci Technol 44:8144–8149. 39. Kim KT, Edgington AJ, Klaine SJ, Cho JW, Kim SD. 2009. Influence of multiwalled carbon nanotubes dispersed in natural organic matter on speciation and bioavailability of copper. Environ Sci Technol 43:8979–8984. 40. Li M, Huang CP. 2011. The responses of Ceriodaphnia dubia toward multi-walled carbon nanotubes: Effect of physical–chemical treatment. Carbon 49:1672–1679. 41. Tao X, Fortner JD, Zhang B, He Y, Chen Y, Hughes JB. 2009. Effects of aqueous stable fullerene nanocrystals (nC60) on Daphnia magna: Evaluation of sub-lethal reproductive responses and accumulation. Chemosphere 77:1482–1487. 42. Petersen EJ, Pinto RA, Mai DJ, Landrum PF, Weber WJ. 2011. Influence of polyethyleneimine graftings of multi-walled carbon nanotubes on their accumulation and elimination by and toxicity to Daphnia magna. Environ Sci Technol 45:1133–1138. 43. Fan W, Cui M, Liu H, Wang C, Shi Z, Tan C, Yang X. 2011. Nano-TiO2 enhances the toxicity of copper in natural water to Daphnia magna. Environ Pollut 159:729–734. 44. Tan C, Fan WH, Wang W-X. 2012. Role of titanium dioxide nanoparticles in the elevated uptake and retention of cadmium and zinc in Daphnia magna. Environ Sci Technol 46:469–476.