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Developmental toxicity of oxidized multi-walled carbon nanotubes on Artemia salina cysts and larvae: Uptake, accumulation, excretion and toxic responses
Environmental Pollution 229 (2017) 679e687
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Developmental toxicity of oxidized multi-walled carbon nanotubes on Artemia salina cysts and larvae: Uptake, accumulation, excretion and toxic responses* Song Zhu, Fei Luo, Xiao Tu, Wei-Chao Chen, Bin Zhu, Gao-Xue Wang* College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 April 2017 Received in revised form 25 June 2017 Accepted 7 July 2017
Using Artemia salina (A. salina) cysts (capsulated and decapsulated) and larvae [instar I (0e24 h), II (24 e48 h) and III (48e72 h)] as experimental models, developmental toxicity of oxidized multi-walled carbon nanotubes (O-MWCNTs) was evaluated. Results revealed that hatchability of capsulated and decapsulated cysts was significantly decreased (p < 0.01) following exposure to 600 mg/L for 36 h. Mortality rates were 33.8, 55.7 and 40.7% for instar I, II and III larvae in 600 mg/L. The EC50 values for swimming inhibition of instar I, II and III were 535, 385 and 472 mg/L, respectively. Instar II showed the greatest sensitivity to O-MWCNTs, and followed by instar III, instar I, decapsulated cysts and capsulated cysts. Effects on hatchability, mortality and swimming were accounted for O-MWCNTs rather than metal catalyst impurities. Body length was decreased with the concentrations increased from 0 to 600 mg/L. OMWCNTs attached onto the cysts, gill and body surface, resulting in irreversible damages. Reactive oxygen species, malondialdehyde content, total antioxidant capacity and antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) activities were increased following exposure, indicating that the effects were related to oxidative stress. O-MWCNTs were ingested and distributed in phagocyte, lipid vesicle and intestine. Most of the accumulated O-MWCNTs were excreted by A. salina at 72 h, but some still remained in the organism. Data of uptake kinetics showed that O-MWCNTs contents in A. salina were gradually increased from 1 to 48 h and followed by rapidly decreased from 48 to 72 h with a range from 5.5 to 28.1 mg/g. These results so far indicate that O-MWCNTs have the potential to affect aquatic organisms when released into the marine ecosystems. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Carbon nanotube Developmental toxicity Brine shrimp Oxidative stress Uptake
1. Introduction Carbon nanotubes (CNTs) possess exceptional electrical, chemical, and physical properties, which enable them utilized in various applications, such as manufacturing (De Volder et al., 2013), medicine (Heister et al., 2013) and other industries (Baughman et al., 2002; Eatemadi et al., 2014). Moreover, advances in CNTs synthesis, purification and chemical modification make them suitable for more commercial applications. In this regard, multi-walled carbon nanotubes (MWCNTs) are the largest product segment, accounting for majority market volume. However, along with the rapid increase of related products and applications, CNTs are inevitably
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This paper has been recommended for acceptance by Baoshan Xing. * Corresponding author. Northwest A&F University, Xinong Road 22nd, Yangling, Shaanxi 712100, China. E-mail address: [email protected] (G.-X. Wang). http://dx.doi.org/10.1016/j.envpol.2017.07.020 0269-7491/© 2017 Elsevier Ltd. All rights reserved.
released into the aquatic environment from: 1) the direct entry to waterbodies from bioremediation; 2) rainwater and runoff from contaminated air and soils; 3) aerial and tyres deposition; 4) emissions from wastewater treatment plants (Boxall et al., 2007; Yang et al., 2008). Eventually, most of the CNTs enter into marine ecosystems (Klaine et al., 2008; Wei et al., 2010). Sun et al. (2016) predicted the environmental emissions of engineered nanomaterials using dynamic probabilistic modeling, and suggested that the environmental concentration of CNTs was at the “mg/kg” level. In addition, most of the CNTs-related products are “durable” products (such as tyres and other polymer composites) that have almost no CNTs release during use. A mass of CNTs will release when the products come to the disposal phase, resulting in a higher CNTs concentration in the future (Sun et al., 2016). Therefore, it is imperative to investigate the potential hazards posed by CNTs at high concentration prior to the wide use of them. Such knowledge will be useful in utilization of CNTs and managing risk in the future.
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In recent years, using aquatic invertebrates as models to assess the potential toxicity of nanomaterials has become prevalent (Ates et al., 2013; Petersen et al., 2010; Zhu et al., 2017). As an invertebrate zooplankton found in various seawater systems from lakes to oceans, Artemia salina (A. salina) plays a key role in the energy flow of food chain (Sorgeloos et al., 1986). The intrinsic characteristics and physiological features of A. salina make it as a suitable model for toxicology testing (Nunes et al., 2006; Rajabi et al., 2015). For example, its cysts are commercially available, and have been widely used in toxicology tests (Caldwell et al., 2003; Rotini et al., 2015). Moreover, many stages are divided along the development process of A. salina, and larvae in a uniform physiological condition can be hatched synchronously (Persoone et al., 1989; Sorgeloos et al., 1979). Recently, several studies have investigated the effects of nanomaterials on A. salina (Gambardella et al., 2014; Mesaric et al., 2015; Zhu et al., 2017). It is currently accepted that one of the major mechanisms for toxicity of nanomaterials is oxidative stress (Ates et al., 2013; Liu et al., 2012; Zhu et al., 2017). For example, Ates et al. (2013) investigated the impacts of Zn and ZnO nanoparticles on A. salina. They demonstrated that the nanoparticles showed significant toxicity to A. salina after exposure for 96 h, and the effects were related to oxidative stress. Furthermore, toxicity of graphene oxide (GO) to A. salina was evaluated in our previous study. Results showed that GO induced significant changes in hatchability, mortality, and morphological, ethological and physiological parameters (Zhu et al., 2017). Accumulation and depuration behaviors of CNTs in organisms are critical factors for risk assessment, which impact the overall toxicity of CNTs (Edgington et al., 2013; Guo et al., 2013; Petersen et al., 2009, 2010). It was reported that MWCNTs could be significantly accumulated in the gut of Daphnia magna, but the accumulated MWCNTs were not entirely eliminated from the daphnia body (Petersen et al., 2009, 2010). Besides, we investigated the uptake and toxic effects of MWCNTs on Saccharomyces cerevisiae, and showed that MWCNTs were clearly visible in lysosome, vacuole, endosome, mitochondria, multivesicular body and perinuclear region (Zhu et al., 2016). Mesari c et al. (2015) evaluated the effects of three different carbon-based nanomaterials on A. salina larvae following exposure for 48 h. They suggested that the nanomaterials were ingested and concentrated in the gut, and attached onto the body surface of A. salina (Mesari c et al., 2015). To date, information about the accumulation, depuration and developmental toxicity of MWCNTs on A. salina is limited. In the study, toxicity of oxidized MWCNTs (O-MWCNTs) to A. salina cysts (capsulated and decapsulated) and larvae (instar I, II and III) were evaluated. Based on previous data, it was hypothesized that: 1) hatchability, mortality, and ethological, morphological and biochemical parameters would be significantly changed; 2) effects would be accounted for O-MWCNTs rather than metal catalyst impurities, and were mediated by oxidative stress; 3) O-MWCNTs would be ingested, accumulated and excreted by A. salina. The study contributes to better understandings of the MWCNTs toxicity, and lay foundations for their future exploitation and application. 2. Materials and methods 2.1. Preparation and characterization of MWCNTs MWCNTs were purchased from Chengdu Organic Chemicals Co., Ltd., Chinese Academy of Sciences (Chengdu, China), and the structural parameters are listed in Table S1. They were oxidized (OMWCNTs) and labeled with fluorescein isothiocyanate (FITCMWCNTs) according to the previous study (Zhu et al., 2016) (as described in the Supplementary material). To prepare the suspensions with nominal concentrations of 25, 50, 100, 200, 400 and
600 mg/L, O-MWCNTs were weighed on aluminum foil and placed in 1 L beakers containing 900 mL of filtered natural seawater (FNSW; 30‰ m/v; pH 8.6). The beakers were placed in an ice bath and then probe sonicated with an ultrasonic processor (Scientz-IID, China). The suspensions were sonicated for 1 h at 100 W using a 50% on/off cycle and left overnight at room temperature, and sonicated again. O-MWCNTs were characterized by scanning electron microscope (SEM, Hitachi S-4800, Japan) and transmission electron microscope (TEM, JEM1200EX, Japan) with accelerating voltages of 15 kV and 100 kV, respectively. To estimate the hydrodynamic size distribution of O-MWCNTs in FNSW, a dynamic light scattering (DLS, Brookhaven BI200SM, USA) was used. An X-ray photoelectron spectroscopy (XPS; PHI-5600, Russia) was used to analyze elemental compositions and chemical states. Raman spectra were recorded on a HR800 spectrophotometer (Longjumeau Cedex, France) with an excitation wavelength of 785 nm. The contents of residual metal catalyst impurities in pristine and oxidized MWCNTs were measured using ICP-MS (Thermo Elemental X7). 2.2. Model organism Commercially available dehydrated cysts (Tianjin, China) of A. salina were used and kept at 4 C until required. Decapsulated cysts and instar I, II and III larvae were obtained as described previously (Zhu et al., 2017) (as described in the Supplementary material). 2.3. Hatching assay Cysts were treated with O-MWCNTs suspensions (0, 25, 50, 100, 200, 400 and 600 mg/L) to study the effects on hatchability. In order to measure the contribution of metal catalyst impurities to the effect, cysts were cultivated in FeCl3 solutions. The concentrations of Fe3þ were 0, 0.022, 0.044, 0.088, 0.176, 0.352, 0.528 mg/L, as the same amount of Fe3þ as 0, 25, 50, 100, 200, 400, 600 mg/L MWCNTs suspensions, respectively (Table S2). Hatching tests were carried out according to the previous study (Zhu et al., 2017). A parallel set of experiments was performed to determine the OMWCNTs settling behavior with A. salina cysts. Briefly, triplicate suspensions (0.5 mL) were sampled after exposure for 0, 6, 12, 18 and 24 h from each treatment. The samples were pipetted into a square quartz groove (side length: 5 mm; height: 1 mm) and then dried in vacuum at 80 C. Contents of O-MWCNTs were quantitatively assessed using a HR800 Raman spectrophotometer (Longjumeau Cedex, France) with an excitation wavelength of 785 nm, and calculated by a standard curve. 2.4. Acute toxicity test Acute toxicity test was performed by adding 10 larvae (instar I, II and III) to each well of 24-well plates. Each well contained 1 mL of O-MWCNTs suspensions (0, 25, 50, 100, 200, 400 and 600 mg/L) or FeCl3 solutions. All plates were incubated with shaking at 28 C under a 16:8 h light/dark management. Larvae were not fed during the test. After exposure for 24 h, the numbers of dead larvae were counted using a microscope (Olympus Optical Co., Ltd., Tokyo, Japan). All of the tests were carried out in octuplicate. For each treatment, instar I, II and III larvae (approximately 1000) were also randomly added into beakers containing 100 mL OMWCNTs suspensions or FeCl3 solutions, and cultured at 28 C with shaking. After exposure for 24 h, samples were randomly took and then immediately performed morphological and behavioral analysis, and ROS measurement. Samples for SEM and TEM analysis were fixed in 2.5% glutaraldehyde at 4 C. Specimens for MDA
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content, T-AOC and enzyme activity measurements were frozen in liquid nitrogen and stored at 80 C. The settling behavior of OMWCNTs with A. salina was quantified during the exposure period. 2.5. Morphological and behavioral analysis After exposure for 24 h, 20 surviving larvae were randomly selected from each treatment for morphological and behavioral analysis (Zhu et al., 2017) (as described in the Supplementary material). 2.6. ROS, MDA content, T-AOC and antioxidant enzymes activities Measurements of ROS, MDA content, T-AOC and antioxidant enzymes activities were performed as described previously (Zhu et al., 2017). Moreover, the potential interferences of O-MWCNTs with the measurements were investigated (as described in the Supplementary material). 2.7. Uptake and excretion of MWCNTs The newly hatched larvae were treated with 50 mg/L OMWCNTs/FITC-MWCNTs for 48 h and then transferred in FNSW to excrete for another 24 h. During the exposure, 5 larvae were randomly selected at 0, 12, 24, 48, 60 and 72 h, and thoroughly washed with FNSW. Observation was performed on a microscope (Leica, Germany) and fluorescence stereomicroscope (Leica MZFL III, Germany), and images were taken by a digital camera (Nikon, Japan). The FITC fluorescence was visualized by using excitation and emission at 485 and 530 nm, respectively. Meanwhile, the uptake and distribution of O-MWCNTs in larvae were also observed by TEM (JEOL, Tokyo, Japan). Larvae TEM samples were prepared according to Bartolomaeus et al. (2009). 2.8. Uptake kinetics Briefly, the newly hatched larvae were treated with 50 mg/L OMWCNTs for 72 h. Then, larvae were sampled at 1, 3, 5, 7, 9, 12, 15, 18, 24, 30, 36, 48, 60 and 72 h, and thoroughly washed with distilled water. Larvae were homogenized using a homogenizer. The homogenates were analyzed using the HR800 Raman spectrophotometer as described in previous section on hatching assay. After exposure, O-MWCNTs suspensions (0.5 mL) were also sampled at each sampling time to check the settling behavior. 2.9. Statistical analysis All of the treatments were carried out at least three times, and the data were expressed as mean ± standard deviation (SD). The EC50 values were calculated using the Probit method. The SPSS Version 11.0 software package (SPSS Inc., Chicago, IL) was used to perform statistical analysis. Significant differences between the controls and treatments were examined using one-way ANOVA followed by Tukey's test. Significance was accepted at p < 0.05 and extremely significant at p < 0.01. 3. Results and discussion 3.1. Characterization of MWCNTs Data showed that some parameters have considerable impact on the toxicity of MWCNTs, such as purity, size, agglomeration state and surface chemistry (Belyanskaya et al., 2009; Johnston et al., 2010; Wang et al., 2009). In this study, the related parameters were measured. Fig. 1A and B show the SEM and TEM images of O-
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MWCNTs, indicating that they were fibrous with varying lengths. According to statistical analysis of the TEM images, the length of MWCNTs was ranged from 25 to 575 nm (average length: 102 nm; Fig. 1C), and shorter than the pristine MWCNTs (between 10 and 30 mm; Table S1). DLS data show that the hydrodynamic size of OMWCNTs was ranged from 117 nm to 121 mm with a mean size of 42 mm (Fig. 1D). The size is obviously larger than that estimated by TEM, indicating that O-MWCNTs were rapidly aggregated in the FNSW. Aggregation is inevitable due to the hydration and reduction of electrostatic repulsion in the aqueous solution (Johnston et al., 2010). Nevertheless, the DLS data cannot reveal the exact size of O-MWCNTs in FNSW due to the anisotropic and fibrous morphology. Fig. 1E shows XPS spectra of pristine and oxidized MWCNTs. The photoelectron peaks reveal that pristine MWCNTs surface consisted mainly of carbon (284 eV), as well as small amounts of oxygen (532 eV), iron [711 (Fe 2p1/2) and 725 (Fe 2p3/2) eV], cobalt (782 eV), nickel (850 eV), zinc (1020 eV) and magnesium (1305 eV). For the oxidized MWCNTs, only the peaks of carbon, oxygen and iron can be identified. Raman spectra of pristine and oxidized MWCNTs are showed in Fig. 1F. Typical G band (around 1580 cm1) and D band (around 1303 cm1) are identified from the spectra. Intensity ratio between of D band (ID) and G band (IG) can be used to evaluate the defect density of the MWCNTs walls, it is increased with the defect density increased (Osswald et al., 2007). The ID/IG ratios of pristine and oxidized MWCNTs were 0.65 and 1.35, indicating that the defect density of MWCNTs walls was increased after oxidation. Furthermore, some peaks were observed between 100 and 400 cm1 from the spectrum of pristine MWCNTs which were not identified from the spectrum of O-MWCNTs. Santangelo et al. (2006) demonstrated that peaks below 500 cm1 are related to the presence of iron catalyst in MWCNTs. ICP-MS data showed that some iron catalysts (Fe, Co, Ni, Zn and Mg) were residual in the pristine MWCNTs, and only Fe was found in the OMWCNTs (Table S2). The result consisted with the XPS analysis. All the related parameters indicated that the oxidation of MWCNTs was sufficient and the impurities were obviously reduced. 3.2. Effects of O-MWCNTs and impurities on hatchability As shown in Fig. 2A and B, for both capsulated and decapsulated cysts at 12, 18, 24 and 36 h, the hatching rates were substantially decreased with the concentrations increased from 0 to 600 mg/L. Hatching rates were significantly decreased (p < 0.01) in 600 mg/L at 36 h for capsulated cysts and in 600 mg/L at 24 and 36 h for decapsulated cysts compared with the controls. Decapsulated cysts showed a higher sensibility to O-MWCNTs than capsulated cysts. Moreover, decapsulated cysts also showed a higher hatchability than capsulated cysts. Sorgeloos et al. (1986) demonstrated that the hatchability was improved by decapsulation due to a lower energy requirement to break out of decapsulated cysts. Several studies reported that the toxic effects of MWCNTs were related to the metal catalyst impurities (Haniu et al., 2010; Liu et al., 2012). In the study, effect of metal catalyst impurities on hatchability was evaluated. As shown in Fig. S1, no obvious influence (p > 0.05) was observed, indicating that the effect on hatchability is accounted for O-MWCNTs rather than impurities. O-MWCNTs settling behaviors with capsulated (Fig. S2A) and decapsulated cysts (Fig. S2B) were measured. Result showed that the initial O-MWCNTs concentrations were lower than the nominal concentrations. Moreover, the results also showed that concentrations of O-MWCNTs in supernatants were decreased with the settling time increased and related to the initial concentration. Substantial settling occurred during the first 24 h and relatively stable states were reached from 24 to 36 h. Ratios (CR/CI) between the residual O-MWCNTs concentrations (CR) and the initial O-
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Fig. 1. Characterization of MWCNTs. SEM (A) and TEM (B) images of O-MWCNTs. Size distributions of O-MWCNTs measured using TEM (C) and DLS (D). XPS (E) and Raman (F) spectra of pristine and oxidized MWCNTs.
MWCNTs concentrations (CI) for 600 mg/L at 36 h were 14.7% (with capsulated cysts) and 14.2% (with decapsulated cysts). Similar decreasing trends were also reported in other studies (Guo et al., 2013; Hu et al., 2012; Petersen et al., 2009). 3.3. Effects of O-MWCNTs and impurities on mortality As shown in Fig. 3A, for instar I, II and III larvae in the controls, mortality rates ranged from 2% to 5%, indicating that no lethal effects on larvae were induced without feeding even up to 72 h. Mean mortality rates in 600 mg/L were 33.8, 55.7 and 40.7% for instar I, II and III, respectively. Toxicity of GO to A. salina was assessed in our previous study (Zhu et al., 2017). The mean mortality rates were 47.3%, 74.4% and 69.9% for instar I, II and III following exposure to 600 mg/L, indicating that O-MWCNTs possess a lower toxicity file than GO. Mesari c et al. (2015) studied the effects of MWCNTs on
A. salina after exposure for 48 h, and demonstrated that no significant lethal effect was observed up to a concentration 100 mg/L. The disagreement may be induced by the shorter length of MWCNTs used in the study that could cause more severe toxicity (Johnston et al., 2010). As shown in Fig. S3A, no obvious influence (p > 0.05) of impurities on mortality was observed, indicating that the effect is accounted for O-MWCNTs rather than impurities. Settling behaviors of O-MWCNTs with instar I, II and III larvae are presented in Fig. S4. Concentrations of O-MWCNTs were decreased with the settling time increased, and substantial settling occurred during the first 18 h. CR/CI for 600 mg/L at 24 h were 14.8% (instar I), 15.5% (instar II) and 13.8% (instar III), which were smaller than that with cysts (~21%; Fig. S2). Presence of larvae would increase the settling of O-MWCNTs from solutions due to enhanced aggregation during passage through the organism guts.
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Fig. 2. Hatching rates of capsulated (A) and decapsulated (B) cysts exposed to different O-MWCNTs concentrations. Values are presented as mean ± SD.
Fig. 3. Mortality (A), swimming inhibition (SI; B) and body length (C) for instar I, II and III larvae exposure to different concentrations of O-MWCNTs. Values are presented as mean ± SD.
3.4. Swimming speed and body length As shown in Fig. 3B, swimming was inhibited in a concentrationedependent manner and decreased as much as 52.3, 69.3 and 57.7% in 600 mg/L for instar I, II and III, respectively. The EC50 values for swimming inhibition of instar I, II and III were 535, 385 and 472 mg/L, respectively. Body length (Fig. 3C) was significantly decreased (p < 0.01) in 600 mg/L for instar III. Effect of impurities on the swimming is showed in Fig. S3B. There was no obvious influence (p > 0.05) on swimming, indicating that the effect is accounted for O-MWCNTs rather than impurities. Effects of pollutants on different stages of A. salina have been investigated and verified that discrepant sensitivity in relation to stages was exhibited (Barahona and S anchezfortún, 1996; Caldwell et al., 2003; Zhu et al., 2017). Caldwell et al. (2003) demonstrated that hatching assay showed a lower sensitivity to algal extracts and short chain aldehydes compared with the mortality assays. Besides, we verified that instar II larvae showed the greatest sensitivity to GO in our previous study (Zhu et al., 2017). In this study, based on the results obtained above, it can be concluded that the sensitivity of A. salina to O-MWCNTs is in the order of instar II > instar III > instar I > decapsulated cysts > capsulated cysts. The conclusion is somewhat similar to the results of previous studies (Barahona nchezfortún, 1996; Caldwell et al., 2003; Zhu et al., 2017). and Sa 3.5. O-MWCNTs attachment and surface damages Interactions of MWCNTs with A. salina can be external, such as attachment, which cause direct damage to the body surface.
Mesari c et al. (2015) studied the effects of carbon-based nanomaterials on A. salina. They demonstrated that MWCNTs extensively attached onto the gill and body surface, causing gill branches to fuse together. In addition, they also reported that high surface adsorption properties were responsible for mortality, swimming inhibition, and biochemical responses in A. salina (Mesaric et al., 2015). In the study, attachment of O-MWCNTs and surface damage to A. salina were checked under a SEM. Body surfaces of instar I (Fig. 4A), II (Fig. 4B) and III (Fig. 4C) larvae in the controls were clean and undamaged. After exposure, O-MWCNTs attached onto the cysts (Fig. 4D), gill (Fig. 4E) and body surface (Fig. 4F) of A. salina. The attachment may be responsible for the mortality and swimming inhibition, as demonstrated by Mesari c et al. (2015). Some irreversible damages to the lipid membranes were induced by the attachment, such as created “holes” in the labrum (Fig. 4G), abdomen (Fig. 4H) and notum (Fig. 4I) regions, causing the surface to wither. Decrease of body length may be due to the damages and abnormal segmentation processes (Go et al., 1990). 3.6. Biochemical responses ROS formation seems to be a key event of the toxic responses following nanomaterials exposure, and the imbalance between ROS formation and T-AOC results in oxidative stress. MDA is derived from lipid peroxidation, and has been widely used as an indicator of oxidative damages to membranes and oxidative stress (Ates et al., 2013). Antioxidant enzymes (such as CAT, SOD and GPx) catalyze the decomposition of ROS and prevent organisms from adverse
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Fig. 4. SEM images of instar I (A), II (B) and III larvae (C) in the controls. Attachment of O-MWCNTs (red arrows) onto the cysts (D), gill (E) and body surface (F) of A. salina. Damages (black arrows) to the labrum (G), abdomen (H) and notum (I) regions of A. salina. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
effects of oxidative stress. In the study, biomarkers of oxidative stress (ROS, MDA, T-AOC,
CAT, SOD and GPx) were measured to elucidate the toxic mechanism. As shown in Fig. 5, all the biomarkers were substantially
Fig. 5. ROS (A), MDA content (B), T-AOC (C) and the changes in CAT (D), SOD (E) and GPx (F) activities in A. salina larvae (instar I, II and III) following exposure to different concentrations of O-MWCNTs. Values are presented as mean ± SD.
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increased following O-MWCNTs exposure, indicating that the toxic effects were related to oxidative stress. Elevation of MDA contents verified the surface damages to A. salina. Increases of CAT, SOD and GPx activities were due to responses to the ROS. Similar result was reported by other studies (Mesari c et al., 2015; Wei et al., 2010). Interestingly, the GPx activity showed a gradual increase and then followed by a decrease. Increase of GPx activity may be due to a response to the superoxide, high GPx activity can efficiently degrade superoxide. The decrease may be due to the elevation of ROS level, high concentration of ROS is able to inhibit the antioxidant enzymes activities. Petersen et al. (2014) reviewed the potential artifacts and misinterpretations that can occur in the nanomaterial ecotoxicity testing and provided ways to avoid or minimize them. They demonstrated that interferences of nanomaterials with assays, residual impurities in nanomaterials and settling of nanomaterials during exposure period have potential to induce misinterpretations of toxicity results (Petersen et al., 2014). Nanomaterials can interfere with assays directly, such as effects on reagent stability, fluorescence and absorbance readings independently of organismreagent interactions. Besides, indirect interference can be induced whereby the effects on organism-reagent interactions, for example adsorption of reagents to nanomaterials surfaces inhibits their transport across the cell membrane (Horst et al., 2013; Tong et al., 2013). In this study, both direct and indirect interferences of OMWCNTs with the biochemical responses were investigated (as described in the Supplementary material). For direct interference, no fluorescence/absorbance signal was detected after O-MWCNTs reaction with reagents without A. salina (data not shown). For indirect interference, no significant differences were induced compared with the controls (Fig. S5). These results indicated that the biochemical assays did not suffer from direct or indirect interference with O-MWCNTs. As shown in Fig. S6, the residual Fe3þ showed no obvious influences (p > 0.05) on biochemical responses, suggesting that under conditions described here, the residual impurities did not lead to misinterpretations. Moreover, these interferences were subtracted from the biochemical responses to make the results reliable. Settling of O-MWCNTs with instar I, II and III larvae (Fig. S7) showed a similar behavior as that in acute toxicity test (Fig. S4), indicating that the volume of exposure solution has no obvious effects (p > 0.05) on the O-MWCNTs settling velocity. Similar results were reported in other studies (Guo et al., 2013; Petersen et al., 2009). 3.7. Uptake, excretion and distribution of MWCNTs Interactions of MWCNTs with A. salina also can be internal, such as uptake of MWCNTs, which cause damages to body tissues. A. salina is a non-selective filter-feeder as daphnia that can ingest particles smaller than 50 mm (Ates et al., 2013). Several studies reported that nanomaterials can be ingested and deposited inside the guts of A. salina (Ates et al., 2013; Mesari c et al., 2015). In this study, the uptake of MWCNTs by A. salina was checked, and representative images are shown in Fig. 6. Gut of newly hatched larva in the control (Fig. 6A) was hazy due to the digestive tract is not fully formed. After exposure for 12 h, O-MWCNTs gradually accumulated in the gut (Fig. 6B). The guts were almost entirely filled with O-MWCNTs at 24 (Fig. 6C) and 48 h (Fig. 6D), manifested by a dark line inside the guts. After transferred in FNSW, the accumulated O-MWCNTs were excreted by A. salina (Fig. 6E). However, the accumulated O-MWCNTs were not completely excreted at 72 h (exposure for 24 h in FNSW; Fig. 6F). For exposure to FITC-MWCNTs, A. salina in the control showed faint selffluorescent in the head (Fig. 6G). After exposure, fluorescence intensity increased markedly over time until 48 h (Fig. 6HeJ).
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Fig. 6. Uptake and excretion of O-MWCNTs/FITC-MWCNTs by A. salina. (A) Gut of newly hatched larva in the control is hazy due to the digestive tract is not fully formed. (BeD) After exposure, O-MWCNTs are gradually accumulated in the guts. (E) The accumulated O-MWCNTs are excreted by A. salina following transfer to FNSW. (F) OMWCNTs are not completely excreted at 72 h (exposure for 24 h in FNSW). (G) A. salina in the control show faint self-fluorescent in the head. (HeJ) After exposure to FITCMWCNTs, fluorescence intensity increases markedly over time until 48 h. After transfer to FNSW, the accumulated FITC-MWCNTs are excreted by A. salina (K), and a small quantity of FITC-MWCNTs is residual in A. salina (L). Scale bars: 300 mm.
Interestingly, as shown in Fig. 6H, the gut was half-baked. That was because that the mouth and anus of larvae are not yet completely opened, and the digestive tract is not fully formed at the early stage of instar I (Sorgeloos et al., 1979). After transferred in FNSW, similar excretion (Fig. 6K) and residual (Fig. 6L) were observed as OMWCNTs. The results indicated that MWCNTs can move in and out of A. salina in accordance to the external concentration. Besides, the accumulated O-MWCNTs were not completely excreted at 72 h most likely due to the formation of larger aggregates inside the guts (Ates et al., 2015). Similar results were reported in previous studies (Edgington et al., 2013; Petersen et al., 2009, 2010). For example, Petersen et al. (2009) investigated the accumulation and elimination of MWCNTs by Daphnia magna, and demonstrated that the accumulated MWCNTs were not entirely eliminated from the daphnia body (Petersen et al., 2009). A TEM was used to check the distribution of O-MWCNTs in
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Fig. 7. Distribution of O-MWCNTs (red arrows) in A. salina checked using a TEM. (AeC) O-MWCNTs are attached on the cortical layer (cl) of cyst. Localization of O-MWCNTs in phagocyte (p; D and E), lipid vesicle (li; D and F) and intestine (in; J-L). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
A. salina. As shown in Fig. 7AeC, O-MWCNTs attached on but not entered into the cysts, indicating that cortical layer was an effective protective barrier to O-MWCNTs. For larvae, O-MWCNTs were clearly visible within the phagocyte (p; Fig. 7D and E), lipid vesicle (li; Fig. 7D and F) and intestine (in; Fig. 7GeI). In our previous study, we investigated the ingestion and distribution of GO in A. salina. Result showed that GO was visible within the primary body cavity, yolk and intestine, and no GO was found in cells (Zhu et al., 2017). The difference may be caused by the shapes of GO and MWCNTs. GO was composed of sheet monolayers, while MWCNTs were fibrous that enable them penetrate through the membrane more easily (Zhu et al., 2016).
3.8. Uptake kinetics Several methods have been performed to quantify CNTs in biological tissues, and one of selection standards for the methods is the limits of detection (LOD) (Petersen et al., 2016). Published studies verified that the raman spectroscopy is a sensitive, nondestructive and reliable method for quantification of CNTs (Bertulli et al., 2013; Salzmann et al., 2007). The method has a low detection limit in biological tissues (0.1e1 mg/kg) (Petersen et al., 2016). Moreover, it has been well performed in our privious study (Zhu et al., 2016). In the study, O-MWCNTs contents were quantificationally measured based on the dry weight of A. salina using raman spectroscopy. As shown in Fig. 8, the average O-MWCNTs contents were ranged from 5.5 to 28.1 mg/g. The contents were gradually increased from 1 to 48 h followed by rapidly decreased from 48 to 72 h. During the first 12 h, the content was showed a slower increase compared with that from 12 to 48 h. That may be due to the mouth of instar I larvae is not yet completely opened, and the gut is not fully formed. Therefore, a slower uptake was observed. Decrease from 48 to 72 h is probably due to the settling of OMWCNTs (Guo et al., 2013; Hu et al., 2012; Petersen et al., 2009). As shown in Fig. S8, the O-MWCNTs concentration in supernatants decreased with the settling time increased, indicating that most OMWCNTs settled over time. The decrease in O-MWCNTs accumulation could cause by the reduction of their concentration in suspensions, which may lead to the release of accumulated O-
Fig. 8. O-MWCNTs contents in A. salina at different time points. Values are presented as mean ± SD.
MWCNTs. Similar result was reported in other studies (Guo et al., 2013; Hu et al., 2012; Petersen et al., 2009). For example, Petersen et al. (2009) investigated the uptake and depuration of MWCNTs by Daphnia magna, and demonstrated that the decrease in body burdens was likely as a result of MWCNTs settling. The maximum content was reached at 48 h (instar II). As mentioned before, instar II larvae showed the greatest sensitivity to OMWCNTs. Therefore, the high content may be responsible for the strong toxic responses. 4. Conclusion In summary, acute exposure of A. salina to O-MWCNTs results in significant effects on hatchability, mortality, and ethological, morphological and biochemical parameters. The effects were accounted for O-MWCNTs rather than metal catalyst impurities, and were mediated by oxidative stress. Instar II showed the greatest sensitivity to O-MWCNTs, indicating that it would be a suitable candidate for nanotoxicological assessment. O-MWCNTs were ingested, accumulated and excreted by A. salina, and distributed in phagocyte, lipid vesicle and intestine. The maximum O-MWCNTs content (28.1 mg/g) was reached at 48 h. Our study so far revealed the short-term effects of O-MWCNTs on A. salina. For safe and commercial purposes, chronic exposure with environmentally realistic concentrations is necessary. Acknowledgements This work is supported by the National Natural Science Foundation of China (Program No. 31602204) and Special Funds for Talents in Northwest A&F University to B. Zhu (Program No. Z111021510). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2017.07.020. References Ates, M., Daniels, J., Arslan, Z., Farah, I.O., Rivera, H.F., 2013. Comparative evaluation of impact of Zn and ZnO nanoparticles on brine shrimp (Artemia salina) larvae:
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Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Developmental toxicity of oxidized multi-walled carbon nanotubes on Artemia salina cysts and larvae: Uptake, accumulation, excretion and toxic responses* Song Zhu, Fei Luo, Xiao Tu, Wei-Chao Chen, Bin Zhu, Gao-Xue Wang* College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 April 2017 Received in revised form 25 June 2017 Accepted 7 July 2017
Using Artemia salina (A. salina) cysts (capsulated and decapsulated) and larvae [instar I (0e24 h), II (24 e48 h) and III (48e72 h)] as experimental models, developmental toxicity of oxidized multi-walled carbon nanotubes (O-MWCNTs) was evaluated. Results revealed that hatchability of capsulated and decapsulated cysts was significantly decreased (p < 0.01) following exposure to 600 mg/L for 36 h. Mortality rates were 33.8, 55.7 and 40.7% for instar I, II and III larvae in 600 mg/L. The EC50 values for swimming inhibition of instar I, II and III were 535, 385 and 472 mg/L, respectively. Instar II showed the greatest sensitivity to O-MWCNTs, and followed by instar III, instar I, decapsulated cysts and capsulated cysts. Effects on hatchability, mortality and swimming were accounted for O-MWCNTs rather than metal catalyst impurities. Body length was decreased with the concentrations increased from 0 to 600 mg/L. OMWCNTs attached onto the cysts, gill and body surface, resulting in irreversible damages. Reactive oxygen species, malondialdehyde content, total antioxidant capacity and antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) activities were increased following exposure, indicating that the effects were related to oxidative stress. O-MWCNTs were ingested and distributed in phagocyte, lipid vesicle and intestine. Most of the accumulated O-MWCNTs were excreted by A. salina at 72 h, but some still remained in the organism. Data of uptake kinetics showed that O-MWCNTs contents in A. salina were gradually increased from 1 to 48 h and followed by rapidly decreased from 48 to 72 h with a range from 5.5 to 28.1 mg/g. These results so far indicate that O-MWCNTs have the potential to affect aquatic organisms when released into the marine ecosystems. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Carbon nanotube Developmental toxicity Brine shrimp Oxidative stress Uptake
1. Introduction Carbon nanotubes (CNTs) possess exceptional electrical, chemical, and physical properties, which enable them utilized in various applications, such as manufacturing (De Volder et al., 2013), medicine (Heister et al., 2013) and other industries (Baughman et al., 2002; Eatemadi et al., 2014). Moreover, advances in CNTs synthesis, purification and chemical modification make them suitable for more commercial applications. In this regard, multi-walled carbon nanotubes (MWCNTs) are the largest product segment, accounting for majority market volume. However, along with the rapid increase of related products and applications, CNTs are inevitably
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This paper has been recommended for acceptance by Baoshan Xing. * Corresponding author. Northwest A&F University, Xinong Road 22nd, Yangling, Shaanxi 712100, China. E-mail address: [email protected] (G.-X. Wang). http://dx.doi.org/10.1016/j.envpol.2017.07.020 0269-7491/© 2017 Elsevier Ltd. All rights reserved.
released into the aquatic environment from: 1) the direct entry to waterbodies from bioremediation; 2) rainwater and runoff from contaminated air and soils; 3) aerial and tyres deposition; 4) emissions from wastewater treatment plants (Boxall et al., 2007; Yang et al., 2008). Eventually, most of the CNTs enter into marine ecosystems (Klaine et al., 2008; Wei et al., 2010). Sun et al. (2016) predicted the environmental emissions of engineered nanomaterials using dynamic probabilistic modeling, and suggested that the environmental concentration of CNTs was at the “mg/kg” level. In addition, most of the CNTs-related products are “durable” products (such as tyres and other polymer composites) that have almost no CNTs release during use. A mass of CNTs will release when the products come to the disposal phase, resulting in a higher CNTs concentration in the future (Sun et al., 2016). Therefore, it is imperative to investigate the potential hazards posed by CNTs at high concentration prior to the wide use of them. Such knowledge will be useful in utilization of CNTs and managing risk in the future.
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In recent years, using aquatic invertebrates as models to assess the potential toxicity of nanomaterials has become prevalent (Ates et al., 2013; Petersen et al., 2010; Zhu et al., 2017). As an invertebrate zooplankton found in various seawater systems from lakes to oceans, Artemia salina (A. salina) plays a key role in the energy flow of food chain (Sorgeloos et al., 1986). The intrinsic characteristics and physiological features of A. salina make it as a suitable model for toxicology testing (Nunes et al., 2006; Rajabi et al., 2015). For example, its cysts are commercially available, and have been widely used in toxicology tests (Caldwell et al., 2003; Rotini et al., 2015). Moreover, many stages are divided along the development process of A. salina, and larvae in a uniform physiological condition can be hatched synchronously (Persoone et al., 1989; Sorgeloos et al., 1979). Recently, several studies have investigated the effects of nanomaterials on A. salina (Gambardella et al., 2014; Mesaric et al., 2015; Zhu et al., 2017). It is currently accepted that one of the major mechanisms for toxicity of nanomaterials is oxidative stress (Ates et al., 2013; Liu et al., 2012; Zhu et al., 2017). For example, Ates et al. (2013) investigated the impacts of Zn and ZnO nanoparticles on A. salina. They demonstrated that the nanoparticles showed significant toxicity to A. salina after exposure for 96 h, and the effects were related to oxidative stress. Furthermore, toxicity of graphene oxide (GO) to A. salina was evaluated in our previous study. Results showed that GO induced significant changes in hatchability, mortality, and morphological, ethological and physiological parameters (Zhu et al., 2017). Accumulation and depuration behaviors of CNTs in organisms are critical factors for risk assessment, which impact the overall toxicity of CNTs (Edgington et al., 2013; Guo et al., 2013; Petersen et al., 2009, 2010). It was reported that MWCNTs could be significantly accumulated in the gut of Daphnia magna, but the accumulated MWCNTs were not entirely eliminated from the daphnia body (Petersen et al., 2009, 2010). Besides, we investigated the uptake and toxic effects of MWCNTs on Saccharomyces cerevisiae, and showed that MWCNTs were clearly visible in lysosome, vacuole, endosome, mitochondria, multivesicular body and perinuclear region (Zhu et al., 2016). Mesari c et al. (2015) evaluated the effects of three different carbon-based nanomaterials on A. salina larvae following exposure for 48 h. They suggested that the nanomaterials were ingested and concentrated in the gut, and attached onto the body surface of A. salina (Mesari c et al., 2015). To date, information about the accumulation, depuration and developmental toxicity of MWCNTs on A. salina is limited. In the study, toxicity of oxidized MWCNTs (O-MWCNTs) to A. salina cysts (capsulated and decapsulated) and larvae (instar I, II and III) were evaluated. Based on previous data, it was hypothesized that: 1) hatchability, mortality, and ethological, morphological and biochemical parameters would be significantly changed; 2) effects would be accounted for O-MWCNTs rather than metal catalyst impurities, and were mediated by oxidative stress; 3) O-MWCNTs would be ingested, accumulated and excreted by A. salina. The study contributes to better understandings of the MWCNTs toxicity, and lay foundations for their future exploitation and application. 2. Materials and methods 2.1. Preparation and characterization of MWCNTs MWCNTs were purchased from Chengdu Organic Chemicals Co., Ltd., Chinese Academy of Sciences (Chengdu, China), and the structural parameters are listed in Table S1. They were oxidized (OMWCNTs) and labeled with fluorescein isothiocyanate (FITCMWCNTs) according to the previous study (Zhu et al., 2016) (as described in the Supplementary material). To prepare the suspensions with nominal concentrations of 25, 50, 100, 200, 400 and
600 mg/L, O-MWCNTs were weighed on aluminum foil and placed in 1 L beakers containing 900 mL of filtered natural seawater (FNSW; 30‰ m/v; pH 8.6). The beakers were placed in an ice bath and then probe sonicated with an ultrasonic processor (Scientz-IID, China). The suspensions were sonicated for 1 h at 100 W using a 50% on/off cycle and left overnight at room temperature, and sonicated again. O-MWCNTs were characterized by scanning electron microscope (SEM, Hitachi S-4800, Japan) and transmission electron microscope (TEM, JEM1200EX, Japan) with accelerating voltages of 15 kV and 100 kV, respectively. To estimate the hydrodynamic size distribution of O-MWCNTs in FNSW, a dynamic light scattering (DLS, Brookhaven BI200SM, USA) was used. An X-ray photoelectron spectroscopy (XPS; PHI-5600, Russia) was used to analyze elemental compositions and chemical states. Raman spectra were recorded on a HR800 spectrophotometer (Longjumeau Cedex, France) with an excitation wavelength of 785 nm. The contents of residual metal catalyst impurities in pristine and oxidized MWCNTs were measured using ICP-MS (Thermo Elemental X7). 2.2. Model organism Commercially available dehydrated cysts (Tianjin, China) of A. salina were used and kept at 4 C until required. Decapsulated cysts and instar I, II and III larvae were obtained as described previously (Zhu et al., 2017) (as described in the Supplementary material). 2.3. Hatching assay Cysts were treated with O-MWCNTs suspensions (0, 25, 50, 100, 200, 400 and 600 mg/L) to study the effects on hatchability. In order to measure the contribution of metal catalyst impurities to the effect, cysts were cultivated in FeCl3 solutions. The concentrations of Fe3þ were 0, 0.022, 0.044, 0.088, 0.176, 0.352, 0.528 mg/L, as the same amount of Fe3þ as 0, 25, 50, 100, 200, 400, 600 mg/L MWCNTs suspensions, respectively (Table S2). Hatching tests were carried out according to the previous study (Zhu et al., 2017). A parallel set of experiments was performed to determine the OMWCNTs settling behavior with A. salina cysts. Briefly, triplicate suspensions (0.5 mL) were sampled after exposure for 0, 6, 12, 18 and 24 h from each treatment. The samples were pipetted into a square quartz groove (side length: 5 mm; height: 1 mm) and then dried in vacuum at 80 C. Contents of O-MWCNTs were quantitatively assessed using a HR800 Raman spectrophotometer (Longjumeau Cedex, France) with an excitation wavelength of 785 nm, and calculated by a standard curve. 2.4. Acute toxicity test Acute toxicity test was performed by adding 10 larvae (instar I, II and III) to each well of 24-well plates. Each well contained 1 mL of O-MWCNTs suspensions (0, 25, 50, 100, 200, 400 and 600 mg/L) or FeCl3 solutions. All plates were incubated with shaking at 28 C under a 16:8 h light/dark management. Larvae were not fed during the test. After exposure for 24 h, the numbers of dead larvae were counted using a microscope (Olympus Optical Co., Ltd., Tokyo, Japan). All of the tests were carried out in octuplicate. For each treatment, instar I, II and III larvae (approximately 1000) were also randomly added into beakers containing 100 mL OMWCNTs suspensions or FeCl3 solutions, and cultured at 28 C with shaking. After exposure for 24 h, samples were randomly took and then immediately performed morphological and behavioral analysis, and ROS measurement. Samples for SEM and TEM analysis were fixed in 2.5% glutaraldehyde at 4 C. Specimens for MDA
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content, T-AOC and enzyme activity measurements were frozen in liquid nitrogen and stored at 80 C. The settling behavior of OMWCNTs with A. salina was quantified during the exposure period. 2.5. Morphological and behavioral analysis After exposure for 24 h, 20 surviving larvae were randomly selected from each treatment for morphological and behavioral analysis (Zhu et al., 2017) (as described in the Supplementary material). 2.6. ROS, MDA content, T-AOC and antioxidant enzymes activities Measurements of ROS, MDA content, T-AOC and antioxidant enzymes activities were performed as described previously (Zhu et al., 2017). Moreover, the potential interferences of O-MWCNTs with the measurements were investigated (as described in the Supplementary material). 2.7. Uptake and excretion of MWCNTs The newly hatched larvae were treated with 50 mg/L OMWCNTs/FITC-MWCNTs for 48 h and then transferred in FNSW to excrete for another 24 h. During the exposure, 5 larvae were randomly selected at 0, 12, 24, 48, 60 and 72 h, and thoroughly washed with FNSW. Observation was performed on a microscope (Leica, Germany) and fluorescence stereomicroscope (Leica MZFL III, Germany), and images were taken by a digital camera (Nikon, Japan). The FITC fluorescence was visualized by using excitation and emission at 485 and 530 nm, respectively. Meanwhile, the uptake and distribution of O-MWCNTs in larvae were also observed by TEM (JEOL, Tokyo, Japan). Larvae TEM samples were prepared according to Bartolomaeus et al. (2009). 2.8. Uptake kinetics Briefly, the newly hatched larvae were treated with 50 mg/L OMWCNTs for 72 h. Then, larvae were sampled at 1, 3, 5, 7, 9, 12, 15, 18, 24, 30, 36, 48, 60 and 72 h, and thoroughly washed with distilled water. Larvae were homogenized using a homogenizer. The homogenates were analyzed using the HR800 Raman spectrophotometer as described in previous section on hatching assay. After exposure, O-MWCNTs suspensions (0.5 mL) were also sampled at each sampling time to check the settling behavior. 2.9. Statistical analysis All of the treatments were carried out at least three times, and the data were expressed as mean ± standard deviation (SD). The EC50 values were calculated using the Probit method. The SPSS Version 11.0 software package (SPSS Inc., Chicago, IL) was used to perform statistical analysis. Significant differences between the controls and treatments were examined using one-way ANOVA followed by Tukey's test. Significance was accepted at p < 0.05 and extremely significant at p < 0.01. 3. Results and discussion 3.1. Characterization of MWCNTs Data showed that some parameters have considerable impact on the toxicity of MWCNTs, such as purity, size, agglomeration state and surface chemistry (Belyanskaya et al., 2009; Johnston et al., 2010; Wang et al., 2009). In this study, the related parameters were measured. Fig. 1A and B show the SEM and TEM images of O-
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MWCNTs, indicating that they were fibrous with varying lengths. According to statistical analysis of the TEM images, the length of MWCNTs was ranged from 25 to 575 nm (average length: 102 nm; Fig. 1C), and shorter than the pristine MWCNTs (between 10 and 30 mm; Table S1). DLS data show that the hydrodynamic size of OMWCNTs was ranged from 117 nm to 121 mm with a mean size of 42 mm (Fig. 1D). The size is obviously larger than that estimated by TEM, indicating that O-MWCNTs were rapidly aggregated in the FNSW. Aggregation is inevitable due to the hydration and reduction of electrostatic repulsion in the aqueous solution (Johnston et al., 2010). Nevertheless, the DLS data cannot reveal the exact size of O-MWCNTs in FNSW due to the anisotropic and fibrous morphology. Fig. 1E shows XPS spectra of pristine and oxidized MWCNTs. The photoelectron peaks reveal that pristine MWCNTs surface consisted mainly of carbon (284 eV), as well as small amounts of oxygen (532 eV), iron [711 (Fe 2p1/2) and 725 (Fe 2p3/2) eV], cobalt (782 eV), nickel (850 eV), zinc (1020 eV) and magnesium (1305 eV). For the oxidized MWCNTs, only the peaks of carbon, oxygen and iron can be identified. Raman spectra of pristine and oxidized MWCNTs are showed in Fig. 1F. Typical G band (around 1580 cm1) and D band (around 1303 cm1) are identified from the spectra. Intensity ratio between of D band (ID) and G band (IG) can be used to evaluate the defect density of the MWCNTs walls, it is increased with the defect density increased (Osswald et al., 2007). The ID/IG ratios of pristine and oxidized MWCNTs were 0.65 and 1.35, indicating that the defect density of MWCNTs walls was increased after oxidation. Furthermore, some peaks were observed between 100 and 400 cm1 from the spectrum of pristine MWCNTs which were not identified from the spectrum of O-MWCNTs. Santangelo et al. (2006) demonstrated that peaks below 500 cm1 are related to the presence of iron catalyst in MWCNTs. ICP-MS data showed that some iron catalysts (Fe, Co, Ni, Zn and Mg) were residual in the pristine MWCNTs, and only Fe was found in the OMWCNTs (Table S2). The result consisted with the XPS analysis. All the related parameters indicated that the oxidation of MWCNTs was sufficient and the impurities were obviously reduced. 3.2. Effects of O-MWCNTs and impurities on hatchability As shown in Fig. 2A and B, for both capsulated and decapsulated cysts at 12, 18, 24 and 36 h, the hatching rates were substantially decreased with the concentrations increased from 0 to 600 mg/L. Hatching rates were significantly decreased (p < 0.01) in 600 mg/L at 36 h for capsulated cysts and in 600 mg/L at 24 and 36 h for decapsulated cysts compared with the controls. Decapsulated cysts showed a higher sensibility to O-MWCNTs than capsulated cysts. Moreover, decapsulated cysts also showed a higher hatchability than capsulated cysts. Sorgeloos et al. (1986) demonstrated that the hatchability was improved by decapsulation due to a lower energy requirement to break out of decapsulated cysts. Several studies reported that the toxic effects of MWCNTs were related to the metal catalyst impurities (Haniu et al., 2010; Liu et al., 2012). In the study, effect of metal catalyst impurities on hatchability was evaluated. As shown in Fig. S1, no obvious influence (p > 0.05) was observed, indicating that the effect on hatchability is accounted for O-MWCNTs rather than impurities. O-MWCNTs settling behaviors with capsulated (Fig. S2A) and decapsulated cysts (Fig. S2B) were measured. Result showed that the initial O-MWCNTs concentrations were lower than the nominal concentrations. Moreover, the results also showed that concentrations of O-MWCNTs in supernatants were decreased with the settling time increased and related to the initial concentration. Substantial settling occurred during the first 24 h and relatively stable states were reached from 24 to 36 h. Ratios (CR/CI) between the residual O-MWCNTs concentrations (CR) and the initial O-
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Fig. 1. Characterization of MWCNTs. SEM (A) and TEM (B) images of O-MWCNTs. Size distributions of O-MWCNTs measured using TEM (C) and DLS (D). XPS (E) and Raman (F) spectra of pristine and oxidized MWCNTs.
MWCNTs concentrations (CI) for 600 mg/L at 36 h were 14.7% (with capsulated cysts) and 14.2% (with decapsulated cysts). Similar decreasing trends were also reported in other studies (Guo et al., 2013; Hu et al., 2012; Petersen et al., 2009). 3.3. Effects of O-MWCNTs and impurities on mortality As shown in Fig. 3A, for instar I, II and III larvae in the controls, mortality rates ranged from 2% to 5%, indicating that no lethal effects on larvae were induced without feeding even up to 72 h. Mean mortality rates in 600 mg/L were 33.8, 55.7 and 40.7% for instar I, II and III, respectively. Toxicity of GO to A. salina was assessed in our previous study (Zhu et al., 2017). The mean mortality rates were 47.3%, 74.4% and 69.9% for instar I, II and III following exposure to 600 mg/L, indicating that O-MWCNTs possess a lower toxicity file than GO. Mesari c et al. (2015) studied the effects of MWCNTs on
A. salina after exposure for 48 h, and demonstrated that no significant lethal effect was observed up to a concentration 100 mg/L. The disagreement may be induced by the shorter length of MWCNTs used in the study that could cause more severe toxicity (Johnston et al., 2010). As shown in Fig. S3A, no obvious influence (p > 0.05) of impurities on mortality was observed, indicating that the effect is accounted for O-MWCNTs rather than impurities. Settling behaviors of O-MWCNTs with instar I, II and III larvae are presented in Fig. S4. Concentrations of O-MWCNTs were decreased with the settling time increased, and substantial settling occurred during the first 18 h. CR/CI for 600 mg/L at 24 h were 14.8% (instar I), 15.5% (instar II) and 13.8% (instar III), which were smaller than that with cysts (~21%; Fig. S2). Presence of larvae would increase the settling of O-MWCNTs from solutions due to enhanced aggregation during passage through the organism guts.
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Fig. 2. Hatching rates of capsulated (A) and decapsulated (B) cysts exposed to different O-MWCNTs concentrations. Values are presented as mean ± SD.
Fig. 3. Mortality (A), swimming inhibition (SI; B) and body length (C) for instar I, II and III larvae exposure to different concentrations of O-MWCNTs. Values are presented as mean ± SD.
3.4. Swimming speed and body length As shown in Fig. 3B, swimming was inhibited in a concentrationedependent manner and decreased as much as 52.3, 69.3 and 57.7% in 600 mg/L for instar I, II and III, respectively. The EC50 values for swimming inhibition of instar I, II and III were 535, 385 and 472 mg/L, respectively. Body length (Fig. 3C) was significantly decreased (p < 0.01) in 600 mg/L for instar III. Effect of impurities on the swimming is showed in Fig. S3B. There was no obvious influence (p > 0.05) on swimming, indicating that the effect is accounted for O-MWCNTs rather than impurities. Effects of pollutants on different stages of A. salina have been investigated and verified that discrepant sensitivity in relation to stages was exhibited (Barahona and S anchezfortún, 1996; Caldwell et al., 2003; Zhu et al., 2017). Caldwell et al. (2003) demonstrated that hatching assay showed a lower sensitivity to algal extracts and short chain aldehydes compared with the mortality assays. Besides, we verified that instar II larvae showed the greatest sensitivity to GO in our previous study (Zhu et al., 2017). In this study, based on the results obtained above, it can be concluded that the sensitivity of A. salina to O-MWCNTs is in the order of instar II > instar III > instar I > decapsulated cysts > capsulated cysts. The conclusion is somewhat similar to the results of previous studies (Barahona nchezfortún, 1996; Caldwell et al., 2003; Zhu et al., 2017). and Sa 3.5. O-MWCNTs attachment and surface damages Interactions of MWCNTs with A. salina can be external, such as attachment, which cause direct damage to the body surface.
Mesari c et al. (2015) studied the effects of carbon-based nanomaterials on A. salina. They demonstrated that MWCNTs extensively attached onto the gill and body surface, causing gill branches to fuse together. In addition, they also reported that high surface adsorption properties were responsible for mortality, swimming inhibition, and biochemical responses in A. salina (Mesaric et al., 2015). In the study, attachment of O-MWCNTs and surface damage to A. salina were checked under a SEM. Body surfaces of instar I (Fig. 4A), II (Fig. 4B) and III (Fig. 4C) larvae in the controls were clean and undamaged. After exposure, O-MWCNTs attached onto the cysts (Fig. 4D), gill (Fig. 4E) and body surface (Fig. 4F) of A. salina. The attachment may be responsible for the mortality and swimming inhibition, as demonstrated by Mesari c et al. (2015). Some irreversible damages to the lipid membranes were induced by the attachment, such as created “holes” in the labrum (Fig. 4G), abdomen (Fig. 4H) and notum (Fig. 4I) regions, causing the surface to wither. Decrease of body length may be due to the damages and abnormal segmentation processes (Go et al., 1990). 3.6. Biochemical responses ROS formation seems to be a key event of the toxic responses following nanomaterials exposure, and the imbalance between ROS formation and T-AOC results in oxidative stress. MDA is derived from lipid peroxidation, and has been widely used as an indicator of oxidative damages to membranes and oxidative stress (Ates et al., 2013). Antioxidant enzymes (such as CAT, SOD and GPx) catalyze the decomposition of ROS and prevent organisms from adverse
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Fig. 4. SEM images of instar I (A), II (B) and III larvae (C) in the controls. Attachment of O-MWCNTs (red arrows) onto the cysts (D), gill (E) and body surface (F) of A. salina. Damages (black arrows) to the labrum (G), abdomen (H) and notum (I) regions of A. salina. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
effects of oxidative stress. In the study, biomarkers of oxidative stress (ROS, MDA, T-AOC,
CAT, SOD and GPx) were measured to elucidate the toxic mechanism. As shown in Fig. 5, all the biomarkers were substantially
Fig. 5. ROS (A), MDA content (B), T-AOC (C) and the changes in CAT (D), SOD (E) and GPx (F) activities in A. salina larvae (instar I, II and III) following exposure to different concentrations of O-MWCNTs. Values are presented as mean ± SD.
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increased following O-MWCNTs exposure, indicating that the toxic effects were related to oxidative stress. Elevation of MDA contents verified the surface damages to A. salina. Increases of CAT, SOD and GPx activities were due to responses to the ROS. Similar result was reported by other studies (Mesari c et al., 2015; Wei et al., 2010). Interestingly, the GPx activity showed a gradual increase and then followed by a decrease. Increase of GPx activity may be due to a response to the superoxide, high GPx activity can efficiently degrade superoxide. The decrease may be due to the elevation of ROS level, high concentration of ROS is able to inhibit the antioxidant enzymes activities. Petersen et al. (2014) reviewed the potential artifacts and misinterpretations that can occur in the nanomaterial ecotoxicity testing and provided ways to avoid or minimize them. They demonstrated that interferences of nanomaterials with assays, residual impurities in nanomaterials and settling of nanomaterials during exposure period have potential to induce misinterpretations of toxicity results (Petersen et al., 2014). Nanomaterials can interfere with assays directly, such as effects on reagent stability, fluorescence and absorbance readings independently of organismreagent interactions. Besides, indirect interference can be induced whereby the effects on organism-reagent interactions, for example adsorption of reagents to nanomaterials surfaces inhibits their transport across the cell membrane (Horst et al., 2013; Tong et al., 2013). In this study, both direct and indirect interferences of OMWCNTs with the biochemical responses were investigated (as described in the Supplementary material). For direct interference, no fluorescence/absorbance signal was detected after O-MWCNTs reaction with reagents without A. salina (data not shown). For indirect interference, no significant differences were induced compared with the controls (Fig. S5). These results indicated that the biochemical assays did not suffer from direct or indirect interference with O-MWCNTs. As shown in Fig. S6, the residual Fe3þ showed no obvious influences (p > 0.05) on biochemical responses, suggesting that under conditions described here, the residual impurities did not lead to misinterpretations. Moreover, these interferences were subtracted from the biochemical responses to make the results reliable. Settling of O-MWCNTs with instar I, II and III larvae (Fig. S7) showed a similar behavior as that in acute toxicity test (Fig. S4), indicating that the volume of exposure solution has no obvious effects (p > 0.05) on the O-MWCNTs settling velocity. Similar results were reported in other studies (Guo et al., 2013; Petersen et al., 2009). 3.7. Uptake, excretion and distribution of MWCNTs Interactions of MWCNTs with A. salina also can be internal, such as uptake of MWCNTs, which cause damages to body tissues. A. salina is a non-selective filter-feeder as daphnia that can ingest particles smaller than 50 mm (Ates et al., 2013). Several studies reported that nanomaterials can be ingested and deposited inside the guts of A. salina (Ates et al., 2013; Mesari c et al., 2015). In this study, the uptake of MWCNTs by A. salina was checked, and representative images are shown in Fig. 6. Gut of newly hatched larva in the control (Fig. 6A) was hazy due to the digestive tract is not fully formed. After exposure for 12 h, O-MWCNTs gradually accumulated in the gut (Fig. 6B). The guts were almost entirely filled with O-MWCNTs at 24 (Fig. 6C) and 48 h (Fig. 6D), manifested by a dark line inside the guts. After transferred in FNSW, the accumulated O-MWCNTs were excreted by A. salina (Fig. 6E). However, the accumulated O-MWCNTs were not completely excreted at 72 h (exposure for 24 h in FNSW; Fig. 6F). For exposure to FITC-MWCNTs, A. salina in the control showed faint selffluorescent in the head (Fig. 6G). After exposure, fluorescence intensity increased markedly over time until 48 h (Fig. 6HeJ).
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Fig. 6. Uptake and excretion of O-MWCNTs/FITC-MWCNTs by A. salina. (A) Gut of newly hatched larva in the control is hazy due to the digestive tract is not fully formed. (BeD) After exposure, O-MWCNTs are gradually accumulated in the guts. (E) The accumulated O-MWCNTs are excreted by A. salina following transfer to FNSW. (F) OMWCNTs are not completely excreted at 72 h (exposure for 24 h in FNSW). (G) A. salina in the control show faint self-fluorescent in the head. (HeJ) After exposure to FITCMWCNTs, fluorescence intensity increases markedly over time until 48 h. After transfer to FNSW, the accumulated FITC-MWCNTs are excreted by A. salina (K), and a small quantity of FITC-MWCNTs is residual in A. salina (L). Scale bars: 300 mm.
Interestingly, as shown in Fig. 6H, the gut was half-baked. That was because that the mouth and anus of larvae are not yet completely opened, and the digestive tract is not fully formed at the early stage of instar I (Sorgeloos et al., 1979). After transferred in FNSW, similar excretion (Fig. 6K) and residual (Fig. 6L) were observed as OMWCNTs. The results indicated that MWCNTs can move in and out of A. salina in accordance to the external concentration. Besides, the accumulated O-MWCNTs were not completely excreted at 72 h most likely due to the formation of larger aggregates inside the guts (Ates et al., 2015). Similar results were reported in previous studies (Edgington et al., 2013; Petersen et al., 2009, 2010). For example, Petersen et al. (2009) investigated the accumulation and elimination of MWCNTs by Daphnia magna, and demonstrated that the accumulated MWCNTs were not entirely eliminated from the daphnia body (Petersen et al., 2009). A TEM was used to check the distribution of O-MWCNTs in
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Fig. 7. Distribution of O-MWCNTs (red arrows) in A. salina checked using a TEM. (AeC) O-MWCNTs are attached on the cortical layer (cl) of cyst. Localization of O-MWCNTs in phagocyte (p; D and E), lipid vesicle (li; D and F) and intestine (in; J-L). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
A. salina. As shown in Fig. 7AeC, O-MWCNTs attached on but not entered into the cysts, indicating that cortical layer was an effective protective barrier to O-MWCNTs. For larvae, O-MWCNTs were clearly visible within the phagocyte (p; Fig. 7D and E), lipid vesicle (li; Fig. 7D and F) and intestine (in; Fig. 7GeI). In our previous study, we investigated the ingestion and distribution of GO in A. salina. Result showed that GO was visible within the primary body cavity, yolk and intestine, and no GO was found in cells (Zhu et al., 2017). The difference may be caused by the shapes of GO and MWCNTs. GO was composed of sheet monolayers, while MWCNTs were fibrous that enable them penetrate through the membrane more easily (Zhu et al., 2016).
3.8. Uptake kinetics Several methods have been performed to quantify CNTs in biological tissues, and one of selection standards for the methods is the limits of detection (LOD) (Petersen et al., 2016). Published studies verified that the raman spectroscopy is a sensitive, nondestructive and reliable method for quantification of CNTs (Bertulli et al., 2013; Salzmann et al., 2007). The method has a low detection limit in biological tissues (0.1e1 mg/kg) (Petersen et al., 2016). Moreover, it has been well performed in our privious study (Zhu et al., 2016). In the study, O-MWCNTs contents were quantificationally measured based on the dry weight of A. salina using raman spectroscopy. As shown in Fig. 8, the average O-MWCNTs contents were ranged from 5.5 to 28.1 mg/g. The contents were gradually increased from 1 to 48 h followed by rapidly decreased from 48 to 72 h. During the first 12 h, the content was showed a slower increase compared with that from 12 to 48 h. That may be due to the mouth of instar I larvae is not yet completely opened, and the gut is not fully formed. Therefore, a slower uptake was observed. Decrease from 48 to 72 h is probably due to the settling of OMWCNTs (Guo et al., 2013; Hu et al., 2012; Petersen et al., 2009). As shown in Fig. S8, the O-MWCNTs concentration in supernatants decreased with the settling time increased, indicating that most OMWCNTs settled over time. The decrease in O-MWCNTs accumulation could cause by the reduction of their concentration in suspensions, which may lead to the release of accumulated O-
Fig. 8. O-MWCNTs contents in A. salina at different time points. Values are presented as mean ± SD.
MWCNTs. Similar result was reported in other studies (Guo et al., 2013; Hu et al., 2012; Petersen et al., 2009). For example, Petersen et al. (2009) investigated the uptake and depuration of MWCNTs by Daphnia magna, and demonstrated that the decrease in body burdens was likely as a result of MWCNTs settling. The maximum content was reached at 48 h (instar II). As mentioned before, instar II larvae showed the greatest sensitivity to OMWCNTs. Therefore, the high content may be responsible for the strong toxic responses. 4. Conclusion In summary, acute exposure of A. salina to O-MWCNTs results in significant effects on hatchability, mortality, and ethological, morphological and biochemical parameters. The effects were accounted for O-MWCNTs rather than metal catalyst impurities, and were mediated by oxidative stress. Instar II showed the greatest sensitivity to O-MWCNTs, indicating that it would be a suitable candidate for nanotoxicological assessment. O-MWCNTs were ingested, accumulated and excreted by A. salina, and distributed in phagocyte, lipid vesicle and intestine. The maximum O-MWCNTs content (28.1 mg/g) was reached at 48 h. Our study so far revealed the short-term effects of O-MWCNTs on A. salina. For safe and commercial purposes, chronic exposure with environmentally realistic concentrations is necessary. Acknowledgements This work is supported by the National Natural Science Foundation of China (Program No. 31602204) and Special Funds for Talents in Northwest A&F University to B. Zhu (Program No. Z111021510). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2017.07.020. References Ates, M., Daniels, J., Arslan, Z., Farah, I.O., Rivera, H.F., 2013. Comparative evaluation of impact of Zn and ZnO nanoparticles on brine shrimp (Artemia salina) larvae:
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